universidad complutense de madrid - digital

UNIVERSIDAD COMPLUTENSE DE MADRID
Facultad de Ciencias Químicas
Dpto. de Bioquímica y Biología Molecular I
Marcadores de activación
alternativa de macrófagos:
DC-SIGN y FRβ
Tesis Doctoral
Elena Sierra Filardi
Madrid, 2010
UNIVERSIDAD COMPLUTENSE DE MADRID
Facultad de Ciencias Químicas
Dpto. de Bioquímica y Biología Molecular I
Marcadores de activación
alternativa de macrófagos:
DC-SIGN y FRβ
Este trabajo ha sido realizado por Elena Sierra Filardi para optar al grado de Doctor,
en el Centro de Investigaciones Biológicas de Madrid (CSIC), bajo la dirección del
Dr. Ángel Luis Corbí López.
Fdo. Dr. Ángel Luis Corbí López
A mis padres,
Rafael y María
“La felicidad humana generalmente no se logra con grandes golpes de suerte,
que pueden ocurrir pocas veces, sino con pequeñas cosas que ocurren todos los días”
Benjamin Franklin
Agradecimientos
Pues sí, aunque parece mentira, por fin ha llegado el momento... Y lo primero que quiero es dar las
gracias a todas las personas que de una forma u otra me han ayudado a llegar hasta aquí.
Ante todo quiero dar las gracias a Ángel, por darme la oportunidad de formar parte de su equipo, de
realizar este trabajo y por toda su ayuda. A Vicente y a Luis, que me ayudaron en mis primeros contactos con el
mundo de la biología. Y a María, porque gracias a ella pude empezar este trabajo.
Y siguiendo el orden cronológico, tengo que dar las gracias a las personas que me ayudaron en mis
comienzos en el laboratorio. Esther, gracias por recibirme cada mañana con una sonrisa, así todo era más fácil.
Diego, gran experto en DC-SIGN, gracias por tu ayuda y tu compañía en tantos y tantos experimentos. Amaya,
gracias por tu ayuda y colaboración durante todos estos años.
Ahora toca el turno de mis compañeras actuales, por las que creo que he sido capaz de llegar hasta el
final. Ángeles, con la que llevo desde el principio y a la que he visto doctorarse, casarse y ahora ser mamá,...
cómo pasa el tiempo! Gracias por tu ayuda. Laura, gracias por tu apoyo y por tus ánimos, y por esas risas, que
al final es con lo que hay que quedarse. Ya pronto te toca a tí! Noemí, gracias por todo, por aguantarme en los
buenos y malos momentos, has sido un gran apoyo para mí. Sonia, gracias por tu ayuda en esta última etapa,
por tu paciencia y tus valiosos consejos.
También quiero dar las gracias a las personas con las que he compartido menos tiempo, a los llegados
recientemente, Mateo, Concha y María, y a los que han ido pasando durante estos años por el laboratorio, Idoia
y Rocío. Carmen, gracias por tus consejos y por tu dulzura. Tilman, gracias por poner siempre una sonrisa a la
vida aún en los momentos más duros, por escucharme y confiar en mí, y por todos los buenos momentos de las
comidas. Aún se te echa de menos!
A los vecinos, las Cristinas, Ángela y Miguel, por sus consejos y críticas en los seminarios. José Luis,
muchas gracias por estar siempre dispuesto a escuchar, por tus ánimos y por hacerme ver la parte positiva de
las cosas. Pilar, gracias por todo tu apoyo, tus ánimos y tus gestos de cariño cuando más se necesitan.
Y ahora a los de siempre, a los viejos amigos del CIB. Luque, qué decir de tí. Muchas gracias por todo,
por escucharme, por intentar hacerme ver que las cosas no son tan malas y, sobre todo, gracias por hacerme
reír tanto. José, poco tiempo pero valioso. Gracias también por esos momentos tan divertidos. A mis grandes
compañeras de gymkana, Marta y Leo, gracias por estar ahí todos estos años. No sé si os prefiero de pitufos,
de pulpo, de pez amarillo, o mejor tal y como sois. Y gracias a todas las personas que en estos años han
compartido alguna hora de comida conmigo (sois tantos que se me olvidaría algún nombre). Gracias a todos.
Y ahora a los últimos y no menos importantes. A mis padres, Rafael y María, gracias a vosotros he llegado
hasta aquí, no sólo en lo profesional, sino también y más importante, en lo personal. Esto es una pequeña
forma de agradeceros todo lo que habéis y seguís haciendo por mí. A mi hermana Mª Luisa y a mis sobrinos
Alejandro y Aitana, por todas las sonrisas que me habéis regalado. Y a Luis, el gran sufridor de mis gruñidos.
Gracias por escucharme y por intentar aconsejarme siempre, y sobre todo, gracias por tu paciencia!
Y a todos lo que están o estarían orgullosos de que haya llegado hasta aquí.
Gracias
Índice
ABREVIATURAS ......................................................................................................................... 1
INTRODUCCIÓN ......................................................................................................................... 5
1. El sistema inmunitario y sus componentes celulares .............................................................. 7
2. Monocitos ................................................................................................................................. 8
3. Células dendríticas.................................................................................................................... 9
4. Macrófagos ............................................................................................................................. 11
4.1 Diferenciación de macrófagos ................................................................................... 12
4.1.1 Citoquinas implicadas ................................................................................... 12
4.1.1.1 Macrófagos generados en presencia de GM-CSF y M-CSF .......... 13
4.1.1.2 Fenotipo y función de macrófagos M1 y M2 ................................... 14
4.1.2 Tejido-especificidad ...................................................................................... 16
4.1.2.1 Macrófagos intestinales ................................................................... 16
4.1.2.2 Macrófagos peritoneales ................................................................. 16
4.2 Activación de macrófagos .......................................................................................... 17
4.2.1 Activación clásica vs. activación alternativa ................................................. 17
4.2.2 Características fenotípicas de macrófagos activados .................................. 19
4.2.3 Macrófagos asociados a tumores ................................................................. 21
4.3 Estudios de expresión génica en diferenciación y activación de macrófagos ........... 23
5. El receptor de patógenos DC-SIGN ....................................................................................... 24
5.1 Expresión y localización tisular .................................................................................. 25
5.2 Estructura y dominios funcionales ............................................................................. 25
5.3 Estructura génica, isoformas y polimorfismos ........................................................... 26
5.4 Función y señalización ............................................................................................... 28
OBJETIVOS ............................................................................................................................... 31
RESULTADOS ........................................................................................................................... 35
1. El receptor de folato β se expresa en macrófagos asociados a tumores y constituye un
marcador de macrófagos anti-inflamatorios/reguladores M2 ..................................................... 39
2. Activina A previene la adquisición de marcadores anti-inflamatorios/M2 y sesga la
secreción de citoquinas por los macrófagos .............................................................................. 53
3. Requerimientos estructurales para la multimerización del receptor de patógenos DCSIGN (CD209) en la superficie celular ....................................................................................... 79
4. Identificación de epítopos en la molécula de DC-SIGN ......................................................... 99
Índice
DISCUSIÓN .............................................................................................................................. 111
El receptor de folato β es un marcador de macrófagos anti-inflamatorios M2 y TAM, cuya
expresión es regulada por activina A ....................................................................................... 113
Requerimientos estructurales de DC-SIGN para su multimerización. Influencia de la
presencia de variantes con menor tamaño en la región del cuello .......................................... 125
Identificación epítopos en la molécula de DC-SIGN ................................................................. 130
CONCLUSIONES ..................................................................................................................... 133
BIBLIOGRAFÍA ........................................................................................................................ 137
ANEXO ..................................................................................................................................... 157
Abreviaturas
Abreviaturas
Gran parte de las abreviaturas y acrónimos empleados en esta Tesis Doctoral proceden del
inglés y como tal se han mantenido:
AAMØ Alternative activated macrophage
APC Antigen presenting cell
CAMØ Classical activated macrophage
CD Cluster of differentiation
CEA Carcinoembryonic antigen
CEACAM Carcinoembryonic antigen-related cell adhesion molecule
CLEC C-type lectin-like receptor
CLP Common lymphoid progenitor
CLR C-type lectin receptor
CMP Common myeloid progenitor
CRD Carbohydrate recognition domain
DAMP Danger-associated molecular patterns
DC Dendritic cell
DC-SIGN Dendritic cell-specific ICAM-3 grabbing nonintegrin
DC-SIGNR DC-SIGN related
DNA Deoxyribonucleic acid
ECD Extracellular domain
FR Folate receptor
FSH Follicle-stimulating hormone
G-CSF Granulocyte colony-stimulating factor
GM-CSF Granulocyte-macrophage colony-stimulating factor
HIV Human immunodeficiency virus
HSC Hematopoietic stem cells
HTLV-1 Human T-cell lymphotropic virus type 1
ICAM Intracellular adhesion molecule
IFN Interferon
IL Interleukin
iNOS Inducible nitric oxide synthase
ITIM Tyrosine-based inhibitory motif
a
b
x
y
Le , Le , Le , Le LewisA, LewisB, LewisX, LewisY
LPS Lipopolysaccharide
M-CSF Macrophage colony-stimulating factor
MDDC Monocyte-derived dendritic cell
MDM Monocyte-derived macrophage
MHC Major histocompatibility complex
MPS Mononuclear phagocyte system
3
Abreviaturas
MyD88 Myeloid differentiation primary response gene (88)
NFκB Nuclear factor-kappaB
NK Natural killer
NO Nitric oxide
NOD Nucleotide-binding oligomerization domain
PAMP Pathogen-associated molecular patterns
PBMC Peripheal blood-mononuclear cells
PPARγ Peroxisome proliferator-activated receptor gamma
PRR Pattern recognition receptor
RA Rheumatoid arthritis
RNA Ribonucleic acid
RNS Reactive nitrogen species
ROS Reactive oxygen species
SARS Severe acute respiratory syndrome
SCF Stem cell factor
TAM Tumor-associated macrophages
TCR T cell receptor
TGFβ Transforming growth factor-beta
Th T helper
TLR Toll-like receptor
TNFα Tumor necrosis factor-alpha
4
Introducción
Introducción
1. El sistema inmunitario y sus componentes celulares
La función esencial del sistema inmunitario es proteger al organismo de agentes infecciosos y
microorganismos presentes en el ambiente. Para ser eficaz, el sistema inmunitario debe detectar
una gran variedad de patógenos, y distinguirlos de las células y tejidos del propio organismo. En
vertebrados, en este sistema de defensa colaboran el sistema inmunitario innato y el sistema
inmunitario adaptativo [1].
El sistema inmunitario innato constituye la primera línea de defensa que limita la infección tras
la exposición a microorganismos, y proporciona una respuesta inmediata e inespecífica, pues
reconoce y responde a los patógenos de forma genérica y sin conferir inmunidad duradera contra
ellos [2]. Este sistema de defensa incluye componentes celulares (células epiteliales, células
dendríticas, macrófagos, neutrófilos y células NK), moléculas del sistema del complemento y
citoquinas. Sus células están equipadas con receptores de reconocimiento de patrones (PRR), que
reconocen patrones moleculares asociados a patógenos (PAMP) y señales endógenas asociadas a
daño tisular (DAMP). El sistema inmunitario innato es capaz de “activarse” únicamente frente a estas
“señales de peligro” detectadas por los PRR de forma específica [3]. Por contra, el sistema
inmunitario adaptativo genera respuestas antígeno-específicas y confiere memoria inmunológica tras
el primer contacto con el antígeno. La respuesta inmunitaria adaptativa está mediada por
componentes celulares (linfocitos T y B) y humorales (anticuerpos). Las células presentadoras de
antígeno (APC), y en especial células dendríticas y macrófagos, juegan un papel fundamental en la
conexión entre la inmunidad innata y la inmunidad adaptativa, ya que son las responsables de
procesar y presentar antígenos a los linfocitos T en el contexto de las moléculas del complejo de
histocompatibilidad (MHC) presentes en su superficie [1]. En consecuencia, el sistema de defensa
innato tiene como segunda función estimular y polarizar la respuesta inmunitaria adaptativa con
objeto de optimizar la eliminación del patógeno y minimizar los daños tisulares colaterales [4].
El sistema inmunitario de los vertebrados superiores está compuesto por gran variedad de
células funcionalmente diferentes que derivan de células madre hematopoyéticas (HSC) [5]. Las
HSC se renuevan a sí mismas y dan lugar a células progenitoras mieloides (CMP) y linfoides (CLP),
con potencial más limitado y que dan origen a granulocitos, monocitos, macrófagos, células
dendríticas y mastocitos [6], o linfocitos B y T, y células NK [7], respectivamente.
7
Introducción
2. Monocitos
Los monocitos se originan en la médula ósea a partir de un precursor mieloide y se liberan
posteriormente al torrente sanguíneo, donde constituyen un 10% de los leucocitos circulantes en
humanos [8]. Los monocitos de sangre periférica tienen una vida media relativamente corta (24-72
horas) [9], y contribuyen a la renovación de los macrófagos y células dendríticas tisulares [10]. Los
monocitos son heterogéneos en términos de morfología, marcadores de superficie y capacidad
fagocítica [11], y exhiben una elevada plasticidad en su proceso de diferenciación, que es tejido y/o
estímulo dependiente [12]. Como consecuencia, el fenotipo y las funciones efectoras de los
macrófagos residentes en los diferentes tejidos (macrófagos alveolares, células de Kupffer,
microglía, osteoclastos) varían considerablemente. La plasticidad del sistema de diferenciación
mieloide se refleja en la capacidad de “transdiferenciación” que exhiben los distintos tipos celulares
derivados de monocitos. Así, por ejemplo, los macrófagos pueden ser inducidos a adquirir
propiedades fenotípicas y funcionales de células dendríticas, mientras que las células dendríticas
derivadas de monocitos (MDDC) in vitro pierden sus funciones efectoras al retirar las citoquinas que
promueven su generación [13] (Figura 1). Dicha plasticidad también se refleja en procesos
fisiológicos como la resolución de la inflamación, donde la presencia de células apoptóticas facilita la
transformación de macrófagos citotóxicos/pro-inflamatorios en macrófagos promotores de
crecimiento/anti-inflamatorios encargados de reparar y limitar el daño tisular asociado al proceso
inflamatorio [14].
Monocito
GM-CSF
M-CSF
IL-4
GM-CSF + IL-4/IL-13/IFNα
IL-3 + IL-4
IL-6 / IL-10 / IFNγ
GM-CSF + IL-4
“cytokine remove” + M-CSF
Macrófago
Célula dendrítica
Figura 1.- Diferenciación in vitro de monocitos. Esquema ilustrativo de la plasticidad y la estímulodependencia de la diferenciación de monocitos de sangre periférica.
Las citoquinas son el estímulo crítico para que los monocitos progresen hacia cada una de sus
alternativas de diferenciación. De hecho, la primera citoquina con la que los monocitos entran en
8
Introducción
contacto determina su programa de diferenciación y su perfil de respuesta a otras citoquinas [15]. La
diferenciación in vitro de monocitos a macrófagos o células dendríticas es un ejemplo de dicha
dependencia (Figura 1). Las citoquinas comúnmente empleadas para generar MDDC in vitro son
GM-CSF e IL-4 [16-18], mientras que los macrófagos se diferencian en presencia de GM-CSF o MCSF [19]. En humanos, IL-4 favorece la diferenciación a células dendríticas e impide la generación
de macrófagos [16, 20], mientras que la presencia de IL-6 limita la generación de estas células y
promueve la diferenciación a macrófagos de manera dependiente de M-CSF [21]. Por otra parte, el
entorno celular y la presencia de estímulos externos también condiciona la diferenciación del
monocito inducida por citoquinas [10].
Por lo que se refiere a factores de transcripción, el factor PU.1 junto con C/EBPα, RUNX1 y AP1, es crítico en la diferenciación monocítica, ya que ratones deficientes en PU.1 carecen de linaje
mielomonocítico, lo que es debido fundamentalmente a su papel esencial en la regulación de los
genes que codifican los receptores de GM-CSF, M-CSF y G-CSF [22].
3. Células dendríticas
En 1973 Ralph M. Steinman y Zanvil A. Cohn describieron un tipo celular presente en los
órganos linfoides periféricos de ratón y al que denominaron “célula dendrítica” (DC) [23].
Posteriormente las DC fueron identificadas como un componente minoritario de las células
mononucleares de sangre periférica (PBMC) en humanos [24], y se caracterizaron por ser las
células estimuladoras más potentes en cultivos leucocitarios mixtos y en la activación de linfocitos
citotóxicos [25, 26]. En la actualidad, las DC se consideran centinelas del sistema inmunitario y APC
“profesionales”, ya que son las únicas APC eficaces en la activación de linfocitos T naive, debido a
su elevada expresión de moléculas de MHC, coestimuladoras y de adhesión en su superficie. Las
DC son capaces de presentar antígenos exógenos en el contexto de MHC-II y MHC-I (“crosspriming”), lo que justifica su capacidad de inducción de respuestas inmunitarias primarias [27].
En función de su linaje o de su estado de activación, las DC tienen la capacidad de iniciar una
respuesta inmunitaria o promover tolerancia [28]. Aún más, las DC determinan el tipo de respuesta
inmunitaria que se genera frente a un antígeno, pues son ellas quienes determinan la polarización
de los linfocitos Th naive hacia Th1 (productores de IFNγ y eficaces en la eliminación de patógenos
intracelulales), Th2 (productores de IL-4 y efectivos en la eliminación de patógenos extracelulares),
Th17 (productores de IL-17 e implicados en respuestas autoinmunes) o Treg (células T reguladoras
implicadas en procesos inmunosupresores) [29].
9
Introducción
Las DC humanas son una población heterogénea en cuanto a fenotipo, localización anatómica
y función, y se clasifican en dos grupos según su grado de parentesco con linajes celulares bien
establecidos: DC mieloides y DC plasmacitoides [30]. Las DC mieloides (CD11c+ CD123-) se
distribuyen prácticamente en todos los tejidos y se denominan de formas diversas dependiendo de
su localización tisular: células de Langerhans (en epidermis y mucosas), DC dérmicas, DC tímicas,
DC intersticiales (en casi la totalidad de órganos), etc. [29]. Las DC mieloides circulantes
representan sólamente un 0.5% de las PBMC totales [31]. Por el contrario, las DC plasmacitoides o
linfoides (CD11c- CD123+) proceden de progenitores distribuidos en el timo y en áreas T de los
órganos linfoides secundarios [32], y residen en nódulos linfáticos, bazo, timo, médula ósea y sangre
periférica [33]. Las DC plasmacitoides son importantes mediadores de la inmunidad anti-viral,
produciendo grandes cantidades de IFNα al ser estimuladas [34].
Sistema circulatorio
DIFERENCIACIÓN
DC de sangre periférica
DC inmaduras
Señales
de peligro
Progenitores de
médula ósea
Antígeno
soluble
Tej
ejido
Médula
ósea
MADURACIÓN
MIGRACIÓN
Célula T
efectora
Célula T naive
Nódulo linfático
Vía linfática
DC maduras
Figura 2.- “Ciclo vital” de las células dendríticas. Las células dendríticas se diferencian a partir de
progenitores de médula ósea que llegan a los tejidos a través del sistema circulatorio, y donde residen como
DC inmaduras hasta que reciben señales que promueven su migración y maduración. Las DC maduras migran
a los ganglios linfáticos, donde activan y polarizan a los linfocitos T naive hacia los diferentes tipos de células
Th.
10
Introducción
Las DC mieloides se originan a partir de progenitores de médula ósea, que generan
precursores circulantes cuya extravasación a los tejidos da lugar a las DC inmaduras residentes
(Figura 2). La elevada capacidad fagocítica de estas células les permite captar y procesar
contínuamente antígenos que son cargados en moléculas de MHC [30]. La detección de “señales de
peligro” a través de los receptores tipo Toll (TLR) y proteínas NOD hace que las DC “maduren” y
migren hacia los órganos linfoides secundarios. Durante ese trayecto, estas células disminuyen su
capacidad de captura y procesamiento de antígenos, y aumentan los niveles de expresión de
moléculas coestimulatorias y MHC en membrana. En las áreas T de los nódulos linfáticos, las DC
acaban interaccionando con linfocitos T que portan TCR específicos para los antígenos que las DC
capturaron en los tejidos de origen, iniciando así la respuesta inmunitaria adaptativa [35]. Las DC
maduras presentan antígenos a los linfocitos T CD8+ y CD4+, y estos últimos a su vez regulan a
otras células del sistema inmunitario, como células T citotóxicas CD8 y células B específicas de
antígeno, o células no específicas de antígeno como macrófagos, eosinófilos y células NK [36].
Como se ha comentado anteriormente, las DC están especializadas en la presentación de
antígeno a células T naive, y se diferencian de los macrófagos por su eficiente capacidad de
presentación de antígeno. Recientemente se ha planteado que las DC no constituyen una población
celular diferente de los macrófagos, ya que prodecen de un mismo precursor común, son sensibles
a los mismos factores de crecimiento, y no existen marcadores específicos ni funciones efectoras
únicas de las DC que justifiquen su distinción de los macrófagos [37].
4. Macrófagos
Metchnikoff, Premio Nobel de Fisiología y Medicina en el año 1908 por sus trabajos sobre el
sistema inmunitario, identificó células capaces de digerir partículas exógenas en el tubo digestivo de
las larvas de peces. A estas células las llamó “fagocitos”, y más tarde las definió como glóbulos
blancos integrantes de la primera línea de defensa contra las infecciones en los seres vivos [38]. El
término “macrófago” (MØ; del griego makros "grande" y phago "comer") fue asignado en 1924 por
Aschoff a un conjunto de células del sistema retículo-endotelial, que incluía monocitos, macrófagos,
histiocitos, fibroblastos, células endoteliales y células reticulares [39]. Posteriormente se reemplazó
este término por el de sistema fagocítico mononuclear (MPS), que comprende monoblastos y
promonocitos de médula ósea, monocitos de sangre periférica y macrófagos tisulares.
Los macrófagos juegan un papel crítico en el desarrollo de la respuesta inmunitaria, debido a
que actúan como primera barrera de defensa, al detectar y eliminar partículas “extrañas”
(microorganismos, macromoléculas tóxicas, células propias dañadas o muertas) mediante
11
Introducción
fagocitosis o secreción de enzimas, citoquinas o producción de especies reactivas de oxígeno
(ROS) y nitrógeno (RNS) [40]. Durante la respuesta inmunitaria adaptativa los macrófagos presentan
antígenos a los linfocitos T en el contexto de MHC-II y/o MHC-I, y colaboran con la respuesta
humoral en la eliminación de agentes extraños [41]. Además, los macrófagos tienen un papel
importante en procesos de reparación de heridas y resolución de la inflamación, promoviendo el
reclutamiento de otras células inflamatorias hacia los focos de inflamación, así como a remodelación
de matriz extracelular y angiogénesis. En consecuencia, el término “macrófago” agrupa una
multiplicidad de células cuya finalidad es el mantenimiento de la homeostasis y la integridad tisular
[12].
4.1 Diferenciación de macrófagos
Los macrófagos se originan a partir de HSC, y derivan en su mayoría de monocitos circulantes
que se extravasan a los tejidos por el influjo de citoquinas y quimioquinas [19]. A pesar de ello, un
pequeño porcentaje de macrófagos (aprox. 5%) derivan de la división local de fagocitos
mononucleares en los tejidos [42]. Como se comentó anteriormente, el fenotipo de los macrófagos
residentes en tejidos está determinado por el microambiente tisular, la matriz extracelular y los
productos de secreción y moléculas de superficie de las células próximas [8].
4.1.1 Citoquinas implicadas
Las principales citoquinas que determinan la supervivencia, diferenciación y quimiotaxis de los
macrófagos son GM-CSF, M-CSF e IL-3 [12] [43]. El M-CSF es sintetizado constitutivamente por
numerosos tipos celulares (macrófagos, células endoteliales, fibroblastos, osteoblastos, células del
estroma, etc.), y su concentración en suero oscila entre de 3-8 ng/ml [44]. Además, su producción es
inducida por la activación de células hematopoyéticas y fibroblastos con GM-CSF, TNFα [45], IL-1 e
IFNγ [46]. La síntesis de M-CSF es regulada de manera tejido-específica [43] y sus niveles son
elevados en estados de inmunosupresión (embarazo, tumores), siendo su papel importante en el
establecimiento de la tolerancia materna hacia el embrión [47]. A diferencia del GM-CSF, esta
citoquina juega un papel fundamental en el desarrollo mieloide, ya que ratones M-CSF-/- exhiben una
generación deficiente de macrófagos [48], mientras que los ratones GM-CSF-/- sólo muestran
alterada la maduración de macrófagos alveolares [49]. El receptor de M-CSF de alta afinidad (CSF1R, M-CSFR, c-fms, CD115) se expresa principalmente en células del linaje monocítico, como
monocitos, DC, macrófagos y sus precursores [43, 50].
12
Introducción
Por otro lado, el GM-CSF es producido por diferentes tipos celulares, incluyendo linfocitos T y
B, macrófagos, mastocitos, eosinófilos, neutrófilos y células endoteliales [43]. En condiciones
fisiológicas el GM-CSF se encuentra en suero a una concentración de 20-100 pg/ml y, aunque
puede ser producida constitutivamente por células tumorales, en la mayoría de los casos se requiere
activación de las células productoras [18]. El GM-CSF promueve viabilidad, proliferación y
maduración de precursores de neutrófilos, eosinófilos y macrófagos, y sus funciones dependen de
su concentración, ya que efectos en viabilidad celular requieren menores concentraciones que las
precisas para afectar a la proliferación celular [18]. Los efectos biológicos del GM-CSF están
mediados por el receptor de GM-CSF que, a diferencia del receptor homodimérico del M-CSF (MCSFR), está compuesto por una cadena α de unión a GM-CSF, y una cadena β necesaria para la
transducción de señales [51].
4.1.1.1 Macrófagos generados en presencia de GM-CSF y M-CSF
GM-CSF y M-CSF presentan una modulación cruzada de sus respectivas actividades
funcionales: mientras que el M-CSF aumenta la generación de macrófagos en presencia de bajos
niveles de GM-CSF [52], altas concentraciones de esta última impiden el desarrollo de macrófagos
mediado por M-CSF, debido a la acción inhibitoria de GM-CSF sobre la expresión de M-CSFR [53,
54]. Aunque los macrófagos humanos derivados de monocitos (MDM) diferenciados en presencia de
GM-CSF o M-CSF in vitro se consideran equivalentes a los macrófagos residentes en los tejidos en
condiciones homeostáticas [19], ambas citoquinas se usan indistíntamente en la generación in vitro
de MDM, dando lugar a poblaciones fenotípica y funcionalmente diferentes [19] (Figura 3). Así, en
presencia de GM-CSF se generan macrófagos, denominados M1, que producen citoquinas proinflamatorias (IL-23, IL-12, IL-1β, IL-6, TNFα) en respuesta a Mycobacterium y promueven
inmunidad de tipo Th1 (pro-Th1) [55, 56]. Por contra, los macrófagos inducidos por M-CSF o M2
secretan IL-10 en respuesta a estímulos externos, inhiben respuestas Th1, y se han implicado en la
inducción de tolerancia [55-57]. Los macrófagos M2 actúan como moduladores de autoinmunidad,
ya que inducen células Treg e inhiben la diferenciación de linfocitos Th1 y Th17 [58]. Por todo ello,
los macrófagos M1 y M2 juegan papeles opuestos durante la respuesta inmunitaria, y son
considerados como macrófagos pro- y anti-inflamatorios, respectivamente (Figura 3). Del mismo
modo, GM-CSF y M-CSF se emplean para la generación in vitro de macrófagos a partir de
precursores de médula ósea de ratón, y sus propiedades pro- y anti-inflamatorias se ajustan a las de
los macrófagos M1 y M2 derivados de monocitos humanos [59, 60].
13
Introducción
GM-CSF
M-CSF
Macrófago pro-inflamatorio
Macrófago anti-inflamatorio
(M1)
(M2)
IL-23, IL-12, IL-1β, IL-6, TNFα
IL-10
Th1
Figura 3.- Macrófagos diferenciados en presencia GM-CSF y M-CSF. Esquema ilustrativo de los
macrófagos generados en presencia de GM-CSF (M1 o pro-inflamatorios) o M-CSF (M2 o anti-inflamatorios) y
sus diferencias en la respuesta inmunitaria.
4.1.1.2 Fenotipo y función de macrófagos M1 y M2
Además de diferencias en la producción de citoquinas en respuesta a LPS o Mycobacterium,
los macrófagos generados en presencia de GM-CSF (M1) y M-CSF (M2) tienen características
fenotípicas diferentes (Tabla 1). Los macrófagos M2 presentan una morfología elongada en forma
de huso, mientras los macrófagos M1 son más redondeados [19]. Por otro lado, los macrófagos M2
presentan mayor expresión de CD14, M-CSFR y del receptor “scavenger” CD163, mientras los
macrófagos M1 expresan mayores niveles de HLA-DQ y HLA-DR [19, 56]. Respecto a la expresión
de PRR, ambos tipos de macrófagos expresan niveles similares de TLR2 y TLR4, y la expresión de
DC-SIGN es baja pero significativa en macrófagos M1 y mayor en macrófagos M2 [56].
Desde el punto de vista funcional, ambas poblaciones de macrófagos también se comportan de
forma diferente (Tabla 1). Los macrófagos M2 presentan mayor capacidad de fagocitosis mediada
por receptores de Fcγ [61], mayor actividad fungicida debida a la producción de ROS [62], y mayor
producción de H2O2 en respuesta a estímulos fagocíticos [63]. Por su parte, los macrófagos
generados en presencia de GM-CSF tienen mayor capacidad de presentación de antígeno que los
macrófagos M2 [56]. Aunque ambos tipos de macrófagos son diana para la infección inicial por HIV1, convirtiéndose en reservorios virales, los macrófagos M2 tienen mayor capacidad de producción
de partículas virales, mientras que los macrófagos M1 inhiben la replicación viral a nivel posttranscripcional [64].
14
Introducción
Características
M1
M2
CD11b
++
++
CD11c
++
++
CD14
-
++
CD71
+
-
CD163
-
+
CD209
-
+
HLA-DR
++
+
HLA-DQ
+
-
710F
+
-
FcγR I (CD64)
+
+
FcγR II (CD32)
+
+
FcγR III (CD16)
-
+
Receptor “scavenger” tipo A
+
+
Antígenos de superficie
Receptores
M-CSFR (c-fms)
+
+++
αvβ3
αvβ5
Fagocitosis mediada por FcγR
Débil
Fuerte
Producción de H2O2
Débil
Fuerte
Sensibilidad a H2O2
Resistente
Sensible
Alta
Baja
Susceptibilidad a HIV-1
Resistente
Susceptible
Susceptibilidad a M. tuberculosis
Susceptible
Resistente
Débil
Fuerte
Integrinas
Funciones
Actividad catalasa
Producción de IL-10
Tabla 1.- Características fenotípicas y funcionales de los macrófagos generados in vitro en presencia de
GM-CSF (M1) o M-CSF (M2). [19, 56].
Otra de las diferencias existentes entre los macrófagos generados en presencia de GM-CSF y
M-CSF es la secreción de quimioquinas. Los macrófagos M2 sólo son capaces de producir CCL18
(PARC) tras estimulación, mientras que los macrófagos M1 secretan niveles constitutivos de CCL22
(MDC), CCL17 (TARC) y CCL18, que mantienen al ser estimulados [56]. A pesar de que los
macrófagos M2 producen niveles bajos de citoquinas pro-inflamatorias y altos niveles de IL-10 tras
estimulación, son capaces de secretar quimioquinas atrayentes de otros tipos celulares (neutrófilos,
monocitos y linfocitos T), lo contribuye a su fenotipo anti-inflamatorio/regulador. En ese sentido,
CXCL8 (IL-8) es producida tanto por macrófagos M1 como M2, mientras que sólo los macrófagos
M2 secretan constitutivamente CCL2 (MCP-1). A su vez, ambos tipos de macrófagos son capaces
de secretar CXCL10, CCL3 (MIP-1α), CCL4 (MIP-1β) y CCL5 (RANTES) tras estimulación con LPS
[56].
15
Introducción
4.1.2 Tejido-especificidad
La heterogeneidad y plasticidad funcional de los macrófagos se refleja en su especialización en
las diferentes localizaciones anatómicas [65]. Los macrófagos localizados en tejidos en contacto con
el entorno exterior (pulmón, placenta, mucosas intestinales) se encuentran continuamente expuestos
a patógenos y desafíos ambientales. Por ello existen mecanismos de inhibición “temporal” de las
funciones de estos macrófagos, lo que evita daños colaterales en el tejido y permite que sólo se
generen reacciones pro-inflamatorias cuando son absolutamente requeridas. Los macrófagos
peritoneales y los situados en el intestino son ejemplos de macrófagos que han desarrollado
estrategias para regular a la baja sus funciones efectoras [66].
4.1.2.1 Macrófagos intestinales
Los macrófagos del tracto digestivo se encuentran estratégicamente localizados en la lámina
propia [67], y en tejidos linfoides secundarios asociados al sistema digestivo, como amígdalas y
placas de Peyer [68]. Funcionalmente, los macrófagos intestinales carecen de actividad
presentadora de antígeno y actividad “respiratory burst”, pero poseen gran capacidad fagocítica y
bactericida [69]. Estas células tienen reducida la producción de citoquinas pro-inflamatorias debido a
la inhibición de NFκB por el TGFβ liberado por las células del estroma [70]. Este estado de falta
parcial de respuesta a estímulos externos ha sido definido como “anergia inflamatoria”, y explica la
incapacidad de los macrófagos intestinales de mediar en la inflamación de la mucosa [71]. De
hecho, en pacientes con enfermedad inflamatoria intestinal se han descrito alteraciones en la vía de
señalización de TGFβ, lo que hace que un gran porcentaje de macrófagos sean capaces de liberar
citoquinas pro-inflamatorias [72, 73]. En consecuencia, los macrófagos intestinales son un claro
ejemplo de macrófagos anti-inflamatorios in vivo [70].
4.1.2.2 Macrófagos peritoneales
En humanos, la concentración de M-CSF en el fluido peritoneal es muy elevada y se
correlaciona con el número de macrófagos peritoneales [74]. Estudios realizados con macrófagos
aislados de muestras de diálisis peritoneal han mostrado que dichas células son fenotípica y
funcionalmente similares a los macrófagos anti-inflamatorios generados in vitro, por cuanto exhiben
alta capacidad de fagocitosis, endocitosis y macropinocitosis, producción de elevadas cantidades de
IL-10 tras estimulación, y una disminuida capacidad de estimulación de células T [75].
16
Introducción
4.2 Activación de macrófagos
4.2.1 Activación clásica vs. activación alternativa
La variedad de estímulos de activación/desactivación de macrófagos [43], combinado con la
heterogeneidad y plasticidad de los macrófagos residentes en tejidos en condiciones homeostáticas,
permite la existencia de numerosos estados de activación de macrófagos [8]. Así, el IFNγ producido
por células Th1, T citotóxicas CD8+ y células NK, convierte a los macrófagos en células con elevada
capacidad citotóxica, microbicida (especialmente de patógenos intracelulares) y anti-proliferativa. La
adquisición de estas propiedades es debida a la producción de mediadores tóxicos (ROS, RNS) y
citoquinas pro-inflamatorias [75]. Este tipo de activación, denominada clásica (CAMØ, M1) [76], da
lugar a macrófagos que secretan altos niveles de IL-12 e IL-23 y muy bajos niveles de IL-10 en
respuesta a Mycobacterium [77], y promueven fuertes respuestas inmunitarias Th1 (Figura 4).
IL-12
IL-10
IFNγ
IL-4
NK
IL-13
Th2
Th1
Activación clásica
Activación alternativa
(CAMØ)
(AAMØ)
Eosinófilo
Basófilo
NK
Figura 4.- Tipos de activación de macrófagos. Representación esquemática de la activación de macrófagos
mediante estimulación con IFNγ (activación clásica) o citoquinas Th2 como IL-4 e IL-13 (activación alternativa).
Las funciones inflamatorias y citotóxicas de los macrófagos activados contribuyeron a la
percepción de que sólo citoquinas Th1 promovían activación de macrófagos, mientras que
citoquinas de tipo Th2 las bloqueaban o desactivaban [78]. Sin embargo, además de inhibir
respuestas Th1, las citoquinas Th2 provocan un aumento de las funciones de los macrófagos como
presentación de antígeno, reparación tisular y capacidad endocítica [77]. Por ello, los factores que
inhiben la generación y actividad de los CAMØ (citoquinas Th2 como IL-4 e IL-13, citoquinas
desactivadoras como IL-10 y TGFβ, hormonas como glucocorticoides y la vitamina D3), e incluso las
células apoptóticas, han sido agrupados como inductores de una forma “alternativa” de activación de
macrófagos (AAMØ, M2) [77] (Figura 4). Los AAMØ producen grandes cantidades de IL-10 y TGFβ
y niveles muy bajos de IL-12 bajo estimulación [79], y pueden presentar funciones
inmunosupresoras e inhibir la proliferación de células T [80].
17
Introducción
Las diferencias en las funciones de CAMØ y AAMØ han sido demostradas en numerosos
ensayos in vitro, donde los AAMØ inducen mayor proliferación celular y deposición de colágeno de
células fibroblásticas [81], e inhiben la proliferación de linfocitos inducida por mitógenos [82]. Al
mismo tiempo, los AAMØ contribuyen a la vascularización in vivo y exhiben actividad angiogénica in
vitro [83], similar a la de MDDC maduras en presencia de citoquinas como IL-10, TGFβ, o
glucocorticoides [84]. Por otro lado, existen numerosos estudios que ponen de manifiesto que los
AAMØ activados con IL-4 son esenciales en la eliminación y control de la infección por patógenos
extracelulares [77].
Aunque el término AAMØ fue inicialmente propuesto para identificar exclusivamente a
macrófagos activados por IL-4/IL-13 [85], la variedad de estímulos anti-inflamatorios que provocan
una activación “no clásica” de macrófagos ha hecho necesario establecer una nomenclatura más
precisa. Mantovani y colaboradores han clasificado estas formas de activación alternativa de
acuerdo con el estímulo inductor: los macrófagos estimulados por las citoquinas Th2 IL-4/IL-13 son
denominados M2a, los activados por complejos inmunes y ligandos de TLR son denominados M2b,
y los macrófagos activados en presencia de IL-10 son denominados M2c [86] (Figura 5, izquierda).
Recientemente se ha propuesto otra clasificación de macrófagos activados de acuerdo con sus
funciones en el mantenimiento de la homeostasis: macrófagos involucrados en la defensa del
organismo, en reparación de heridas y en regulación inmunitaria. Sin embargo, es preciso enfatizar
que además de estos tres grupos es posible definir numerosos estados funcionales intermedios, lo
que avala la existencia de un amplio rango de estados de activación de macrófagos [87] (Figura 5,
derecha).
M1
M2a
Defensa
IL-13
IL-4
INFγ
Reguladores
LPS
TGFβ
M2b
Inmunocomplejos
IL-10
Reparación
de heridas
M2c
Figura 5.- Propuestas de clasificación de macrófagos activados. Los macrófagos polarizados se pueden
clasificar en función del estímulo de activación (izquierda) [86] o de su función efectora primordial (derecha)
[87]. Los tres colores primarios (rojo, amarillo, azul) representan las tres poblaciones de macrófagos definidas,
mientras que los colores secundarios representan macrófagos con funciones intermedias.
18
Introducción
4.2.2 Características fenotípicas de macrófagos activados
Los macrófagos activados presentan diferentes propiedades fenotípicas y funcionales en
función del estímulo de activación (Figura 6). Aunque los macrófagos M2a y M2b exhiben niveles de
expresión de moléculas de adhesión (CD11a, CD54, CD58) y coestimuladoras (CD40, CD80, CD86)
similares a los CAMØ, la activación alternativa de macrófagos en respuesta a IL-4 e IL-13 va unida a
la adquisición de un repertorio de receptores fagocitarios característicos. Estos receptores dotan a
los macrófagos M2a de potentes actividades endocitotóxicas y fagocíticas, y entre ellos son
destacables: 1) el receptor de manosa (MR1, CD206), cuya señalización intracelular está asociada a
la producción de IL-10, la expresión de IL-1Rα, y a la inhibición de la producción IL-12 en respuesta
a endotoxina [79, 88]; 2) el receptor “scavenger” 1 de macrófagos (MSR1, CD204), con un claro
papel en el reconocimiento y eliminación de lipoproteínas [89]; 3) el receptor de β-glucanos Dectin-1,
con especificidad por glucanos β-1,3 y β-1,6, típicos de hongos y algunas bacterias, y que colabora
funcionalmente con TLR2 en la respuesta inflamatoria anti-fúngica [88, 90]; y 4) DC-SIGN, con un
amplio espectro de reconocimiento de patógenos [88, 91, 92] (Figura 6). Otros marcadores de
activación alternativa de macrófagos humanos son DCIR y DCL-1 [93-95], CD23 [77] y el receptor
“scavenger” CD163 [85].
M1
CCL2-5
CCL11
CCL17
CCL22
CXCL1-5
CXCL8-11
CXCL16
Resistencia al tumor
Respuesta Th1
Eliminación de patógenos intracelulares
IL-1
IL-6
IL-12
IL-23
TNFα
CD80
CD86
MHCII
Crecimiento tumoral
Respuesta Th2
Alergia
Eliminación de parásitos
Encapsulación de parásitos
CD23 CD163
M2a
IL-10
CD206
MSR1
CD16
CD32
CD64
CCL11
CCL17
CCL18
CCL22
CCL24
Dectin-1
DC-SIGN
TLR2
TLR4
CCL1
CCL11
CCL17
CCL22
CCL24
M2b
IL-1
IL-6
IL-10
TNFα
IL-10
TGFβ
CD163
Respuesta Th2
Inmunoregulación
Crecimiento tumoral
Inmunoregulación
Deposición de matriz
Remodelación de tejido
CCL16
CCL18
CXCL13
M2c
Figura 6.- Características fenotípicas y funcionales de macrófagos polarizados en función del estímulo
de activación. En el dibujo se representan las principales características fenotípicas (secreción de
quimioquinas (cuadro gris) y citoquinas (cuadro amarillo), y expresión de receptores de membrana), así como
las características funcionales que diferencian los diversos estados de polarización de macrófagos.
19
Introducción
La expresión de genes que controlan el metabolismo celular también se utiliza para discernir
entre los diferentes tipos de macrófagos activados. Así, la expresión de genes que participan en el
metabolismo de la arginina, permite diferenciar CAMØ y AAMØ en ratón, pero no en macrófagos
humanos [96, 97]. La arginasa 1 (Arg1) es un marcador prototípico de activación alternativa, ya que
su expresión es dependiente de IL-4/IL-13, mientras que la óxido nítrico sintasa (iNOS) es inducida
por IFNγ. Los CAMØ metabolizan arginina vía iNOS, generando óxido nítrico, que posee elevada
actividad microbicida. Por el contrario, la expresión de Arg1 permite a los AAMØ producir poliaminas
y prolina, que son esenciales para la proliferación celular y la producción de colágeno,
respectivamente [98]. Otros marcadores de AAMØ en ratón, y que carecen de homólogos en
humanos, son los miembros de la familia quitinasa Ym1 y Ym2 (Chi3l3 y Chi3l4), y Fizz1,
involucrado en el metabolismo de lípidos [99].
Por otro lado, la polarización del macrófago hacia un fenotipo alternativo lleva asociada un
aumento en la expresión de genes relacionados con el metabolismo de lípidos, especialmente de
aquellos implicados en la captación y oxidación de ácidos grasos [100]. Así, además de Fizz1, Stab1 y la lipoxigenasa ALOX15 presentan mayor expresión en AAMØ [77, 93]. A diferencia de AAMØ,
los CAMØ sobre-expresan genes involucrados en el metabolismo del colesterol como ABCA1 y
apolipoproteínas L (APOL1-3,6), involucrados en su transporte y en el desarrollo de aterosclerosis
[93, 101]. A su vez, genes que codifican para las enzimas implicadas en el metabolismo de
mediadores lipídicos (eicosanoides, leucotrienos, esfingosina y ceramida) también se expresan
diferencialmente entre CAMØ y AAMØ. Más concretamente, la expresión de COX-2 está asociada
con el metabolismo de ácido araquidónico en CAMØ, mientras que las enzimas esfingosina y
ceramida quinasas, que catalizan el equilibrio ceramida-esfingosina, están más expresadas en
CAMØ y AAMØ, respectivamente [93].
El receptor PPARγ, y alguno de sus genes diana (FABP4), también se incluyen dentro de los
genes con mayor expresión en AAMØ, ya que IL-4 es un inductor de este receptor y de sus
activadores metabólicos [102]. Los ratones deficientes en PPARγ tienen disminuidos los niveles de
mRNA y la actividad de Arg1, no presentan macrófagos con fenotipo alternativo y, dado su papel en
el metabolismo de ácidos grasos, tienen mayor tendencia a la obesidad [103]. Además, se ha
descrito a PPARγ como regulador negativo de la activación clásica del macrófago [104]. Por tanto,
PPARγ regula las respuestas dependientes de IL-4, y es requerido para la adquisición y
mantenimiento del fenotipo alternativo en macrófagos activados [103].
Mientras que PPARγ es un factor crítico para la activación alternativa inducida por IL-4, la
activación de los factores de transcripción NFκB, STAT-1 y AP-1 son esenciales para la polarización
clásica del macrófago [105]. Estímulos inflamatorios como LPS, activan rutas de señalización
dependientes de MyD88, que llevan a la activación de NFκB y AP-1, y rutas independientes de este
adaptador intracelular, con la activación de IRF3 y STAT-1 [106]. Por el contrario, la IL-10 liberada
20
Introducción
por algunos AAMØ inhibe la activación de NFκB y mantiene su fenotipo inmunosupresor [107-109].
De hecho, la pérdida de expresión de IRF3, STAT-1 y NFκB en macrófagos derivados de médula
ósea de ratón está asociada a la supresión de la polarización pro-inflamatoria [110].
4.2.3 Macrófagos asociados a tumores
Los macrófagos asociados a tumores (TAM) constituyen un ejemplo paradigmático de la
plasticidad del proceso de activación de macrófagos y de su repercusión fisiológica y patológica. En
los tumores existe una gran infiltración de leucocitos inflamatorios [111], cuyo estado de maduración
y localización espacial determina su influencia sobre el tumor. Los macrófagos son el componente
mayoritario de dicho infiltrado tumoral [112], y constituyen un claro ejemplo de activación alternativa
patológica de macrófagos.
Los TAM se originan a partir de monocitos de sangre periférica reclutados hacia el tumor, en su
fase inicial de formación, por factores como M-CSF, MCP-1, VEGF y Angiopoietina-2 [113-117]
(Figura 7). La diferenciación intratumoral da lugar a macrófagos con niveles reducidos de receptores
de quimioquinas, lo que evita su migración desde los tejidos tumorales. Los TAM regulan varios
pasos clave en el desarrollo del tumor, y su abundancia se correlaciona con la progresión tumoral,
remodelación de matriz extracelular, estimulación de la proliferación, migración e invasión de las
células cancerosas, e inhibición de la inmunidad adaptativa (inmunosupresión) [117]. La elevada
densidad de macrófagos en zonas metastáticas, como los nódulos linfoides regionales, favorece el
crecimiento del tumor [113].
Angiogénesis y
remodelación de matriz
VEGF, FGF, TGFβ
Quimioquinas
M-CSF, VEGF, MCP-1
Reclutamiento/
supervivencia
Factores de crecimiento
MMP-9, uPA
TAM
Célula tumoral
IL-10, TGFβ
IL-10, TGFβ
Anergia, supresión, respuesta Th2
Figura 7.- Interacción entre macrófagos y células tumorales. Las células tumorales secretan factores que
atraen y determinan la polarización de los macrófagos en los tumores. A su vez, los TAM producen factores de
crecimiento que promueven angiogénesis y remodelación del tejido, y contribuyen a la progresión y
diseminación del tumor [118].
21
Introducción
El fenotipo y función de los TAM está determinado por los factores microambientales presentes
en el tumor [118, 119] (Figura 7). Citoquinas y factores de crecimiento derivadas del tumor (IL-10,
TGFβ, M-CSF, VEGF, MCP-1) aumentan la generación de macrófagos y reducen la diferenciación
de DC y, en consecuencia, determinan los niveles relativos de APC en el tumor y en los tejidos
cercanos [21]. Junto con TGFβ, M-CSF es el mayor responsable del ambiente inmunosupresor
intratumoral [111]. De hecho, en un modelo de carcinoma mamario espontáneo, los ratones M-CSF-/presentan una progresión tumoral más lenta que los ratones normales [120]. La IL-10 presente en el
tumor induce en los TAM la adquisición de funciones asociadas a macrófagos M2 [121]. Por ello, los
TAM tienen reducida la capacidad de producir moléculas anti-tumorales (TNFα, IL-1, ROS, NO) y
citoquinas inflamatorias (IL-12, IL-1β, TNFα, IL-6) [122], y no presentan activación de NFκB [111].
La producción de mediadores inmunosupresores (prostaglandinas, IL-10 y TGFβ) permite a los
TAM inducir la diferenciación de células Treg, que suprimen la actividad de los linfocitos T efectores
y de otras células inflamatorias [111], favoreciendo por tanto el crecimiento tumoral [123, 124]. La
actividad angiogénica del tumor está asimismo favorecida por la acumulación de TAM en regiones
de hipoxia poco vascularizadas, a las que se adaptan por la activación de factores como HIF-1 y
HIF-2 [125]. Los TAM también promueven angiogénesis a través de la liberación de factores de
crecimiento (VEGF, FGF y HGF), metalo-proteasas (MMP-9) y el activador de plasminógeno (uPA),
todos los cuales contribuyen a degradar la matriz extracelular, facilitando por tanto la migración e
invasión de células tumorales [126] (Figura 7).
La expresión de marcadores típicos de macrófagos M2 de ratón como Arg1, Ym1, Fizz1 y Mgl2
se observa en TAM procedentes de fibrosarcoma y de linfoma T BW-Sp3, lo que corrobora el
fenotipo alternativo de estos macrófagos [127, 128]. Sin embargo, en ese mismo modelo se
observan también altos niveles de quimioquinas Th1 como CCL5, CXCL9 y CXCL10, lo que sugiere
la desviación de las características típicas de macrófagos M2 [127]. Aunque los TAM son
considerados macrófagos con fenotipo anti-inflamatorio por su secreción de citoquinas y la deficiente
activación de NFκB, también contribuyen a la angiogénesis y crecimiento tumoral mediante la
secreción de mediadores típicos de macrófagos M1 y reguladores de NFκB, como TNFα, IL-1β y
MMP-9. Por otro lado, en un estado tumoral avanzado, los TAM de ratón expresan constitutivamente
NOS2 y Arg1 que, implicados en el metabolismo de la arginina, producen liberación de NO y
aumento en la producción de ROS (O2- y H2O2) y RNS (ONOO-), deteniendo la proliferación y,
eventualmente, provocando la muerte de células T [116]. En consecuencia, los TAM son capaces de
expresar características pro-inflamatorias y supresoras, existiendo un equilibrio en su polarización
entre un fenotipo M1 y M2. Esta versatilidad en el fenotipo de los TAM es posiblemente debida al
cambio dinámico existente en el microambiente tumoral desde eventos tempranos hasta los estados
avanzados del tumor, y está regulada por mecanismos moleculares, como la modulación de la
actividad de NFκB o las vías de señalización activadas por hipoxia [129]. En los casos que la
22
Introducción
presencia de TAM se correlaciona con un buen pronóstico del tumor, el GM-CSF podría ser
responsable de la adquisición de un fenotipo citotóxico por los macrófagos intratumorales [130].
4.3 Estudios de expresión génica en diferenciación y activación de
macrófagos
La identificación de genes diferencialmente expresados en distintas poblaciones de macrófagos
activados permite determinar su papel en la adquisición de un fenotipo de polarización concreto, y
su posible participación en determinados procesos celulares o fisiológicos [131-133]. En este
sentido, estudios realizados en macrófagos peritoneales tratados con IL-4 han permitido identificar
marcadores de activación alternativa de macrófagos en ratón, como Ym1 y Arg1 [134]. La expresión
diferencial de estos genes dependientes de IL-4 se ha corroborado en un modelo de infección con el
nematodo Brugia malayi [135]. La identificación de genes asociados a los diferentes estados de
polarización de macrófagos puede proporcionar nuevas dianas terapéuticas en patologías
inflamatorias y/o autoinmunes.
Respecto a los estudios realizados en macrófagos humanos polarizados en presencia de
citoquinas, Mantovani y colaboradores han determinado los cambios génicos inducidos en la
+
diferenciación de monocitos CD14 en presencia de M-CSF, y las diferencias existentes entre
macrófagos polarizados por LPS e IFNγ o IL-4 [93]. Posteriormente, se han identificado genes cuya
expresión se modifica en monocitos expuestos a GM-CSF o GM-CSF e IL-4 [136], o a estímulos
“alternativos” como IL-13 [101] o IL-10 [137]. Por otro lado, Hamilton y colaboradores han analizado
macrófagos de ratón generados en presencia de GM-CSF (M1) o M-CSF (M2), y han evidenciado la
contribución de IFN de tipo I en las diferencias fenotípicas de ambas poblaciones [138]. Según estos
autores, la expresión diferencial de citoquinas y quimioquinas en respuesta a LPS se justifica porque
la señalización desde TLR4 se lleva a cabo de forma distinta en ambos tipos de macrófagos, por la
ruta MyD88-independiente (caso de los M2) o MyD88-dependiente (en los M1) [138].
Estos estudios de expresión génica han permitido identificar marcadores moleculares asociados
a respuestas inmunitarias frente a infecciones bacterianas [139, 140], patologías como la
enfermedad pulmonar obstructiva crónica (EPOC), y el desarrollo de tumores [141, 142]. Por otro
lado, estudios realizados sobre la interacción macrófago-patógeno han identificado estrategias de
defensa del hospedador y de evasión por parte del patógeno [143]. En consecuencia, todas estas
aproximaciones han hecho posible diseccionar la polarización de macrófagos frente a estímulos
patogénicos concretos, lo que ha permitido establecer que los procesos de activación/polarización
de macrófagos y de maduración de MDDC son específicos del estímulo que los provoca [140, 141,
144].
23
Introducción
5. El receptor de patógenos DC-SIGN
Las lectinas son proteínas que reconocen de manera específica carbohidratos presentes en
antígenos propios y patógenos [145]. En vertebrados, las lectinas se clasifican en diferentes
subgrupos, siendo los receptores lectina de tipo C (CLR) uno de los mejor estudiados. Los CLR se
caracterizan por tener al menos un dominio de reconocimiento de carbohidratos (CRD) a través del
cual unen carbohidratos de forma dependiente de Ca2+ [146]. Los CLR pueden ser proteínas
solubles o proteínas transmembrana, y se han definido siete sub-grupos en función de su homología
de secuencia, estructura y disposición del CRD respecto al resto de la molécula [147] (Tabla 2). Los
grupos I, III y VII engloban lectinas solubles, mientras que el resto de grupos corresponden a
lectinas de membrana, que a su vez pueden ser proteínas de tipo I, como el receptor de manosa
(MR), o de tipo II, como DC-SIGN [148].
Grupo
Moléculas representativas
Características
I
Agrecano, versicano, neurocano
Proteoglicanos, glicoproteínas de matriz
extracelular
II
Receptor de asialoglicoproteína, CD23,
DC-SIGN, LSECtin
Receptores de membrana tipo II
III
Proteína de unión a manosa, SP-A, SP-D
Colectinas. Oligómeros asociados por un dominio
tipo colágeno. Extracelulares y solubles
IV
Selectinas L, P y E
Glicoproteínas de membrana de tipo I, implicadas
en adhesión leucocitaria
V
NKG2, LY49, CD69
Antígenos linfocitarios de tipo II
VI
Receptor de manosa, DEC-205
Receptores de membrana de tipo I con varios
CRD extracelulares
VII
Proteína asociada a
pancreatitis/hepatoma
Extracelulares y solubles
Tabla 2.- Clasificación de las lectinas de tipo C.
DC-SIGN (“Dendritic cell-specific ICAM-3 grabbing nonintegrin”, CD209, CLEC4L) fue descrito
en 1992 por Curtis y colaboradores como una lectina de tipo C que reconoce la proteína gp120 de la
envuelta del HIV-1 [149]. Posteriormente se caracterizó como un receptor presente en MDDC que
participa en la interacción DC-célula T mediante el reconocimiento de la molécula de adhesión
intracelular ICAM-3 [150]. En la actualidad y, como se ha mencionado anteriormente, DC-SIGN
constituye un marcador de macrófagos anti-inflamatorios M2 y AAMØ [56, 151].
24
Introducción
5.1 Expresión y localización tisular
Aunque descrita como específica de células dendríticas, DC-SIGN no sólo se expresa in vivo en
DC de tejidos periféricos y linfoides [152], sino que también se expresa en poblaciones CD14+ de
sangre periférica [153] y en determinadas subpoblaciones de macrófagos presentes en sinusoides
medulares de nódulos linfáticos [151], intestino [154], pulmón [152], placenta [152, 155, 156] y
macrófagos sinoviales [157]. La expresión de DC-SIGN se induce in vitro por IL-4 en monocitos [92,
150], en macrófagos [91, 158] y en la línea celular mieloide THP-1 [91], y sus niveles de expresión
son controlados por el factor de transcripción PU.1 [159]. Estudios de localización subcelular han
situado a DC-SIGN en “lipid rafts”, microdominios de membrana ricos en colesterol y esfingolípidos,
lo que puede favorecer a su capacidad de unión e internalización de partículas víricas, así como a
su capacidad señalizadora tras el reconocimiento de ligandos [160, 161].
5.2 Estructura y dominios funcionales
Estructuralmente, DC-SIGN es una proteína transmembrana tipo II de 404 aminoácidos, cuya
región extracelular incluye un CRD, un cuello o “stalk” que le separa de la zona transmembrana, y
con una corta región citoplásmica de 42 aminoácidos [162] (Figura 8).
DOMINIO
ESTRUCTURAL
FUNCIÓN
Ct
Dominio
lectina
Interacción
con ligandos
Cuello
(dominios
repetidos)
Multimerización
NLT
NLT
EEE
Dominio
citoplásmico
LL
Y
EEE
LL
Y
NLT
EEE
LL
Y
Internalización,
tráfico y
señalización
Nt
Figura 8.- Estructura y función de los dominios de DC-SIGN. Ct, extremo carboxilo-terminal; Nt, extremo
amino-terminal; NLT, motivo de glicosilación; EEE, dominio triacídico; LL, motivo dileucina; Y, tirosina del motivo
YKSL.
25
Introducción
El CRD de DC-SIGN es una estructura globular que consta de 12 cadenas β, 2 hélices α y 3
puentes disulfuro, además de 2 sitios de unión a Ca2+ [162] . Uno de esos sitios es esencial para la
conformación del CRD, mientras que el otro participa en la interacción con los ligandos
carbohidratados y determina su especificidad. La secuencia de aminoácidos de este segundo sitio
contiene un motivo EPN que confiere a DC-SIGN especificidad por manosa. El cuello de DC-SIGN
está compuesto por 8 dominios repetidos de 23 aminoácidos ricos en leucinas, el primero de los
cuales contiene un motivo de glicosilación (NLT) (Figura 8) [163]. Esta región es fundamental para
la formación de estructuras multiméricas, más concretamente tetrámeros, lo que incrementa
considerablemente la avidez de interacción de DC-SIGN por sus ligandos [164-166]. La región
43
transmembrana comprende desde Leu
a Ser61 [163]. La zona amino-terminal constituye la cola
citoplásmica, que posee un motivo dileucina (LL) que promueve la rápida internalización de DCSIGN tras interaccionar con ligandos solubles, un motivo triacídico (EEE), que determina que los
complejos DC-SIGN-ligando sean dirigidos a compartimentos lisosomales [163, 167], y un motivo
basado en tirosina (ITIM-like), que capacita a esta lectina para transmitir señales intracelulares [161]
(Figura 8).
5.3 Estructura génica, isoformas y polimorfismos
El gen de DC-SIGN mapea en la región p13 del cromosoma 19, y consta de 7 exones [168]
(Figura 9). Los exones 1a y 1c codifican la cola citoplásmica, el exón 3 codifica la región del cuello,
y los exones 4, 5 y 6 codifican el CRD [163]. En ratón no existe un gen ortólogo de DC-SIGN
humano, aunque existen moléculas homólogas dentro de la familia SIGN: mDC-SIGN (murine DCSIGN) o SIGNR5, SIGNR1 (SIGN related), SIGNR2, SIGNR3, SIGNR4, el pseudogen SIGNR6,
SIGNR7 y SIGNR8 [169, 170].
El gen de DC-SIGN está sometido a un complejo sistema de “splicing” alternativo, que origina
un gran número de transcritos con estructuras diferentes de la prototípica [168]. Entre estas
variantes se incluyen isoformas con una cola citoplásmica alternativa, isoformas sin región
transmembrana e isoformas con CRD incompletos, así como una gran variedad de transcritos con
un número variable de repeticiones en la región del cuello. El patrón de isoformas y los niveles de
expresión de cada una de ellas es variable, tanto en individuos de una población como en los
distintos estadios de diferenciación de un mismo tipo celular [168].
26
Introducción
+ 46
206
(100)
Ib
Ia
981 1052 1425
II
II
3293 3405
2420 2571
(425)
1
I
Ic
1994
(372)
(774)
(721)
4272
4470
(866)
2 3 4 5 6 7 8
III
III
IV
IV
V
V
VI
Figura 9.- Estructura génica de DC-SIGN. En el esquema se representan los exones que codifican para cada
una de las regiones que forman DC-SIGN (números romanos) y su tamaño (números arábigos), así como la
localización y tamaño de los intrones (números romanos y arábigos en gris).
La variabilidad estructural del gen de DC-SIGN a nivel poblacional puede tener importantes
repercusiones patológicas, ya que se han descrito polimorfismos en la región codificante y
reguladora que se asocian con susceptibilidad alterada a infecciones como tuberculosis o HIV-1
[171, 172]. Existen discrepancias entre el posible papel protector de las variantes génicas de DCSIGN, que pueden ser debidas a las diferentes poblaciones estudiadas, e incluso a la existencia de
otros polimorfismos. La mayoría de estos estudios se han centrado en un cambio en el nucleótido
-336 (variante G o A) en la región promotora de esta lectina, que afecta al sitio de unión del factor de
transcripción Sp1 [173]. Martin y colaboradores asocian la presencia de la variante DC-SIGN-336G
con una mayor susceptibilidad a la infección por HIV-1 por vía parenteral pero no por vía mucosa
[174], mientras que otros autores encuentra asociación únicamente entre la variante DC-SIGN-139C
y una progresión acelerada del SIDA en individuos hemofílicos japoneses infectados por HIV-1
[175].
Respecto a la infección por M. tuberculosis, las variantes DC-SIGN-336A y -871G se asocian a
una protección frente a la infección en una población en el sur de África [176], mientras que en la
población sub-Sahariana el alelo -336G está asociado a una mayor protección [177]. Sin embargo,
otros trabajos posteriores en pacientes colombianos [178], tunecinos [179] y africanos [180], no han
observado asociación entre los polimorfismos en la posición DC-SIGN-336 y la susceptibilidad a
tuberculosis. Recientemente se ha analizado la frecuencia de la variante DC-SIGN-336G en
individuos de India infectados con HIV-1 y/o tuberculosis. Al ser menos frecuente en individuos
infectados por HIV-1, se especula que la presencia de esta variante protege frente a la infección por
HIV-1 y, sin embargo, aumenta la susceptibilidad a tuberculosis [181].
La presencia de polimorfismos en la región promotora de DC-SIGN también se ha asociado con
susceptibilidad alterada frente a otras infecciones y patologías. De hecho, la variante DC-SIGN336G está asociada con mayor protección frente a la fiebre del Dengue, pero no frente a la fiebre
hemorrágica del Dengue en individuos de Tailandia [182]. Por otro lado, no se ha encontrado
27
Introducción
asociación entre la variante DC-SIGN-336A/G y la susceptibilidad a la enfermedad celiaca, aunque
la variante DC-SIGN-336G sí está asociada a dicha enfermedad dentro del grupo de pacientes HLADQ2(-) [183]. La enorme variabilidad en el gen de DC-SIGN se puso de manifiesto en un estudio que
analizó la presencia de variantes en las posiciones -336, -332, -201 y -139 en cuatro grupos étnicos
de Brasil, y su posible correlación con la infección por HTLV-1 [184]. Según este estudio, las
variantes -336A y -139A son más comunes en individuos asiáticos, y la variante -201T no se
observa en caucásicos, asiáticos ni amerindios. Por otro lado, la variante -336A es más frecuente en
pacientes infectados por HTLV-1 y el alelo -139A está asociado con la protección frente a la
infección por este virus.
De
todos
estos
estudios
se
concluye
que
DC-SIGN
puede
contribuir
a
la
susceptibilidad/transmisibilidad de las infecciones provocadas por numerosos patógenos. Además
de estas variantes en la región reguladora, existen polimorfismos en la región codificante que se
localizan principalmente en el exón 3 que codifica el cuello de DC-SIGN. De ellos y de las
discrepancias sobre su posible asociación con susceptibilidad a infecciones en diferentes grupos
étnicos, se profundizará en el apartado de “Discusión”.
5.4 Función y señalización
DC-SIGN es, probablemente, la lectina con el mayor rango de ligandos descrito, siendo capaz
de actuar como receptor de adhesión celular y de reconocer estructuras de carbohidratos presentes
en antígenos propios y en patógenos (Tabla 3). DC-SIGN presenta una alta afinidad por
carbohidratos con dimanosas terminales y estructuras internas de manosas ramificadas
(manotriosas α1→3, α1→6) [185, 186], y por carbohidratos que contienen fucosa, en concreto por
los trisacáridos que constituyen los antígenos de los grupos sanguíneos de Lewis (Lex, Ley, Lea, Leb)
[187-189].
Como receptor de patógenos, DC-SIGN interacciona con sus PAMP y el complejo DC-SIGNpatógeno se internaliza, promoviendo el procesamiento y la posterior presentación de antígenos a
los linfocitos T, para acabar induciendo respuestas inmunitarias frente a dichos microorganismos
[167, 190]. Dentro del amplio rango de patógenos reconocidos por DC-SIGN [191], se encuentran
bacterias [192-194], hongos [195, 196], parásitos [197] y virus [149, 198, 199]. Recientemente
incluso se ha descrito la interacción de DC-SIGN con alérgenos comunes [200] (Tabla 3).
28
Introducción
Patógeno
Ligando de DC-SIGN
HIV-1
gp120
CMV
gB
Ébola
GP de la envuelta
Margburg
GP de la envuelta
Dengue
gE
HCV
gE1/gE2
SARS
proteína S
Herpesvirus humano
?
H5N1 (cepa del virus de la gripe aviar)
?
cepas patogénicas de Mycobacterium
ManLAM
Helicobacter pilori
LPS
Klebsiella pneumonia
LPS
Neisseria meningitidis
LPS
Neisseria gonorrhoeae
LPS
Lactobacillus acidophilus NCFM
SlpA
Leishmania
LPG?
Schistosoma mansoni
SEA
Candida albicans
?
Aspergillus fumigatus
Galactomanano
Virus
Bacterias
Parásitos
Hongos
Tabla 3.- Patógenos y ligandos que se unen a DC-SIGN. HIV: virus de la inmunodeficiencia humana; CMV:
citomegalovirus; HCV: virus de la hepatitis C; SARS: síndrome respiratorio agudo severo; gB, gE, gE1, gE2:
glicoproteínas B, E, E1, E2; GP: glicoproteína; LPG: lipofosfoglicano; LPS: lipopolisacárido; ManLAM:
lipoarabinomanano recubierto de manosas; SEA: antígeno soluble de los huevos; SlpA: proteína A de la capa
x
x
y
y
superficial; Le : Lewis ; Le : Lewis .
Por su capacidad de reconocer ligandos endógenos, DC-SIGN también puede mediar procesos
de adhesión intercelular (Figura 10). Así, DC-SIGN podría intervenir en la migración transendotelial
de DC gracias a la interacción con ICAM-2 presente en células endoteliales [153]. La unión de DC a
neutrófilos tiene lugar a través del reconocimiento por DC-SIGN de los carbohidratos ricos en Lex de
la integrina Mac-1 (CD11b/CD18) [201, 202] y de CEACAM-1 [202-204]. DC-SIGN también reconoce
el antígeno carcinoembrionario (CEA) de células de cáncer colorrectal, caracterizado por una mayor
presencia de Lex y Ley [191]. Otro de los ligandos endógenos propuestos para DC-SIGN es ICAM-3.
Aunque en un principio se propuso que la adhesión inicial entre DC y linfocitos T vírgenes estaba
mediada por la interacción DC-SIGN/ICAM-3 [150], esta hipótesis no ha podido ser corroborada por
otros autores [151, 205, 206].
29
Introducción
Célula endotelial
Célula T
ICAM-2
ICAM-3
CEACAM-1
Mac-1
CEA
Neutrófilo
Célula tumoral
Figura 10.- Ligandos endógenos de DC-SIGN. Representación esquemática de las interacciones de DCSIGN con sus ligandos endógenos: ICAM-2 de células endoteliales, ICAM-3 de células T, CEA de células
tumorales, y las moléculas CEACAM-1 y Mac-1 en neutrófilos.
Como se ha comentado anteriormente, DC-SIGN es capaz de transmitir señales intracelulares
específicas tras su interacción con carbohidratos presentes en patógenos, señales que a su vez se
interrelacionan con las señales procedentes de TLR [160]. En función de la naturaleza del
carbohidrato reconocido por DC-SIGN, las MDDC secretan un patrón diferente de citoquinas [207].
Así, la unión de patógenos que expresan manosas en su superficie, como M. tuberculosis o HIV-1,
conduce a un aumento en la producción de IL-10, IL12 e IL-6 de forma dependiente de Raf-1 [207].
Sin embargo, la unión de ligandos que contienen fucosa, como Ley de H. pilori, disminuye la
secreción de IL-12 e IL-6 de manera dependiente de Raf-1 mientras que se incrementa la
producción de IL-10 de forma independiente de Raf-1. El mecanismo molecular responsable del
aumento en la producción de IL-10 de forma Raf-1-dependiente implica la posterior acetilación de
p65 de NFκB, que conlleva a un incremento en la actividad transcripcional de IL-10 [208]. Por otro
lado, la activación de ERK en la ruta de señalización de DC-SIGN parece ser dependiente del
ligando involucrado. Así, la activación de DC-SIGN con anticuerpos específicos frente al CRD, la
unión de gp120 de HIV-1, o la unión del alergeno Ara h1, induce fosforilación de ERK1/2 [160, 209,
210]. Sin embargo, otros estudios han demostrado que la unión de ligandos patogénicos a DCSIGN, como ManLAM de M. tuberculosis o la proteína Salp15 de Ixodes scapularis, no provoca
activación de ERK [208, 211]. En consecuencia, DC-SIGN es considerado un modulador de la
respuesta inmune al ser capaz de alterar el balance Th1/Th2 y de modificar las señales procedentes
de otros PRR como TLR4 [212].
30
Objetivos
Objetivos
El objetivo general de esta Tesis Doctoral consistió en la identificación y caracterización
de marcadores de macrófagos activados con un fenotipo anti-inflamatorio/alternativo, y en
concreto el estudio de dos esos marcadores, el receptor de folato β (FRβ) y DC-SIGN:
1. Análisis de la expresión del FRβ en macrófagos anti-inflamatorios M2 y macrófagos
asociados a tumores.
2. Búsqueda de factores que regulan la expresión y función del FRβ en macrófagos M2.
3. Caracterización estructural y funcional de isoformas y polimorfismos de DC-SIGN en células
dendríticas derivadas de monocitos.
4. Identificación de epítopos estructurales y funcionales en la molécula de DC-SIGN mediante el
empleo de anticuerpos monoclonales.
33
Resultados
Resultados
Esta Tesis Doctoral se presenta en formato de artículos publicados. La sección de
resultados incluye los artículos que dan respuesta a los objetivos planteados:
1. Los resultados del análisis de la expresión del FRβ en macrófagos anti-inflamatorios y
macrófagos asociados a tumores se presentan en el siguiente artículo:
Sierra-Filardi E, et al. Folate receptor beta is expressed by tumor-associated macrophages
and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res,
2009 Dec 15;69(24):9395-403.
2. Los resultados obtenidos de la búsqueda de factores que regulan la expresión y función del
FRβ en macrófagos M2 se recogen en el siguiente artículo:
Sierra-Filardi E, et al. Activin prevents the acquisition of M2/anti-inflammatory markers and
skews the macrophage cytokine profile. Manuscrito en preparación.
3. Los resultados generados tras la caracterización de isoformas y polimorfismos de DC-SIGN
se publicaron en el artículo:
Sierra-Filardi E, et al. Structural requirements for multimerization of the pathogen receptor
DC-SIGN (CD209) on the cell surface. J Biol Chem, 2008 Feb 15;283(7):3889-903.
4. Los resultados obtenidos tras el análisis estructural de la molécula de DC-SIGN se recogen
en el siguiente artículo:
Sierra-Filardi E, et al. Epitope mapping on the dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN) pathogen-attachment factor. Mol Immunol, 2010 Jan;47(4):840-848.
37
Resultados
1. El receptor de folato β se expresa en macrófagos asociados a tumores y
constituye un marcador de macrófagos anti-inflamatorios/reguladores M2
La activación de macrófagos comprende un amplio espectro de estados funcionales
dependientes del microambiente de citoquinas. Los macrófagos activados se han agrupado
funcionalmente según su respuesta a estímulos pro-Th1/pro-inflamatorios (LPS, IFNγ, GM-CSF) (M1)
o pro-Th2/anti-inflamatorios (IL-4, IL-10, M-CSF) (M2). En el presente manuscrito demostramos que
el receptor de folato β (FRβ), codificado por el gene FOLR2, es un marcador de macrófagos
generados en presencia de M-CSF (M2), pero no de GM-CSF (M1), y que su expresión se
correlaciona con un aumento de la captación de folato. La capacidad de captar folato por los
macrófagos es promovida por M-CSF, mantenida por IL-4, prevenida por GM-CSF y reducida por
IFNγ, lo que indica una relación entre la expresión del FRβ y la polarización M2. De acuerdo con los
datos in vitro, la expresión del FRβ se detecta en macrófagos asociados a tumores (TAM), que
exhiben un perfil funcional de tipo M2 y ejercen potentes funciones inmunosupresoras dentro del
ambiente tumoral. El FRβ se expresa y media la captación de folato por TAM CD163+ CD14+ IL-10+,
y su expresión es inducida de una manera dependiente de M-CSF por líquido ascítico tumoral y por
el medio condicionado de fibroblastos y líneas tumorales. Estos resultados definen al FRβ como un
marcador de la polarización M2 de macrófagos, e indican que los conjugados de folato con drogas
terapéuticas son una potente herramienta en inmunoterapia frente a los TAM.
39
Immunology
Folate Receptor β Is Expressed by Tumor-Associated Macrophages
and Constitutes a Marker for M2 Anti-inflammatory/
Regulatory Macrophages
Amaya Puig-Kröger,1,2 Elena Sierra-Filardi,1 Angeles Domínguez-Soto,1 Rafael Samaniego,3
María Teresa Corcuera,4 Fernando Gómez-Aguado,4 Manohar Ratnam,5
Paloma Sánchez-Mateos,2 and Angel L. Corbí1
1
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas; 2Unidad de Inmuno-Oncología and 3Unidad de
Microscopía Confocal, Hospital General Universitario Gregorio Marañón; 4Servicio de Anatomía Patológica, Hospital Carlos III, Madrid,
Spain; and 5University of Toledo College of Medicine, Toledo, Ohio
Abstract
Macrophage activation comprises a continuum of functional
states critically determined by cytokine microenvironment.
Activated macrophages have been functionally grouped
according to their response to pro-Th1/proinflammatory
stimuli [lipopolysaccharide, IFNγ, granulocyte macrophage
colony-stimulating factor (GM-CSF); M1] or pro-Th2/antiinflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2].
We report that folate receptor β (FRβ), encoded by the FOLR2
gene, is a marker for macrophages generated in the presence
of M-CSF (M2), but not GM-CSF (M1), and whose expression
correlates with increased folate uptake ability. The acquisition
of folate uptake ability by macrophages is promoted by M-CSF,
maintained by IL-4, prevented by GM-CSF, and reduced by
IFNγ, indicating a link between FRβ expression and M2 polarization. In agreement with in vitro data, FRβ expression is
detected in tumor-associated macrophages (TAM), which
exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRβ is
expressed, and mediates folate uptake, by CD163+ CD68+ CD14+
IL-10–producing TAM, and its expression is induced by tumorderived ascitic fluid and conditioned medium from fibroblasts
and tumor cell lines in an M-CSF–dependent manner. These
results establish FRβ as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of
therapeutic drugs are a potential immunotherapy tool to
target TAM. [Cancer Res 2009;69(24):9395–403]
Introduction
Macrophages exhibit a continuum of functional activation
states under homeostatic and pathologic conditions (1, 2). Depending on the stimulus, activated macrophages acquire microbicidal, pro-inflammatory, and antitumor activities, but might
also contribute to tissue repair, resolution of inflammation,
and tumor cell growth and metastasis (1). These two extremes
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
A. Puig-Kröger and E. Sierra-Filardi are co-first authors. P. Sánchez-Mateos and
A.L. Corbí contributed equally to this work. The order of authors should be considered
arbitrary.
Requests for reprints: Amaya Puig-Kröger, Laboratorio de Inmuno-Oncología,
Hospital General Universitario Gregorio Marañón, Doctor Esquerdo 46, 28007
Madrid, Spain. Phone: 34-91-5868750; Fax: 34-91-5868052; E-mail: apuig.
[email protected].
©2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-09-2050
www.aacrjournals.org
of the spectrum of macrophage activation have been coined as
“classic”/M1 and “alternative”/M2 (3) and play opposing roles
during immune and inflammatory responses. Although granulocyte macrophage colony-stimulating factor (GM-CSF) and M-CSF
contribute to macrophage differentiation, each cytokine promotes
the acquisition of distinct pathogen susceptibility (4) and inflammatory functions (5–8). GM-CSF–derived macrophages (M1) are proinflammatory and potentiate Th1 responses, whereas M-CSF–driven
macrophages (M2) secrete IL-10 in response to pathogens and do
not activate Th1 responses (8).
Tumor-associated macrophages (TAM) are abundant immunosuppressive cells recruited into the tumor microenvironment by
cytokines such as M-CSF and CCL2 (9). The relevance of M-CSF
and TAM in tumor progression and metastasis is now well established (10, 11). TAM represent a unique type of M2-polarized
macrophages, as they promote angiogenesis, tissue remodeling,
and repair (2, 12). In fact, clinical studies have revealed a correlation between high tumor macrophage content and poor patient
prognosis. Because TAM are potential targets for anticancer therapy (13, 14), identification of TAM-specific markers constitutes a
very active area of research.
The folate receptor gene family includes four members (FRα or
FOLR1, FRβ or FOLR2, FRγ or FOLR3, and FRδ or FOLR4), whose encoded products bind folic acid with high affinity (15). FOLR1 and
FOLR2 encode glycosyl phosphatidylinositol–anchored endocytic receptors expressed in certain epithelial tissues and various tumors
(FOLR1; refs. 16, 17) or in normal myeloid cells and acute myelogenous leukemias (FOLR2; refs. 18–20). Within the myeloid lineage, folate receptor β (FRβ) is expressed in a nonfunctional state in CD34+
bone marrow cells (21, 22) and neutrophils (18), whereas it mediates
folate binding in activated synovial macrophages from rheumatoid
arthritis (23) and in ovarian cancer–associated murine macrophages
(24). The high affinity of FRα and FRβ for folate binding, their endocytic capacity, and their restricted expression have prompted the
evaluation of the potential therapeutic value of folate-drug conjugates in cancer and inflammatory pathologies (25, 26).
In the present article, we describe that functional FRβ is specifically expressed by M-CSF–polarized (M2) macrophages as well as
by ex vivo isolated TAM, and that tumors induce its expression in an
M-CSF–dependent manner, thus supporting folate-drug conjugates
as valuable tools to target TAM in tumor immunotherapy protocols.
Materials and Methods
Cell culture and treatments. Human monocytes were purified by magnetic cell sorting using CD14 microbeads (Miltenyi Biotech) as described (27).
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Figure 1. FOLR2 mRNA and FRβ protein expression and function in M1 and M2 macrophages. A, FOLR2, JDP2, SLC11A1, and IL10 are differentially expressed
in M1 and M2 macrophages, as determined by microarray DNA analysis and quantitative RT-PCR. B, right, FRβ expression in cell membrane extracts, as determined
by Western blot using an antihuman FRβ polyclonal antiserum (18). As a control, CD29 expression levels were determined in parallel. Left, cell surface expression
of FRβ on M1 and M2 macrophages, determined by flow cytometry using a polyclonal antiserum against human FRβ (ref. 18; empty histogram). As a control
(filled histogram), a previously described rabbit preimmune antiserum (29) was used. C, FRβ function in M1 and M2 macrophages, as shown by binding (4°C) and
uptake (37°C) of folate-FITC (empty histogram, black line). Transferrin-FITC internalization (empty histogram, gray line) was determined in parallel on both macrophage
types. Each experiment was done three times, and a representative experiment is shown. D, binding (4°C) and internalization (37°C) of folate-FITC by M2
macrophages, in the absence (empty histograms, black line) or the presence (empty histograms, gray line) of a 100 mol/L excess of folic acid. The experiment was
done four times, and one of the experiments is shown. Representative confocal sections of M2 macrophages incubated with folate FITC for 1 h at 37°C, and their
corresponding differential interference contrast images, are shown. The percentage of marker-positive cells and the mean fluorescence intensity (in parentheses)
are indicated in flow cytometry experiments (B–D), and filled histograms indicate cell autofluorescence (C and D).
M1 or M2 monocyte-derived macrophages were generated in the presence of
GM-CSF (1,000 units/mL, ImmunoTools GmbH) or M-CSF (10 ng/mL),
respectively. When indicated, macrophages were treated for 72 h with IL-6
or IL-10 (50 ng/mL), and anti-M-CSF blocking monoclonal antibody
(Abingdon) was used at 0.5 μg/mL. For activation, macrophages were treated with IL-4 (1,000 units/mL), IL-10 (50 ng/mL), IFNγ (500 units/mL), or
lipopolysaccharide (LPS; 50 ng/mL; E. coli 055:B5, Sigma) for 48 h. Human
tumor cell lines (JAR, JEG-3, NIH-OVCAR-3, and Colo320) were cultured in
DMEM containing 10% FCS. Cultures of tumor-associated fibroblasts were
established from primary melanoma according to standard procedures.
Human TAM were obtained from melanoma and breast adenocarcinoma patients after obtaining written informed consent and following Medical Ethics committee procedures (Hospital General Universitario Gregorio
Marañón). Histopathologic diagnosis was confirmed for each specimen.
TAM were isolated by Ficoll gradient cell separation and subsequent magnetic cell sorting using CD14 microbeads. Phenotypic analysis was carried
out by indirect immunofluorescence (28) using rabbit polyclonal antisera
anti-human FRβ (18). Folate-FITC binding and endocytosis assays were
done as reported (26). Flow cytometry on permeabilized ex vivo isolated
TAM was done using phycoerythrin (PE)-labeled anti-CD68 monoclonal antibody (clone Y1/82A, Biolegend), Alexa Fluor 647–labeled anti-CD163
monoclonal antibody (clone RM3/1, Biolegend), and a polyclonal antiserum
against human FRβ followed by incubation with FITC-labeled goat antirabbit affinity-purified antibody. The presence of Tie2-positive FRβ-positive
macrophages was evaluated using a PE-labeled anti-Tie2 monoclonal antibody (clone 33.1, Biolegend). Isotype-matched monoclonal antibodies (PE-
Cancer Res 2009; 69: (24). December 15, 2009
42
Control, Alexa 647-Control) and a preimmune rabbit antiserum (29) were
used as negative controls.
Western blot. Western blot was carried out with 10 μg of lysates from
crude plasma membranes (30). Protein detection was done with a polyclonal antisera against FRβ (18) or a monoclonal antibody against CD29. For
control purposes, a previously described rabbit pre-immune antiserum was
used (29).
PCR. Total RNA from solid tumor tissue and TAM was extracted
(RNAeasy kit, Qiagen), retrotranscribed, and amplified using standard procedures. Oligonucleotides specific for FOLR2, MAFB, IL10, ESR1, MAGEA3,
and GAPDH were as follows: FRBs, 5′-AGAAAGACATGGTCTGGAAATGGATG-3′, and FRBas, 5′-GACTGAACTCAGCCAAGGAGCCAGAGTT-3′
(21); Maf-Bs, 5′-CCCGGCTGGCCCGCGAGAGAC-3′, and Maf-Bas, 5′-CTAGGAGGCGGCGCTGGCGT-3′ (31); IL10s, 5′-ATGCCCCAAGCTGAGAACCAAGACCCA-3, and IL10as, 5-TCTCAAGGGGCTGGGTCAGCTATCCCA-3;
ESR1s, 5′-TCAGATAATCGACGCCAGG-3′, and ESR1as, 5′-GGCTCAGCATCCAACAAGG-3′; MAGEA3s, 5′-GAAGCCGGCCCAGGCTCG-3′, and MAGEA3as, 5′-GGAGTCCTCATAGGATTGGCTCC-3′; and GAPDHs,
5′-GGCTGAGAACGGGAAGCTTGTCA-3′, and GAPDHas, 5′-CGGCCATCACGCCACAGTTTC-3′. Amplified fragments (783 bp for FOLR2, 347 bp
for MAFB, 352 bp for IL10, 511 bp for ESR1, 457 bp for GAPDH, and 423
bp for MAGEA3) were resolved by agarose gel electrophoresis. For quantitative reverse transcription-PCR (RT-PCR), oligonucleotides for FOLR1,
FOLR2, FOLR3, JDP2, NRAMP1, and IL10 were designed according to the
Roche software for quantitative real-time PCR, and RNA was amplified using the Universal Human Probe Roche library (Roche Diagnostics). Assays
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Folate Receptor β Is an M2 Macrophage Marker
were made in triplicates and results normalized according to the expression levels of 18S RNA and GAPDH. Results were obtained using the ΔΔCT
method for quantitation and expressed as normalized fold expression.
Confocal microscopy and immunohistochemistry. Human melanoma tissues (subcutaneous tissue, lymph node, and lung metastasis) were
obtained from patients with primary and metastatic lesions undergoing
surgical treatment. Thick sections (4 μm in depth) of cryopreserved tissue
were first blocked for 10 min with 1% human immunoglobulins and then
incubated for 1 h with a rabbit polyclonal antiserum against human FRβ
(18), anti-CD163 or HMB-45 monoclonal antibodies, or isotype-matched
control antibodies. All primary antibodies were used at 1 to 5 μg/mL, followed by incubation with FITC-labeled antimouse and Texas red–labeled
antirabbit secondary antibodies. Samples were imaged using a confocal
scanning inverted AOBS/SP2 microscope (Leica Microsystems) with a
63× PL-APO NA 1.3 immersion objective. Image processing and colocalization analyses (scatter plots) were assessed with the Leica Confocal Software
LCS-15.37. Tissue microarrays (TMAH-MTC-01, RayBiotech) were processed according to the manufacturer's recommendations.
Results
FRβ is expressed in macrophages generated in the presence
of M-CSF. Gene expression profiling on macrophages generated in
the presence of GM-CSF (M1) or M-CSF (M2) resulted in the identification of more than 250 differentially expressed genes (>2-fold
differences, P < 0.05; data not shown). Among them, FOLR2, which
codes for FRβ, was preferentially expressed in M2 macrophages
(P = 1.3 × 10−7; Fig. 1A). The JDP2 gene, which encodes an activator
protein-1 repressor, also showed higher expression in M2 macrophages (P = 0.02), whereas SLC11A1, which encodes the NRAMP1
protein associated with classic macrophage activation, was expressed at higher levels in M1 macrophages (P = 0.029; Fig. 1A).
Interestingly, and in agreement with their anti-inflammatory activity, the expression of IL10 was considerably higher in M-CSF–
primed macrophages (P = 1.2 × 10−4). The differential expression
of FOLR2, JDP2, SLC11A1, and IL10 in both types of macrophages
was confirmed by real-time RT-PCR on mRNA from independent
donors (Fig. 1A). Besides, FRβ expression was exclusively detected
in membrane lysates and on the cell surface of M2 macrophages
(Fig. 1B), thus validating the transcriptome data.
Because FRβ binds folic acid and folate conjugates (32), the ability of FRβ to mediate folate-FITC uptake by M2 macrophages was
assessed. Whereas both macrophage types endocytosed transferrinFITC, M-CSF–polarized macrophages displayed folate binding and
internalization ability, and GM-CSF–induced macrophages showed
no folate uptake capacity, in agreement with their lack of FRβ expression (Fig. 1C). Folate binding and uptake by M-CSF macrophages were specific, as both were inhibited by a 100 mol/L
excess of folic acid (Fig. 1D). Moreover, folate conjugates entered
cells by endocytosis because most of the folate-FITC fluorescence
could not be stripped from the cell surface by an acid wash step
(Supplementary Fig. S1). Considering that neither FOLR1 nor
FOLR3 was expressed by M-CSF macrophages (Supplementary
Fig. S2), FOLR2-encoded FRβ protein must be responsible for
the folate binding ability of M2 macrophages. Kinetic studies revealed that FOLR2 mRNA and FRβ protein are initially detected 48
to 72 hours after M-CSF addition, and that their levels dramatically increase at later incubation times (Fig. 2A and B). Acquisition of
folate uptake ability correlated with protein expression at all time
points and showed its highest level at the end of the culture period
(Fig. 2C). Therefore, M-CSF promotes the expression of a functional FRβ protein, which constitutes a marker of M-CSF–polarized
M2 macrophages.
Expression of FRβ in TAM. TAM are an M2-skewed macrophage population that exhibits immunosuppressive activity within
the tumor microenvironment, and whose recruitment and differentiation is influenced by M-CSF (9). Given the preferential expression of FRβ in M-CSF–polarized M2 macrophages, its presence
was evaluated in TAM. Immunohistochemistry revealed that FRβ
is frequently coexpressed with CD163 in TAM from primary and
metastatic melanoma (Figs. 3A and 4A) but is absent from melanoma HMB-45+ cells (Figs. 3A and 4A). In fact, FOLR2 mRNA could
be detected in three melanoma samples (Fig. 3B). Ex vivo isolated
CD14 + TAM from the pleural fluid of a metastatic melanoma
Figure 2. Acquisition of FRβ expression
on monocyte treatment with M-CSF.
A, FOLR2 mRNA expression levels
along M-CSF–induced polarization of
macrophages, as determined by
quantitative RT-PCR. Columns, mean
normalized fold expression (relative to 18S
rRNA levels) from triplicate determinations;
bars, SD. B, FRβ expression along M1
and M2 macrophage polarization, as
determined by Western blot at the indicated
time points. As a control, CD29
expression levels were also determined.
C, internalization of folate-FITC during
M-CSF–induced macrophage polarization
(empty histograms), as determined by flow
cytometry at the indicated time points.
Filled histograms, cell autofluorescence.
The percentage of marker-positive cells
and the mean fluorescence intensity
(in parentheses) are indicated in each
case.
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Cancer Research
Figure 3. Expression and function of FRβ in TAM isolated from melanoma. A, confocal sections of infiltrating macrophages on a subcutaneous primary melanoma
tissue sample, as determined by double immunofluorescence analysis of FRβ (green) and the macrophage marker CD163 (red; top), or FRβ and the melanoma
marker HMB-45 (red; bottom). The corresponding scatter plots are shown, and colocalizing pixels (blue rectangles) are displayed on the merge images as white
masks. Magnification of a FRβ/CD163 colocalizing area appears enlarged in the top image, and the enlarged area is depicted in white. In the top image, note the
coexpression of FRβ by tumor-infiltrating macrophages (CD163+), whereas nonstained areas correspond to tumor cells (CD163−). Nuclei were counterstained with
4′,6-diamidino-2-phenylindole (DAPI). B, detection of FOLR2, MAFB, IL10, MAGEA3, and GAPDH mRNA by RT-PCR on RNA from two different primary melanoma
tissues (lanes 2 and 3) and from CD14+ cells isolated from the pleural fluid of a metastatic melanoma (lane 4). Control RT-PCR reactions were loaded in lane 1,
next to the lane containing the molecular size markers. C, expression of CD68, CD163, Tie2, and FRβ in CD14+ TAM isolated from a melanoma pleural fluid, as
determined by three-color flow cytometry analysis on permeabilized cells. Isotype-matched monoclonal antibodies and a preimmune rabbit antiserum were used as
negative controls. The percentages of single- and double-positive cells are indicated. D, binding (4°C) and internalization (37°C) of folate-FITC by CD14+ TAM
isolated from a metastatic melanoma (empty histograms). Filled histograms, cell autofluorescence. The percentage of marker-positive cells and the mean fluorescence
intensity (in parentheses) are indicated.
expressed mRNA for FOLR2, IL10, and the macrophage-specific
MAFB (31), whereas they lacked expression of the melanomaspecific marker MAGEA3 mRNA (Fig. 3B, lanes 4) and were devoid of FOLR1 and FOLR3 mRNA (Supplementary Fig. S3).
Three-color analysis on isolated melanoma TAM indicated that
all FRβ+ macrophages are CD68+, and that the percentage of
FRβ+ CD163+ macrophages (87%) is similar to that of CD163+
CD68+ cells (88%; Fig. 3C). Thus, most melanoma TAM from
the analyzed sample coexpress CD163, CD68, and FRβ and exhibit folate-FITC internalization ability (Fig. 3D). It is also worth
noting that a percentage of FRβ+ macrophages coexpress Tie2
(36%; Fig. 3C). Altogether, these results indicate that FRβ is
functionally expressed on IL10 mRNA–expressing CD14+ CD68+
CD163+ melanoma TAM.
Evaluation of FRβ expression on other tumor tissues indicated
that FRβ is detected in the stroma of lung, ovary, colon, gastric,
and breast cancers, where numerous CD68+ TAM were also present (Fig. 4B). Analysis of ex vivo isolated CD14+ TAM from a metastatic breast adenocarcinoma also revealed the coexpression of
CD68 and FRβ, and that 80% of the cells exhibited a CD163 +
FRβ+ phenotype (Fig. 5A). Importantly, primary and metastatic
breast adenocarcinoma tissues were found to contain both FOLR2
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44
and MAFB mRNA (Fig. 5B), and ex vivo isolated CD14+ metastatic
breast adenocarcinoma TAM expressed FOLR2, IL10, and MAFB
mRNA (Fig. 5B, lane 5). CD14+ CD163+ TAM also exhibited specific
folate-FITC binding and uptake (Fig. 5C) and produced IL-10 in response to LPS stimulation (Fig. 5D). Because FOLR2 and IL10
mRNA are coexpressed in M2 macrophages in vitro (Fig. 1), and
TAM from metastatic breast adenocarcinoma express functional
FRβ and produce IL-10, these results indicate that FRβ activity
marks anti-inflammatory M2-like TAM.
Parameters affecting FRβ expression on human macrophages. GM-CSF and M-CSF are tumor-derived factors that modulate myeloid cell differentiation (33). Unlike M-CSF, GM-CSF
abrogated the aquisition of FOLR2 mRNA during in vitro monocyte-tomacrophage differentiation, even in the presence of M-CSF (Fig. 6A).
This result explains the differential expression of FRβ on both types
of macrophages, and suggests that the relative levels of tissue
GM-CSF and M-CSF determine macrophage FRβ expression. Other
cytokines commonly released by tumors (33) also affected FOLR2
mRNA; IL-6 alone and IL-10 in combination with M-CSF upregulated FOLR2 mRNA expression (Fig. 6B). Therefore, tumor-derived
cytokines (M-CSF, GM-CSF, IL-6, and IL-10) modulate FRβ
expression in human macrophages.
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Folate Receptor β Is an M2 Macrophage Marker
Expression of FRβ on TAM led us to analyze the nature of the
stimuli that might control its presence in the tumor microenvironment. FOLR2 mRNA was variably upregulated by supernatants
from tumor cell lines, with placenta choriocarcinoma JAR and
JEG-3 cells and ovary carcinoma NIH-OVCAR-3 cells promoting
the highest level of upregulation (Fig. 6C). In contrast, conditioned
media from colon carcinoma Colo320 cells had no effect (Fig. 6C).
More importantly, ascitic fluid from the breast carcinoma analyzed
in Fig. 5 promoted a strong upregulation of FOLR2 mRNA (Fig. 6C),
confirming that tumor cells release factors that upregulate human
macrophage FRβ expression. The addition of a blocking anti–
M-CSF monoclonal antibody greatly reduced the upregulation
of FOLR2 mRNA promoted by ascitic fluid from breast carcinoma (Fig. 6D) or by conditioned medium from tumor-associated
fibroblasts or JEG-3 tumor cells (Fig. 6D). Therefore, M-CSF is a
major determinant for FRβ expression on human macrophages
and contributes, alone or in combination with other cytokines,
to FRβ cell surface expression on TAM.
Discussion
GM-CSF and M-CSF contribute to the generation of different
macrophage subsets and enhance myeloid cell survival and proliferation (9). However, GM-CSF promotes the generation of myeloid
cells with potent antigen presentation activity, whereas M-CSF
leads to the generation of macrophage cells with regulatory properties (9). Gene expression profiling allowed us to identify FRβ as
preferentially expressed by macrophages generated under the influence of M-CSF, which display FRβ-dependent folate binding
ability. FRβ expression on in vitro differentiating macrophages
was enhanced by M-CSF and by tumor cell-conditioned medium
in an M-CSF–dependent manner. Conversely, GM-CSF prevented
the acquisition of FRβ expression. Importantly, FRβ was detected
in TAM, where FRβ-mediated folate binding activity correlates with
the presence of IL10 mRNA. Therefore, FRβ constitutes a marker for
M-CSF–primed IL-10–expressing M2-polarized macrophages, providing a molecular basis for the value of folate-conjugated drugs
in cancer therapy approaches.
Figure 4. Expression of FRβ in TAM from primary and metastatic melanoma. A, expression of FRβ in melanoma-infiltrating macrophages on a primary melanoma
(#130, bottom) or two metastatic melanomas (#98 and #146, top and middle), as determined by double immunofluorescence analysis of FRβ and the macrophage
marker CD163 (top rows) or FRβ and the melanoma tumor marker HMB-45 (bottom rows). B, expression of FRβ in tumors of distinct tissue origins. Light microscopy
images of the macrophage marker CD68 (middle) and FRβ (right) staining of tumor tissue from lung squamous cancer (1; magnification, ×20), ovarian
cystadenoma-mucous (2; magnification, ×20), rectal colon adenocarcinoma (3; magnification, ×20), gastric adenocarcinoma (4; magnification, ×40), and breast
invasive ductal cancer (5; magnification, ×40). Left, staining yielded by normal rabbit serum, used as a control.
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Because macrophage polarization is stimulus dependent (1), alternatively activated M2 macrophages have been further classified
as M2a, M2b, or M2c in an effort to link genetic markers to specific
macrophage-activating stimuli (34). The expression of FRβ in
M-CSF–generated macrophages indicates that it is preferentially
expressed by IL-10–producing M2 macrophages and, therefore,
identifies a population of macrophages with anti-inflammatory/
regulatory properties. The presence of FRβ in M-CSF–primed
in vitro macrophages is in agreement with its upregulation in human decidual macrophages, which exhibit an immunosuppressive
phenotype and whose gene expression profile closely corresponds
to that of M2-polarized macrophages (35). Further supporting its
presence on M2 macrophages, FRβ has been detected on F4/80+
CD68+ murine peritoneal macrophages (36), where its mRNA levels
can be further upregulated by IL-4 (37). Therefore, FRβ expression
seems not to be restricted to anti-inflammatory/regulatory IL-10–
producing M2 macrophages and marks a wider range of alternatively activated macrophages in the human and murine systems.
However, the functional state of FRβ on murine peritoneal macrophages is still not clear because folate binding ability is only detected after stimulation with inflammatory stimuli (26).
The expression of FRβ on TAM from primary and metastatic
melanoma and breast carcinoma (Figs. 3–5) is also in agreement
with a previous report describing the presence of FRβ in CD68+
CD163+ cells within human and rat glioblastoma (36). In an apparent contradiction, gene expression profiling has revealed downregulated FRβ mRNA levels in murine fibrosarcoma TAM relative to
the levels detected in thioglycollate-elicited peritoneal macrophages (12). However, because the latter exhibit functional characteristics of M-CSF–driven M2 macrophages (38), these results do
not rule out the presence of detectable levels of FRβ in murine
TAM. Besides, it is also possible that FRβ is expressed by TAM
in a tumor-dependent manner, a phenomenon which would be
in agreement with its differential upregulation by distinct tumorconditioned media (Fig. 6) and the variable levels of FRβ in TAM
from a variety of human tumors (Fig. 4). Finally, it is also possible
that differences might exist between murine and human TAM,
as it is already evident that paradigmatic M2 murine macrophage
markers (Arginase and Ym1) are not useful to identify human alternatively activated macrophages (39). Whether the acquisition
of FRβ expression by tumor-infiltrating macrophages is detrimental for the tumor (e.g., by removing folate) or favors tumor
cell growth is a matter that deserves further investigation. Regardless of the precise role of FRβ on TAM, the presence of
FRβ on their cell surface provides an opportunity for depletion
of TAM through the use of folate-conjugated drugs. As an example, and while this article was being completed, Nagai and coworkers have shown the feasibility of reducing tumor growth by
Figure 5. Expression and function of FRβ in TAM isolated from breast adenocarcinoma. A, expression of FRβ, CD68, and CD163 in permeabilized CD14+ TAM
from a metastatic breast adenocarcinoma, as determined by flow cytometry using PE-labeled anti-CD68, Alexa Fluor 647–labeled anti-CD163, and a polyclonal
antiserum against human FRβ (18), followed by FITC-labeled goat anti-rabbit antibodies. Isotype-matched monoclonal antibodies and a preimmune rabbit antiserum
(29) were used as negative controls (top). The percentages of single-positive and double-positive cells are indicated. B, detection of the indicated mRNA by
RT-PCR on RNA from three different primary breast adenocarcinoma tissues (lanes 2–4) and from CD14+ cells isolated from ascitic fluid from a metastatic breast
adenocarcinoma (lane 5). Control RT-PCR reactions were loaded in lane 1, next to the lane containing the molecular size markers. C, binding (4°C; top) and
internalization (37°C; bottom) of folate-FITC by CD14+ TAM isolated from a metastatic breast adenocarcinoma, in the absence (empty histograms, black line) or the
presence (empty histograms, gray line) of a 100 mol/L excess of folic acid. Filled histograms, cell autofluorescence. The percentage of marker-positive cells and
the mean fluorescence intensity (in parentheses) are indicated. The experiment was done two times, and one of the experiments is shown. Representative confocal
sections and differential interference contrast microscopy images of macrophages incubated with folate-FITC. D, CD14+ TAM isolated from a metastatic breast
adenocarcinoma were either untreated or stimulated with LPS (50 ng/mL) for 24 h, and IL-10 release was determined by ELISA. Columns, mean of triplicate
determinations; bars, SD.
Cancer Res 2009; 69: (24). December 15, 2009
46
9400
www.aacrjournals.org
Folate Receptor β Is an M2 Macrophage Marker
Figure 6. Parameters affecting FRβ expression on human macrophages. A and B, FOLR2 mRNA expression in macrophages exposed for 72 h to the indicated
cytokines, as determined by quantitative RT-PCR. Results are expressed as normalized fold expression relative to 18S rRNA levels and the FOLR2 RNA levels in
peripheral blood monocytes (Mon.). Columns, mean of triplicate determinations; bars, SD. C, FOLR2 mRNA expression in macrophages exposed for 72 h to conditioned
media from the ascitic fluid of a breast carcinoma (ABC) or tumor cell lines, as determined by quantitative RT-PCR. Results are expressed as normalized fold
expression (relative to 18S rRNA levels). Columns, mean of triplicate determinations from three independent macrophage preparations; bars, SD. D, inhibitory effect of
anti–M-CSF on FOLR2 mRNA levels induced by ascitic fluid from metastatic breast carcinoma (ABC) or conditioned-medium from JEG-3 placenta choriocarcinoma
(JEG-3) or tumor-associated fibroblasts (Fibroblast). The results are depicted as the FOLR2 mRNA levels detected in the presence of the anti–M-CSF antibody relative
to the levels seen in untreated cells (set to 100 in the three cases).
targeting an immunotoxin to TAM using an antimouse FRβ
monoclonal antibody (36).
Given the tumor influence on macrophage functions (40), FRβ
might specifically mark tumor-infiltrating human macrophages
whose effector functions have been already skewed by tumor-derived
factors. In this regard, our data also suggest that tumor-derived
M-CSF, which recruits and shapes macrophage functions (33),
would be the primary determinant for FRβ expression. However,
FRβ expression is also detected in resident macrophages within
nontumor tissue (data not shown). This fact, together with the increase in FRβ expression during the in vitro macrophage differentiation that takes place in the absence of exogenous cytokines,
might indicate that FRβ could be a macrophage differentiation
marker under homeostatic conditions, and whose levels could be
maintained or upregulated by anti-inflammatory cytokines and
downregulated by pro-inflammatory stimuli. In this regard, cytokines such as IL-4, IL-13, and IL-10, which promote macrophage
alternative activation, trigger a transient increase of FOLR2 mRNA
www.aacrjournals.org
levels in M2 macrophages (Supplementary Fig. S4A and B). By contrast, LPS greatly downregulates FOLR2 mRNA levels (Supplementary Fig. S4A and B) although it does not lead to a great decrease in
cell surface FRβ (Supplementary Fig. S4C). This divergence might
be explained by the fact that FOLR2 is an endocytic receptor whose
protein levels are higher than those present on the cell surface. In
fact, flow cytometry on permeabilized cells showed that a large
proportion of FRβ is located intracellularly in both in vitro M2
macrophages and TAM (Supplementary Fig. S5).
The presence of functional FRβ on M-CSF–primed macrophages
and the detection of FRβ mRNA in other types of M2-polarized
macrophages (12, 35, 37) are difficult to reconcile with its expression (25) and function (26) in synovial macrophages from rheumatoid arthritis patients, which are embedded in a inflammatory
pro-M1 environment. It could be speculated that synovial macrophages might exhibit a mixed M1/M2 phenotype, similar to what
occurs with myeloid populations within tumors (41), an explanation that would be compatible with the high levels of M-CSF
9401
Cancer Res 2009; 69: (24). December 15, 2009
47
Cancer Research
present in rheumatoid arthritis synovia (42). M-CSF is produced by
synovial fibroblasts, and administration of M-CSF is known to exacerbate arthritis in some settings (9, 43). Therefore, the levels of
M-CSF within the synovia of rheumatoid arthritis might suffice to
promote FRβ expression on surrounding macrophages, although
the concomitant presence of extremely high levels of tumor necrosis factor α might override its immunosuppressive actions. Alternatively, because M-CSF contributes to macrophage recruitment, FRβ
expression might mark macrophages newly recruited into the
arthritic synovia, whose later levels of FRβ expression would be
determined by the pro-inflammatory environment. In this regard,
it is worth noting that (a) FRβ+ macrophages are more prominently
detected at early stages during development of animal models of
atherosclerosis and muscle injury and in rheumatoid arthritis in
humans;6 (b) in vitro acute (48 hours) exposure of FRβ-expressing
M2 macrophages to M1-polarizing stimuli (e.g., LPS, GM-CSF,
and IFNγ) does not result in loss of FRβ expression, which is only
moderately downregulated by IFNγ (Supplementary Fig. S4C).
Therefore, folate-targeted killing of FRβ+ macrophages in inflammatory disease murine models might contribute to inflammation resolution by preferentially eliminating newly recruited macrophages.
6
P. Low, personal communication.
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 6/4/09; revised 10/9/09; accepted 10/15/09; published OnlineFirst 12/8/09.
Grant support: Ministerio de Educación y Ciencia (grant BFU2008-01493-BMC),
Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (Spanish Network for
the Research in Infectious Diseases, REIPI RD06/0008), and Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36663/07; A.L. Corbí); grant
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49
Supplementary Figures
Folate-FITC
Folate-FITC + Acidic wash
1.0 (4.7)
94 (35)
74 (13.5)
Supplementary Figure 1.- Folate-FITC internalization ability of M-CSF-primed M2 macrophages.
Internalization was done for 1 hour at 37ºC and cells were subsequently either untreated (empty histogram,
black line) or subjected to an acidic cold wash with PBS 50 mM Glycine pH 3.2 (empty histograms, grey line)
to eliminate cell surface-bound fluorecence. Autofluorescence of cells is indicated (grey histogram). In all
cases, the percentage of fluorescence-positive cells and the mean fluorescence intensity (between parenthesis)
are indicated.
A
B
20
2.0
FOLR1
FOLR2
FOLR3
Normalized Fold Expression
Normalized Fold Expression
HeLa
M2 (MCSF)
Monocytes
15
10
5
FOLR1
FOLR2
FOLR3
1.5
1.0
0.5
Donor
#1
Donor
#2
Donor
#3
Supplementary Figure 2.- A. FOLR1, FOLR2 and FOLR3 mRNA expression levels determined by qRT-PCR
in HeLa cells, M2 (MCSF) macrophages and peripheral blood monocytes, and expressed as Normalized Fold
Expression (relative to 18S rRNA levels). Shown is the mean and standard deviation of triplicate
determinations for each gene. B. FOLR1, FOLR2 and FOLR3 mRNA expression levels determined by qRTPCR in three different M2 macrophage preparations, and expressed as Normalized Fold Expression (relative to
18S rRNA levels). Shown is the mean and standard deviation of triplicate determinations for each gene.
51
16
14
B
FOLR2
12
10
8
6
4
2
MCSF LPS
C
Folate
capture
FRβ
expression
M2
IL-4
Normalized Fold Expression
Normalized Fold Expression
A
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
IL-13 IL-10
1.0 (3)
74 (14)
3.0 (4.3)
1.0 (3.4)
82 (13.5)
1.0 (3.3)
44 (9.2)
MCSF LPS
M2 + IL-4
M2 + IFN-γ
2.0 (3)
96 (33)
6.0 (5.6)
FOLR2
M2 + GM-CSF
5.0 (4)
87 (34)
23 (7)
1.0 (3.7)
34 (8.7)
IL-4
IL-13 IL-10
M2 + LPS
3.0 (4.3)
95 (33)
24 (7)
1.0 (3.5)
78 (15)
5 (5.5)
1.0 (3.6)
62 (12)
1.0 (4.3)
71 (15)
Folate-FITC
Folate-FITC
+ 100X Folic Acid
Supplementary Figure 3.- A-B. FOLR2 mRNA expression in day-7 M2 macrophages exposed for 24 (A) or 48
(B) hours to LPS, IL-4, IL-13 or IL-10, as determined by qRT-PCR. Results are expressed as Normalized Fold
Expression (relative to GAPDH mRNA levels and the FOLR2 mRNA levels in macrophages exposed to MCSF). Shown is the mean and standard deviation of triplicate determinations. C. Folate-FITC capture ability
(upper panels) and FRβ cell surface expression (lower panels) in M-CSF-primed M2 macrophages exposed to
the indicated cytokines for the last 48 hours of the 7-day differentiation process. Internalization was done either
in the absence (empty histograms, black line) or the presence (empty histograms, grey line) of a 100-molar
excess of folic acid. Filled histograms (thin line) indicate cell autofluorescence in each case. Cell surface
expression was determined by flow cytometry using a polyclonal antiserum against human FRβ (empty
histograms with thick lines) and a previously reported pre-immune rabbit antiserum as negative control (filled
histograms with thin lines). The percentage of marker-positive cells and the mean fluorescence intensity
(between parenthesis) are indicated. Each experiment was performed twice, and a representative one is shown.
M2
CD14+ TAMs
(Breast Adenocarcinoma)
2 (3,5)
93 (14)
Control
FRβ
2 (4,4)
67 (24)
Non-permeabilized
2 (4)
100 (84)
11 (4)
99 (76)
Permeabilized
Supplementary Figure 4.- FRβ expression in M-CSF-primed M2 macrophages (left panels) and isolated
CD14+ TAM from a breast adenocarcinoma (right panels) as determined by flow cytometry using a polyclonal
antiserum against human FRβ (empty histograms with thick lines) and a previously reported pre-immune rabbit
antiserum as negative control (filled histograms with thin lines). In both cases cells were analyzed before
(upper panels) or after permeabilization (lower panels), to detect only cell surface or total content of FRβ. The
percentage of marker-positive cells and the mean fluorescence intensity (between parenthesis) are indicated in
each case.
52
Resultados
2. Activina A previene la adquisición de marcadores anti-inflamatorios/M2 y
sesga la secreción de citoquinas por los macrófagos.
La progresión tumoral está favorecida por el cambio en la polarización de los macrófagos
asociados a tumores hacia la adquisición de funciones efectoras inmunoreguladoras y antiinflamatorias. A diferencia del GM-CSF que polariza los macrófagos hacia un fenotipo inflamatorio
M1, el M-CSF genera macrófagos inmunosupresores M2 que expresan el receptor de folato β (FRβ)
y producen IL-10. Debido a que la depleción de macrófagos FRβ+ ha sido utilizado en terapia frente
a tumores, hemos buscado factores que controlan la expresión del FRβ en macrófagos. En este
sentido, hemos identificado a la activina A como una citoquina producida por los macrófagos M1
(GM-CSF), y cuya presencia limita la adquisición de la expresión del FRβ y otros marcadores M2 (MCSF). De hecho, el GM-CSF promueve la expresión de activina A, mientras que es inhibida por MCSF incluso en macrófagos M1 (GM-CSF). La activina A secretada por los macrófagos M1 (GMCSF) realza la actividad de los promotores génicos dependientes de Smad, explicando así la
activación diferencial de Smad2 en los macrófagos M1 (GM-CSF) y M2 (M-CSF), lo que contribuye a
la inhibición del crecimiento de células tumorales por el medio condicionado de macrófagos M1 (GMCSF). Además, la activina A modula la producción de citoquinas por los macrófagos M2, ya que
reduce la producción de IL-10, aunque no modifica la secreción de TNFα, en respuesta a LPS. Por
lo tanto, la activina A sesga la polarización del macrófago contribuyendo a la generación de
macrófagos inflamatorios en respuesta a GM-CSF y limitando la generación de macrófagos antiinflamatorios.
53
Activin prevents the acquisition of M2/anti-inflammatory markers and skews the
macrophage cytokine profile
Running head: Activin shapes macrophage polarization
Elena Sierra-Filardi
∗,‡
, Amaya Puig-Kröger
∗,‡
, Francisco J. Blanco*, Carmelo Bernabéu*,
Miguel A. Vega ∗ and Angel L. Corbí ∗
∗
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
‡
Both authors contributed equally and the order of authors should be considered arbitrary.
Corresponding author: Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, CSIC. Ramiro de
Maeztu, 9. Madrid 28040; Phone: 34-91-8373112, ext. 4376; FAX: 34-91-5627518.
E-mail: [email protected]
1
This work was supported by the Ministerio de Ciencia e Innovación (Grant BFU2008-01493-BMC),
Instituto de Salud Carlos III (Spanish Network for the Research in Infectious Diseases REIPI
RD06/0008, and Red de Investigación en SIDA RIS RD06/0006/1016), Fundación para la
Investigación y Prevención del SIDA en España (FIPSE 36663/07), and Fundación Mutua Madrileña,
to ALC, and grant PI08/1208 from Instituto de Salud Carlos III to APK. APK is supported by
Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (CP06/00199).
2
Address correspondence and reprint requests to: Dr. Angel L. Corbí, Centro de Investigaciones
Biológicas, CSIC. Ramiro de Maeztu, 9, 28040 Madrid. Tel. +34-91-8373112 + 4376; FAX: 34-915627518; E-mail addresses: [email protected]
55
ABSTRACT
Tumor progression is favored by the shift in the polarization state of tumor-associated macrophages
towards the acquisition of immunoregulatory and anti-inflanmatory effector functions. Unlike GMCSF, which polarizes macrophages towards a pro-inflammatory M1 phenotype, M-CSF generates IL10-producing Folate Receptor β (FRβ)-expressing immunosuppressive M2 macrophages. Since
depletion of FRβ-expressing macrophages has proven successful in cancer therapy strategies, we
sought to identify the factors controlling macrophage FRβ expression. The search identified Activin
A as a cytokine produced by M1 (GM-CSF) macrophages, and whose presence limits the acquisition
of FRβ and other M2 (M-CSF)-specifc markers. In fact, GM-CSF promotes Activin A expression,
whereas M-CSF downregulates its expression even in fully polarized M1 (GM-CSF) macrophages.
M1 (GM-CSF) macrophage-derived Activin A enhances the activity of Smad-dependent gene
promoters, thus explaining the differential Smad2 activation of M1 (GM-CSF) and M2 (M-CSF)
macrophages, and contributes to the tumor cell growth inhibitory activity of M1 (GM-CSF)
Macrophage-conditioned medium. Besides, Activin A modulates cytokine production by M2
macrophages, as it reduces their LPS-induced IL-10 production and it had no effect on the TNFα
release in response to LPS. Therefore, Activin A skews macrophage polarization by contributing to
the generation of pro-inflammatory macrophages in response to GM-CSF and limiting the generation
of anti-inflammatory macrophages.
56
INTRODUCTION
Tissue resident macrophages are phenotypically and functionally heterogeneous under homeostatic
conditions because of their extreme sensitivity to the extracellular cytokine millieu 1-3. Although GMCSF and M-CSF contribute to cell survival, proliferation and macrophage development, they exert
distinct actions during macrophage differentiation in vivo and in vitro. Deficiency of M-CSF alters
the development of various macrophage populations 4, whereas GM-CSF-deficient mice only exhibit
altered maturation of alveolar macrophages 5. Along the same line, both cytokines promote the in
vitro differentiation of macrophages with distinct morphology, pathogen susceptibility
6
and
inflammatory function 7-10. GM-CSF gives rise to monocyte-derived macrophages with high antigenpresenting properties and which produce pro-inflammatory cytokines in response to LPS, whereas
M-CSF leads to the generation of macrophages with high phagocytic activity and IL-10-producing
ability in response to pathogens 10,11. Based on their respective cytokine profiles, human macrophages
generated in the presence of GM-CSF or M-CSF are considered as representative of the classically
(M1) or alternatively activated (M2) macrophage polarization states, respectively
10,12,13
. Moreover,
since they might play opposite roles during immune and inflammatory responses, M1 (GM-CSF) and
M2 (M-CSF) macrophages are now considered as pro- and anti-inflammatory macrophages
10,12
,
respectively.
Activins are pluripotent and ubiquitous growth and differentiation factors, which are structurally
composed of two β subunits (activin-A, βAβA; activin-AB, βAβB; activin-B, βBβB) linked by a
single covalent disulfide bond
14,15
. Activin biological activities are mediated by signal transduction
molecules shared by TGFβ (Smad2,3)
16
. Like TGFβ, activins exert both immunostimulatory and
immunosuppressive functions at the T cell level
promotion of macrophage alternative activation
16
, and their effects on myeloid cells include
17
and inhibition of CD40L-induced cytokine
18
production by monocyte-derived dendritic cells . Activin A expression has been detected in many
immune cell types 16, and is upregulated upon activation and in response to inflammatory mediators
both in vitro
18
and in vivo
19
, what has led to the suggestion that it functions as a modulator of
inflammatory responses by limiting cytokine and chemokine release.
We have recently dissected the differences in gene expression between M1 (GM-CSF) and M2 (MCSF) macrophages, and described the preferential expression of Folate Receptor β (FRβ) on in vitro
derived M2 (M-CSF) macrophages and ex vivo Tumor-Associated Macrophages (TAM)
20
. To
identify factors mediating the acquisition of their respective profiles, we analyzed whether M1derived factors influenced the acquisition of M2-specific markers. M1 macrophages were found to
secrete large amounts of functional Activin A, whose presence conditions the activation state of the
57
TGFβ signaling system, impairs the acquisition of M2 (M-CSF) markers, and modulates the
production of IL-10. These results place Activin A as a factor that contributes to macrophage
polarization and shapes the inflammatory behaviour of macrophages. Moreover, given the
macrophage ability for re-polarization under appropriate cytokine conditions
21
, Activin A might
function, in an autocrine or paracrine manner, by halting macrophage switch between polarization
states.
58
MATERIALS AND METHODS
Cell culture and flow cytometry.- Human peripheral blood mononuclear cells (PBMC) were isolated
from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway)
gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell
sorting using CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes (>95%
CD14+ cells) were cultured at 0.5 x 106 cells/ml for 7 days in RPMI supplemented with 10% fetal
calf serum (FCS) (completed medium), at 37ºC in a humidified atmosphere with 5% CO2, and
containing 1000U/ml GM-CSF or M-CSF (10 ng/ml, ImmunoTools GmbH, Friesoythe, Germany) to
generate M1 and M2 monocyte-derived macrophages, respectively. Cytokines were added every two
days. When indicated, recombinant human Activin A (2.5-25 ng/ml, Miltenyi Biotech, Bergisch
Gladbach, Germany) was added together with the indicated cytokine. To generate monocyte-derived
dendritic cells (MDDC), monocytes were cultured at 0.7 x 106 cells/ml in complete medium
containing GM-CSF (1000 U/ml) and IL-4 (1000U/ml, ImmunoTools GmbH, Friesoythe, Germany)
for 5-7 days, with cytokine addition every second days. When indicated, M1 macrophages were
treated with IL-4 (1000U/ml) or IFNγ (500U/ml) for 48 hours. The mink lung epithelial cell line
Mv1Lu
22
was maintained in DMEM supplemented with 10% fetal calf serum. Phenotypic analysis
was carried out by flow cytometry as previously reported 20, and using rabbit polyclonal antisera antihuman FRβ 23 or a previously described preimmune serum 24, and FITC-labelled Fab goat anti-rabbit
IgG. All incubations were done in the presence of 50 µg/ml of human IgG to prevent binding through
the Fc portion of the antibodies.
Western blot.- Cell lysates were obtained in 10mM Tris-HCl pH 8, 150mM NaCl, 1%NP-40 (NP-40
lysis buffer) containing 2 mM Pefabloc and 2 µg/ml of aprotinin, antipain, leupeptin and pepstatin.
Ten µg of cell or membrane lysates was subjected to SDS-PAGE and transferred onto an Immobilon
polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking of the unoccupied
sites with 5% non-fat dry milk in 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20, protein
detection was performed using the Supersignal West Pico Chemiluminescent system (Pierce,
Rockford, IL). Protein detection was carried out using polyclonal antisera against phosphorylated
Smad2 (pSmad2 (Ser465/467), clone A5S, Millipore), Smad2 (anti-Smad2/3, Millipore), GAPDH
(sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin (Sigma-Aldrich, UK ).
ELISA.- Supernatants from M1 and M2 macrophages were tested for the presence of cytokines and
growth factors using commercially available ELISA for TNF-α (ImmunoTools), IL-6
(Immunotools), IL-12p40 (OptEIATM IL-12p40 set, BD Pharmingen, San Diego, CA), IL-10
59
(ImmunoTools), and Activin A (R&D Systems, Inc, Minneapolis, USA) following the protocols
supplied by the manufacturers.
Reporter Gene Assays.- The effect of macrophage culture supernatants on the Activin signaling
pathway was analyzed by transfecting 0.5 μg of the p3TP-Lux reporter construct 25 in Mv1Lu cells, a
well stablished cellular model to study the signaling of the TGF-β superfamily
22
, using Superfect
(Qiagen). After transfections, cells were washed, cultured in DMEM plus 0.2% FCS, and treated with
undiluted condition media from M1 (GM-CSF) or M2 (M-CSF) macrophages, 25 ng/ml rhActivin A
(Miltenyi Biotec) or 10 ng/ml TGF-β1 (R&D Systems) for 24 hours. When indicated, cells were
preincubated for 30 minutes with 10 μM SB431542 (Sigma), an ALK4, 5 and 7 inhibitor, before
treatment. Activin A activity in M1 supernatants was neutralized using 0.1 μg/ml of a blocking
antibody (R&D Systems). In some experiments, cells were cotransfected with 0.4 μg of expression
vectors for a dominant negative mutant of either Smad2
26
or Smad3
27
. To normalize transfection
efficiency, cells were co-transfected with an SV40 promoter-based β-galactosidase expression
plasmid (RSV-βgal). Measurement of relative luciferase units and β-galactosidase activity were
performed using the Dual-Glo Luciferase Assay System (Promega) and the Galacto-Ligth kit
(Tropix), respectively, in a Varioskan Flash spectral scanning multimode reader (Thermo Scientific).
Quantitative real-time RT-PCR.- Oligonucleotides for selected genes were designed according to
the Roche software for quantitative real time PCR. Total RNA from M1 and M2 macrophages was
extracted using the RNAeasy kit (Qiagen), retrotranscribed and amplified using the Universal Human
Probe Roche library (Roche Diagnostics). Assays were made in triplicates and results normalized
according to the expression levels of 18S RNA and GAPDH.
the ΔΔCT method for quantitation.
60
Results were expressed using
RESULTS
M1 (GM-CSF)-conditioned medium prevents acquisition of Folate Receptor β (FRβ) expression.Expression of cell surface FRβ, encoded by the FOLR2 gene, identifies IL-10-producing ex vivo
isolated Tumor-Associated Macrophages (TAM) 20, as well as M2 (M-CSF), but not M1 (GM-CSF),
in vitro polarized macrophages (Figure 1A). FRβ mediates the capture of Folate-FITC by M2 (MCSF) polarized macrophages, but not GM-CSF-polarized (M1) macrophages (Figure 1B). That the
differential expression FRβ in M1 and M2 macrophages is due to the opposite effect of GM-CSF and
M-CSF on FOLR2 gene expression was indicated by the dramatic downregulation of FOLR2 RNA
levels in FRβ-positive M2 (M-CSF) macrophages after exposure to GM-CSF (Figure 1C), and by the
inhibitory effect of M1 (GM-CSF) macrophage-conditiones medium on the FOLR2 RNA induction
that takes place in cytokine-free medium
20
(Figure 1D). In fact, this inhibitory activity was evident
even after a 1/10 dilution of the M1 (GM-CSF)-conditioned medium, which reduced FOLR2 RNA
induction by more than 90% (Figure 1D). Therefore, M1 (GM-CSF) macrophages secrete factor(s)
that prevent the acquisition of the M2 (M-CSF)-specific marker FRβ.
1% (3)
1% (4)
1% (3)
82% (13)
M2
M1
B
D
Transferrin-Texas Red
Folate-FITC
M1
Relative mRNA levels
C
FRβ
Control
M2
1.2
FOLR2
GM-CSF
1.0
M-CSF
0.8
0.6
0.4
0.2
Relative mRNA levels
A
10
FOLR2
8
6
4
2
Mo. 0 10 50 100
% SN M1
Figure 1.- M1 (GM-CSF)-conditioned medium inhibits the M-CSF-induced Folate Receptor β (FRβ) expression.- A.
Cell surface expression of FRβ on M1 and M2 macrophages, as determined by flow cytometry using a polyclonal antiserum
against human FRβ 23 (empty histogram). As a control, a previously described rabbit pre-immune antiserum 24 was used
(filled histogram). The percentage of marker-positive cells and the mean fluorescence intensity (in parenthesis) are
indicated. B. FRβ function in M1 and M2 macrophages, as demonstrated by confocal microscopy on cells incubated at 37ºC
with Folate-FITC (green fluoresence) and Transferrin-Texas red (red fluorescence). C. FOLR2 mRNA expression levels, as
determined by qRT-PCR on M2 (M-CSF) macrophages after replacement of the culture supernatant by either M-CSF- (grey
histograms) or GM-CSF-containing complete medium (empty histograms) for 24 hours. Results are expressed as Relative
mRNA levels (relative to 18S rRNA levels and referred to the RNA levels in cells maintained in M-CSF-containing
medium). Mean and standard deviation of triplicate determinations are shown. D. FOLR2 mRNA expression levels
determined by qRT-PCR on monocytes (Mo.) and macrophages exposed to M-CSF for 7 days and either in the absence (0)
61
or in the presence of different concentrations of M1 (GM-CSF) macrophage-conditioned media (% SN M1). Results are
expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to levels detected in peripheral blood
monocytes). Mean and standard deviation of triplicate determinations are shown.
M1 (GM-CSF) Macrophages secrete Activin A, which downregulates FOLR2 gene expression.- To
identify M1 (GM-CSF)-derived factors that prevent FRβ induction, we searched for soluble factors
preferentially produced by M1 macrophages. Gene expression profiling 20 revealed that expression of
the INHBA gene, which codes for the Inhibin βA subunit 14,15, is >30-times higher in M1 than in M2
macrophages (log2 M1/M2 = 6.1; p = 5.3 x 10-8, Figure 2A), a difference further verified by qRTPCR on independent samples (Figure 2A). In fact, and unlike FOLR2, INHBA RNA expression was
induced in M2 macrophages after exposure to GM-CSF (Figure 2B), and abrogated in fully polarized
M1 (GM-CSF) macrophages upon replacement of their conditioned medium by M-CSF (Figure 2C).
In agreement with RNA data and its inducibility by GM-CSF
28
, Activin A protein levels were
considerably higher in M1 (GM-CSF)-conditioned media (Figure 2D), where Activin A levels
continuously increased from the initial stages of the M1 differentiation/polarization process (Figure
19
2E). Moreover, and although LPS increases circulating Activin A levels in vivo
, the differential
production of Activin A by both types of macrophages was maintained after LPS stimulation (Figure
2F). Altogether, these results indicate that Activin A expression is differentially regulated by GM-
10
8
6
4
2
70
60
50
40
30
20
10
C
INHBA
Microarray
GM-CSF
qPCR
M-CSF
1.0
0.8
0.6
0.4
0.2
4.0
M1
3.0
M2
2.0
1.0
#1
Addition Replace
M-CSF
#2
#3
#4
#5
#6
Donor
GM-CSF
F
6
Activin A (ng/ml)
E
Activin A (ng/ml)
D
INHBA
Activin A (ng/ml)
B
INHBA
Relative mRNA levels
log 2 M1 / M2
A
Relative mRNA levels
CSF and M-CSF, and that Activin A expression inversely correlates with FOLR2 gene expression.
M1
M2
2
1
0
1
3
Donor # 1
7
0
1
3
Donor # 2
7 days
16
M1
12
M2
8
4
M1 M1 M2 M2
+LPS
+LPS
Figure 2.- Activin A is differentially produced by M1 (GM-CSF) and M2 (M-CSF) polarized macrophages.- A. Relative
INHBA gene expression in M1 and M2 macrophages, as determined by microarray DNA analysis (empty histograms) and
quantitative RT-PCR (grey histograms). B. INHBA mRNA expression levels as determined by qRT-PCR on M2 (M-CSF)
macrophages after replacement of the culture supernatant by either M-CSF- (grey histograms) or GM-CSF-containing
complete medium (empty histograms) for 24 hours. Results are expressed as Relative mRNA levels (relative to 18S rRNA
62
levels and referred to the RNA levels in cells maintained in M-CSF-containing medium). Mean and standard deviation of
triplicate determinations are shown. C. INHBA mRNA expression levels as determined by qRT-PCR on M1 (GM-CSF)
macrophages treated with M-CSF (grey histograms) or GM-CSF (empty histograms) for 24 hours and either in their own
conditioned medium (Addition) or after replacement of the culture supernatant (Replace). Results are expressed as Relative
mRNA levels (relative to 18S rRNA levels and referred to the RNA levels determined in cells cultured with GM-CSF). D.
Determination of Activin A levels released by M1 (GM-CSF) and M2 (M-CSF) macrophages generated from peripheral
blood monocytes of six independent donors, as determined by ELISA. Each determination was performed in triplicate, and
mean and standard deviations are shown. E. Determination of Activin A release during the differentiation of M1 (GM-CSF)
and M2 (M-CSF) macrophages from two independent donors, as determined by ELISA on culture supernatants removed at
the indicated time points. Each determination was performed in triplicate, and mean and standard deviations are shown. F.
Determination of Activin A levels released by M1 (GM-CSF) and M2 (M-CSF) macrophages either untreated or stimulated
with 10 ng/ml LPS for 24 hours. Each determination was performed in triplicate, and mean and standard deviations are
shown.
To determine whether INHBA-encoded Activin A affects FOLR2 gene expression, M2 polarization
was accomplished in the presence of recombinant human Activin A. The M-CSF-dependent
acquisition of FOLR2 RNA expression was dose-dependently reduced in the presence of Activin A,
an effect that could be observed both during (3 days) and at the end (7 days) of the
macrophage/polarization process (Figure 3A,B). Moreover, Activin A also reduced the FOLR2 RNA
upregulation that takes place in the absence of exogenous M-CSF (Figure 3B). Therefore, Activin A
inhibits the acquisition of FOLR2 RNA expression in differentiating macrophages. Consequently, it
can be concluded that the differential expression of FRβ on M1 and M2 macrophages is a
FOLR2
1.2
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
3 days
M-CSF
FOLR2
GM-CSF
7 days
M-CSF+Act. A
B
Relative mRNA levels
A
Relative mRNA levels
consequence of the opposite actions of GM-CSF and M-CSF on INHBA gene expression.
FOLR2
200
150
100
50
Mo.
M-CSF
GM-CSF
Act. A 2.5 ng/ml
Act. A 25 ng/ml
Figure 3.- Activin A inhibits Folate Receptor β (FRβ) expression.- A. FOLR2 mRNA expression levels as determined by
qRT-PCR on macrophages differentiated for 3 days (left panel) or 7 days (right panel) in the presence of M-CSF, GM-CSF
or M-CSF plus Activin A (Act. A). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and
referred to the expression level observed in the presence of M-CSF), and shown is the mean and standard deviation of
triplicate determinations. B. FOLR2 mRNA expression levels determined by qRT-PCR on monocytes (Mo.) or
macrophages differentiated in the presence of the indicated cytokine combinations. Results are expressed as Relative
mRNA levels (relative to 18S rRNA levels and referred to the expression level in monocytes), and shown is the mean and
standard deviation of triplicate determinations.
63
Activin A contributes to M1 (GM-CSF) Macrophage polarization.- Considering the above results,
we assessed the influence of Activin A on genes whose expression, like FOLR2, is significantly
higher in M2 (MCSF) than in M1 (GM-CSF) macrophages
20
(Figure 4A). After a 7-day M-CSF-
driven polarization, Activin A was capable of inhibiting the M-CSF-dependent induction of MAF,
HTR2B, SEPP1, IGF1 and F13A1, and abrogating that of SERPINB2, whereas it had no inhibitory
effect on HS3ST1 gene expression (Figure 4B). Therefore, Activin A inhibits the expression of genes
preferentially upregulated during M-CSF-dependent polarization. On the other hand, and since the
upregulation of other M2 (M-CSF)-specific genes like MAFB or HMOX1 is not affected by Activin
A but prevented by GM-CSF (Supplementary Figure 1), it is tempting to conclude that the combined
action of both GM-CSF and Activin A limits the acquisition of M2 (M-CSF) macrophage markers
during the M-CSF-dependent polarization.
A 10
log2 M1 / M2
8
6
4
2
1.2 x 10-2
Microarray
qPCR
4.4 x 10-7 3.9 x 10-12 2.0 x 10-2
4.2 x 10-4
3.6 x 10-7
SEPP1 SERPINB2 F13A1
HTR2B
5.4 x 10-3
-2
-4
-6
-8
IGF1
Relative mRNA levels
Relative mRNA levels
B
MAF
HTR2B
SEPP1
1.0
1.0
1.0
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
IGF1
SERPINB2
F13A1
1.0
1.0
1.0
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
M-CSF
MAF SERPINE1
GM-CSF
HS3ST1
2.0
1.5
1.0
0.5
M-CSF+Act. A
Figure 4.- Effect of Activin A on the acquisition of M2 (M-CSF)-specific markers.- A. Relative expression of the
indicated genes in M1 (GM-CSF)- and M2 (M-CSF)-polarized macrophages, as determined by microarray DNA analysis
(empty histograms) and quantitative RT-PCR (grey histograms). B. MAF, HTR2B, SEPP1, IGF1, SERPINB2, F13A1 and
HS3ST1 mRNA expression levels as determined by qRT-PCR on macrophages differentiated for 7 days in the presence of
M-CSF, GM-CSF or M-CSF plus Activin A (Act. A). Results are expressed as Relative mRNA levels (relative to 18S
rRNA levels and referred to the expression level observed in the presence of M-CSF), and shown is the mean and standard
deviation of triplicate determinations.
64
Because of its continuous presence during GM-CSF-mediated polarization, we hypothesized that
Activin A could shape the phenotypic and functional polarization state of macrophages in an
autocrine/paracrine manner. It has been proposed that Activin A is a Th2-polarizing cytokine 17 and,
therefore, its effects on the expression of genes preferentially found in both M1 (GM-CSF) and IL-4activated macrophages
29
was evaluated. Activin A did not modify the expression of TM4SF1,
MMP12 and CCL17, which are preferentially expressed by M1 (GM-CSF) macrophages
(Supplementary Figure 2A), or ILR1N, a known Actinin A target gene 30 (Supplementary Figure 2B).
By contrast, the presence of Activin A enhanced SERPINE1 RNA levels, which are significantly
higher in M1 (GM-CSF) macrophages (Figure 5A). Therefore Activin A contributes to shaping the
phenotypic polarization of M1 (GM-CSF) macrophages.
Next, conditioned medium from M1 (GM-CSF) macrophages was analyzed for Activin A-dependent
functions. Activin A has tumour suppressive properties and contributes to cancer cell growth arrest
31,32
, and results indicated that M1 (GM-CSF) macrophage-conditioned medium inhibits the growth
of K562 leukemic cells (Figure 5B) and promotes their differentiation into Hemoglobin-expressing
cells (Figure 5C). Moreover, both activities were reduced in the presence of a blocking anti-Activin
A monoclonal antibody (Figure 5B,C). Therefore, M1 (GM-CSF) macrophages release functional
Activin A, what might endow them with the tumor-resistance capability that characterizes M1polarized macrophages 33.
GM-CSF
M-CSF
Activin A
Donor # 1
Donor # 2
106
100
80
60
40
20
81
73
80
51
-
M1 M1 M2 ActA ActA
+
+
antiActA
antiActA
B enzid ine+ ce lls
1.0
0.8
0.6
0.4
0.2
C
B
SERPINE1
R elativ e ce ll
P ro lifera tio n (9 6 h)
R elativ e m R N A lev els
A
30
20
10
-
M1 M1 M2 M1 M2 ActA ActA
+
+
antiActA
antiActA
Donor # 1 Donor # 2
Figure 5.- Effect of Activin A on the acquisition of M1 (M-CSF)-specific markers and effector functions.- A. SERPINE1
mRNA expression levels as determined by qRT-PCR on macrophages from two independent donors, and treated for 7 days
with M-CSF, GM-CSF or Activin A. Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and
referred to the expression level observed in the presence of GM-CSF), and shown is the mean and standard deviation of
triplicate determinations. B. Proliferation of K562 cells exposed for 96 hours to the indicated conditioned media (M1 or
M2) or Activin A, and either in the absence or presence or a blocking anti-Activin A monoclonal antibody. Results are
expressed relative to the proliferation observed in untreated cells (Relative cell proliferation). C. Differentiation of K562
cells into Hemoglobin-containing cells (Benzidine+ cells) after exposure for 96 hours to Activin A or the indicated
conditioned media (M1 or M2) from two independent donors, and either in the absence or presence or a blocking antiActivin A monoclonal antibody.
65
Activin A modulates the cytokine-producing profile of M2 (M-CSF) macrophages.- The above
results imply that Activin A actively participates in shaping macrophage polarization. Therefore, the
influence of Activin A on the paradigmatic effector functions of M1 (GM-CSF) and M2 (M-CSF)
macrophages was studied. As reported
10,12
, M1 and M2 macrophages differ in terms of T cell
stimulayory activity and cytokine/chemokine release in response to pathogenic stimulation
9,10
.
Although M1 (GM-CSF) induced considerable higher T cell proliferation than M2 (M-CSF) in
allogeneic MLR, exposure of the latter to Activin A did not modify their T cell stimulatory ability
(Supplementary Figure 3), indicating that M1 (GM-CSF)-derived Activin A is not responsible for the
high T cell stimulary activity of M1 (GM-CSF) macrophages. Regarding cytokine release, and in
agreement with previous reports
10
, LPS stimulation led to production of significant levels of pro-
inflammatory cytokines (TNFα, IL-12p40, IL-6) and acquisition of dendritic cell maturation ability
(Supplementary Figure 4) by M1 (GM-CSF) macrophages, whereas M2 (M-CSF) produced high
levels of IL-10 and low (TNFα) or undetectable (IL-6, IL12p40) levels of pro-inflammatory
cytokines (Figure 6A). However, the presence of Activin A significantly reduced the release of IL-10
from LPS-stimulated M2 (M-CSF) macrophages, although it had no effect on the production of
TNFα (Figure 6B). This result indicates that Activin A negatively regulates IL-10 production from
M2 (M-CSF) macrophages, and suggests that Activin A contributes to the potent pro-inflammatory
nature of M1 (GM-CSF) macrophages by inhibiting IL-10 production. Moreover, macrophages
exposed to Activin A during the M-CSF-driven polarization process exhibited a highly diminished
production of IL-10 in response to LPS (Figure 6B). Therefore, Activin A also interferes with the
acquisition of the IL-10-producing ability by M2 (M-CSF) macrophages. This result further supports
the involvement of Activin A in shaping macrophage polarization by impairing the acquisition of an
anti-inflammatory phenotype and cytokine profile.
66
IL-12p40 (pg/ml)
3000
2500
2000
1500
1000
500
TNFα(pg/ml)
700
600
500
400
300
200
100
7000
6000
5000
4000
3000
2000
1000
B
p=0.003
Relative levels of
Relative levels of
LPS-induced TNFα LPS-induced IL-10
IL-10 (pg/ml)
300
250
200
150
100
50
IL-6 (pg/ml)
A
p=0.01
100
80
60
40
20
200
160
120
80
40
LPS
LPS
ActA
M2
LPS
LPS
ActA
M2 ActA
M1 M1/LPS M2 M2/LPS
Figure 6.- Effect of Activin A on the LPS-induced cytokine profile of polarized macrophages.- A. Determination of IL12p40, IL-6, IL-10 and TNFα release by ELISA in culture supernatants of M1 and M2 macrophages either untreated or
stimulated with LPS (10 ng/ml) for 24 hours. Each determination was performed in triplicate, and mean and standard
deviations are shown. B. Determination of IL-10 and TNFα release by ELISA in culture supernatant of M2 macrophages
differentiated in the absence (M2) or presence of Activin A (M2 ActA), and either unstimulated or stimulated with LPS (10
ng/ml) for 24 hours in the presence or absence of Activin A. Each determination was performed in triplicate, and mean and
standard deviations are shown.
Activin A-initiated signaling during macrophage polarization.- Since Activin A activates Smad2 16,
Smad2 phosphorylation was determined in unstimulated and LPS-stimulated macrophages. Smad2
was constitutively phosphorylated in M1 (GM-CSF) macrophages, and LPS treatment did not modify
its phosphorylation state (Figure 7A). By contrast, no Smad2 phosphorylation was detected in either
untreated or after LPS-stimulated M2 (M-CSF) macrophages (Figure 7A). The absence of Smad2
activation in M2 macrophages was not due to a defective Activin/Smad signaling pathway, as
Activin A treatment of M2 macrophages readily led to overt Smad2 phosphorylation (Figure 7B). A
definitive support for an Activin A role in shaping M1 (GM-CSF) macrophage polarization was
obtained through evaluation of the transcriptional effects of M1 (GM-CSF)-derived Activin A. Like
TGFβ and recombinant Activin A, M1 (GM-CSF)-conditioned medium transactivated the Smad2dependent p3TP-Lux reporter construct (p = 0.0008), whereas supernatants from M2 (M-CSF)
macrophages had no effect (Figure 7C). Importantly, the transactivation ability of the M1 (GMCSF)-conditioned medium was abolished (p = 0.015) by SB431542, an inhibitor of ALK4, ALK5
67
and ALK7 receptors which prevents Smad2 phosphorylation
34
(Figure 7C), by cotransfection of a
dominant negative form of Smad2 (Figure 7C) and by a blocking antibody against human Activin A
(p = 0.0001) (Figure 7C). Altogether, this set of results demonstrate that M1 (GM-CSF) macrophagederived Activin A activates Smad2, and modulates gene expression in macrophages and other cell
types, thus providing a molecular basis for its macrophage polarization shaping ability.
n.s.
M2/LPS
0 0.5 2
C
hours
pSmad2
Smad2
β-actin
B
-
M2
+Act.A
pSmad2
p=0.0001
16
14
12
10
8
6
4
2
Act. A
M1 SN
M2 SN
GAPDH
p=0.015
p=0.0008
TGF-β1
M1/LPS
0 0.5 2
RLU (Luc/β-Gal)x100
A
SB431542 anti.Act.A
Smad2
(d.n.)
Smad3
(d.n.)
Figure 7.- Smad2 is constitutively phosphorylated in M1 (GM-CSF) macrophages, whose Activin A release activates
Smad-dependent reporter genes.- A. Detection of activated and total Smad2 on lysates of untreated or LPS-treated M1 and
M2 macrophages, as determined by Western blot. β-actin expression levels were determined in parallel as a loading control.
B. Detection of activated Smad2 on lysates of untreated or Activin A-treated M2 macrophages, as determined by Western
blot. GAPDH expression levels were determined in parallel as a loading control. C. Transcriptional activity of the p3TPLux reporter construct in Mv1Lu cells either unstimulated or exposed to 10 ng/ml TGFβ1, 25 μg/ml Activin A, or
conditioned medium from M1 (M1 SN) or M2 (M2 SN) macrophages. When indicated, cells were either preincubated for
30 minutes with 10 μM SB431542 before treatment, maintained in culture medium with 0.1 μg/ml of an anti-Activin A
(anti-Act.A) or cotransfected with expression vectors coding for dominant negative mutants of either Smad2 (Smad2 d.n.)
or Smad3 (Smad3 d.n.). For normalization purposes, cells were co-transfected with the RSV-β-gal expression plasmid, and
results are presented as RLU (Relative Light Units), which indicate the units of luciferase activity per unit of βgalactosidase activity for each assay condition. Experiments were performed in triplicate, and shown are the mean and
standard deviation.
Tumor-conditioned media modulates Activin A expression.- Since INHBA mRNA levels proinflammatory macrophages and tumors-derived factors polarize macrophages towards an
alternative/anti-inflammatory state
35
, we hypothesized that the expression of INHBA and FOLR2
mRNA could be also oppositely modulated in the presence of tumor-conditioned media. To test this
hypothesis, Activin A-expressing M1 (GM-CSF) macrophages were exposed to ascitic fluid from
three independent metastatic gastric carcinomas or a metastatic colon carcinoma. As shown in Figure
8, the presence of the tumor-derived media caused a great reduction in the levels of INHBA mRNA
expression, that is compatible with tumor-derived factors promoting a shift in macrophage
polarization. In fact, and in agreement with its preferential expression in anti-inflammatory
macrophages
20
, FOLR2 mRNA levels exhibited a concomitant increase in those M1 (GM-CSF)
macrophages that had been exposed to the ascitic fluid with a more potent inhibitory action on
68
INHBA mRNA expression (Figure 8). Altogether these results confirm the modulatory action that
tumor-derived factors have on macrophage polarization, and indicate that both INHBA and FOLR2
mRNA levels are adequate markers for pro-inflammatory M1 and anti-inflammatory M2
Normalized relative mRNA levels
macrophages.
INHBA
1.0
1
0,47
0,07
0,1
11,02
10
0,56
FOLR2
7,46
5
1.0
1
#1
#2
0,03
0,16
#3
Colon
Gastric carcinoma
Figure 8.- Tumor-conditioned media oppositely modulates INHBA and FOLR2 mRNA in pro-inflammatory M1 (GMCSF) macrophages.- INHBA and FOLR2 mRNA levels were determined in M1 (GM-CSF) macrophages exposed for 24
hours to a 1:1 dilution of ascitic fluid from the indicated metastatic tumors. Results are expressed as Normalized Relative
mRNA levels according to GAPDH mRNA levels and relative to the respective mRNA levels present in M1 (GM-CSF)
macrophages maintained under normal culture conditions. Shown is the mean and standard deviation of triplicate
determinations.
69
DISCUSSION
Macrophage differentiation and polarization are critically determined by the cellular environment,
which also dictates cytokine responsiveness 36. The search for the mechanisms underlying GM-CSFand M-CSF-driven macrophage differentiation and the acquisition of anti-inflammatory M2 (M-CSF)
macrophage markers has led to the identification of Activin A as a factor that shapes macrophage
polarization in response to GM-CSF, and whose presence limits the production of IL-10 and prevents
the expression of genes associated to M2 macrophage polarization. The relevance of the Activin A
expression by macrophages is underscored by its downregulation in the presence of tumorconditioned media. Since tumors skew macrophage polarization towards the acquisition of
alternative/M2 phenotypic and functional characteristics
37
, the downmodulation of Activin A by
tumor-derived ascitic fluids strongly suggests that Activin A constitutes a useful marker for the
identification of TAM whose polarization state has not yet been fully modulated by the tumor
microenvironment. Whereas the identity of the tumor-derived factors that downmodulate Activin A
is currently unknown, it is tempting to speculate that M-CSF might be a contributing factor, specially
considering its negative regulatory effect on INHBA mRNA expression in vitro (Figure 2) and its the
positive regulatory action on FOLR2 mRNA levels.
The ability of Activin A to trigger Arginase-1 expression and inhibit IFNγ-induced NO synthase
expression has led to the suggestion that it functions as a Th2 cytokine that promotes alternative
murine macrophage activation 17. However, although M1 macrophages release high levels of Activin
A, they do not display any of the phenotypic makers that characterize alternatively activated human
macrophages
29
(Puig-Kröger, Sierra-Filardi, Vega and Corbí, unpublished). Activin exhibits both
pro- or anti-inflammatory activities 19, and is synthesized by monocytes/macrophages in response to
inflammatory stimuli. Although it does not elicit TNF-α release by itself, Activin A stimulates the
production of IL-1, IL-6 and TNF-α by human monocytes and macrophages, inhibits IL-10 effects
on prostatic epithelial cells 38, and its inhibition by follistatin leads to reduced levels of LPS-induced
IL-1 and TNF-α 19. Activin A production by activated monocytes/macrophages is PKC-dependent 39,
and is promoted by LPS
19
and pro-inflammatory agents like TNFα 40 or IL1β 41. The link between
pro-inflammatory macrophage polarization and Activin A is further illustrated by the fact that
Activin A expression is inhibited by anti-inflammatory agents like glucorticoids and retinoic acid 28.
Since factors promoting M1/classical macrophage polarization enhance Activin A production, its
expression might be a common parameter of M1-polarized macrophages, as well as a critical
contributor to their phenotype and effector functions. In this regard, since M1 (GM-CSF)
macrophages express type I (ALK4, ACR1B) and type II (ACVR2A, ACVR2B) Activin A receptors
mRNA (data not shown), it can be predicted that Activin A influences the gene expression profile of
70
M1 polarized macrophages in a paracrine/autocrine manner. This appears to be true, at least
considering the state of phosphorylation od Smad2 in M1 (GM-CSF) macrophages. Smad2
phosphorylation is induced upon Activin A binding to its cell surface receptors 14, and Smad2 was
found to be phosphorylated in unstimulated M1 macrophages, whereas no Smad2 activation was
observed in M2 macrophages (Figure 6C). The constitutive activation of Smad2 in M1 (GM-CSF)
macrophages undoubtfully suggests that a prominent role for Activin A (and/or TGFβ family factors)
in shaping the inflammatory response of M1 macrophages to exogenous stimuli.
The identity of the genes specifically upregulated by Activin A in M1 (GM-CSF) macrophages
remains to be determined, although we have alredy shown that SERPINE1 expression is clearly
enhanced when macrophage polarization takes place in the presence of Activin A. Conversely,
various M2 (M-CSF)-specific markers have already been identified whose expression is either
prevented or downmodulated by Activin A (Figure 4), thus leading to the conclusion that Activin A
negatively affects the acquisition of genes preferentially expressed by anti-inflammatory
macrophages. This effect is particularly relevtant in the case of IL-10, whose release constitutes a
hallmark of stimulated M2 (M-CSF) macrophages 10. In the specific case of IL-10, it is worth noting
that its transcription in macrophages is dependent on the cMaf transcription factor42, that also
suppresses IL-12p70 production
43
. The expression of the cMaf transcription factor is significantly
higher in M2 (M-CSF) than in M1 (GM-CSF) macrophages, both the RNA (Figure 4) and protein
level (data not shown), and, like in the case of Th2 lymphocyte polarization 44, its expression seems
to mark the acquisition of the anti-inflammatory phenotype by M2 macrophages. Importantly,
Activin A inhibits the M-CSF-dependent acquisition of MAF RNA expression, what might constitute
the molecular basis for its negative effect on the production of IL-10 by LPS-stimulated M2 (M-CSF)
macrophages.
In summary, the present manuscript identifies Activin A as a relevant contributor to the differential
gene expression profile exhibited by pro-inflammatory and anti-inflammatory macrophages. The
importance of Activin A in macrophage polarization is supported by its ability to reduce IL-10
production by anti-inflammatory macrophages, and to inhibit the acquisition of the IL-10-producing
ability during M-CSF-driven polarization. The identification of a set of M2 (M-CSF) macrophagespecific genes whose expression is found in Tumor-Associated Macrophages 20 (and data not shown)
and negatively affected by Activin provides potential therapeutic targets for the modulation of the
macrophage inflammatory response under pathological conditions.
71
ACKNOWLEDGMENTS
The autors gratefully acknowledge Dr. Carmen Sánchez-Torres for valuable discussions and
suggestions.
72
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75
Relative mRNA levels
Supplementary Figures
MAF
MAFB
HMOX1
M-CSF
M-CSF+GM-CSF
M-CSF+Act. A
1.2
1.0
1.2
1.0
0.8
0.6
0.4
0.2
1.2
1.0
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
Supplementary Figure 1.- MAF, MAFB and HMOX1 mRNA expression levels as determined by qRT-PCR on
macrophages exposed for 3 days to M-CSF, M-CSF plus GM-CSF or M-CSF plus Activin A (Act. A). Results
are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level
observed in the presence of M-CSF). Shown is the mean and standard deviation of triplicate determinations.
log2 M1 / M2
A 12 TM4SF1 MMP12 CCL17
10
8
Microarray
qPCR
6
4
B
Relative mRNA levels
2
CCL17
8000
2000
TM4SF1
300
60
30
6000
4000
MMP12
IL1RN
20
10
200
100
40
RPMI
GM-CSF
Activin A
20
Supplementary Figure 2.- A. Relative expression of the indicated genes in M1 and M2 macrophages, as
determined by microarray analysis (empty histograms) and quantitative RT-PCR (grey histograms). B. CCL17,
IL1RN, MMP12 and TM4SF1 mRNA expression levels determined by qRT-PCR on macrophages
differentiated for 3 days in the absence or in the presence of GM-CSF or Activin A. Results are expressed as
Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the
absence of cytokines), and shown is the mean and standard deviation of triplicate determinations.
77
B
1:200
1:40
1:8
40
30
20
10
M1
20
18
16
14
12
10
8
6
4
2
Proliferation (cpm x 10-3)
Proliferation (cpm x 10-3)
A
1:40
1:8
M1
M2
M2
M2+Act.A
Supplementary Figure 3.- M1 or M2 macrophages (A,B), or M2 macrophages differentiated with M-CSF in
the presence of Activin A (Act. A) (B) were irradiated and used to stimulate 2 x 105 allogeneic peripheral blood
T lymphocytes at the indicated Macrophage/T cell ratios. After a 5 day co-culture, 3H-thymidine was added to
the culture for 16 hours and T cell proliferation determined by measuring the incorporated thymidine. Each
experiment was performed in triplicate, and mean and standard deviations are shown.
P3X63
CD83
CD86
3.6
(0.4)
11.7
(0.5)
33.4
(0.8)
8.9
(0.4)
11.2
(0.5)
28.5
(0.7)
M1/LPS
5.3
(0.4)
53.3
(1.2)
64.7
(1.9)
M2
8.0
(0.4)
13.4
(0.5)
26.0
(0.6)
M2/LPS
5.1
(0.4)
16.4
(0.5)
36.3
(0.9)
M1
Supplementary Figure 4.- Immature monocyte-derived dendritic cells (MDDC) were exposed to conditioned
media from either untreated or LPS (10 ng/ml)-treated M1 and M2 macrophages. After 48 hours, MDDC cell
surface expression of CD83 and CD86 was determined by flow cytometry (P3X63, isotype-matched control).
The percentage of marker-positive cells (upper number) and the mean fluorescence intensity (lower number)
are indicated in each case.
78
Resultados
3. Requerimientos estructurales para la multimerización del receptor de
patógenos DC-SIGN (CD209) en la superficie celular
DC-SIGN (Dendritic cell-specific ICAM3- grabbing non-integrin, CD209) es una lectina tipo C
que reconoce oligosacáridos presentes en patógenos con gran relevancia clínica (HIV,
Mycobacterium, Aspergillus). Mediante “splicing” alternativo y polimorfismos genéticos se generan
variantes de DC-SIGN que se detectan a nivel de mRNA en los sitios de entrada y transmisión de
patógenos. En este estudio se demuestra que las células mieloides expresan variantes de DC-SIGN
con diferentes tamaños de cuello, y que la multimerización de DC-SIGN en el contexto celular
depende del dominio lectina y del número y disposición de las repeticiones de la región del cuello,
cuya glicosilación afecta negativamente a la formación de oligómeros. Variantes de la región del
cuello de DC-SIGN que ocurren de forma natural difieren en su capacidad de mediar multimerización
en la membrana celular, exhiben una habilidad alterada de unión de azúcares, y conservan su
capacidad de interacción con patógenos. En consecuencia, se puede concluir que la formación de
agregados de moléculas de DC-SIGN inducida por patógenos predomina sobre la capacidad de
multimerización basal. El análisis de polimorfismos en el cuello de DC-SIGN indica que el número de
variantes alélicas en la población es mayor de lo esperado, y que la multimerización de la molécula
prototípica es modulada por la presencia de variantes alélicas con una estructura de cuello diferente.
Estos resultados demuestran que la presencia de variantes alélicas o la expresión de isoformas de
“splicing” del dominio del cuello pueden influir en la presencia y estabilidad de multímeros de DCSIGN en la superficie celular, lo que proporciona una explicación molecular para la asociación entre
polimorfismos de DC-SIGN y la susceptibilidad alterada a HIV y otros patógenos.
79
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 7, pp. 3889 –3903, February 15, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Structural Requirements for Multimerization of the Pathogen
Receptor Dendritic Cell-specific ICAM3-grabbing
Non-integrin (CD209) on the Cell Surface*□
S
Received for publication, July 23, 2007, and in revised form, November 26, 2007 Published, JBC Papers in Press, December 11, 2007, DOI 10.1074/jbc.M706004200
Diego Serrano-Gómez‡1,2, Elena Sierra-Filardi‡1, Rocı́o T. Martı́nez-Nuñez‡, Esther Caparrós‡, Rafael Delgado§,
Mari Angeles Muñoz-Fernández¶, Marı́a Antonia Abad储, Jesús Jimenez-Barbero‡, Manuel Leal储, and Angel L. Corbı́‡3
From the ‡Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientı́ficas, Ramiro de Maeztu 9, Madrid 28040,
§
Laboratorio de Microbiologı́a Molecular, Hospital Doce de Octubre, Madrid 28041, the ¶Servicio de Inmunologı́a, Hospital
General Universitario Gregorio Marañón, Madrid 28007, and the 储Servicio de Enfermedades Infecciosas,
Hospital Universitario Virgen del Rocı́o, Sevilla 41013, Spain
* This work was supported in part by the Ministerio de Educación y Ciencia
(Grants SAF2005-0021, AGL2004-02148-ALI, and GEN2003-20649-C06-01/
NAC), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (Spanish Network for the Research in Infectious Diseases, Grant REIPI
RD06/0008), and Fundación para la Investigación y Prevención del SIDA en
España (Grant FIPSE 36422/03) (to A. L. C.). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1 and S2.
1
Both authors contributed equally to this work.
2
Supported by a Formación de Profesorado Universitario predoctoral grant
(AP2002-2151) from Ministerio de Educación y Ciencia (Spain).
3
To whom correspondence should be addressed. Tel.: 34-91-837-3112 (ext.
4376); Fax: 34-91-562-7518; E-mail: [email protected].
FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7
Dendritic cells (DCs)4 link the innate and adaptive branches
of the immune response by virtue of their capacity to recognize
pathogen-specific structures (1) via pathogen-associated
molecular pattern receptors (2). Immature DCs express a number of lectins and lectin-like molecules, which endow them with
a broad capacity for pathogen recognition, as they mediate the
specific recognition of parasitic, bacterial, yeast, and viral
pathogens (3, 4). Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) is a type II membrane C-type lectin (5, 6) abundantly expressed in vivo on myeloid DC and macrophage subpopulations (5–12), as well as on in vitro generated
monocyte-derived dendritic cells (MDDCs) and alternatively
activated macrophages (12–14). DC-SIGN binds a large array
of pathogens, including HIV (15), Ebola (16), hepatitis C (17–
19), and Dengue virus (20) and Leishmania amastigotes and
promastigotes (21, 22), Mycobacterium tuberculosis (23, 24),
Aspergillus fumigatus (25), and Candida albicans (26) via mannan- and Lewis oligosaccharides-dependent interactions (27,
28). In addition, DC-SIGN appears to mediate DC contacts
with naı̈ve T lymphocytes through its recognition of ICAM-3
(6), DC trafficking through interactions with endothelial
ICAM-2 (8), and DC-neutrophil interactions by interacting
with the CD11b/CD18 integrin (29).
Structurally, DC-SIGN contains a carbohydrate-recognition
domain, a neck region composed of eight 23-residue repeats,
and a transmembrane region followed by a cytoplasmic tail
containing recycling and internalization motifs (5, 30 –32).
Analysis of recombinant molecules has revealed that the monomeric lectin domain has low affinity for carbohydrates, whereas
full-length DC-SIGN molecules form tetramers through their
neck domain, thus allowing high affinity recognition of specific
ligands (33–35). In addition to this prototypical structure, alternative splicing events generate DC-SIGN isoform transcripts
whose presence exhibits inter-individual variations (36). The
numerous DC-SIGN isoform transcripts reported to date
include an alternative cytoplasmic tail, an absent transmembrane region, truncated lectin domains, and a variable number
4
The abbreviations used are: DC, dendritic cell; MDDC, monocyte-derived
dendritic cell; DC-SIGN, dendritic cell-specific ICAM-3-grabbing non-integrin; DC-SIGNR, DC-SIGN-related; HIV, human immunodeficiency virus;
FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; DTSSP,
dithiobis(succinimidylpropionate); MFI, mean fluorescence intensity.
JOURNAL OF BIOLOGICAL CHEMISTRY
3889
81
Downloaded from www.jbc.org at CSIC - Centro de Investigaciones Biológicas, on June 10, 2010
The myeloid C-type lectin dendritic cell-specific ICAM3grabbing non-integrin (DC-SIGN, CD209) recognizes oligosaccharide ligands on clinically relevant pathogens (HIV, Mycobacterium, and Aspergillus). Alternative splicing and genomic
polymorphism generate DC-SIGN mRNA variants, which have
been detected at sites of pathogen entrance and transmission.
We present evidence that DC-SIGN neck variants are expressed
on dendritic and myeloid cells at the RNA and protein levels.
Structural analysis revealed that multimerization of DC-SIGN
within a cellular context depends on the lectin domain and the
number and arrangement of the repeats within the neck region,
whose glycosylation negatively affects oligomer formation. Naturally occurring DC-SIGN neck variants differ in multimerization competence in the cell membrane, exhibit altered sugar
binding ability, and retain pathogen-interacting capacity,
implying that pathogen-induced cluster formation predominates over the basal multimerization capability. Analysis of DCSIGN neck polymorphisms indicated that the number of allelic
variants is higher than previously thought and that multimerization of the prototypic molecule is modulated in the presence of
allelic variants with a different neck structure. Our results demonstrate that the presence of allelic variants or a high level of
expression of neck domain splicing isoforms might influence
the presence and stability of DC-SIGN multimers on the cell
surface, thus providing a molecular explanation for the correlation between DC-SIGN polymorphisms and altered susceptibility to HIV-1 and other pathogens.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
Isolation and Structural Characterization of Alternatively
Spliced DC-SIGN Isoforms
EXPERIMENTAL PROCEDURES
Three DC-SIGN allelic variants (-D3, -D5, and -D7) were
identified by PCR on genomic DNA from 300 independent
donors. Amplification of the DC-SIGN neck domain-encoding
exon was carried out on 300 ng of genomic DNA using oligonucleotides CD209-4F, (5⬘-GGGATTAACCAAGACCTTGGCTC-3⬘) and CD209-4R, (5⬘-CCCAACTTCTCCTAGTCTGGAGG-3⬘). After 35 cycles of denaturation (95 °C, for 45 s),
annealing (61 °C, for 30 s), and extension (72 °C, for 90 s), followed by a 10-min extension step at 72 °C, PCR-generated fragments were resolved in agarose gels, purified, cloned into
pCR4-TOPO vector (Invitrogen), and sequenced.
Swapping of the neck domains between the allelic variants
and the prototypic form of DC-SIGN was done after introduction of silent mutations creating restriction sites at Val63 (KpnI)
and Ala247/Ala248 (SacII) in pCDNA3.1-DC-SIGN 1A and the
allelic variants in pCR4-TOPO. Oligonucleotides used for
mutagenesis included: DC-SIGN-Val63s (5⬘-TGTCCAAGTGTCCAAGGTACCCAGCTCCATAAGTCAG-3⬘), DC-SIGNVal63as (5⬘-CTGACTTATGGAGCTGGGTACCTTGGACACTTGGACA-3⬘), DC-SIGN-247/248s (5⬘-GCTGACCCAGCTGAAGGCCGCGGTGGAACGCCTGTGCCAC-3⬘), and
DC-SIGN-247/248as (5⬘-GTGGCACAGGCGTTCCACCGCGGCCTTCAGCTGGGTCAGC-3⬘). The resulting plasmids
(pcDNA3.1-DC-SIGN-D3, pcDNA3.1-DC-SIGN-D5, and
pcDNA3.1-DC-SIGN-D7) were verified by sequencing.
An expression vector for N-terminal-His epitope-containing
DC-SIGN 1A (pCDNA3.1-DC-SIGN 1A-His) was created by
PCR on pCDNA3.1-DC-SIGN 1A using oligonucleotides
Generation of MDDCs
Human peripheral blood mononuclear cells were isolated
from buffy coats from healthy donors over a Lymphoprep
(Nycomed, Norway) gradient according to standard procedures. Monocytes were purified from peripheral blood mononuclear cells by magnetic cell sorting using CD14 microbeads
(Miltenyi Biotech, Bergisch Gladbach, Germany), and immediately subjected to the dendritic cell differentiation protocol
(40). Monocytes were cultured for 5–7 days in complete
medium with 1000 units/ml granulocyte macrophage-colony
stimulating factor (Schering-Plough, Kenilworth, NJ) and 1000
units/ml interleukin-4 (PreProtech, Rocky Hill, NJ) cytokine
addition every second day, to obtain a population of immature
MDDCs.
Cells
The acute monocytic leukemia cell line THP-1, and the
erythroleukemic K562 were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum (complete medium).
COS-7 and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium 10% fetal calf serum. THP-1 differentiation was induced by treatment with phorbol 12-myristate
13-acetate (10 ng/ml), Bryostatin (10 nM), either alone or in
combination with interleukin-4 (1000 units/ml), as
described before (14).
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DC-SIGN isoforms were isolated by reverse transcriptionPCR on RNA from MDDCs of a healthy donor. Reverse transcription-PCR was performed essentially as described previously (41). DC-SIGN mRNA was optimally amplified after 35
cycles of denaturation (95 °C, for 45 s), annealing (62 °C, for
45 s), and extension (72 °C, for 1 min), followed by a 10-min
extension step at 72 °C. Oligonucleotides used for amplification
of the coding region of the prototypical DC-SIGN isoform 1A
(DC-SIGN 1A) mRNA were CD209s (5⬘-GGGAATTCAGAGTGGGGTGACATGAGTGAC-3⬘) and CD209as (5⬘-CCCCAAGCTTGTGAAGTTCTGCTACGCAGGAG-3⬘) (6, 36).
Amplification of DC-SIGN isoforms was accomplished
using the primer pairs CD209s/CD209as, CD209soluble/
CD209as, and CD209Ib/CD209as. The oligonucleotide
CD209soluble (5⬘-GATACAAGAGCTTAGCAGTGTCCA3⬘) spans through the exon Ic/exon III junction previously
described for potentially soluble transmembrane-lacking DCSIGN isoforms. The oligonucleotide CD209Ib (5⬘-GGGAATTCTGGCCAGCCATGGCCTCAGC-3⬘) includes the alternative translation initiation site found in exon Ib, which originates
the DC-SIGN 1B isoforms (Fig. 1A). PCR-generated fragments
were resolved in agarose gels, purified, sequenced, and cloned
into pCDNA3.1(⫺) vector.
Identification of DC-SIGN Polymorphic Isoforms and
Generation of His- and FLAG-containing DC-SIGN
Expression Vectors
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of repeats within the neck domain (36). Moreover, the 23-residue repeat region of DC-SIGN is polymorphic at the genomic
level (37, 38). Five different alleles for the DC-SIGN neck
domain have been identified to date, whose presence correlates
with altered susceptibility to HIV-1 transmission (38). The
functional relevance of the DC-SIGN neck variants has been
further suggested by their detection at mucosal HIV transmission sites (39). Given the involvement of the neck domain in
recombinant DC-SIGN multimerization, we hypothesized that
the existence of this large array of polymorphic variants might
have an impact on the repertoire of pathogen recognition by
dendritic cells, as well as on the establishment of interactions
between dendritic cells and other cell types. We have characterized naturally occurring alternative splicing isoforms, allelic
variants and mutant isoforms of DC-SIGN in terms of surface
receptor multimerization and adhesive and pathogen-recognition capabilities, and found that the lectin domain contributes
to DC-SIGN multimerization on the cell surface, that glycosylation of the neck domain negatively regulates formation of
multimers, and that a neck domain with a single 23-residue
repeat is sufficient to mediate DC-SIGN multimerization on
the cell surface. Functional comparison of the distinct constructs revealed that the basal multimerization of DC-SIGN
does not correlate with enhanced binding to endogenous or
pathogenic ligands, indicating that pathogen-induced cluster
formation predominates over the basal multimerization capability of the DC-SIGN molecule and is the driving force for the
DC-SIGN-dependent pathogen capture and internalization.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
CD209His (5⬘-GGGAATTCGCCACCATGCATCATCATCATCATCATAGTGACTCCAAGGAACCAAGAC-3⬘) and
CD209as. Generation of expression vectors for DC-SIGN-D3,
-D5, and -D7 with FLAG epitope at the N terminus
(pCDNA3.1-DC-SIGN-D3-FLAG, pCDNA3.1-DC-SIGN-D5FLAG, and pCDNA3.1-DC-SIGN-D7-FLAG) was done by PCR
using oligonucleotides CD209FLAG (5⬘-GGGAATTCGCCACCATGGACTACAAGGACGACGATGACAAGAGTGACTCCAAGGAACCAAGAC-3⬘) and CD209as.
Stable and Transient Transfection of DC-SIGN Mutants and
Isoforms
Site-directed Mutagenesis and Generation of DC-SIGN
Chimeric Molecules
Site-directed mutagenesis was carried out using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla,
CA) on the pCDNA3-DC-SIGN 1A expression plasmid (13)
according to the manufacturer’s instructions. Oligonucleotides
used for mutagenesis included: DC-SIGN-C/Ss (5⬘-GAGCTTAGCAGGGTCTCTTGGCCATGGTC-3⬘) and DC-SIGN-C/
Sas (5⬘-GACCATGGCCAAGAGACCCTGCTAAGCTC-3⬘),
for mutation of Cys37 to Ser, with the resulting plasmid termed
pCDNA3.1-(DC-SIGN C/S); and DC-SIGN-N/Qs (5⬘-GACGCGATCTACCAGCAGCTGACCCAGCTTAAAG-3⬘) and
DC-SIGN-N/Qas (5⬘-CTTTAAGCTGGGTCAGCTGCTGGTAGATCGCGTC-3⬘), for mutation of Asn80 to Gln, with the
resulting plasmid termed pCDNA3.1-(DC-SIGN N/Q). Each
mutant construct was verified by DNA sequencing.
To generate DC-SIGN expression vectors lacking the lectin
domain, PCR was performed on the pCDNA3.1-DC-SIGN 1A
using oligonucleotides CD209s and CD209⌬Lectin (5⬘-CCCCAAGCTTGTCACAGGCGTTCCACTGCAGC-3⬘). PCR-generated fragments were resolved in agarose gels, purified, and
sequenced. Fragments containing either the full-length (8d⌬L)
and a 7- and 6-repeat neck regions (repeats 1 through 7, 7d⌬L;
repeats 1 through 6, 6d⌬L) were cloned into pCDNA3.1(⫺) to
yield pCDNA3.1-DC-SIGN 8d⌬L, pCDNA3.1-DC-SIGN
7d⌬L, and pCDNA3.1-DC-SIGN 6d⌬L plasmids.
Flow Cytometry and Antibodies
Cellular phenotypic analysis was carried out by indirect
immunofluorescence, using FITC-labeled goat anti-mouse
antibody (Serotec, Oxford, UK). Monoclonal antibodies used
for cell surface staining included MR1 (directed against the lectin domain of DC-SIGN), and the supernatant from the mouse
FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7
Immunofluorescence
Cells were resuspended in PBS and allowed to adhere onto
poly-L-lysine-coated coverslips for 60 min at 37 °C. After a brief
washing step with PBS, cells were fixed and permeabilized in a
1:1 solution of acetone:methanol for 10 min at ⫺20 °C, washed,
and stained with the MR1 monoclonal antibody (13) followed
by an incubation with an FITC-labeled goat anti-mouse antibody. Coverslips were mounted in fluorescent mounting
medium (DakoCytomation, Carpinteria, CA), and representative fields were photographed through an oil immersion lens on
a Nikon Eclipse E800 microscope equipped for epifluorescence
or by confocal microscopy.
Cell Surface Protein Labeling and Precipitation
For labeling, immature MDDCs were washed with PBS 1 mM
EDTA, resuspended in PBS, pH 8.0, and incubated in 0.5 mg/ml
biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester
sodium salt (Pierce) for 30 min at 4 °C. Cells were extensively
washed in PBS and lysed using 10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.025% sodium azide, 1% Brij 58 (Sigma-Aldrich), 1 mM
iodoacetamide, 2 mM Pefabloc (Alexis Biochemicals, Lausen,
Switzerland), and 2 ␮g/ml aprotinin, antipain, leupeptin, and
pepstatin. For precipitation of biotin-labeled proteins, Streptavidin-agarose (Sigma-Aldrich) was added to the lysates, and the
mixture incubated for 1 h at 4 °C. After centrifugation, beads
were extensively washed in 10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.025% sodium azide, 0.1% Brij 58, resuspended in 3⫻
Laemmli sample buffer (2% SDS, 6.25 mM Tris base, 10% glycerol), and boiled. Eluted material was resolved by SDS-PAGE
under reducing or non-reducing conditions and subsequent
Western blot with polyclonal antibodies specific for DC-SIGN.
Coprecipitation of DC-SIGN 1A/DC-SIGN 8d⌬L or DC-SIGN
1A-His/DC-SIGN-D3/-D5-FLAG hetero-oligomers was performed on lysates from transiently transfected COS-7 with
MR1 antibody as previously described (42), and precipitated
material was detected with specific polyclonal antibodies, or
using anti-His antibody for precipitation and anti-FLAG-HRP
antibody for detection of precipitated material, respectively.
Cross-linking Experiments
Cross-linking experiments were performed using the watersoluble cross-linking agent dithiobis(succinimidylpropionate)
(DTSSP) according to the manufacturer’s instructions (Pierce).
Briefly, immature MDDC was washed with PBS 1 mM EDTA,
resuspended in 1 ml of PBS, and incubated in the presence of
100 ␮l of 10 mM DTSSP in sodium citrate 5 mM, pH 5.0, for 30
min at room temperature. Stop solution (20 mM Tris-HCl, pH
7.5) was added (15 min at room temperature), and cells were
washed twice with PBS. Total cell lysates were obtained in 10
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.025% sodium azide, 0.5%
Nonidet P-40, 1 mM iodoacetamide, 2 mM Pefabloc (Alexis Biochemicals), and 2 ␮g/ml aprotinin, antipain, leupeptin, and
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For transient transfections, COS-7 or HEK293T cells were
transfected with SuperFect (Qiagen) using pCDNA3.1-based
expression plasmids containing the distinct isoforms or
mutants of the DC-SIGN cDNA. To generate stable transfectants, K562 cells were transfected with pCDNA3.1-based constructs using SuperFect and cultured in complete medium supplemented with 300 ␮g/ml G418 (Invitrogen). Stable DC-SIGN
expression of the selected population was verified using the
anti-DC-SIGN MR1 monoclonal antibody (13). Isolation of
K562-DC-SIGN 1A expressing different levels of DC-SIGN was
accomplished by cell sorting after staining with the MR1 monoclonal antibody (13).
myeloma P3-X63Ag8 (X63) was used as the control. All incubations were done in the presence of 50 ␮g/ml human IgG to
prevent binding through the Fc portion of the antibodies. Flow
cytometry analysis was performed with an EPICS-CS (Coulter
Cientı́fica, Madrid, Spain) using log amplifiers.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
pepstatin (Nonidet P-40 lysis buffer). 10 ␮g of each lysate was
subjected to SDS-PAGE as described for Western blot experiments. For cleaving the cross-linker agent, lysates were incubated with 5% ␤-mercaptoethanol in Laemmli sample buffer.
Generation of Polyclonal Antisera against DC-SIGN Structural
Domains
Functional Characterization of DC-SIGN Isoforms and Mutants
C. albicans and A. fumigatus Binding Assays—Conidia were
labeled with 0.1 mg/ml FITC for 1 h at room temperature and
extensively washed. For conidia-binding assays (25), cells were
washed, resuspended in complete medium, and pretreated for
20 min at room temperature with anti-DC-SIGN (MR1) or an
isotype-matched irrelevant antibody (X63). Then cells were
incubated with FITC-labeled A. fumigatus or C. albicans
conidia at the indicated ratios for 30 min at room temperature.
After extensive washing, cells were fixed with 2% paraformaldehyde in PBS for 1 h at 4 °C, washed, and analyzed by flow
cytometry.
DC-SIGN-dependent Adhesion Assays—DC-SIGN-dependent
adhesion was evaluated using Saccharomyces cerevisiae mannan
as specific ligand. 96-well microtiter EIA II-Linbro plates were
coated overnight with mannan at 50 ␮g/ml in PBS at 4 °C, and
the remaining sites were blocked with 0.5% bovine serum albumin for 2 h at 37 °C. Cells were labeled in RPMI 0.5% bovine
serum albumin with the fluorescent dye 2⬘,7⬘-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester
(Molecular Probes, The Netherlands) at 37 °C and then preincubated for 20 min with either the isotype-matched control
X63 or the function-blocking MR1 antibodies. Cells were then
allowed to adhere to each well for 15 min at 37 °C. Unbound
cells were removed by three washes with RPMI 0.5% bovine
serum albumin, and adherent cells were quantified using a fluorescence analyzer. Where specified, results are presented as
“DC-SIGN-dependent binding,” defined as: DC-SIGNdependent binding ⫽ (% bound cells in the presence of
P3X63 ⫺ % bound cells in the presence of MR1).
Leishmania Amastigote Binding Assays—Cells were washed
in PBS 1 mM EDTA, resuspended in complete medium and
5,6-carboxyfluorescein succinimidyl ester-labeled parasites
were added onto the cells at a 10:1 (amastigotes:cell) ratio, and
incubated at room temperature for 30 min. Afterward, cells
were fixed and analyzed by flow cytometry. For inhibition
assays, cells were preincubated for 10 min at room temperature
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NMR Experiments
Binding of soluble glucomannan from Candida utilis (IF) to
DC-SIGN transfectants was done by basic Saturation Transfer
Difference, as previously described (43).
Western Blot
Total cell lysates were obtained in Nonidet P-40 lysis buffer,
and 10 ␮g of each lysate was subjected to SDS-PAGE under
reducing or non-reducing conditions and transferred onto an
Immobilon polyvinylidene difluoride membrane (Millipore,
Bedford, MA). After blocking of the unoccupied sites with 5%
nonfat dry milk in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1%
Tween 20, protein detection was performed using the SuperSignal West Pico chemiluminescent system (Pierce). Detection
of DC-SIGN was carried out using polyclonal antiserum against
the C-terminal 20-residue peptide of DC-SIGN (C-20,
sc-11038, Santa Cruz Biotechnology, Santa Cruz, CA), amino
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Peptides based on the sequence of the sixth repeated domain
of the DC-SIGN neck region (GELPEKSKQQEIYQELTRLKAAV), and the region between residues 6 and 33 of the cytoplasmic tail (EPRLQQLGLLEEEQLRGLGFRQTRGYKS), were
synthesized by the multiple antigen peptide system (13). New
Zealand White rabbits were immunized by subcutaneous injection of each peptide (for DSG-1 and DSG-2 antisera) or the
recombinant DC-SIGN lectin domain (for DSG-4 antiserum)
expressed in bacteria (0.5 ml of a 1 mg/ml solution in PBS) in
complete Freund’s adjuvant (1:1) on day 0 and in incomplete
Freund’s adjuvant (1:1) on days 21 and 42. Rabbits were bled on
day 49, and serum was assayed for DC-SIGN recognition in
Western blot experiments.
with either MR1 antibody or an irrelevant antibody (X63) in
complete medium before parasite addition.
DC-SIGN Internalization—Cells were washed, resuspended
in complete medium, and incubated with MR1 antibody (13)
for 1 h at 4 °C to prevent DC-SIGN internalization. After extensive washing, cells were placed at 37 °C to allow internalization
to occur. At the indicated time points, internalization was
stopped by adding of cold PBS, and cells were immediately
placed at 4 °C. To detect the remaining membrane-bound MR1
antibody, an FITC-labeled goat anti-mouse antibody was
added, incubated for 30 min at 4 °C, and analyzed by flow
cytometry. All incubations were done in the presence of 50
␮g/ml human IgG to prevent binding through the Fc portion of
the antibodies.
Ebola GP1-Fc Binding Assays—Cells were washed in PBS and
1 mM EDTA, resuspended in complete medium, and incubated
with GP1-Fc either in the presence of a monoclonal antibody
against DC-SIGN (MR1) or an irrelevant antibody (X63) for 20
min at 4 °C. Then, cells were incubated with a phycoerythrinlabeled polyclonal antiserum against human IgG Fc (Beckman
Coulter), and analyzed by flow cytometry.
Sugar-coated Fluorescent Bead Binding to DC-SIGN—Synthetic fluorescein-labeled fucose- or Lewisx-containing polyacrylamide beads (FITC-PAA-NAc-Gal, FITC-PAA-Fuc, and
FITC-PAA-Lex) were obtained from Lectinity (Moscow, Russia). After washing with PBS and 1 mM EDTA, transiently transfected HEK293T cells were resuspended in complete medium,
and sugar-PAA-FLU beads were added to a final concentration
of 20 ␮g/ml and incubated at 37 °C for 30 min. After extensive
washing, cells were fixed for 1 h at room temperature, and analyzed by flow cytometry. For inhibition assays, cells were preincubated for 10 min at room temperature with either MR1 antibody or an irrelevant antibody (X63) in complete medium
before beads addition. Results from binding assays were
expressed as “Binding Index,” which represents the DC-SIGNdependent binding relative to DC-SIGN expression levels
according to the formula: Binding index ⫽ (mean fluorescent
intensity (MFI) of cells plus beads ⫺ MFI of cells plus beads in
the presence of MR1)/(MFI after MR1 staining/MFI after staining with X63).
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
acids 61–200 (H-200, sc-20081, Santa Cruz Biotechnology), or
polyclonal antisera raised against peptides based on the sixth
23-residue repeats within the DC-SIGN neck region (DSG-1),
against a 28-residue peptide from the DC-SIGN cytoplasmic
tail (DSG-2), or against the whole lectin domain (DSG-4).
Carbohydrate Affinity Precipitations
For precipitation of mannan- and N-acetylgalactosaminebinding proteins, transiently transfected HEK293T or COS-7
cells (3 ⫻ 106) were lysed in Nonidet P-40 lysis buffer. Then, 200
␮l of each lysate was taken to 1 ml with Nonidet P-40 lysis buffer
and incubated with 50 ␮l of mannan- or N-acetylgalactosamine-agarose (Sigma-Aldrich) for 12 h at 4 °C. After extensive washing in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.025%
sodium azide, 0.05% Nonidet P-40, bound proteins were eluted
by boiling the agarose beads in 3⫻ Laemmli sample buffer.
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SDS-eluted and non-bound materials were resolved by SDSPAGE and DC-SIGN detection accomplished with specific
polyclonal antibodies.
RESULTS
Range of DC-SIGN Alternatively Spliced Isoforms—MDDCs
express a high number of alternatively spliced DC-SIGN
mRNA species (36), which are also found at mucosal HIV transmission sites (39). To determine the range of DC-SIGN mRNA
species found in MDDC from a single donor, three different set
of primers were designed to specifically amplify the prototypical DC-SIGN mRNA (DC-SIGN 1A), or species encoding
either an alternative cytoplasmic domain (DC-SIGN 1B) or
lacking the transmembrane domain (DC-SIGN ⌬TM) (Fig. 1A).
Sequencing of the amplified fragments resulted in the identification of DC-SIGN mRNA species encoding for variants with
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FIGURE 1. Detection of DC-SIGN isoforms on monocyte-derived dendritic cells. A, schematic representation of the DC-SIGN mRNA and the position of
oligonucleotides used to amplify DC-SIGN 1A isoforms (sense plus antisense), DC-SIGN 1B isoforms (Ib plus antisense), or DC-SIGN ⌬TM isoforms without the
transmembrane region (soluble plus antisense). (Exons I–VI, genomic organization; ATG, translational start sites; CYT, cytoplasmic domain; TM, transmembrane
region). B, schematic structure of the major PCR fragments obtained from RNA of immature MDDCs from a single donor. C–F, lysates from COS-7 cells
transiently transfected with expression vectors for DC-SIGN 1A, a chimeric construct lacking the lectin domain (8d⌬L) or an empty vector (Mock) (C), precipitated material from surface (biotin-labeled) immature MDDCs (D), lysates from THP-1 cells differentiated with Bryostatin (Bryo) in the presence or absence of
interleukin-4 (E), or lysates from immature MDDCs either untreated or incubated with the cross-linking agent DTSSP (F) were resolved by SDS-PAGE under
non-reducing or reducing conditions (in the presence of ␤-mercaptoethanol, ␤-MSH). The gels were then subjected to Western blot using polyclonal antisera
against the neck domain (DSG-1 in C and F; H-200 in E), against the cytoplasmic tail of DC-SIGN (DSG-2) (C, D, and F) or against the C-terminal 20-amino acid
peptide of DC-SIGN (C-20) (C). The specificity of the distinct antisera is indicated in each panel. Thin lines indicate the position of bands with higher mobility than
the full-length DC-SIGN isoform. In D, the biotin-labeled proteins precipitated with streptavidin-agarose (SP) were analyzed in parallel with whole cell extracts
(WE) and proteins in the supernatant or non-precipitated (SN).
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
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greatly reduced multimerization ability (Fig. 2B, right panel),
which indicates that, although DC-SIGN multimerization
might be mediated by the neck region (34, 35, 45), it requires or
is stabilized by the lectin domain of the molecule.
Along this line, the presence of lectin domain-lacking constructs (8d⌬L, 7d⌬L, and 6d⌬L) had a negative impact on the
degree of multimerization of DC-SIGN 1A, as we observed a
lower level of DC-SIGN 1A multimers in the presence of these
deletion constructs (Fig. 2C). This could be explained by an
increased formation of heteromultimers (formed by DC-SIGN
1A and constructs lacking the lectin domain), which might
exhibit lower stability in the presence of denaturing detergent,
thus precluding its detection. If so, the existence of heteromultimers could be demonstrated by coprecipitation experiments
on lysates from cells cotransfected with DC-SIGN 1A and
8d⌬L. The fact that the 8d⌬L isoform was pulled down after
immunoprecipitation of lectin domain-containing molecules
with the MR1 monoclonal antibody (Fig. 2D) confirms that lectin domain-lacking constructs associate with the prototypic
DC-SIGN 1A isoform, suggests that heteromultimers of DCSIGN 1A and 8d⌬L are more sensitive to the presence of denaturing agents than DC-SIGN 1A homomultimers and confirms
a role for the lectin domain of DC-SIGN in the formation of
stable oligomers.
Structural Requirements of the Neck Domain for DC-SIGN
Multimerization—Although the neck domain is absolutely
required for the formation of multimers of recombinant nonglycosylated DC-SIGN and DC-SIGNR (34, 35, 45), its role in
DC-SIGN multimerization on the cell membrane remains
unclear. To address this issue, we analyzed the pattern of multimerization of the prototypic full-length molecule (1A), naturally occurring (4d, 4d⬘, 2d, and 1d) or in vitro generated (3d)
isoforms differing in the number and order of the neck region
repeats, and constructs mutated at the N-linked glycosylation
site (1AN/Q and 1dN/Q) (Fig. 3A). Transient transfection
revealed that the distinct DC-SIGN isoforms differed in their
ability to form oligomers. A high proportion of full-length DCSIGN 1A appeared as multimers, whereas deletion of half the
neck region (4d) resulted in a considerable reduction of high
order multimers (Fig. 3A). By contrast, isoforms 3d and 2d,
whose neck regions are composed of three and two repeats,
exhibited an oligomerization ability roughly similar to that of
the full-length molecule, whereas isoform 1d showed the weakest oligomerization (Fig. 3A). These results indicate that the
presence of at least two repeats within the neck region is sufficient for DC-SIGN multimerization. On the other hand, the
lower multimerization of 4d suggests that there is no direct
correlation between the length of the neck region and oligomerization, and that the distinct repeats within the neck
region might not be functionally equivalent. This hypothesis
was confirmed when comparing the low multimerization capability of 4d (composed of neck repeats 1, 6, 7, and 8) with the
normal (similar to 1A) oligomerization pattern of 4d⬘ isoform,
whose neck region is composed of repeats 1, 2, 3, and 8 (Fig. 3A),
thus confirming that multimerization capability of DC-SIGN
on the cell membrane is dependent not only on the number of
neck repeats but also on their arrangement, and that the repeats
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alternative cytoplasmic tails and potentially soluble isoforms,
with each group including transcripts differing in the neck
domain or the carbohydrate-binding region (Fig. 1B). Therefore, and in agreement with previous reports (14, 36, 39), the
DC-SIGN gene gives rise to a large number of alternatively
spliced mRNA species, most of which differ in the number of
23-residue repeats within the neck domain, previously demonstrated to mediate multimerization of recombinant DC-SIGN
(34).
Next we generated polyclonal antisera specific for either the
neck domain (DSG-1) or the prototypic cytoplasmic tail of DCSIGN 1A (DSG-2). Both DSG-1 and DSG-2 specifically
detected the 44-kDa band of the prototypic full-length isoform
DC-SIGN 1A, as well as a deletion mutant lacking the lectin
domain (8d⌬L), whereas a polyclonal antiserum against the 20
C-terminal residues of the lectin domain (C-20) only detected
the full-length molecule (Fig. 1C).
To determine the degree of DC-SIGN multimerization on
MDDC, cell surface proteins were biotin-labeled, and streptavidin pulled-down material was analyzed for the presence of
DC-SIGN. Under non-reducing conditions, the DSG-2 antiserum detected distinct several bands corresponding to DCSIGN monomers, dimers, trimers, tetramers, and high order
multimers either in the whole extracts and the pull-down (Fig.
1D, left panel, lanes WE and SP, respectively), which suggests
that DC-SIGN multimers are found on the cell surface of
MDDCs. Analysis of cell surface DC-SIGN molecules from
MDDC under reducing conditions also revealed the presence of
additional higher mobility bands that were also recognized by
the DSG-2 antiserum (Fig. 1D, right panel, lane SP). The same
pattern was detected in total lysates of dendritic-like THP-1
cells (14) using a polyclonal antiserum against the whole neck
region of the molecule (Fig. 1E), and similar bands could be
detected in MDDC lysates with both DSG-1 and DSG-2 antisera (Fig. 1F). Therefore, DC-SIGN isoforms can be detected on
the cell surface of monocyte-derived dendritic cells, although to
a lower extent than the full-length DC-SIGN 1A.
Contribution of the Lectin Domain to DC-SIGN Multimerization on the Cell Membrane—DC-SIGN multimer formation in
MDDC could be readily identified by SDS-PAGE (Fig. 1D) (33,
44). In fact, although treatment with the membrane-impermeable cross-linker DTSSP enhanced the formation of high order
multimers, DC-SIGN monomers, dimers, and multimers were
readily detected by C-20, DSG-1, and DSG-2 antisera under
non-reducing conditions (Fig. 1F). The detection of DC-SIGN
multimers was almost completely prevented in the presence of
reducing agents (Fig. 1, D and F), indicating that disulfide
bridges contribute to multimerization. Because all Cys residues
within the lectin domain are engaged in intramolecular disulfide bridges (27), we determined the effect of mutating Cys37,
the only DC-SIGN cysteine residue outside of the lectin domain
and located within the cytoplasmic tail. Mutation of Cys37 had
no effect on the degree of formation of DC-SIGN multimers
(Fig. 2B, left panel), suggesting that multimerization could be
dependent on cysteine residues within the lectin domain. The
lectin domain was then removed from either the DC-SIGN prototypic isoform (8d⌬L) or from an isoform with only six repeats
(6d⌬L) (Fig. 2A). Both 8d⌬L and 6d⌬L constructs displayed
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
within the neck region of DC-SIGN are not functionally
interchangeable.
In agreement with the results obtained after transient transfection, the 4d isoform also exhibited a greatly reduced proportion of DC-SIGN multimers when stably expressed in K562
cells, whereas multimerization of 2d isoform was similar to that
of DC-SIGN 1A (Fig. 3B), a finding also observed after transfection in T lymphoblastoid Jurkat cell (data not shown). Functional analysis of the three isoforms in K562 transfectants
revealed that 1A, 4d, and 2d bound soluble C. utilis glucomannan (IF), as determined by one-dimensional saturation transfer
difference (43, 46) (Fig. 3C), and were internalized after MR1mediated engagement (Fig. 3D). Therefore, it can be concluded
that the degree of multimerization of functional DC-SIGN isoforms on the cell surface is cell-type independent and does not
influence the ligand-induced internalization of the molecule.
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The inability of the first repeat to mediate multimerization
(1d in Fig. 3A), and the fact that it contains the only potential
N-glycosylation site of DC-SIGN, prompted us to determine
the contribution of glycosylation to DC-SIGN oligomerization.
Replacement of Asn80 for Gln in the context of the full-length
molecule (1AN/Q) greatly increased the proportion of
DC-SIGN multimers (compare 1A and 1AN/Q in Fig. 3A) and
suggests that glycosylation of the first neck repeat negatively
affects DC-SIGN multimerization. The negative influence of
glycosylation on multimerization was even more evident upon
analysis of the 1dN/Q mutant, whose neck domain is formed
only by the first repeat with the Asn80/Gln replacement. Unlike
the 1d isoform, oligomers (and even high order multimers) of
the 1dN/Q mutant could be easily detected (Fig. 3A). In fact,
and like in the case of 1AN/Q, no 1dN/Q monomers were
observed under non-reducing conditions (Fig. 3A). Therefore,
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FIGURE 2. Determination of structural requirements for DC-SIGN multimerization. A, schematic representation of the DC-SIGN alternatively spliced
isoforms (1A, 4d, 4d⬘, 3d, 2d, and 1d), mutants (1AC/S, 1AN/Q, and 1dN/Q) and chimeric molecules (8d⌬L, 7d⌬L, and 6d⌬L) used throughout the study. The
Cys37 residue is indicated by a cross (†) on the transmembrane region, and the presence of potential N-glycosylation sequence is indicated by a black dot on the
first repeat of the neck domain. B and C, COS-7 cells transiently transfected with the indicated DC-SIGN constructs were lysed, resolved by SDS-PAGE, and
subjected to Western blot using DSG-2 (B), or DSG-1 and DSG-4 (C) polyclonal antisera. D, lysates from COS-7 cells transiently transfected with the indicated
DC-SIGN constructs were immunoprecipitated with a monoclonal antibody against the DC-SIGN lectin domain (MR1) or a control antibody (CNT), and
immunoprecipitated proteins were resolved by SDS-PAGE and subjected to Western blot using DSG-1 polyclonal antiserum. Immunoprecipitated proteins
were analyzed in parallel with whole cell lysates (WE).
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
glycosylation of the first repeat in the neck region impairs multimerization of DC-SIGN molecules.
Influence of Multimerization on DC-SIGN Pathogen
Recognition—Despite the differences in their ability to multimerize, transient transfection of the whole range of constructs
previously assayed revealed that all of them are capable of
binding Candida yeasts and Leishmania amastigotes to a
similar extent (supplemental Fig. S1A). To rule out the subtle
differences in pathogen binding among the distinct constructs
we evaluate Candida and L. pifanoi amastigotes binding by cells
expressing decreasing levels of three naturally occurring isoforms (1A, 4d, and 2d), and no significant difference was
observed when comparing binding by cells expressing similar
levels of the three constructs (supplemental Fig. S1B and not
shown). However, the results showed that the binding ability of
the distinct DC-SIGN isoforms correlate with their expression
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level (supplemental Fig. S1B). Therefore, because the multimerization ability of the 4d isoform is considerably lower than
that of 1A and 2d (see Fig. 3A), these results indicate that the
recognition of C. albicans yeasts or L. infantum amastigotes by
distinct DC-SIGN isoform/mutants does not correlate with
their multimerization degree. Consequently, the multimerization degree of an isoform does not predict its pathogen-binding
ability.
Influence of Multimerization on Sugar Recognition by
DC-SIGN—The lack of correlation between multimerization
degree and pathogen-binding ability of DC-SIGN isoforms
could be explained by the large amount of DC-SIGN ligands
immobilized on the pathogen surface, which would drive the
formation of DC-SIGN-containing clusters (47, 48) and
might obscure the contribution of the affinity/avidity of
individual molecules/oligomers to the whole interaction. To
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FIGURE 3. Multimerization capacity of DC-SIGN isoforms and constructs. A and B, lysates from transiently transfected HEK293T cells (A) or K562 cells
stably transfected (B) with the indicated DC-SIGN constructs (see upper drawing) or a mock construct were analyzed by SDS-PAGE and subjected to
Western blot with the DSG-2 polyclonal antiserum. In B, two different clones of K562-DC-SIGN 1A were analyzed, whose relative level of DC-SIGN
expression is indicated by a dark triangle. C, binding of C. utilis glucomannan (IF) to K562 cells stably transfected with the indicated DC-SIGN isoforms by
means of one-dimensional saturation transfer difference NMR. The lower profile represents the 1H NMR spectrum of IF in PBS at 298 K. For comparative
purposes, two subpopulations of K562-DC-SIGN 1A, which differ in their DC-SIGN cell surface expression level, were assayed. The graph illustrates the
signal intensity yielded by each transfectant (y-axis) and the chemical shift (␦) in parts per million (ppm). D, monoclonal antibody-induced internalization of DC-SIGN isoforms in K562 cells stably transfected with the 1A, 4d, or 2d isoforms. DC-SIGN expression at different time points is shown relative
to the initial cell surface expression (100%, upper panel), and was determined by flow cytometry. For the three stable transfectants, the MFI (lower
number) and the percentage of positive cells (upper number) at time zero are shown.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
avoid pathogen-induced clustering effects on the membrane, we assess the ability of the distinct DC-SIGN constructs to be retained by sugars after membrane solubilization. As shown in Fig. 4A (upper panels), except 1d, all
DC-SIGN constructs were specifically retained by mannan
(a polysaccharide that blocks most DC-SIGN interaction).
However, analysis of molecules not retained by mannan
(supernatant) revealed that constructs 1A, 1AN/Q, 4d, and
1dN/Q are retained with higher efficiency than the 4d⬘, 3d,
and 2d constructs (Fig. 4A, lower panels). Monomers were
preferentially retained by mannan within the strong mannan-binding and N-glycosylation-containing constructs (1A
and 4d) (lanes 1A and 4d in the left panels of Fig. 4A). By
contrast, those exhibiting lower binding to mannan (4d⬘, 3d,
and 2d) were preferentially retained as multimers, as monomers were almost exclusively detected in the supernatant
(lanes 4d⬘, 3d, and 2d in left panels of Fig. 4A). Furthermore,
and in agreement with the negative effect of N-glycosylation
on DC-SIGN multimerization, the 1AN/Q and 1dN/Q constructs were preferentially retained as multimers. Therefore,
functional analysis of detergent-solubilized cellular DC-SIGN
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demonstrates that multimer formation compensates for the
lower mannan-binding affinity of certain DC-SIGN constructs after membrane solubilization, an effect that
becomes even more evident when less-than-optimal sugar
ligands (NAc-Gal) were used, which only retained lectin
multimers (Fig. 4B). Therefore, this set of data indicates that
the number and arrangement of the repeats within the neck
domain directly influences the specificity and the sugarbinding ability of the DC-SIGN lectin domain.
To further evaluate the relevance of DC-SIGN cell surface
multimerization on ligand binding, cell surface expressed 1d
and 1dN/Q constructs were compared in their ability to bind
FITC-PAA-Fucose and Lewisx beads. 1dN/Q, which appears
almost exclusively as multimers, displayed a stronger beadbinding activity than 1d, whose multimers can barely be
detected, and the same finding was observed at three distinct
cell surface expression levels (Fig. 4C). These results further
support the involvement of cell surface DC-SIGN multimerization in ligand binding, and establish N-linked-glycosylation as a
critical parameter for the DC-SIGN ligand-binding activity on
the cell surface.
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FIGURE 4. Sugar recognition by DC-SIGN isoforms. A and B, lysates of HEK293T cells transiently transfected with the indicated DC-SIGN variants were
precipitated with mannan- (A) or N-acetylgalactosamine (NAc-Gal)-agarose (B). Eluted proteins (upper panels) and non-bound proteins (supernatant, lower
panels) were resolved by SDS-PAGE under reducing (right panels in A) or non-reducing (left panels in A, both panels in B) conditions, and subjected to Western
blot with DSG-2 antiserum. C, HEK293T cells transiently transfected with the indicated DC-SIGN variants (1A, 1d, and 1dN/Q) were incubated with either
FITC-PAA-fucose or FITC-PAA-Lewisx beads (20 ␮g/ml) in the presence of MR1 blocking antibody or an irrelevant antibody, and the percentage of cells with
bound beads was determined by flow cytometry. The percentage (upper number) and MFI (lower number) of cells stained with either a MR1 (black text) or an
isotype-matched antibody (gray text) are indicated in each case. The results from three independent experiments on cells with different DC-SIGN cell surface
levels (high, left panel; middle, middle panel; and low, right panel) are shown.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
Structural and Functional Characterization of Polymorphic
Variants of DC-SIGN—The above results demonstrate that the
neck region is an important determinant in the ligand-binding
activity of DC-SIGN on the cell surface. It has been reported
that, among the polymorphisms in the DC-SIGN gene (38,
49 –52), those affecting the length of the neck domain correlate
with altered susceptibility to HIV-1 infection (38). In fact, similar findings have been reported in the case of the related DCSIGNR and susceptibility to HIV-1 and severe acute respiratory
syndrome infection (53–55). To evaluate the functional significance of polymorphic DC-SIGN neck domains, three distinct
allelic variants, whose neck domains contain only seven repeats,
were identified at the genomic DNA and RNA level (Fig. 5, A
and B) and functionally characterized. These polymorphic variants lack repeats 3, 5, or 7, but their multimerization ability (Fig.
5B), cell surface expression (Fig. 5C), and ligand-induced internalization capability (Fig. 5D) were found to be indistinguishable from that of the prototypic molecule. Moreover, the three
variants displayed unaltered capacity for recognition of Leish-
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mania and Aspergillus (supplemental Fig. S2A) and mediated
cellular binding to Ebola GP1-Fc and Mannan (supplemental
Fig. S2B) and were retained by agarose-bound mannan after
membrane solubilization (supplemental Fig. S2C), and mediated binding of C. utilis glucomannan to cells as determined by
one-dimensional saturation transfer difference NMR (supplemental Fig. S2D). Therefore, DC-SIGN polymorphic variants
lacking a single neck domain repeat (3, 5, or 7) exhibit functional activities that are similar to those exhibited by the prototypic DC-SIGN molecule.
Because altered susceptibility to infections has been mostly
observed in individuals with heterozygosity at the DC-SIGN (or
DC-SIGNR) gene (38, 53–55), we next evaluated the influence
of DC-SIGN polymorphic variants (-D3, -D5, and -D7, Fig. 5B)
on the expression, multimerization, and functional capability of
the prototypic molecule when expressed on the same cell.
Transient transfection experiments demonstrated that the
expression of any of the polymorphic variants had no influence
on the DC-SIGN 1A total or cell surface expression (Fig. 6, A
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FIGURE 5. Structural and functional characterization of DC-SIGN polymorphic variants. A, schematic representation of the DC-SIGN gene and the position
of oligonucleotides used to amplify the DC-SIGN neck domain-polymorphic variants (upper panel). Examples of the amplification of genomic DNA (left lower
panel) and RNA (right lower panel) are shown, indicating the genotype of each donor (1A/1A, homozygote for DC-SIGN, with two 8-neck repeat alleles; 1A/-D3,
1A/-D7, heterozygotes, with a full-length neck domain in one allele and a second allele coding for a neck with 7 repeats, missing either repeat 3 (-D3) or repeat
7 (-D7), respectively). B, upper panel: schematic structure of the prototypic DC-SIGN variant (1A) and the three polymorphic alleles identified (-D3, -D5, and -D7).
Lower panel: lysates from K562 cells stably transfected with the indicated constructs were lysed, subjected to SDS-PAGE under reducing and non-reducing
conditions, and analyzed by Western blot with the DSG-2 polyclonal antiserum. C, cell surface expression of DC-SIGN polymorphic variants on stable transfectants on K562 cells, as determined by flow cytometry (upper panels) and immunocytofluorescence (lower panels). The middle panels show the corresponding
phase contrast images. The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGN MR1 antibody (black text and profile) or
the X63 control antibody (gray text and profile) are indicated. D, monoclonal antibody-induced internalization of DC-SIGN in K562 cells stably transfected with
the indicated polymorphic variants. Flow cytometry expression is expressed relative to the level of DC-SIGN in each transfectant maintained at 4 °C (arbitrarily
considered as 100).
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
and B). Like in the case of the 1A/8d⌬L cotransfectants, heterooligomers might have an increased sensitivity to denaturing
agents. However, coimmunoprecipitation experiments with
epitope-tagged molecules demonstrated that the DC-SIGN 1A
molecules preferentially formed homo-oligomers, and associate weakly to seven repeat-containing polymorphic variants
(⬍1% of the prototypic DC-SIGN molecules are engaged in
hetero-oligomer formation) (Fig. 6C). Therefore, the shorter
polymorphic variants can be expressed on the cell surface but
tend to form homo-oligomers and associate very weakly with
the prototypic DC-SIGN 1A full-length isoform. This result
would imply that cells heterozygous at the DC-SIGN gene
might almost exclusively express homo-oligomers on the cell
surface. We tested this hypothesis by analyzing the DC-SIGN
isoform expression and multimerization state in MDDC from a
donor previously identified as a CD209 heterozygote (see Fig.
5A). The prototypic and shorter variant of DC-SIGN were
expressed to a similar extent in heterozygous dendritic cells,
but no evidence was found of hetero-oligomer formation (Fig.
FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7
6D), confirming that DC-SIGN multimerization takes place
preferentially among variants whose neck region has identical
structure.
The low percentage (or impaired stability) of lectin heterooligomers might explain the reduced pathogen-binding capacity exhibited by cells coexpressing allelic variants of DC-SIGNR
(53) and the correlation between DC-SIGN/DC-SIGNR neck
region heterozygosity and susceptibility to viral infection (38,
53–55). Consequently, DC-SIGN-dependent activities of cells
coexpressing different DC-SIGN allelic variants were evaluated
on transiently transfected cells. As shown in Fig. 6B, coexpression of the DC-SIGN-D7 isoform (which lacks the seventh neck
domain repeat) did not significantly affect the ability of the
prototypic DC-SIGN 1A isoform to bind immobilized mannan.
Along the same line, capture of fucose- or Lewisx-coated polyacrylamide beads by DC-SIGN 1A was not affected by the coexpression of the DC-SIGN-D3 isoform (which lacks the third
neck domain repeat) (Fig. 7A and not shown). These results
indicated that expression of polymorphic variants with shorter
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FIGURE 6. Influence of DC-SIGN polymorphic variants on the expression, multimerization, and functional capability of the prototypic molecule.
A, lysates from COS-7 cells transiently transfected with the indicated DC-SIGN constructs were subjected to Western blot with the DSG-2 polyclonal antiserum.
The mobility of monomers and trimers is indicated. The right panel shows the same experiment after a longer electrophoretic separation for increased
resolution of the DC-SIGN trimers. B, adhesion to immobilized mannan of HEK293T cells transiently transfected with the indicated DC-SIGN variants in the
presence of either a blocking antibody (MR1) or an irrelevant antibody (X63) (lower panel). The DC-SIGN expression levels of the distinct transfectants was
determined by flow cytometry and is indicated in the upper panel. The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGN
MR1 antibody (black text and profile) or the X63 control antibody (gray text and profile) are indicated. C, lysates from COS-7 cells transiently transfected with the
indicated constructs were immunoprecipitated with an anti-5xHis monoclonal antibody, and immunoprecipitates were subjected to Western blot using either
an anti-FLAG monoclonal antibody (upper panel) or the DSG-2 polyclonal antiserum (lower panel). Whole cell lysates were also analyzed as a transfection
control. D, lysates from MDDCs generated from donors characterized as homozygote for DC-SIGN with 8 neck repeats (1A/1A) or heterozygote, with alleles with
neck domain of 8 and 7 repeats (1A/-D7), were separated by SDS-PAGE under reducing and non-reducing conditions, and then subjected to Western blot with
the DSG-2 polyclonal antiserum. For control purposes, lysates from K562 cells stably transfected and COS-7 cells transiently transfected with the indicated
constructs were included in the experiment.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
neck domains does not significantly alter the pathogen recognition ability of cells expressing the prototypic DC-SIGN 1A
isoform.
Finally, because sugar precipitation had previously allowed
the identification of functional differences among alternatively
spliced isoforms (Fig. 4), lysates from MDDCs coexpressing
DC-SIGN 1A and DC-SIGN-D7 were subjected to precipitation with mannan-agarose. Whereas a single band (corresponding to DC-SIGN 1A) was specifically retained from the
1A/1A dendritic cells, both DC-SIGN 1A and DC-SIGN-D7
isoforms were equally retained by mannan-agarose when the
dendritic cell lysate from the 1A/-D7 donor was used (Fig. 7B).
Therefore, DC-SIGN 1A and DC-SIGN-D7 are retained by
mannan to a similar extent, confirming that shorter neck polymorphic variants of DC-SIGN retain their sugar-recognition
ability, showing no differences from that of the prototypic fulllength DC-SIGN 1A molecule.
DISCUSSION
DC-SIGN-dependent binding and uptake of clinically relevant pathogens by dendritic cells relies on the lectin ability
to bind mannose- and fucose-containing glycans (56). Studies on recombinant molecules have demonstrated that the
avidity of such interactions is mediated through multimerization of the lectin, which is accomplished through intermolecular associations mediated by the neck domain of the
molecule (27, 34). The neck region of DC-SIGN is composed
of eight 23-amino acid repeats, which are encoded in a single
exon whose polymorphism has been already demonstrated
(38, 51). In fact, DC-SIGN alleles with 4 –9 repeats within the
neck region-coding exon have been described (51), and heterozygosity at this specific exon correlates with altered susceptibility to HIV-1 infection (38). Besides, numerous DCSIGN alternatively spliced isoforms have been described at
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the mRNA level (14, 36). The combination of alternative
splicing and genomic polymorphism predicts that a large
repertoire of DC-SIGN protein isoforms might exist, most of
which would differ in the size of the neck domain (14, 36, 38,
51). However, to date, the functional characterization of DCSIGN isoforms and allelic variants on the cell membrane had
not been addressed. In the present manuscript we present
evidences that 1) DC-SIGN alternatively spliced mRNA species give rise to proteins that are expressed at the cell membrane on monocyte-derived dendritic cells, cell lines, and
transfectants; 2) DC-SIGN alternatively spliced isoforms differ in their multimerization capability and sugar-binding
ability; 3) the presence of two repeats within the neck
domain is sufficient for DC-SIGN multimerization; 4) the
neck domain repeats are not functionally interchangeably,
because the number and arrangement of repeats within the
neck domain critically determines the multimerization and
ligand-binding ability; 5) the lectin domain of DC-SIGN stabilizes or contributes to the neck region-dependent multimerization of DC-SIGN, which is negatively influenced by the
N-linked glycosylation of the first neck domain repeat; 6)
basal multimerization of the molecule does not predict the
pathogen-binding ability and does not correlate with ligandinduced internalization; and 7) polymorphic variants differing in neck domain composition can self-associate, but multimerize very poorly with the prototypic full-length
molecule, suggesting that the DC-SIGN molecules on the
cell surface predominantly appear as homo-multimers. The
data here presented constitutes the first demonstration that
alternative splicing and polymorphic variants of DC-SIGN
are expressed on monocyte-derived dendritic cells, where
they exhibit altered multimerization and carbohydratebinding abilities (splicing variants) and tend to segregate
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FIGURE 7. Influence of DC-SIGN polymorphic variants on the functional capability of the prototypic molecule. A, binding of either FITC-PAA-NAcGal or FITC-PAA-fucose beads to HEK293T cells transiently transfected with the indicated DC-SIGN constructs. Expression levels were determined by
flow cytometry (upper panels). The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGN MR1 antibody (black text
and profile) or the X63 control antibody (gray text and profile) are indicated. B, lysates from MDDCs from a homozygote (1A/1A) and a heterozygote
(1A/-D7) donors were incubated with mannan-agarose. Bound proteins (eluted, right panels) or whole cell lysates (whole lysate, left panels) were
resolved by SDS-PAGE under reducing conditions, and subjected to Western blot with the DSG-2 polyclonal antiserum or a monoclonal antibody against
CD45 as control (upper panels).
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7
unique among the repeats and contributes to the multimerization ability of recombinant DC-SIGNR (58). These results demonstrate the critical role of repeats 1 and 2 for DC-SIGN multimerization, because repeat 1 is capable of mediating multimer
formation, and the mere presence of repeat 2 appears sufficient
to overcome the inhibitory effect of the N-glycosylation at
repeat 1. These results are compatible and extend previous data
on the multimerization capability of recombinant DC-SIGN/
DC-SIGNR molecules, and establish neck glycosylation as an
important parameter to limit the degree of DC-SIGN multimerization in the cell.
The combination of genomic polymorphism and alternative
splicing at the DC-SIGN gene results in the generation of a large
number of isoforms/allelic variants of the molecule. Considering their variable multimerization capability, and the higher
avidity displayed by multimers, it is tempting to speculate that
the existence of all these variants might endow macrophages
and dendritic cells with a broader repertoire of ligand-binding
affinity and/or specificity. In fact, the ability of mannan-agarose
to differentially retain the various DC-SIGN splicing isoforms
(Fig. 4) would support this hypothesis. On the other hand, an
alternative function for the numerous DC-SIGN isoforms
could be the modulation of full-length DC-SIGN-dependent
functions. In this regard, and like the lectin domain-lacking
chimeric constructs (Fig. 2), isoforms with truncated lectin
domains might reduce the effective concentration of full-length
DC-SIGN molecules on the cell membrane, thus impairing its
multimerization on the cell surface and, consequently, the
binding and uptake of pathogens/ligands containing limiting
amounts of sugar ligands.
Regarding polymorphic variants, our results indicate that the
number of DC-SIGN allelic variants is greater than previously
thought. The study of Barreiro and Liu (38, 51) has defined
polymorphisms within the neck region of DC-SIGN and classified them according to the number of repeats. However, and at
least within the Spanish population, the allelic variants containing only 7 neck repeats are not structurally identical, and three
distinct alleles have been identified which differ in the missing
repeat within the neck domain (D3, D5, and D7). Therefore, it is
likely that most of the previously defined CD209 alleles are
really heterogeneous in terms of the arrangement of the neck
domain repeats they contain. On the other hand, and despite
the association found between neck domain heterozygosity at
the CD209 and CD209L genes and altered susceptibility to
HIV-1 (38, 55), hepatitis C (59, 60), or severe acute respiratory
syndrome infection (53), the polymorphic variants that we have
characterized exhibit similar homo-multimerization capability
and pathogen- and carbohydrate-binding specificity as the fulllength molecule. However, the polymorphic variants containing seven repeats (-D3, -D5, and -D7) exhibit a very weak ability
to assemble into hetero-multimers with the full-length
DC-SIGN 1A prototypic molecule, as hetero-multimers cannot
be observed by Western blot and an extremely low percentage
of the -D3 variant can be coprecipitated with DC-SIGN 1A (Fig.
6). This result is in contrast to the reported ability of recombinant polymorphic forms of DC-SIGNR to engage in stable
homo- and hetero-tetramers (58). However, we feel that this is
only an apparent discrepancy, because the N-linked glycosylaJOURNAL OF BIOLOGICAL CHEMISTRY
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from the prototypic molecule forming homo-multimers
(polymorphic variants with a shorter neck domain).
Whether DC-SIGN and related lectins are bona fide pathogen-recognition receptors or antigen-binding receptors whose
function is subverted by pathogens is still unclear (4, 57). Our
results indicate that the various alternatively spliced isoforms
differ in their ability to be retained by immobilized mannan,
whereas all of them are equally efficient in terms of pathogen
binding. We hypothesize that the large amount of DC-SIGN
ligands on the surface of interacting pathogens compensate for
the distinct affinity and multimerization ability of the isoforms.
If this is the case, pathogen-induced formation of DC-SIGNcontaining clusters on the cell surface would counterbalance
for the diminished multimerization ability of certain isoforms
and would justify the large range of pathogens bound and internalized via DC-SIGN. Therefore, according to this hypothesis,
isoforms would have a physiological role (increasing the range
of soluble antigens bound and internalized by DC-SIGN), but
would not have a major impact on the range of pathogens
bound by DC-SIGN. Further studies are needed to clarify these
issues, because it is currently unknown whether the basal multimerization of DC-SIGN on the cell surface (33, 44) is exclusively mediated by intermolecular interactions or is a soluble
ligand-induced event. In this regard, all the experiments performed in the present study were done after extensive washing
of the cells with EDTA, to prevent any carbohydrate-DC-SIGN
interaction that might affect multimerization of the molecule
on the cell surface.
Sequence analysis has allowed the definition of 23-residue
repeats within the neck region of DC-SIGN, which is sometimes divided into 7.5 repeats to account for the presence of an
unrelated and unique sequence at the N-terminal half of the
first repeat (34). Ultracentrifugation and cross-linking of
recombinant truncated DC-SIGN molecules have established
that removal of the two N-terminal repeats only partially
affected the tetramerization ability, whereas recombinant proteins containing only repeats 7– 8 formed partially dissociating
dimers. This has led to the proposal that repeats close to the
lectin domain mediate dimer formation while the membrane
proximal repeats are required for tetramer formation (34). Our
results with transient and stable transfectants of the naturally
occurring DC-SIGN 4d and 2d isoforms, which include repeats
1, 6, 7, and 8 and 1 and 2 (Fig. 3A), indicate that the two more
N-terminal domains are sufficient for multimerization in a cellular context, a fact further confirmed by the very different multimerization capability of the 4d (1, 6, 7, and 8) and 4d⬘ (1, 2, 3,
and 8) isoforms. The importance of repeats 1 and 2 for the
ability of DC-SIGN to multimerize in the cell membrane is even
more evident when considering that the DC-SIGN 1d isoform
(containing only repeat 1) does not multimerize, and that
removal of the N-glycosylation site (1dN/Q mutant) allows
multimerization within a cellular context. In addition, the 3d
construct, which includes the first repeat followed by the N-terminal half of repeat 2, the C-terminal half of repeat 7 and the
entire repeat 8, also exhibits an efficient multimerization capability within a cellular context. Therefore, essential residues for
multimerization can be mapped to the sequence GELSE at the
beginning of the second repeat, which includes a serine residue
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
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VOLUME 283 • NUMBER 7 • FEBRUARY 15, 2008
Downloaded from www.jbc.org at CSIC - Centro de Investigaciones Biológicas, on June 10, 2010
tion of the N-terminal neck repeat limits the extent of multimerization of the molecular within a cellular context (Fig. 2) and,
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Whether the reduced ability of DC-SIGN polymorphic variants
to associate with the full-length molecule contributes to the
altered susceptibility of heterozygous individuals to various
infections remains to be determined. However, the preferential
formation of homo-multimers in heterozygous individuals
must lead to a reduction (50%) in the number of multimers
containing the full-length DC-SIGN 1A molecule, what might
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and allelic variants influence the presence and stability of DCSIGN multimers on the cell surface, and provide relevant clues
about the underlying molecular mechanisms for the association
between DC-SIGN polymorphisms and altered susceptibility to
clinically relevant pathogens.
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/12/12/M706004200.DC1.html
Expression and Function of DC-SIGN Variants
FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7
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Supplementary Figures
A
Mock
1A
% cells with bound pathogens
7.1%
0.4
1AN/Q
80.2%
9.0
75.5%
4.4
4d
4d’
84.1%
9.0
3d
2d
88.0%
17.9
1d
90.0%
14.5
84.3%
11.4
45
40
1dN/Q
79.9%
6.8
Mock
1A
1AN/Q
4d
4d'
3d
2d
1d
1d-N/Q
35
30
25
20
15
10
5
C. albicans yeasts
B
% cells with bound spores
90.4%
18.0
L. infantum amastigotes
81,8 (9,9)
79,2 (8.0)
74,4 (5,7)
39,4 (1,2)
77,7(5,8)
66,8 (3,0)
32,0 (0,9)
Pathogen
79,3 (7,0)
52,7 (2,2)
29,2 (0,8)
45
40
35
30
25
20
15
10
5
3
2
1 0.5
1A
3 1.5 0.75
4d
3 1.5 0.75
2d
µg
DNA transfected
Supplementary Figure 1.- Pathogen-binding capacity of DC-SIGN isoforms and mutants.- A, B. HEK293T
cells transiently expressing the indicated DC-SIGN isoforms/mutants (A) or decreasing levels of DC-SIGN 1A,
4d or 2d (B), were incubated with fluorescent C. albicans yeasts and L. infantum amastigotes and the
percentage of cells with bound pathogens was determined by flow cytometry. Cell surface expression of each
DC-SIGN construct is indicated (thick lines, DC-SIGN; thin lines, control antibody for (A) and one profile for
each distinct amounts of transfected plasmids in (B) (3 µg, thick line; 2 or 1.5 µg, thin line; 1 µg, dashed line;
0.75 and 0.5 µg, dotted line). In each case, the Mean Fluorescence Intensity and the percentage of positive cells
are shown. The experiment was done three times with similar results, and a representative experiment is shown.
97
A
Mannan-agarose precipitation
C Mock 1A
K562-DC-SIGN
K562
mock
1A
1.2 (0.3)
9.9 (0.5)
12.3 (0.6)
-D3
0.2 (0.3)
72.8 (5.7)
16.4 (0.6)
0.8 (0.3)
60.2 (5.5)
13.4 (0.9)
-D5
-D7
0.1 (0.3)
73.8 (6.4)
20.1 (0.8)
0.3 (0.3)
57.5 (4.0)
10.7 (0.6)
-D3 -D5 -D7 Mock 1A -D3 -D5 -D7
kDa
Leishmania
250
150
100
75
50
37
0.1 (0.3)
38.9 (2.1)
7.6 (0.9)
0.1 (0.3)
44.5 (2.9)
9.2 (1.0)
0.1 (0.3)
44.4 (3.0)
12.5 (1.0)
Aspergillus
25
MOI=0
MOI=10 + X63
MOI=10 + MR1
Eluted
Supernatant
Non-reduced
DSG-2
% Binding to Mannan
D
80
70
60
50
40
30
20
10
GP1-Fc binding (MFI)
B
0.0 (0.3)
29.6 (1.5)
6.2 (0.6)
12
K562 mock
1A low
1A
K562
-D3
DC-SIGN
-D5
-D7
10
DC-SIGN -D7
Signal intensity
0.0 (0.3)
3.9 (0.4)
4.1 (0.5)
DC-SIGN -D5
DC-SIGN -D3
4
2
DC-SIGN 1A
DC-SIGN 1A (low)
K562-Mock
1H mock +IF
δ (ppm)
8
6
4
2
X63
MR1
Supplementary Figure 2.- Pathogen and sugar-binding capacity of DC-SIGN polymorphic variants.- A.
K562 cells stably transfected with the indicated DC-SIGN variants were incubated with fluorescent C. albicans
yeasts and L. infantum amastigotes, in the presence of either X63 or MR1 antibodies as indicated, and the
percentage of cells with bound pathogens was determined by flow cytometry. In each case, the first number
indicates the percentage of cells with bound amastigotes or conidia, and the Mean Fluorescence Intensity of the
whole cell population is indicated in parenthesis. B. Adhesion to immobilized mannan (upper panel) or binding
of Ebola GP1-Fc (lower panel) of K562 cells stably expressing the indicated DC-SIGN variants in the presence
of a blocking (MR1) or an irrelevant antibody (X63) antibody. For comparative purposes, two subpopulations
of K562-DC-SIGN 1A with different DC-SIGN cell surface expression levels were assayed. C. Lysates from
COS-7 cells transiently transfected with the indicated DC-SIGN polymorphic variants were incubated with
mannan-agarose. After extensive washing, bound (Eluted, left panel) and non-bound proteins (Supernatant,
right panel) were resolved by SDS-PAGE under non-reducing conditions and subjected to Western blot with
DSG-2. D. Binding of Candida utilis glucomannan (IF) to K562 cells stably transfected with the indicated DCSIGN polymorphic variants by means of 1D Saturation Transfer Difference NMR. The lower profile represents
the 1H NMR spectrum of IF-S in PBS at 298 K. Graph illustrates the signal intensity yielded by each
transfectant (y-axis) and the chemical shift (d) in parts per million (ppm).
98
Resultados
4. Identificación de epítopos en la molécula de unión a patógenos DC-SIGN
DC-SIGN (Dendritic cell-specific ICAM-3-grabbing non-integrin) es una lectina tipo C que
reconoce oligosacáridos que contienen fucosa y/o manosa, presentes en patógenos con relevancia
clínica. La señalización intracelular iniciada tras la unión de DC-SIGN con el ligando interfiere con
las señales iniciadas por los TLR, y modula la activación y polarización de células T inducida por las
células presentadoras de antígeno. El dominio de reconocimiento de carbohidratos C-terminal (CRD)
de DC-SIGN es precedido por un cuello integrado por ocho repeticiones de 23 residuos, que media
la multimerización de la molécula, y cuyos polimorfismos se correlacionan con una susceptibilidad
alterada a la infección por SARS y HIV. Con el fin de definir epítopos estructurales y funcionales de
DC-SIGN, hemos utilizado isoformas que ocurren de forma natural y moléculas recombinantes
quiméricas. De los tres epítopos identificados en el CRD, uno de ellos está expuesto solamente en
la forma monomérica de DC-SIGN, siendo dependiente de la multimerización de la molécula. Los
epítopos del dominio del cuello son independientes de la conformación e inalterados por la
multimerización de DC-SIGN, pero son diferencialmente afectados por la ausencia de repeticiones
de esta región. Aunque los anticuerpos específicos frente al cuello de DC-SIGN exhiben menor
capacidad de bloqueo funcional, son más eficientes en la inducción de internalización de la molécula.
Por otra parte, la unión de los diversos anticuerpos a sus epítopos correspondientes da lugar a
distintos grados de agrupación de moléculas de DC-SIGN en la superficie celular. La identificación
de epítopos independientes en DC-SIGN podría facilitar el diseño de reactivos que modulen la
capacidad de activación y polarización de células T por las células que expresan DC-SIGN, sin
alterar su capacidad de reconocimiento de antígenos y patógenos.
99
Molecular Immunology 47 (2010) 840–848
Contents lists available at ScienceDirect
Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm
Epitope mapping on the dendritic cell-specific ICAM-3-grabbing non-integrin
(DC-SIGN) pathogen-attachment factor
Elena Sierra-Filardi a , Ana Estecha b , Rafael Samaniego b , Elena Fernández-Ruiz c , María Colmenares a ,
Paloma Sánchez-Mateos b , Ralph M. Steinman d , Angela Granelli-Piperno d , Angel L. Corbí a,∗
a
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Unidad de Inmuno-Oncología, Hospital General Universitario Gregorio Marañón, Madrid, Spain
Unidad de Biología Molecular, Hospital Universitario de la Princesa, Madrid, Spain
d
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY, USA
b
c
a r t i c l e
i n f o
Article history:
Received 7 February 2009
Received in revised form
20 September 2009
Accepted 30 September 2009
Available online 30 October 2009
Keywords:
Human
Dendritic cells
Adhesion molecules
Pathogen recognition
DC-SIGN
a b s t r a c t
DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachment factor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinically
relevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes with
TLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presenting
cells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domain
composed of eight 23-residue repeats which mediate molecule multimerization, and whose polymorphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isoforms
and chimaeric molecules, in combination with established recognition properties, were used to define
seven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which is
multimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domain
were conformation-independent and unaltered upon molecule multimerization, but were differentially
affected by neck domain truncations. Although neck-specific antibodies exhibited lower functionblocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of
the different epitopes resulted in distinct levels of microclustering on the cell surface. The identification
of independent epitopes on the DC-SIGN molecule might facilitate the design of reagents that modulate
the T cell activating and polarizing ability of DC-SIGN-expressing cells without preventing its antigenand pathogen-recognition capacities.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Dendritic cells (DC) express a large array of cell surface lectins
and lectin-like molecules, which endow them with a broad
capacity for recognition of parasitic, bacterial, yeast and viral
pathogens (Cambi and Figdor, 2003; Robinson et al., 2006; van
Kooyk and Geijtenbeek, 2003). Dendritic cell-specific ICAM-3grabbing non-integrin (DC-SIGN, CD209) is a type II membrane
C-type lectin (Curtis et al., 1992; Geijtenbeek et al., 2000c) abundantly expressed in vivo on myeloid DC, macrophages (Bleijs et
al., 2001; Curtis et al., 1992; Geijtenbeek et al., 2000a,c, 2001;
Lee et al., 2001; Soilleux et al., 2001, 2002), and in vitro generated monocyte-derived dendritic cells (MDDC) and alternatively
activated macrophages (Puig-Kroger et al., 2004; Relloso et al.,
2002; Soilleux et al., 2002). DC-SIGN binds HIV (Geijtenbeek and
∗ Corresponding author. Tel.: +34 91 8373112x4376; fax: +34 91 5627518.
E-mail address: [email protected] (A.L. Corbí).
0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2009.09.036
van Kooyk, 2003), Ebola (Alvarez et al., 2002), SARS (Marzi et al.,
2004), Hepatitis C (Lozach et al., 2004, 2003; Pohlmann et al.,
2003) and Dengue virus (Tassaneetrithep et al., 2003), Leishmania amastigotes and promastigotes (Colmenares et al., 2004, 2002),
Mycobacterium tuberculosis (Geijtenbeek et al., 2003; Tailleux et
al., 2003), Aspergillus fumigatus (Serrano-Gomez et al., 2004), and
Candida albicans (Cambi et al., 2003), via mannan- and Lewis
oligosaccharides-dependent interactions (Feinberg et al., 2001;
Frison et al., 2003). Besides, DC-SIGN mediates intercellular adhesion through its recognition of ICAM-3 (Geijtenbeek et al., 2000c;
Gijzen et al., 2007), ICAM-2 (Geijtenbeek et al., 2000a), CEA and
CEACAM1 (van Gisbergen et al., 2005a), and the CD11b/CD18 integrin (van Gisbergen et al., 2005b). The extracellular part of DC-SIGN
contains a carbohydrate-recognition domain (CRD) and a neck
region composed of eight 23-residue repeats (Curtis et al., 1992;
Engering et al., 2002; Geijtenbeek et al., 2000b; Kwon et al., 2002).
Analysis of recombinant molecules indicates that the neck domain
mediates the formation of DC-SIGN tetramers, possibly as a strategy to increase the avidity for ligand binding (Bernhard et al., 2004;
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Feinberg et al., 2005; Mitchell et al., 2001). Alternative splicing
and genetic polymorphisms generate numerous DC-SIGN isoform
transcripts whose presence has been detected at mucosal HIV
transmission sites (Liu et al., 2005, 2004; Mummidi et al., 2001;
Sakuntabhai et al., 2005) and correlates with altered susceptibility
to HIV-1 transmission (Liu et al., 2004).
Engagement of cell surface DC-SIGN by monoclonal antibodies
prevents pathogen-attachment (Geijtenbeek et al., 2000c; GranelliPiperno et al., 2005; Relloso et al., 2002), and also leads to molecule
internalization (Engering et al., 2002) and intracellular signaling
(Caparros et al., 2006; Gringhuis et al., 2007; Hodges et al., 2007),
with the latter being antibody-specific (Hodges et al., 2007). DCSIGN functions as an antigen-capturing molecule which targets
bound molecules to endosomal compartments for subsequent antigen presentation (Engering et al., 2002). In fact, targeting antigens
to human dendritic cells via a humanized anti-DC-SIGN antibody
promotes effective naive and recall T cell responses (Tacken et al.,
2005). Consequently, DC-SIGN-specific monoclonal antibodies can
be useful not only preventing pathogen spreading into myeloid
cells, but also as therapeutic tools. Through the use of previously
described monoclonal antibodies (Granelli-Piperno et al., 2005;
Relloso et al., 2002), we now describe the identification of seven
epitopes within the DC-SIGN molecule, some of which can be used
to distinguish the multimerization state of the molecule. The epitope mapping on the DC-SIGN molecule suggests that anti-DC-SIGN
monoclonal antibodies can block DC-SIGN functions by alternative
mechanisms, including ligand-binding blockade, antibody-induced
internalization and modulation of the multimerization state on the
cell surface.
2. Materials and methods
2.1. Generation of monocyte-derived dendritic cells (MDDC)
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from healthy donors over a Lymphoprep
(Nycomed, Norway) gradient according to standard procedures.
Monocytes were purified from PBMC by magnetic cell sorting using
CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany),
and immediately subjected to the dendritic cell differentiation protocol using 1000 U/ml GM-CSF (Schering-Plough, Kenilworth, NJ)
and 1000 U/ml IL-4 (PreProtech, Rocky Hill, NJ) (Dominguez-Soto
et al., 2007), with cytokine addition every second day.
2.2. Stable and transient transfection of DC-SIGN mutants and
isoforms
Stable transfectants of DC-SIGN 1A in K562 cells have been
previously described (Relloso et al., 2002). For transient transfections, 2 × 105 COS-7 or HEK293T cells were transfected with
Superfect (Qiagen, Hilden, Germany) in 6-well plates, using 2 ␮g
of the distinct pCDNA3.1(−)-based expression plasmids containing
the distinct isoforms, mutants and chimaeric forms of DC-SIGN.
COS-7 and HEK293T cells were routinely grown in DMEM supplemented with 10% Fetal Calf Serum (FCS). DC-SIGN isoforms
and polymorphic variants were isolated by RT-PCR on RNA
from MDDC or genomic DNA, and the constructs were generated using standard molecular biology techniques and verified
by sequencing, as described (Serrano-Gomez et al., 2008). Generation of DC-SIGN expression vectors lacking the lectin domain
PCR was performed on the pCDNA3-DC-SIGN 1A construct (Relloso
et al., 2002) using oligonucleotides CD209 sense and CD209lectin (5 -CCCCAAGCTTGTCACAGGCGTTCCACTGCAGC-3 ). PCRgenerated fragments were resolved in 1.5% agarose gels, purified
and sequenced. Fragments containing either the full-length (8d)
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and 7-, 6-, and 4-repeat neck regions (repeats 1 through 7, 7d;
repeats 1 through 6, 6d; repeats 1 through 4, 4d) were cloned into
EcoRI/HindIII-digested pCDNA3.1- to yield pCDNA3.1-DC-SIGN 8d,
pCDNA3.1-DC-SIGN 7d, pCDNA3.1-DC-SIGN 6d and pCDNA3.1-DCSIGN 4d plasmids.
2.3. Immunoprecipitation and Western blot
For immunoprecipitation, DC-SIGN-transfected HEK293T cells
were collected, washed with PBS 1 mM EDTA, resuspended in 1 ml
PBS pH 8.0 and lysed in 10 mM Tris–HCl pH 8.0, 150 mM NaCl,
0.025% sodium azide, 0.5% NP-40, 1 mM iodoacetamide, 2 mM Pefabloc (Alexis Biochemicals, Lausen, Switzerland), and 2 ␮g/ml of
aprotinin, antipain, leupeptin and pepstatin (NP-40 lysis buffer).
After preclearing, anti-DC-SIGN antibodies (Granelli-Piperno et
al., 2005) were added onto the cell lysate, followed by addition
of sepharose-coupled protein G. Retained molecules were eluted
with 3× Laemmli’s sample buffer (2% SDS, 6.25 mM Tris base,
10% glycerol), and eluates resolved by SDS-PAGE under reducing
or non-reducing conditions, and subjected to Western blot with
polyclonal antiserum raised against a 28-residue peptide from the
DC-SIGN cytoplasmic tail (DSG2) (Serrano-Gomez et al., 2008). For
Western blot, 10 ␮g of total cell lysate in NP-40 lysis buffer was subjected to SDS-PAGE under reducing or non-reducing conditions and
transferred onto Immobilon polyvinylidene difluoride membrane
(Millipore, Bedford, MA). After blocking with 5% non-fat dry milk
in 50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20, protein
detection was performed using the Supersignal West Pico Chemiluminescent system (Pierce, Rockford, IL). Detection of DC-SIGN was
carried out with the distinct anti-DC-SIGN monoclonal antibodies
(Granelli-Piperno et al., 2005), or the DSG2 anti-DC-SIGN polyclonal
antiserum.
2.4. Flow cytometry and antibodies
Indirect immunofluorescence was done using the previously
described anti-DC-SIGN monoclonal antibodies (Granelli-Piperno
et al., 2005), MR1 (directed against the lectin domain of DC-SIGN)
as a positive control (Relloso et al., 2002), or the supernatant of
the P3X63Ag8 myeloma as negative control, and FITC-labeled goat
anti-mouse IgG as a secondary antibody. Where indicated, a polyclonal antiserum against a neck-derived peptide (DSG1) was also
used as a positive control. All incubations were done in the presence of 50 ␮g/ml of human IgG to prevent binding through the Fc
portion of the antibodies. Flow cytometry analysis was performed
with an EPICS-CS (Coulter Científica, Madrid, Spain) using log
amplifiers.
2.5. DC-SIGN-dependent adhesion to S. cerevisiae mannan and
aggregation
96-well microtiter EIA II-Linbro plates were coated overnight
with mannan at 50 ␮g/ml in PBS at 4 ◦ C, and the remaining sites
were blocked with 0.5% BSA for 2 h at 37 ◦ C. Transfected cells
were labeled in DMEM containing 0.5% BSA with the fluorescent
dye 2 ,7 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, The Netherlands) at 37 ◦ C and
preincubated for 20 min in DMEM 0.5% BSA containing either the
isotype-matched control P3X63 or the distinct anti-DC-SIGN antibodies (Granelli-Piperno et al., 2005). Cells were then allowed
to adhere to each well for 15 min at 37 ◦ C. Unbound cells were
removed by three washes with DMEM 0.5% BSA, and adherent cells were quantified using a fluorescence analyzer. Results
were expressed as “% Binding”, which indicates the percentage of
mannan-bound cells relative to the total cellular input. For aggregation assays, K562-DC-SIGN cells were washed with PBS 1 mM
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EDTA, resuspended in RPMI 10% FCS, and placed in 24-well plates
at 105 cells/ml for 20 min at 37 ◦ C and either in the presence or
absence of anti-DC-SIGN antibodies.
bation with a suboptimal concentration of biotin-labeled MR1
antibody and HRP-conjugated streptavidin.
2.7. Immunofluorescence and confocal microscopy
2.6. Specificity of antibody recognition by ELISA
The cDNA region encoding the extracellular portion of DCSIGN was generated by PCR, cloned in-frame downstream of
the hexahistidine sequence of pET100/D-TOPO (Invitrogen), and
transformed into BL21 bacteria to generate HIS-DC-SIGN, which
was purified on Ni2+ -nitrilotriacetic acid-agarose (Qiagen). The
DSG1 peptide sequence (GELPEKSKQQEIYQELTRLKAAV) was based
on the sequence of the sixth repeated domain of the DC-SIGN
neck region. DSG1 peptide was coated onto 96-well Maxisorp
Immunoplates (Nunc) and binding of anti-neck monoclonal antibodies assessed by a standard direct ELISA procedure using
HRP-conjugated anti-mouse IgG rabbit polyclonal antiserum. For
competition experiments, 96-well HIS-DC-SIGN protein-coated
plates were incubated with anti-CRD antibodies, followed by incu-
105 immature MDDC were layered over fibronectin-coated
glass coverslips (5 ␮g/ml) and incubated for 30 min at 37 ◦ C. For
staining of early endosomes, cells were subsequently pulsed during 10 min with A633 conjugated biferric-transferrin (10 ␮g/ml)
(Molecular Probes). Cells were then fixed (4% paraformaldehyde in PBS, 15 min at room temperature) and washed three
times with 25 mM Tris buffer saline. When permeabilization was
required, samples were incubated with 0.3% Triton X-100 (5 min
at room temperature). After a blocking step in Blocking Reagent
(Boheringer Manheim) containing 0.5% sodium azide and 1 ␮g/ml
of human immunoglobulins (10 min at room temperature), cells
were labeled with the indicated antibody (30 min at 37 ◦ C) followed by FITC-labeled anti-mouse IgG. Cells were finally washed
in PBS and water, and mounted with fluorescence mounting
Fig. 1. Identification of CRD- and neck-specific DC-SIGN antibodies. (A) Schematic structure of the naturally occuring and chimaeric DC-SIGN constructs used in the present
study. (B) The indicated DC-SIGN constructs (DC-SIGN 1A, DC-SIGN 8d and DC-SIGN 4d) or an empty vector (Mock) were transient transfected in HEK293T cells, and the
reactivity of the distinct anti-DC-SIGN monoclonal antibodies, the anti-DC-SIGN polyclonal antiserum DSG1 (positive control) or the negative control P3X63 (Control) was
determined by flow cytometry. MFI (lower number) and percentage of positive cells (upper number) are shown in each case. (C) COS-7 cells were transiently transfected
with the indicated expression vectors (DC-SIGN 1A and DC-SIGN 8d), lysed after 48 h, and cell lysates resolved by SDS-PAGE under non-reducing conditions and subjected to
Western blot using the indicated anti-DC-SIGN monoclonal antibodies. (D) Lysates of HEK293T cells transiently transfected with DC-SIGN 1A were immunoprecipitated with
the indicated anti-DC-SIGN antibodies. Immunoprecipitates were resolved by SDS-PAGE under reducing conditions, and subjected to Western blot with the anti-DC-SIGN
polyclonal antiserum DSG2. The position of high-order multimers is indicated by an arrow. (E) HEK293T cells were transfected with expression vectors for the indicated
DC-SIGN constructs (1A, 1AN/Q, 2d and 1d) or an empty vector (Mock), and the reactivity of the indicated anti-DC-SIGN monoclonal antibodies, the anti-DC-SIGN monoclonal
antibody MR1 (positive control) or the negative control P3X63 (Control) was determined by flow cytometry. MFI (lower number) and percentage of positive cells (upper
number) are shown in each case. Each experiment was performed twice with similar results, and one of the experiments is shown.
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medium (DAKO). For induction of patching, cells were incubated
with the indicated anti-DC-SIGN antibody (1 ␮g/ml, 10 min at
37 ◦ C), fixed and subsequently incubated with a FITC-labeled secondary antibody. For microclustering quantification, more than
100 spots were measured from at least 10 randomly chosen
cells.
Laser scanning confocal microscopy was performed with a
confocal scanning inverted AOBS/SP2-microscope (Leica Microsystems, Heidelberg, Germany). All images were acquired with a 63X
PL-APO NA 1.3 glycerol immersion objective. The theoretical x, yresolution of this lens at Airy-1 and 488 nm excitation length is
∼150 nm. Assessment of fluorophore colocalization was performed
with the Leica software, using a global statistic method to perform
intensity correlation analysis. Plots display the intensity distribution and degree of colocalization corresponding to the entire cell,
which is shown next to the scatter plot. Co-localizing events in
the scatter plot were gated (double positive pixels above the dual
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threshold) and visualized as a white overlay on the green and red
merged image.
3. Results and discussion
3.1. Identification of anti-CRD and anti-neck DC-SIGN-specific
monoclonal antibodies
Engagement of cell surface DC-SIGN by monoclonal antibodies results in pathogen recognition blockade (Geijtenbeek et al.,
2000c; Granelli-Piperno et al., 2005; Relloso et al., 2002), internalization (Engering et al., 2002) and intracellular signaling (Caparros
et al., 2006; Gringhuis et al., 2007; Hodges et al., 2007). To define
functional epitopes within the molecule, ten monoclonal antibodies (Granelli-Piperno et al., 2005) were assayed for their ability
to recognize natural and chimaeric variants of DC-SIGN (Fig. 1A).
All antibodies recognized the prototypical DC-SIGN isoform (DC-
Fig. 2. Fine specificity of the neck-specific anti-DC-SIGN monoclonal antibodies. (A and B) HEK293T cells were transiently transfected with expression vectors for DC-SIGN
1A, DC-SIGN chimaeric constructs (1A, 2d and 1d) (A), three different DC-SIGN polymorphic variants which contain only seven repeats but differ in the identity of the absent
repeat (-D3, -D5 and -D7) (B), or an empty vector (Mock) and the reactivity of the neck-specific monoclonal antibodies (2, 4, 7, 10 and 11), the anti-CRD MR1 monoclonal
antibody (positive control) or the negative control P3X63 (Control) was determined by flow cytometry. MFI (lower number) and percentage of positive cells (upper number)
are shown in each case. Each experiment was performed twice, and one of the experiments is shown. (C) DC-SIGN deletion mutants lacking the lectin domain but differing in
the number of the neck region repeats (8d, 7d and 6d) were transient transfected in COS7 cells, and cell lysates subjected to Western blot with a polyclonal antiserum against
the DC-SIGN cytoplasmic domain (DSG2) or the indicated neck-specific monoclonal antibodies. (D) Cross-inhibition experiments. Antibodies 1, 7 and MR1 were purified and
PE labeled (PE-Mab), and their binding to K562-DC-SIGN 1A stable transfectants assayed by flow cytometry in the presence of the indicated competing antibodies (Comp.
Mab). Results are expressed as the percentage of binding in the presence of each competing antibody relative to the binding observed in the presence of an irrelevant isotypematched antibody. (E) Recognition of the DSG1 peptide by the indicated neck-specific anti-DC-SIGN monoclonal antibodies by ELISA. (F) Effect of CRD-specific antibodies on
the recognition of rHIS-DC-SIGN by the MR1 monoclonal antibody, as determined by inhibition ELISA.
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SIGN 1A) on HEK293T cells (Fig. 1B), but only five (antibodies
2, 4, 7, 10 and 11) (numbering according to (Granelli-Piperno et
al., 2005)) bound the CRD-lacking construct (DC-SIGN 8d) and
retained their reactivity against a construct which lacks half the
neck domain (DC-SIGN 4d, Fig. 1B). The same pattern of reactivity
was observed by Western blot on lysates from COS-7 cells overexpressing DC-SIGN 1A or DC-SIGN 8d: antibodies 2, 4, 7, 10 and 11
recognized DC-SIGN 8d, and bound to monomeric and multimeric
forms of DC-SIGN (Fig. 1C), while antibodies 3, 6 and 13 did not
recognize denatured DC-SIGN, and antibodies 1 and 9 exclusively
bound to the monomeric form of full-length DC-SIGN 1A (Fig. 1C).
The differences between both sets of antibodies were also seen
in immunoprecipitation assays, as DC-SIGN high-order multimers
were only brought down by antibodies 2, 4, 7, 10 and 11 (Fig. 1D).
Therefore, five antibodies (2, 4, 7, 10 and 11) recognize the neck
region of DC-SIGN (Neck-specific antibodies), whereas the specificity of the rest (1, 3, 6, 9 and 13) depends on the presence of the
CRD region (CRD-dependent antibodies).
The above experiments also allowed the definition of three distinct specificity groups within the CRD-dependent antibodies. From
the Western blot analysis in Fig. 1C, it is evident that antibodies
1 and 9 bind only denatured DC-SIGN monomers, thus suggesting that, unlike 3, 6 and 13, they recognize linear CRD epitopes
not accessible in DC-SIGN multimers. On the other hand, antibodies 1, 3, 9 and 13 yielded flow cytometry profiles (almost bimodal)
which differ from that of antibody 6 (Fig. 1B). Therefore, three types
of CRD-dependent DC-SIGN antibodies might be defined: Group 1
(antibodies 1 and 9), which recognizes CRD sequential epitope(s)
probably masked in DC-SIGN multimers, Group 2 (antibodies 3 and
13) and Group 3 (antibody 6).
3.2. Comparison of the specificity of neck-specific antibodies
To further define the specificity of the DC-SIGN neck-specific
antibodies, their reactivity was assayed on DC-SIGN constructs
lacking the N-glycosylation site in the first neck domain repeat (DCSIGN 1A N/Q), or whose neck domain included only two repeats
(DC-SIGN 2d) or a single repeat (DC-SIGN 1d). Disruption of the
N-glycosylation site led to a reduction in the reactivity of the
neck-specific antibodies (Fig. 1E and not shown), possibly reflecting the higher degree of multimerization of DC-SIGN glycosylation
mutants (Serrano-Gomez et al., 2008).
Truncation of the neck domain to a single repeat (DC-SIGN
1d) did not have any influence on the binding of CRD-dependent
antibodies (Fig. 1E and not shown), indicating that recognition by
CRD-specific antibodies is independent on the length of the neck
domain. By contrast, neck-specific antibodies displayed a variable
level of reactivity against the DC-SIGN 2d construct, and their binding was completely abolished when the neck region was exclusively
composed of the first repeat (DC-SIGN 1d) (Fig. 2A). Antibodies 4,
10 and 11 bound DC-SIGN 2d to a similar extent as the prototypic
DC-SIGN 1A molecule, implying that their epitope(s) is retained
within the two N-terminal repeats of the neck domain (Fig. 2A).
Conversely, antibody 2 recognized DC-SIGN 2d to a lower extent,
and antibody 7 displayed no binding to DC-SIGN 2d (Fig. 2A). Therefore, the two N-terminal repeats of the DC-SIGN neck retain the
Fig. 3. Function-blocking ability of anti-DC-SIGN monoclonal antibodies. (A) HEK293T cells were transiently transfected with the prototypic isoform DC-SIGN (DC-SIGN 1A)
or an empty vector (Mock), and expression levels determinated by flow cytometry with the anti-DC-SIGN MR1 monoclonal antibody. (B) Transiently transfected HEK293T
cells were labeled with BCECF and adhesion to S. cerevisiae mannan was performed in the presence of the distinct anti-DC-SIGN antibodies (MR1, DSG1, 1, 2, 4, 6, 7 and 9) or
an irrelevant antibody (X63, Control) as negative control. (C) K562-DC-SIGN 1A stable transfectants were seeded in 24-well plates at 105 cells/ml and maintained for 20 min
at 37 ◦ C either in the absence (Untreated) or presence of the indicated anti-DC-SIGN antibodies (10 ␮g/ml). (D) Schematic representation of the epitopes recognized by the
assayed anti-DC-SIGN monoclonal antibodies.
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epitope(s) recognized by antibodies 4, 10 and 11, whereas antibodies 2 and 7 require the presence of additional repeats to exhibit
their maximal binding activity. On the other hand, all the neckspecific antibodies equally recognized three different polymorphic
variants of DC-SIGN which contain only seven repeats but differ in
the identity of the absent repeat (-D3, -D5, -D7) (Fig. 2B and data not
shown), suggesting that they recognize epitopes shared by several
repeats. A similar conclusion was drawn from Western blot analysis of the DC-SIGN 8d, 7d and 6d neck region deletion constructs,
which were recognized to a similar extent by the neck-specific antibodies (Fig. 2C), and from cross-competition experiments, as all the
neck-specific antibodies could inhibit the binding of antibodies 4
or 7 (Fig. 2D and not shown). Therefore, neck-specific antibodies
recognize epitopes shared by repeats 2-to-8, but exhibit differential recognition of the 2-repeat-containing DC-SIGN 2d construct,
allowing the definition of three distinct epitopes on the DC-SIGN
neck domain.
To extend the above findings, neck-specific antibodies were
evaluated for their ability to bind the DSG1 peptide, whose
sequence is based on the DC-SIGN neck repeat 6. Only three antibodies (4, 10 and 11) bound significantly to DSG1-coated plates
(Fig. 2E), indicating that they recognize common or overlapping
epitopes which differ from those recognized by antibodies 2 and
7, in agreement with flow cytometry data. Regarding CRD-specific
antibodies, and based on the cross-inhibition experiments (Fig. 2D),
it was apparent that MR1 recognized an epitope different from
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those defined by antibodies 1/9, 3/13 and 6. To further confirm
this possibility, inhibition ELISA experiments were performed using
the binding of biotin-labeled MR1 to recombinant HIS-DC-SIGN as
a read-out. As shown in Fig. 2F, none of the anti-CRD antibodies
inhibited MR1 binding to recombinant HIS-DC-SIGN, thus demonstrating that MR1 defines a separate epitope on the DC-SIGN CRD
which is distinct from those defined by antibodies 1/9, 3/13 and 6.
3.3. Functional comparison of CRD- and neck-specific monoclonal
antibodies
The analyzed antibodies have been demonstrated to inhibit
HIV-1 transmission from Raji-DCSIGN transfectants to T cells
(Granelli-Piperno et al., 2005). To determine whether a correlation exists between epitope recognition and function-blocking
ability, the antibodies were evaluated for their ability to inhibit
the binding of DC-SIGN 1A-expressing HEK293T cells (Fig. 3A)
to mannan-coated surfaces. Whereas MR1 antibody completely
blocked cell binding to S. cerevisiae mannan, the neck-specific DSG1
polyclonal antiserum had no effect (Serrano-Gomez et al., 2007)
(Fig. 3B). Regarding CRD-dependent antibodies, and in line with
the cell-binding experiments, antibodies 1 and 9 abrogated DCSIGN–mannan interaction, whereas antibody 6 caused a moderate
(50%) inhibition (Fig. 3B). In the case of neck-specific antibodies,
antibodies 4 and 7 partially reduced the binding, and antibody
2 had no effect on the DC-SIGN–mannan interaction (Fig. 3B).
Fig. 4. Immunofluorescence staining and antibody-induced DC-SIGN microclustering of immature MDDC. (A) Immunolocalization of transferin A633-loaded (red fluorescence) fixed and permeabilized fibronectin-bound MDDC with the indicated anti-DC-SIGN antibodies (green fluorescence). In the case of antibody 10 (two right panels),
shown is the colocalization analysis. The scatter plot (most right panel) displays the intensity distribution of each fluorochrome and the degree of colocalization. (B) (Untreated
MDDC panel) Non-permeabilized fibronectin-bound MDDC were fixed and stained with the indicated anti-DC-SIGN antibodies to determine the pattern of membrane staining
at high magnification. (Antibody-treated MDDC panel) Fibronectin-bound MDDC were incubated for 10 min at 37 ◦ C with the indicated anti-DC-SIGN antibodies (1 ␮g/ml),
washed to remove unbound antibodies, and fixed and stained with FITC-labeled anti-mouse IgG polyclonal antiserum. Marked areas are shown below at higher magnification. Microclustering was evaluated by measuring more than 100 spots per antibody in a minimum of 10 randomly chosen cells. (C) Quantitation of the size of the
DC-SIGN-containing microcluster size in MDDC under basal conditions (Untreated) or exposed to the indicated anti-DC-SIGN antibodies for 10 min at 37 ◦ C (Antibody-treated).
Shown are the range, mean and SD of the measurement of 10 individual cells from each experimental condition. Significant difference (p < 0.001) between untreated and
antibody-treated cells was only seen with antibodies 7, 10 and MR1.
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Similar results were seen on the Leishmania-binding ability of DCSIGN, with neck-specific antibodies exhibiting the lowest inhibitory
effect (data not shown). Likewise, the DC-SIGN-dependent homotypic aggregation of K562-DC-SIGN 1A cells was strongly inhibited
by CRD-dependent antibodies (1, 3, 6), but was almost unaffected
by the 7 and 10 neck-specific antibodies (Fig. 3C). Therefore, antiCRD antibodies consistently display a stronger function-blocking
capacities than those directed against the neck domain.
The combination of structural and functional assays so far
described allowed the identification of different structural and
functional epitopes on DC-SIGN (Fig. 3D). At least four epitopes
could be defined within the CRD according to their linear/sequential
architecture and their accessibility on the cell surface, with two of
them differentially involved in DC-SIGN-dependent adhesive functions. The rest of the epitopes were mapped within the neck domain
of the molecule, and their existence is inferred from the distinct
reactivity of the antibodies against truncated forms of the molecule,
a peptide based on the sequence of a single repeat, and their distinct
functional effects in adhesion assays.
3.4. Clustering- and internalization-inducing ability of DC-SIGN
antibodies
Regardless of their specificity, all antibodies equally recognized DC-SIGN on MDDC after cell permeabilization, as they
yielded a spotted staining enriched at the lamellipodium and also
labeled discrete cytoplasmic structures near the plasma membrane
(Fig. 4A and not shown). The identity of these structures as endocytic/sorting compartments was determined after a 10 min pulse
with A633-labeled transferrin, which labels both early and sorting endosomes, as most DC-SIGN cytoplasmic spots co-localized
with transferrin-loaded endosomes (Fig. 4A, two rightmost panels). To determine the influence that each antibody might have
on the cell surface distribution of DC-SIGN, fibronectin-bound
non-permeabilized MDDC were either stained with the distinct
antibodies, or incubated with antibodies at 37 ◦ C for 10 min before
subsequent fixation and staining with secondary antibodies. As
shown in Fig. 4B (upper panel, Untreated MDDCs), all antibodies,
except for #6, yielded an equivalent spotted distribution on the
plasma membrane of intact MDDC, with a generally enriched staining at the lamellipodium. Preincubation with the distinct antibodies
revealed that the antibodies differed in their ability to modify the
size of DC-SIGN-containing microclusters on MDDC (Fig. 4B, lower
panels, Antibody-treated MDDC). Quantitation of the microcluster
size indicated that only antibodies 7, 10 and MR1 significantly
(p < 0.001) enhanced the size of the MDDC DC-SIGN-containing
microclusters when compared to the size of the clusters observed
at 4 ◦ C under basal conditions (Fig. 4C). This result suggests that the
ability to promote DC-SIGN redistribution on the MDDC cell surface
is independent on their recognition specificity, as enhanced microclustering was induced by anti-neck (7, 10) and CRD-dependent
(MR1) antibodies. Given the differential function-blocking ability
of the microclustering-enhancing antibodies (Fig. 3), these results
also suggest that the ability of the antibodies to inhibit DC-SIGN
recognition functions is not related to their ability to induce DCSIGN cell surface redistribution. Whether the clustering-inducing
ability might affect their ability to trigger intracellular signaling
remains to be determined.
The ability of anti-neck antibodies of partially inhibiting DCSIGN adhesive functions could be explained by their ability to alter
the multimeric state of the lectin on the cell surface through disruption of the neck–neck intercellular interactions which mediate
DC-SIGN multimerization. Alternatively, neck-specific antibodies
could also alter DC-SIGN internalization, thus reducing the number
of available cell surface molecules. To determine whether this was
the case, the ability of the different antibodies to promote DC-SIGN
Fig. 5. Internalization-inducing ability of anti-DC-SIGN antibodies. MDDC cells were either kept at 4 ◦ C or treated with the indicated antibodies for 10 min at 37 ◦ C, and
subsequently fixed and incubated with a Cy3-labeled secondary antibody to detect DC-SIGN molecules on the cell surface. Then, cells were permeabilized and incubated with
an FITC-labeled secondary antibody to detect internalized molecules. Representative examples of cells exposed to the different antibodies under both incubation procedures
are shown in the bottom panel.
107
E. Sierra-Filardi et al. / Molecular Immunology 47 (2010) 840–848
internalization in MDDC in suspension was evaluated by comparing the DC-SIGN cell surface expression on MDDC maintained at
4 ◦ C or subjected to a 30 min incubation at 37 ◦ C with the distinct
antibodies (Fig. 5, upper panel). Interestingly, maximal internalization was promoted by antibodies 2, 4, 7 and 10, all of which
recognize neck-specific epitopes (Fig. 5, lower panel). Among the
CRD-specific antibodies, MR1 exhibited the highest internalizationinducing ability (Fig. 5). Therefore, it is tempting to speculate that
the function-blocking ability of neck-specific antibodies derives
from their capacity to diminish the amount of available DC-SIGN
molecules on the cell surface.
DC-SIGN is an antigen-capturing molecule whose ligands
are subsequently taken into the antigen-presentation pathway
(Engering et al., 2002), and functions as a pathogen-attachment
factor (den Dunnen et al., 2009). DC-SIGN nanoscale clusters on the
cell surface are not distributed randomly, as they are preferentially
localized to the leading edge of MDDC lamellipod and excluded
from the ventral plasma membrane (Neumann et al., 2008). This
restricted localization and its clathrin-dependent internalization
(Cambi et al., 2009) appear to be tightly regulated (Neumann et
al., 2008) as a possible strategy to maximize recognition and capturing functions. In fact, the DC-SIGN cytoplasmic tail is known to
interact with the F-actin binding phosphoprotein LSP1 (Smith et al.,
2007), which also contributes to DC-SIGN signaling (Gringhuis et
al., 2009). The identification of reagents that modulate DC-SIGN cell
surface distribution, like the DC-SIGN neck-specific antibodies that
trigger maximal DC-SIGN internalization in MDDC, can therefore be
of great value to identify cytoskeletal or cytoplasmic components
involved in the regulated cell surface distribution of the molecule.
Anti-DC-SIGN antibodies have been valuable tools to put forward the pathogen-binding ability of the receptor and its capacity
to trigger intracellular signaling (Caparros et al., 2006; Gringhuis
et al., 2007; Hodges et al., 2007), that interferes with the NF␬B
activation route and results in increased production of IL-10. In
this regard, the DC-SIGN-initiated signaling has been shown to
be differentially promoted by distinct antibodies (Hodges et al.,
2007), and different intracellular signals appear to be triggered
upon recognition of mannose- or Lewis-containing ligands by DCSIGN (Gringhuis et al., 2009). However, most studies on DC-SIGN
intracellular signaling make use of ligands which are not exclusively recognized by DC-SIGN and might be also sensed by other
lectins and/or pathogen recognition receptors. Thus, the identification of antibodies that detect independent epitopes on the DC-SIGN
molecule might constitute a first step for the design of reagents
to (1) dissect the DC-SIGN-initiated intracellular signaling pathways and (2) target lectin-initiated intracellular signals as a way
to modulate the polarizing ability of DC-SIGN-expressing antigenpresenting cells.
The antibodies whose epitopes have been mapped in the
present manuscript can be also helpful to potentiate the
internalization ability of the molecule without preventing its
pathogen-recognition capacity. The search for reagents specific for
the DC-SIGN related molecule L-SIGN has led to the identification
of monoclonal antibodies that greatly enhance its internalization
rate (Dakappagari et al., 2006). In the case of DC-SIGN, we have
determined that the antibodies that promote the strongest DCSIGN internalization in MDDC are directed against an epitope
located within the neck domain and out of the ligand-recognition
domain (CRD), what suggests that their moderate inhibitory of
DC-SIGN recognition functions is due to either steric hindrance
or to enhanced removal of the molecule from the cell surface.
Conversely, the anti-L-SIGN antibodies that display the most
efficient ligand-blocking effect are also internalized most efficiently in K562 transfectants and liver sinusoidal endothelial
cells (Dakappagari et al., 2006). Since the L-SIGN antibodies have
not been structurally mapped, it is not possible at this time to
108
847
determine whether this difference is due to either cell-specific
or molecule-specific effects. Regardless of the explanation, the
internalization-inducing DC-SIGN neck-specific antibodies appear
to be ideally suited to potentiate the generation of immune
responses against DC-SIGN-interacting ligands without compromising the pathogen-attachment function of the molecule.
Acknowledgements
This work was supported by the Ministerio de Educación
y Ciencia (Grants BFU2008-0149-BMC), Ministerio de Sanidad
y Consumo, Instituto de Salud Carlos III (Spanish Network for
the Research in Infectious Diseases, REIPI RD06/0008, and AIDS
Research Network, RIS RD06/0006), and Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36663/07) to ALC.
We gratefully acknowledge Dr. M.A. Abengózar for performing the
Leishmania-DC-SIGN interaction experiment.
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109
Discusión
Discusión
La plasticidad del proceso de activación de macrófagos se refleja en la existencia de
subpoblaciones de este tipo celular con funciones diferentes e, incluso, opuestas [87]. Con el fin de
determinar las bases moleculares de estas diferencias funcionales, se realizaron estudios de
expresión génica diferencial entre distintas poblaciones de macrófagos generados in vitro a partir de
monocitos de sangre periférica en presencia de GM-CSF (M1), M-CSF (M2), IFNγ (CAMØ) o IL-4
(AAMØ) (Figura 11).
INFγ
Activación clásica
(CAMØ)
GM-CSF
IL-4
Macrófago pro-inflamatorio
(M1)
Monocito
M-CSF
Activación alternativa
(AAMØ)
Macrófago anti-inflamatorio
(M2)
Figura 11.- Diferenciación in vitro de macrófagos. Esquema ilustrativo de la generación in vitro de las
poblaciones de macrófagos utilizadas en los ensayos de expresión génica.
El perfil de expresión génica de cada población de macrófagos se conoce como su “firma genética”,
y permite determinar su presencia en un tejido sano o su implicación en procesos inflamatorios. A su
vez, la firma genética puede ser utilizada para predecir la evolución de procesos patológicos, debido
a que el diferente comportamiento de estas subpoblaciones celulares permite prognosticar el
desarrollo de ciertas enfermedades, como por ejemplo la progresión de tumores (actividad pro-/antitumoral). Por otro lado, la comparación de los perfiles de expresión génica de diferentes poblaciones
celulares permite establecer diferencias y semejanzas fenotípicas entre ellas. De este modo, hemos
identificado genes diferencialmente expresados entre las poblaciones de macrófagos analizadas,
cuya expresión restringida ha sido validada mediante PCR cuantitativa, y que podrían ser utilizados
como “marcadores fenotípicos” específicos de cada una de ellas (Figura 12). En este sentido, se
han identificado 149 genes diferencialmente expresados entre macrófagos generados con GM-CSF
(M1) y M-CSF (M2) (>2 veces de diferencia, p<0.05), 94 de los cuales están significativamente
sobre-expresados en macrófagos M2. Por otro lado, entre AAMØ y CAMØ existen 283 genes
diferencialmente expresados (>2 veces de diferencia, p<0.05), de los cuales 112 están asociados a
la activación alternativa de macrófagos. Entre los genes sobre-expresados en macrófagos M2
113
Discusión
Figura 12.- Representación gráfica de la expresión relativa de genes en las diferentes poblaciones de
macrófagos. La escala de colores representa la expresión relativa de cada gen respecto a la media de
intensidad de fluorescencia de las cuatro poblaciones (negro), desde los genes con menor expresión (verde) a
los genes más expresados que la media (rojo). A la derecha se han seleccionado algunos de los genes con
mayor expresión en macrófagos generados con IL-4 (cuadro verde), M-CSF (cuadro azul) o IFNγ (cuadro
naranja).
114
Discusión
generados en presencia de M-CSF encontramos genes que justifican su fenotipo anti-inflamatorio.
Por ejemplo, genes inducidos o regulados por TGFβ como TGFB1 o SEPP1, genes que promueven
angiogénesis como IGF-1, o genes involucrados en reparación de heridas como F13A1, se
encuentran diferencialmente expresados en macrófagos M2. Del mismo modo, en estas células se
sobre-expresan genes relacionados con la producción de la citoquina anti-inflamatoria IL-10, como el
factor de transcripción MAF, del cual depende su expresión y la inhibición de IL-12p70 [213, 214], o
la enzima HMOX-1 y el receptor “scavenger” CD163, que son inducidos por IL-10 [215]. Por otro
lado, hemos evaluado la expresión de grupos funcionales de genes entre las subpoblaciones de
macrófagos para intentar explicar sus diferentes características. Por ejemplo, la expresión de genes
implicados en la regulación de los niveles de hierro intracelular podría justificar la función microbicida
de los macrófagos M1 (asociada con bajos niveles de hierro intracelular) y el papel permisivo frente
a patógenos de los macrófagos M2 (asociado con un elevado contenido de hierro intracelular)
(Sierra-Filardi et al., Immunobiology, en prensa, 2010). A su vez, los estudios de expresión génica
en las diferentes poblaciones de macrófagos también nos han permitido identificar genes cuya
expresión no se encuentra descrita en dichas células, lo que posibilita el estudio de la regulación de
su expresión y de sus posibles funciones en ellas.
El receptor de folato β es un marcador de macrófagos anti-inflamatorios M2 y TAM,
cuya expresión es regulada por activina A.
El receptor de folato β (FRβ) es uno de los genes que hemos identificado como marcador de
macrófagos anti-inflamatorios generados en presencia de M-CSF (M2). El folato o vitamina B9 es un
nutriente hidrosoluble esencial en las células, ya que se encuentra implicado en numerosas rutas
bioquímicas como el metabolismo de aminoácidos, la síntesis de purinas y pirimidinas, y la
metilación de ácidos nucleicos, proteínas y lípidos [216]. La deficiencia de esta vitamina conlleva a
una serie de anormalidades clínicas [217] como anemia megaloblástica, retraso del crecimiento,
desórdenes neurológicos, depresión [216] y enfermedad de Alzheimer [218]. Por el contrario, su
suplemento óptimo previene defectos en el tubo neural durante el embarazo y reduce el riesgo de
enfermedades cardiovasculares, desarrollo de tumores [219] y la predisposición a aterosclerosis
[220]. La forma biológicamente activa es el tetrahidrofolato (THF), cofactor esencial en reacciones
de metilación entre las que se incluye la formación de metionina a partir de homocisteína en el
conocido como ciclo de la homocisteína/metionina [221], y que se encuentra acoplado a un segundo
ciclo, conocido como ciclo del folato [222] (Figura 13).
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Discusión
MTHFR
Biosíntesis de
nucleótidos
5,10-MTHF
5-MTHF
SHMT
Metionina
sintasa
Homocisteína
THF
CBS
Metionina
Cistationina
Cisteína
SAH
CH3X
DHFR
SAM
X
Metilación de DNA,
proteínas, lípidos
Folato
Figura 13.- Metabolismo del folato y vías relacionadas. Esquema simplificado de la conexión entre los ciclos
del folato y de la homocisteína. Los rectángulos muestran los sustratos y los óvalos las enzimas. DHFR,
dihidrofolato reductasa; THF, tetrahidrofolato; SHMT, serinhidroximetil transferasa; 5,10-MTHF, 5,10metilentetrahidrofolato; MTHFR, 5,10-metilentetrahidrofolato reductasa; 5-MTHF, 5-metiltetrahidrofolato; SAM,
S-adenosilmetionina; SAH, S-adenosilhomocisteína; X, sustrato para metilación; CBS, cistationina-β-sintasa.
El ácido fólico y sus formas reducidas (folatos) son captados a través de la membrana celular
mediante dos principales sistemas de transporte: 1) transporte vía canales o proteínas
transportadoras de membrana con baja afinidad (Kd~μM); y 2) endocitosis vía receptor de folato (FR)
de alta afinidad (Kd~pM) [223]. Además, existe un flujo de transporte asociado al metabolismo
energético de la célula que permite el flujo de folato hacia el exterior celular mediante hidrólisis de
ATP [224]. La expresión de los FR se encuentra restringida a aquellos tejidos que utilizan la
segunda vía de captación de folato. Así, a excepción de riñón y placenta, el resto de tejidos sanos
expresan niveles bajos o no detectables [225], mientras que numerosos tejidos tumorales (ovario,
mama, bronquios, riñón, cerebro) expresan altos niveles de estos receptores [226]. La expresión de
los FR se modula de manera inversa por la concentración extracelular de folato: en cultivos
celulares, niveles bajos de folato en el medio incrementan la expresión del receptor en membrana,
mientras que la adición de ácido fólico al medio revierte este aumento [227]. En humanos, existen 4
isoformas del FR, FRα (FOLR1), FRβ (FOLR2), FRγ (FOLR3) y FRδ (FOLR4) [228], cuyos perfiles
de expresión en tejidos normales y patológicos son diferentes [229]. Así, el FRα es la isoforma más
expresada en tejidos adultos sanos, sobre todo en tejidos epiteliales, y en algunos tipos de tumores
como los ginecológicos (ovario, útero) [230]. A diferencia del resto de isoformas, el FRγ y su forma
truncada (FRγ’) no se encuentran anclados a la membrana plasmática y son secretados por células
hematopoyéticas de algunos tejidos sanos y tumorales, como médula ósea, bazo y timo [231, 232].
Por el contrario, ni la expresión en tejidos adultos ni embrionarios, ni la capacidad de unir folato del
116
Discusión
FRδ ha sido descrita [233]. En ratón se han identificado homólogos de FRα y FRβ [234], y un tercer
FR que se expresa en bazo y timo [233]. Debido al papel del ácido fólico en el desarrollo
embrionario, los ratones Folbp1-/- y Folbp2-/- presentan defectos en la formación del tubo neural [235,
236].
Respecto a la expresión del FRβ, éste se encuentra restringido al linaje mieloide [237], se
detecta en células hematopoyéticas de médula ósea CD34+ [238] y está incrementado durante la
maduración de neutrófilos y la activación de monocitos y macrófagos [239]. En tejidos sanos el FRβ
sólo se expresa significativamente en placenta, bazo y timo [240], mientras que lo hace en el 70%
de los casos de leucemia mieloide aguda (AML), donde se localiza también en células CD34+ [237,
241]. Además, el FRβ se expresa en macrófagos sinoviales de pacientes con artritis reumatoide
(RA) [239] y en un modelo inducido de artritis en rata [242]. Respecto a su capacidad funcional, el
FRβ expresado en células hematopoyéticas CD34+, neutrófilos y monocitos no es capaz de unir
folato [238], mientras que sí lo hace en condiciones patológicas como AML [231, 241] y en
macrófagos sinoviales en RA [243]. Como se ha comentado anteriormente, en nuestros estudios de
expresión génica hemos determinado que el FRβ se expresa en macrófagos anti-inflamatorios M2,
donde su expresión es inducida por M-CSF. Adicionalmente, hemos observado que otras citoquinas
de activación alternativa de macrófagos, como IL-4 e IL-13, mantienen o aumentan su expresión en
dichas células, a diferencia de estímulos pro-inflamatorios, como GM-CSF o LPS, que la inhiben. En
este sentido, el patrón de expresión del FRβ en ratón es similar al que hemos observado en
humano, ya que se expresa en macrófagos peritoneales activados F4/80+ CD68+ que presentan un
fenotipo M2 [244][245], y se induce con IL-4 [128]. Por tanto, el FRβ también podría considerarse un
marcador de macrófagos anti-inflamatorios y de activación alternativa en ratón. Por otro lado, hemos
demostrado que los macrófagos M2 son capaces de captar folato mediante endocitosis vía receptor,
y que es únicamente el FRβ el que media esta unión, ya que ni el FRα ni el FRγ se expresan en
estas células. A diferencia de nuestros resultados, en macrófagos peritoneales de ratón el FRβ sólo
es funcional tras la activación con estímulos inflamatorios [246]. En conclusión, el FRβ constituye un
marcador de macrófagos anti-inflamatorios/alternativos, ya que su expresión es inducida por M-CSF
y citoquinas Th2 in vitro en monocitos y macrófagos M2, y además es el responsable de la captación
de folato por estas células.
Además de la inhibición de la expresión del FRβ por estímulos inflamatorios como GM-CSF,
observamos que el medio condicionado de macrófagos M1 es también capaz de inhibir su
adquisición. Este hecho nos llevó a buscar factores secretados por estas células que pudiesen
regular la expresión de este receptor. Según nuestros estudios de expresión génica, la activina A es
el gen más diferencialmente expresado entre macrófagos M1 y M2, y además hemos determinado
que es un citoquina secretada al medio de cultivo de los macrófagos M1. Según nuestras
observaciones, la expresión y secreción de activina A es inducida por GM-CSF in vitro en la
diferenciación de macrófagos a partir de monocitos de sangre periférica y de células de médula ósea
117
Discusión
de ratón. Además, y de acuerdo con su rápida secreción en procesos inflamatorios [247], la activina
A es liberada al medio de cultivo desde las primeras horas de estimulación con GM-CSF. Por el
contrario, los macrófagos diferenciados en presencia de M-CSF no son capaces de secretar activina
A ni siquiera en presencia de un estímulo inflamatorio como LPS, a diferencia de lo descrito en
macrófagos de ratón [248]. Por tanto, la diferente regulación de la expresión de activina A y FRβ por
GM-CSF y M-CSF, respectivamente, nos hizo pensar que la activina A podría ser un factor regulador
de la expresión del FRβ y otros marcadores de macrófagos M2.
Las activinas son factores de crecimiento y diferenciación pertenecientes a la familia de TGFβ,
que estructuralmente están compuestas por dos subunidades β (activina A, βA-βA; activina B, βBβB; activina AB, βA-βB) unidas por un único enlace disulfuro [249]. La activina A (INHBA) se
identificó inicialmente por su papel en el control de la secreción de la hormona estimuladora del
folículo (FSH) [2], y en la actualidad es conocida por sus numerosas funciones en diferenciación,
proliferación y apoptosis celular, homeostasis, reparación de heridas, inflamación, además de su
función endocrina y su papel en la progresión de tumores [249-253]. Su actividad biológica es
neutralizada por folistatina, debido a la elevada afinidad de esta proteína por las activinas [254-256].
Respecto a la señalización de la activina A, como miembro de la familia de TGFβ, se une a los
receptores transmembrana ActRIB y ActRIIA y señaliza a través de las proteínas Smad2/3 [257,
258]. La activina A es sintetizada por una gran variedad de células, incluyendo monocitos [259, 260],
macrófagos [261], células dendríticas [262, 263], células endoteliales [264], células del estroma de la
médula ósea [265-267] y mastocitos [268], y algunas células linfoides como timocitos [269] y células
esplénicas T CD4+ de ratón [270]. A su vez, la activina A es secretada durante procesos
inflamatorios por lo que está considerada como un factor modulador de la respuesta inmunitaria.
A pesar de su secreción por macrófagos pro-inflamatorios M1, la activina A está descrita como
una citoquina secretada preferentemente por células Th2 y que promueve activación alternativa de
macrófagos en ratón, al inducir la expresión de Arg1 e inhibir la expresión de iNOS [270]. Además,
otra citoquina Th2 como IL-13 aumenta la concentración de activina A en el lavado broncoalveolar e
intraepitelial en ratones naive y durante inflamación alérgica [271], mientras que IFNγ (citoquina Th1)
no es capaz de inducirla [272]. Sin embargo, según nuestras observaciones, la activina A secretada
por macrófagos M1, al igual que la proteína recombinante, ejerce funciones pro-inflamatorias en la
diferenciación de monocitos, ya que previene la adquisición de la expresión del FRβ (FOLR2), y
otros marcadores de macrófagos M2 como SERPINB2 o MAF. Además, hemos determinado que la
activina A regula la función anti-inflamatoria de los macrófagos generados en presencia de M-CSF,
al inhibir la secreción de IL-10 por estas células tras ser estimuladas con LPS. De acuerdo con
nuestro resultado, la activina A inhibe la producción de IL-10 en cultivos de células epiteliales
prostáticas [273]. Por tanto, la activina A es capaz de regular el fenotipo y función de los MDM. Por
otro lado, a pesar de regular la función anti-inflamatoria de los macrófagos M2 en cuanto a la
producción de IL-10, hemos observado que la activina A no afecta a la capacidad presentadora de
118
Discusión
antígeno de estas células. Además, el medio condicionado de macrófagos M1 no induce la
maduración de DC, mientras que sí lo hace después de estimular dichas células con LPS. Es decir,
según nuestras observaciones y de acuerdo con un trabajo previo [263], la activina A secretada
constitutivamente por macrófagos M1 no induce maduración de DC, mientras que sí lo hacen otras
citoquinas pro-inflamatorias secretadas tras estimulación. En conclusión, en papel modulador en la
respuesta inflamatoria de la activina A podría deberse a su capacidad de regular el fenotipo y
función de los macrófagos, ya que inhibe la adquisición de marcadores anti-inflamatorios y la
producción de IL-10 por macrófagos M2, pero no influye en la capacidad de activación de linfocitos T
en estas células o en la maduración de DC.
Respuesta
inmunitaria
TLR
NFκB
MAPK
Activina A
Macrófago
Célula B
IL-1β TNFα
IL-6
iNOS
Célula T
Célula NK
DC
Inflamación
Figura 14.- Funciones de la activina A en procesos inflamatorios. La activina A es capaz de inducir la
producción de mediadores inflamatorios, como IL-1β, TNFα, IL-6 e iNOS, a la vez que, por su función antiinflamatoria, puede ejercer un efecto inhibidor sobre la respuesta inmunitaria [274].
Respecto al papel modulador en la respuesta inmunitaria de la activina A, existe una dicotomía
entre sus acciones pro- y anti-inflamatorias [274] (Figura 14), a diferencia de otro miembro de su
misma familia, como el TGFβ, que presenta siempre funciones supresoras [275]. Por su acción
inflamatoria, la activina A estimula la secreción in vitro de citoquinas pro-inflamatorias como
TNFα, IL-1β e IL-6 [276-278], así como la expresión de iNOS y la producción de NO [277, 279].
Además, la activina A está implicada en la degradación de IκB, la translocación al núcleo de NFκB y
la fosforilación de ERK1/2 y la quinasa p38 [280-282]. Respecto a sus acciones anti-inflamatorias, la
activina A puede bloquear la producción y función de citoquinas inflamatorias como IL-1β e IL-6. En
monocitos humanos, la activina A inhibe la producción de IL-1β, bloqueando la conversión de su
precursor a su forma activa, e induce la expresión del antagonista del receptor IL-1 (IL-1RN), dando
119
Discusión
lugar a una reducción neta en la actividad de IL-1β [282]. Respecto a la IL-6, se ha demostrado que
la activina A inhibe su expresión en respuesta a LPS en microglía de ratón in vitro e in vivo [283] y
en células humanas epiteliales del endometrio [284]. Por otro lado, la activina A inhibe numerosas
funciones pro-inflamatorias de estas citoquinas, incluyendo proliferación de células T y B, o la
fagocitosis de monocitos [282, 285-289] (Figura 14). Esta dualidad en los efectos estimuladores o
inhibitorios de la activina A depende de su concentración. Así, en células amnióticas humanas la
producción IL-6 se estimula a dosis bajas, mientras que a mayores dosis su efecto es inhibitorio
[290]. Por tanto, la actividad pro- o anti-inflamatoria de la activina A depende de su concentración
local en tejidos.
Como ya mencionamos en el apartado de Introducción, los TAM son considerados
fundamentalmente macrófagos anti-inflamatorios/M2 [111, 127, 291], aunque su fenotipo y función
varía durante el desarrollo de los tumores [118]. Por ello, nos planteamos determinar la expresión
del FRβ en TAM como marcador fenotípico de macrófagos M2. Así, hemos observado que este
receptor se expresa en TAM de melanoma primario y metastático, mientras que no lo hace en el
resto de células tumorales. Adicionalmente, hemos determinado que el FRβ se expresa en células
CD14+ aisladas ex vivo de líquido pleural de melanoma y fluido ascítico de adenocarcinoma de
mama metastático, donde es funcional y tiene la capacidad de captar folato. Estos resultados están
de acuerdo con la expresión del receptor en células CD68+ CD163+ en glioblastoma humano y de
rata [244]. Por el contrario, en otro estudio afirman que los TAM de fibrosarcoma de ratón expresa
menores niveles de FRβ que los macrófagos peritoneales elicitados con tioglicolato [127]. Esta
discrepancia puede ser debida a la influencia del microambiente tumoral en la expresión de este
receptor, y que puede variar en función del tipo de tumor o de la fase de desarrollo en la que se
encuentre. En este sentido, hemos determinado que tanto el líquido ascítico de algunos carcinomas
gástricos o los medios condicionados de algunas líneas celulares tumorales, como las citoquinas
anti-inflamatorias liberadas por los tumores como M-CSF e IL-10, inducen la expresión in vitro del
FRβ en monocitos. Además, la exposición conjunta a varias de estas citoquinas, situación similar a
la que ocurriría in vivo, tiene un efecto sinérgico en la inducción de este receptor. Por otro lado, está
descrito que la expresión de activina A es mayor en tejidos tumorales que en tejidos sanos [292,
293], y disminuye con la progresión tumoral, siendo más elevada en tumores primarios que en
carcinomas metastáticos [294]. Según nuestras observaciones, los líquidos ascíticos de algunos
carcinomas gástricos inducen la expresión del FRβ e inhiben la expresión de activina A en
monocitos y macrófagos pro-inflamatorios M1. Por tanto, el microambiente tumoral condiciona el
fenotipo de los TAM y, en consecuencia, el desarrollo tumoral. Además, la activina A secretada en la
primera fase de iniciación del tumor contribuiría a la inhibición de las propiedades supresoras de los
TAM, por lo que la activina A sería un regulador el establecimiento y desarrollo tumoral.
El fenotipo de los TAM es un factor importante en la progresión de los tumores, debido a que los
macrófagos son las células inflamatorias que regulan el inicio y desarrollo tumoral [129, 295, 296].
120
Discusión
En los sitios de inflamación crónica donde se inicia la formación del tumor los macrófagos presentan
un fenotipo inflamatorio M1 con actividad anti-tumoral [297, 298] (Figura 15). Posteriormente cuando
el tumor se establece, los TAM dejan de secretar citoquinas inflamatorias como TNFα, IL-1β e IL-6, y
producen señales de supervivencia para las células tumorales, apoyando el crecimiento,
angiogénesis y metástasis del tumor [120, 299-301]. Este hecho se ha observado en modelos de
cáncer colorectal inducido por colitis y cáncer de hígado inducido químicamente en ratón, donde
existe una inhibición de la producción de citoquinas inflamatorias (IL-6, TNFα) [302-304]. Por tanto,
los macrófagos presentan una función dual en el contexto de la progresión tumoral: mientras que en
las primeras etapas de formación del tumor los macrófagos tienen el potencial de expresar
actividades anti-tumorales (fenotipo M1), en tumores establecidos juegan un papel pro-tumoral
(fenotipo M2) [126] (Figura 15). Una posible explicación a estas diferencias funcionales de los
macrófagos puede ser la eficacia de la respuesta inmunitaria en las distintas etapas de desarrollo
tumoral [305, 306]. Así, en estados tempranos de formación del tumor, la respuesta inmunitaria es
eficaz y los macrófagos ejercen su actividad citotóxica eliminando células tumorales. Una vez
estabilizado el tumor, cuando las células tumorales persistentes han escapado del “ataque”
inmunitario, se crea un ambiente inmunosupresor con una polarización anti-inflamatoria/M2
predominante en los macrófagos. Por ello, una posible estrategia terapéutica frente al avance
tumoral podría consistir en alterar el balance funcional de los macrófagos hacia un fenotipo
tumoricida [307].
INICIACIÓN
ELIMINACIÓN
NK
CD4
CD4
EQUILIBRIO
CD8 CD8
NK
CD8
ESCAPE
CD4
NK
CD4
CD8
Progresión del tumor
M1
M2
Figura 15.- Polarización de los macrófagos en el desarrollo tumoral. El fenotipo de los macrófagos sufre un
cambio progresivo con el avance tumoral, desde un fenotipo tumoricida/M1 en las primeras etapas de iniciación
hasta un fenotipo supresor/M2 cuando el tumor se establece [308].
Por otro lado, la función de los TAM está condicionada por su localización en respuesta a
señales locales [309]. Así, en áreas del tumor poco vascularizadas los TAM son altamente pro-
121
Discusión
angiogénicos en respuesta a hipoxia [310], y su presencia se ha correlacionado con el crecimiento
de tumores de mama [311]. Por el contrario, un elevado número de TAM en áreas vascularizadas
del tumor se correlaciona con buena prognosis en algunos tumores humanos, sugiriendo un posible
fenotipo anti-tumoral M1 en esos sitios [312]. Además, existe un fenotipo “mixto” debido a que los
TAM pueden presentar al mismo tiempo características pro-inflamatorias (M1) y anti-inflamatorias
(M2). Así, en un modelo tumoral en ratón los TAM son capaces de expresar a la vez iNOS y Arg1
[313, 314]. De forma similar, monocitos de pacientes con tumor gástrico avanzado expresan niveles
elevados y mayores de IL-12 e IL-10, citoquinas pro- y anti-inflamatorias respectivamente, que los
monocitos de individuos sanos [315]. En estos casos los TAM pueden exhibir actividades que
previenen el establecimiento y la extensión de las células tumorales y, simultáneamente, contribuir al
crecimiento y diseminación del tumor [316].
Al igual que ocurre en tumores, los macrófagos parecen ser los promotores principales de
enfermedades inflamatorias/autoinmunes como RA, donde existe una correlación directa entre la
actividad de estas células y la inflamación de las articulaciones y la destrucción del hueso [243].
Esto es debido a que los macrófagos del líquido sinovial de RA secretan múltiples mediadores de
inflamación y destrucción de tejido, incluidas citoquinas pro-inflamatorias (IL-1, IL-6, TNFα),
quimioquinas, prostaglandinas, metaloproteasas y ROS [317]. Además, estos macrófagos activados
participan en presentación de antígeno contribuyendo a la activación y proliferación de las células T
y su consecuente actividad destructiva [318]. Por todo ello, los macrófagos presentes en el líquido
sinovial de RA tendrían un fenotipo pro-inflamatorio M1. De acuerdo con esto, la expresión de
activina A en membranas sinoviales y su concentración en el líquido sinovial es elevada en
pacientes con RA [286, 288, 319]. Sin embargo, está descrita la expresión del FRβ en los
macrófagos presentes en el líquido sinovial de RA [243]. La expresión de este marcador de
macrófagos anti-inflamatorios estaría justificada por los elevados niveles de M-CSF existentes en el
líquido sinovial de RA, producidos por los fibroblastos sinoviales [57, 59, 320]. Por tanto, al igual que
ocurre en tumores, los factores presentes en el líquido sinovial de RA pueden condicionar el fenotipo
de los macrófagos, existiendo un cambio entre sus funciones inflamatorias (M1) y supresoras (M2).
Por otro lado, la activina A está involucrada en otras enfermedades inflamatorias como la
enfermedad inflamatoria intestinal, meningitis, asma, etc. En enfermedad inflamatoria intestinal, la
activina A se localiza en la mucosa de tejidos inflamados donde existe una elevada expresión de IL1β [287], y en intestino y plasma en tres modelos diferentes de la enfermedad en ratón [321]. Los
niveles de activina A son elevados en células T CD4+ y en suero en pacientes asmáticos [272, 322]
y en el de lavado broncoalveolar en modelos de asma en ratón [268, 271, 323]. Por tanto, la activina
A secretada en los sitios de inflamación, podría condicionar el fenotipo de los macrófagos allí
presentes, regulando así la respuesta inflamatoria.
La expresión y capacidad de endocitosis selectiva del FRβ en TAM o macrófagos sinoviales de
RA, posibilita el desarrollo de conjugados de folato como agentes terapéuticos frente a tumores y
122
Discusión
enfermedades inflamatorias e autoinmunes [324] (Figura 16). Estos conjugados irían dirigidos de
forma específica frente a las células implicadas en procesos patológicos, evitando el daño colateral
en las células sanas [325]. A su vez, el ácido fólico es un ligando óptimo como agente terapéutico
debido a su facilidad de conjugarse y a su elevada afinidad por su receptor incluso después de su
conjugación [324]. Un ejemplo del uso de terapéutico del FRβ se demuestra en dos modelos de
ratón de lupus eritematoso sistémico, enfermedad autoinmune crónica caracterizada por inflamación
y daño en el tejido conjuntivo, y en la que los macrófagos activados contribuyen a su desarrollo al
secretar mediadores inflamatorios y atraer otras células inflamatorias. En ellos se ha demostrado
que la depleción de macrófagos FRβ+ mediante inmunoterapia frente al receptor reduce los síntomas
de la enfermedad, sin provocar daño en tejidos sanos, y prolonga la supervivencia de los animales
[326]. Por otro lado, un estudio reciente muestra la posibilidad de reducir el crecimiento tumoral por
la unión selectiva a los TAM de una inmunotoxina usando un anticuerpo monoclonal frente al FRβ
[244].
A
B
Macrófago
Célula NK
Conjugado
de folato
Anticuerpo
FcR
H+
FRβ
Endosoma
R-SH
H+
H+
Célula
tumoral
R-SH
H+
R-SH
+
H+ H
H+ H+ H+
H+
H+
H+
Lisosoma
R-SH
Célula tumoral
Figura 16.- Estrategias terapéuticas frente al FRβ. A. Endocitosis de conjugados de folato. Los conjugados
de folato se unen al FRβ con elevada afinidad, se internalizan por invaginación de la membrana, y
posteriormente, se separan y reducen liberando el agente terapéutico. B. Inmunoterapia mediada por el FRβ.
Los haptenos unidos a folato son capaces de estimular una respuesta inmunitaria, produciéndose la destrucción
de células tumorales por macrófagos y células NK.
123
Discusión
DC-SIGN constituye un marcador de macrófagos anti-inflamatorios M2 y AAMØ [56, 151], ya
que su expresión en monocitos y macrófagos es inducida por M-CSF e IL-4 [92, 150], e inhibida por
estímulos inflamatorios como IFNγ [92]. Por ello, DC-SIGN se expresa en macrófagos implicados en
reparación de heridas y en macrófagos alternativos/reguladores M2. Por otro lado, un estudio
reciente realizado en nuestro grupo demuestra que DC-SIGN se expresa en TAM, y al igual que en
el caso del FRβ, su expresión podría contribuir a la adquisición fenotipo inmunosupresor en los
tumores (Domínguez-Soto et. al, J Immunol en revisión, 2010).
Requerimientos estructurales de DC-SIGN para su multimerización. Influencia de la
presencia de variantes con menor tamaño en la región del cuello.
El reconocimiento de PAMP por las células dendríticas (DC) es el primer paso para
desencadenar la respuesta inmunitaria. DC-SIGN es un receptor de DC que interacciona con
proteínas glicosiladas presentes en patógenos, por lo que está implicado en etapas tempranas de
infecciones producidas por virus, hongos y bacterias [149, 193, 196]. Este reconocimiento a través
de DC-SIGN permite la internalización, procesamiento y posterior presentación antigénica a los
linfocitos T [150, 164, 187]. Estructuralmente DC-SIGN es una proteína de membrana cuya región
citoplásmica contiene motivos de reciclaje e internalización [149, 150], y su dominio extracelular
(ECD) consta de un cuello formado por 8 repeticiones de 23 aminoácidos seguido de un CRD [164,
165]. Procesos de “splicing” alternativo y polimorfismos a nivel genético dan lugar a variantes de DCSIGN tanto a nivel de RNA como a nivel proteico [168, 327].
Mummidi y colaboradores identificaron variantes de “splicing” de DC-SIGN en PBMC activados
con PHA, DC maduras derivadas de células hematopoyéticas CD34
+
y células THP-1, que
presentan un tamaño variable en la región del cuello y en el CRD, así como isoformas solubles o
con una cola citoplásmica “alternativa” [168]. En nuestro estudio por primera vez se identificaron
isoformas de “splicing” de DC-SIGN en células dendríticas derivadas de monocitos (MDDC) que,
además, dan lugar a proteínas funcionales que se expresan en membrana. La generación de
construcciones de DC-SIGN con diferente tamaño y disposición de las repeticiones del cuello a partir
de estas isoformas, y su expresión en líneas celulares, ha sido de gran ayuda para determinar los
requerimientos estructurales de la multimerización y función de DC-SIGN. A su vez, para este
estudio hemos generado y utilizado mutantes de DC-SIGN sin CRD y con un número variable de
repeticiones en la región del cuello.
Aunque ya existían evidencias de la capacidad de oligomerización de la proteína recombinante
[166], se ha descrito que DC-SIGN aparece formando tetrámeros en la membrana de MDDC [164],
aumentando así la avidez por sus ligandos [165, 328]. La región del cuello es la responsable de esta
multimerización, mientras que existe controversia en el papel que desempeña el CRD [166, 329]. En
125
Discusión
este sentido, un estudio reciente afirma que el CRD no influye en la formación ni en la estabilidad de
los tetrámeros de DC-SIGN [328]. Sin embargo, y en un contexto celular, nosotros observamos que
el mutante que carece de CRD tiene disminuida su capacidad de multimerización. Por tanto, aunque
la oligomerización de DC-SIGN tiene lugar a través de la región del cuello, las interacciones entre
CRD contribuirían a su estabilización. Así, la ausencia de este dominio podría provocar una
disminución en las interacciones entre las regiones c-terminal de los cuellos, que llevaría a la
pérdida de estabilidad de los tetrámeros. Por otro lado, hemos determinado que la coexpresión de la
forma completa de DC-SIGN (1A) y el mutante sin CRD (8d) disminuye la formación de homomultímeros 1A-1A, debido posiblemente a la formación de hetero-multímeros 1A-8d, y que tendrían
menor estabilidad frente a agentes desnaturalizantes ya que no son detectables mediante Western
blot. El hecho de que la forma prototípica de DC-SIGN sea capaz de asociarse con mutantes sin
CRD confirma que la multimerización tiene lugar a través del cuello, y la menor estabilidad de los
hetero-multímeros 1A-8d vuelve a demostrar que la presencia de CRD estabiliza los oligómeros.
Por otro lado, el estado de oligomerización de DC-SIGN depende en gran medida del pH del
entorno [329]. Así, la elevada acidez existente en los endosomas provoca un cambio de
conformación de los tetrámeros con la consiguiente pérdida de afinidad y liberación de los ligandos
unidos a DC-SIGN [330]. De acuerdo con nuestros resultados, la estabilización de los multímeros de
DC-SIGN por el CRD se observa en ensayos con proteínas recombinantes donde al disminuir el pH,
en ausencia de CRD los tetrámeros se disocian totalmente, mientras que el ECD completo no lo
consigue. Al igual que el pH, otros factores como la concentración de sales estabilizan los
tetrámeros por aumento en las interacciones hidrofóbicas entre las regiones de cuello de DC-SIGN
[331].
Respecto al número de repeticiones de la región del cuello requeridas para la multimerización
de DC-SIGN, un estudio con proteínas recombinantes con el ECD truncado afirma que la presencia
de seis dominios mantiene la formación del tetrámero, mientras que la deleción de sucesivos
dominios (≤ 5.5 repeticiones) lo disocia parcialmente (dímeros) [165]. Además, proteínas de 3.5-2.5
repeticiones son difíciles de purificar por su baja estabilidad, lo que impide la determinación de su
estado de oligomerización; proteínas de menos de 2 repeticiones son más estables y presentan una
parcial disociación de los dímeros (monómeros); y el CRD sólo o con 0.5 repeticiones aparece como
monómero. Por ello y según estos autores, se requiere la presencia de al menos 3 dominios en el
cuello para que DC-SIGN pueda tetramerizar. En nuestro estudio, las construcciones de DC-SIGN
generadas a partir de las isoformas de “splicing” alternativo identificadas en MDDC y que contienen
4, 3, 2 ó 1 dominio repetido en el cuello, difieren en su capacidad de multimerización en líneas
celulares. Así, la isoforma de DC-SIGN con sólo la primera repetición en el cuello aparece como
monómero, mientras que las construcciones con mayor número de dominios (2, 3 y 4) lo hacen de
forma similar a la proteína completa con las 8 repeticiones de esta región. Por tanto, sería necesario
la presencia de al menos 2 repeticiones en el cuello para que DC-SIGN pueda multimerizar. De
126
Discusión
acuerdo con nuestras observaciones, un estudio realizado con su homólogo DC-SIGNR afirma que
la secuencia GELSE es necesaria para la multimerización de la molécula [332], por lo que la
presencia del segundo dominio de DC-SIGN sería necesaria para su multimerización (Figura 17).
DOMINIO
V S K V P S S
G
G
G
G
G
G
G
I
E
E
E
E
E
E
E
S
L
L
L
L
L
L
L
Q
S
P
P
P
P
P
P
E
E
E
E
E
E
E
E
Q
K
K
K
K
K
K
K
S
S
S
S
S
S
S
S
R
K
K
K
K
K
K
K
Q
L
L
L
M
Q
Q
Q
D
Q
Q
Q
Q
Q
Q
Q
A
E
E
E
E
E
E
E
I
I
I
I
I
I
I
I
Y
Y
Y
Y
Y
Y
Y
Y
Q
Q
Q
Q
Q
Q
Q
Q
N
E
E
E
E
E
E
E
L
L
L
L
L
L
L
L
T
T
T
T
T
T
T
T
Q
Q
R
W
R
R
R
Q
L
L
L
L
L
L
L
L
K
K
K
K
K
K
K
K
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
V
V
V
V
V
V
V
V
1
2
3
4
5
6
7
8
Figura 17.- Secuencia de aminoácidos de las repeticiones de la región del cuello de DC-SIGN.
Como puede verse en la Figura 17, las repeticiones que forman parte de la región del cuello de
DC-SIGN difieren en su secuencia de aminoácidos. Los estudios realizados con las isoformas
identificadas en MDDC que presentan el mismo número pero distinta disposición de los dominios del
cuello (4d vs. 4d’), nos permiten afirmar que las repeticiones no son equivalentes funcionalmente.
Así observamos que estas isoformas difieren en su capacidad de multimerización y unión a ligandos.
De nuevo el residuo de serina (S) presente en el segundo dominio y en la isoforma 4d’ parece
contribuir en la multimerización de DC-SIGN. Por otro lado, las repulsiones entre los residuos de
arginina (R) con carga positiva y presentes en los dominios 6 y 7 de la isoforma 4d podrían
desestabilizar los tetrámeros. De acuerdo con esto, las diferencias funcionales existentes entre DCSIGN y DC-SIGNR, como la estabilidad de los oligómeros, la especificidad de unión de azúcares y la
señalización intracelular [188], podrían justificarse en parte por la diferente secuencia de
aminoácidos de las regiones del cuello de ambos receptores. Así, la presencia de un residuo de
leucina (L) en las repeticiones 6 y 7 de DC-SIGNR en lugar de la glutamina (Q) existente en DCSIGN, hace que este receptor sea más estable en estudios desnaturalización [328]. Además, la
sustitución del residuo de leucina (L) por glutamina (Q) en moléculas quiméricas de DC-SIGNR hace
que adquieran el comportamiento de DC-SIGN [328].
Por otro lado y como se ha comentado anteriormente, DC-SIGN presenta variaciones génicas
tanto en la región reguladora como en la región codificante, y cuya presencia se asocia a una
susceptibilidad alterada frente a infecciones por HIV-1 o M. tuberculosis [171, 172]. Los
polimorfismos en el exón 4 de DC-SIGN dan lugar principalmente a variantes con diferente número
de repeticiones en la región del cuello, y su frecuencia entre la población es baja (aprox. 1%) [333].
La presencia de estas variantes puede influir en la función de la molécula debido al papel del cuello
en la multimerización y soporte del CRD. Al igual que ocurría en el caso de los polimorfismos en la
región reguladora de DC-SIGN, existe controversia entre el posible papel protector de las variantes
127
Discusión
del cuello frente a determinadas infecciones. En este sentido, existen estudios que asocian la
presencia de variantes génicas del cuello de DC-SIGN a una resistencia a la infección por HIV-1 en
individuos de EEUU [327] o China [334], mientras que otros estudios no encuentran esta asociación
en la población del norte de Asia [335], norte de India [336, 337], sur de África, Tailandia [337] o
China [333]. Respecto a la susceptibilidad a la infección por M. tuberculosis no se ha encontrado un
papel protector de estas variantes en individuos de Colombia [178], sur de África [338] o Túnez
[179].
Esta discrepancia existente en la presencia de variantes de DC-SIGN con menor tamaño del
cuello y su asociación con susceptibilidad alterada frente a la infección por HIV-1, puede ser debida
al grupo étnico en el que se realiza el estudio, ya que tanto el tipo de polimorfismo como su
frecuencia es diferente entre ellos [180, 339, 340]. Así, las variantes de DC-SIGN son más
numerosas y frecuentes en individuos de China respecto a la población caucásica [333, 334].
Mientras que en individuos de China se han identificado alelos con 4, 5, 6, 7 y 9 repeticiones en el
cuello (genotipos 4/8, 5/8, 6/8, 7/8 y 8/9) [333, 334], en individuos de EEUU, sur de África, norte de
India y Tailandia sólo se han encontrado variantes con 7 y 9 repeticiones (genotipos 7/8 y 8/9) [327,
337], y en individuos de Colombia y Túnez únicamente se han encontrado variantes con 7
repeticiones (genotipos 7/7 y 7/8, y 7/8, respectivamente) [178, 179]. Por el contrario, otro estudio ha
identificado alelos con 5, 6, 7 y 9 dominios repetidos (genotipos 5/8, 6/8, 7/8 y 8/9) en individuos del
sur de África [338]. La discrepancia en las variantes génicas de DC-SIGN encontradas en los
diversos grupos étnicos puede ser debida a la generación de formas “resistentes” del receptor frente
a los patógenos presentes en las diferentes áreas geográficas, constituyendo un mecanismo de
“escape” del sistema inmunitario frente a dichos patógenos. Por ejemplo, un estudio realizado en la
población de un país en vías de desarrollo y un país industrializado, demuestra que la diversidad
genética de DC-SIGN en la población de Zimbawe es mayor (82%) que en individuos de Canadá
(33%) [171].
De acuerdo con los estudios mencionados anteriormente, las variantes de DC-SIGN que hemos
identificado en la población española corresponden a alelos con una repetición menos en el cuello,
encontrando individuos heterocigotos con genotipo 7/8 y un individuo homocigoto con genotipo 7/7.
Además, hemos determinado que estos alelos carecen de las repeticiones 3, 5 ó 7. Con la intención
de justificar su posible papel protector, caracterizamos estructural y funcionalmente estas variantes.
Las variantes génicas de DC-SIGN que carecen del dominio 3, 5 ó 7 expresadas en líneas celulares
de forma estable o transitoria, mantienen respecto a la molécula completa su expresión en
membrana y su capacidad de multimerización, internalización de ligando y unión a ligandos o
patógenos. Por otro lado, demostramos por primera vez que los polimorfismos de DC-SIGN se
expresan a nivel de proteína en MDDC de individuos heterocigotos, y lo hacen de manera similar a
la proteína prototípica y sin influir en su expresión. Además, en MDDC estas variantes son
funcionales y mantienen su capacidad de unión de ligandos, sin existir diferencias funcionales entre
128
Discusión
las MDDC de individuos que no presentan polimorfismos en el exón 4 de DC-SIGN e individuos con
un genotipo 7/8 en esa región. Respecto a la capacidad de asociación de estas variantes con la
forma completa de DC-SIGN, en líneas celulares la formación de homo-oligómeros está favorecida
respecto a la hetero-oligomerización, mientras que en MDDC de individuos heterocigotos no hay
evidencias de asociación entre ellas. Esto confirmaría la asociación preferente de moléculas con un
tamaño de cuello similar, debido posiblemente a que el mayor contacto entre los cuellos de igual
tamaño estabiliza las interacciones homotípicas, mientras que la disposición de CRD a diferente
altura no las favorece.
A pesar de los estudios que relacionan la presencia de variantes alélicas en la región del cuello
de DC-SIGN con una protección frente a la infección por HIV-1, no está muy claro el papel protector
de estas variantes. Según nuestro estudio, la formación preferencial de homo-multímeros y el hecho
de que las variantes con una repetición menos en el cuello mantienen su capacidad de unión de
ligandos, no justificaría su papel protector frente a la infección por HIV-1 en individuos heterocigotos.
Por el contrario, una posible causa de esta protección podría ser que la presencia de algunos
polimorfismos en el cuello de DC-SIGN provoque una menor expresión de moléculas en membrana.
En este sentido, en un estudio reciente identifican variantes de DC-SIGNR a partir de DNA
genómico de individuos polimórficos, las expresan en células Raji, y realizan ensayos de transinfección de HIV-1 [341]. Los polimorfismos estudiados consisten en cuellos de diferente tamaño (6,
8 y 10 dominios) y un cambio de nucleótido (A/G) en el exón 5, y afectan a la susceptibilidad frente a
la infección por HIV-1 [342-344]. Según estos autores, estas variantes de DC-SIGNR afectan a la
cantidad de moléculas expresadas en la superficie celular, lo que se correlaciona con la eficiencia de
trans-infección de DC-SIGNR independientemente del tamaño de la región del cuello o del
aminoácido codificado en el exón 5 (A/G). Por tanto, esta justificación basada en la disminución en
la expresión in vivo de DC-SIGN o DC-SIGNR en membrana estaría de acuerdo con nuestros
resultados donde las variantes de DC-SIGN en MDDC mantienen su expresión y, por tanto, su
funcionalidad. En este mismo sentido, la variante -336G de DC-SIGN que afecta al sitio de unión de
Sp1, modula su actividad transcripcional in vitro disminuyendo su expresión [182], por lo que su
presencia justificaría también una protección frente determinadas infecciones. De acuerdo con esto,
la presencia de isoformas de “splicing” de DC-SIGN también puede influir en el número de
moléculas expresadas en la superficie. En mucosas genitales o intestinales esta descrita la
presencia de isoformas solubles que pueden ser intracelulares o secretadas [168, 345] y que son
más abundantes que la forma prototípica [346], dando lugar a una menor avidez de DC-SIGN por
HIV-1 y, en consecuencia, una protección frente a la infección.
Por otro lado, la cantidad de moléculas de DC-SIGN expresada en la superficie celular se
correlaciona con los diferentes estados de oligomerización y se refleja en su estabilidad y avidez por
los ligandos. Así, la protección de las variantes del cuello de DC-SIGN frente a la infección por HIV-1
estaría también justificada porque el menor número de repeticiones en el cuello disminuye la
129
Discusión
formación y la estabilidad de tetrámeros [171, 330]. A su vez, estas variantes alterarían la posición
del CRD respecto la superficie celular, afectando a su accesibilidad y unión de patógenos [347, 348].
La señalización de DC-SIGN modula la respuesta inmune en función del patógeno involucrado
[207]. En el caso de HIV-1, la señalización de DC-SIGN conlleva la trans-infección del virus a células
T, lo que promueve infección del hospedador siendo beneficioso para el virus [212]. En este sentido,
existen estudios in vitro que dicen que HIV-1 y otros patógenos utilizan DC-SIGN para escapar de la
vigilancia inmunológica y promover su supervivencia [349, 350]. Por ello, el posible papel protector
de las variantes de DC-SIGN quedaría reflejado en la disminución de la unión e internalización del
virus. Por otro lado, la expresión reducida de DC-SIGN por la presencia de polimorfismos en DC y
macrófagos podría tener efectos perjudiciales en la eliminación de patógenos por la disminuida
capacidad de presentación de antígeno de estas células [171]. Así, un estudio reciente en ratones
transgénicos que expresan DC-SIGN humano exhiben un reducido daño tisular y una prolongada
supervivencia [351]. Por tanto, la presencia de polimorfismos de DC-SIGN también puede tener un
efecto indirecto en la modulación de respuestas inmunológicas frente a la infección por diferentes
patógenos.
Identificación epítopos en la molécula de DC-SIGN.
En un segundo estudio hemos empleado las construcciones de DC-SIGN generadas a partir de
las variantes de “splicing” y polimorfismos identificados en MDDC, así como los mutantes sin CRD y
con distinto número de repeticiones en el cuello de la molécula, para identificar siete epítopos
independientes en la molécula de DC-SIGN y confirmar algunas de las observaciones a las que
llegamos en el trabajo anterior. Para ello, empleamos anticuerpos monoclonales frente a DC-SIGN
que son capaces de inhibir trans-infección de HIV-1 por células Raji-DC-SIGN a linfocitos T [151]. Al
igual que ocurría con la unión a ligandos de DC-SIGN, el CRD es reconocido por los anticuerpos
independientemente del tamaño del cuello, y el reconocimiento de la molécula por los anticuerpos
dirigidos frente al cuello es dependiente de su tamaño, siendo necesaria la presencia de al menos
dos dominios para su reconocimiento. Aunque según nuestro anterior estudio el estado de
multimerización de DC-SIGN no predice su capacidad de unión a ligandos o patógenos, los
anticuerpos frente al CRD reconocen mejor las formas de DC-SIGN que se expresan en la
membrana como monómeros (1d) que como multímeros (2d), debido posiblemente a la disposición
espacial de los epítopos que reconocen. Por otro lado y de acuerdo con su capacidad de unión de
ligandos, las variantes polimórficas de DC-SIGN son reconocidas por igual por los anticuerpos
dirigidos frente al cuello y el CRD.
130
Discusión
Respecto a la capacidad de los anticuerpos de inhibir la unión de DC-SIGN a ligandos o
patógenos, los anticuerpos frente al CRD la bloquean total o parcialmente, mientras que los
anticuerpos frente al cuello lo hacen en menor extensión o incluso no lo consiguen. La diferencia en
la capacidad de inhibición por parte de los anticuerpos frente al CRD puede ser debida al epítopo
que reconocen y bloquean en cada caso. En este sentido, existen aminoácidos en el CRD de DCSIGN claves en la unión de ligandos (Glu347, Asn349, Glu354 y Asn365), ya que interactúan con el Ca2+
dictando el reconocimiento específico de carbohidratos, y cuya mutación conlleva a la pérdida total
de capacidad de unión de ligandos [352]. Mientras, la modificación de otros residuos como Val351,
sólo afecta a la interacción con determinados ligandos sin afectar a la unión de otros, lo que indica
que los ligandos de DC-SIGN pueden tener diferentes, pero solapados, sitios de unión [162]. Por
otro lado, el bloqueo funcional que producen los anticuerpos frente al cuello puede deberse a su
habilidad de modificar el estado de multimerización de la molécula, que altera su avidez por
ligandos, o bien a su capacidad de inducir su internalización, disminuyendo así el número de
moléculas en superficie.
En conclusión, la identificación de epítopos independientes en la molécula de DC-SIGN podría
facilitar el diseño de reactivos que modulen parte de las funciones de las células que expresan este
receptor. De este modo, se podría alterar el estado de multimerización de DC-SIGN en membrana,
inducir su internalización o bloquear su unión a patógenos de manera independiente. Por ejemplo,
en este estudio describimos anticuerpos capaces de potenciar la internalización de DC-SIGN en
MDDC, lo que generaría el desarrollo de una respuesta inmunológica, sin bloquear su capacidad de
unión. Debido a que la señalización de DC-SIGN depende del ligando que involucrado [207], el uso
de reactivos específicos frente a diferentes epítopos podrían ser una herramienta útil para el estudio
de las vías de señalización de la lectina, o incluso para modular la polarización de linfocitos T por
DC.
131
Conclusiones
Conclusiones
1. El receptor de folato β es un marcador de macrófagos anti-inflamatorios/M2 in vitro y de
macrófagos asociados a tumores (TAM), en los que media la captación de folato.
2. La expresión del receptor de folato β se induce por factores presentes en el microambiente
tumoral como M-CSF o IL-10, y citoquinas promotoras de la activación alternativa de macrófagos
como IL-4 o IL-13, y es inhibida por estímulos inflamatorios como LPS o IFNγ.
3. La activina A es secretada por macrófagos pro-inflamatorios/M1 y condiciona la adquisición
de los marcadores y las funciones efectoras características de los macrófagos M2, lo que sugiere su
implicación en la progresión tumoral.
4. La multimerización del receptor de patógenos DC-SIGN en la membrana celular depende de
la región del cuello y el dominio de reconocimiento de carbohidratos, y requiere la presencia de al
menos dos dominios en la región del cuello.
5. Las variantes polimórficas de DC-SIGN se expresan en la membrana de células dendríticas
derivadas de monocitos, y mantienen la capacidad de multimerización y unión de ligandos.
6. Al menos se pueden identificar siete epítopos independientes en la molécula de DC-SIGN,
tres de ellos localizados en la región del cuello y cuatro en el dominio de reconocimiento de
carbohidratos.
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Anexo
Anexo
Durante la realización de esta Tesis Doctoral he participado en diferentes proyectos que
han dado lugar a las siguientes publicaciones:
1.- Caparrós E, Muñoz P, Sierra-Filardi E, Serrano-Gómez D, Puig-Kröger A, RodríguezFernández JL, Mellado M, Sancho J, Zubiaur M, Corbí AL. DC-SIGN ligation on dendritic cells results
in ERK and PI3K activation and modulates cytokine production. Blood, 2006 May 15;107(10):39508.
2.- Núñez C, Rueda B, Martínez A, Malvenda C, Polanco I, López-Nevot MA, Ortega E, SierraFilardi E, Gómez de la Concha E, Urcelay E, Martín J. A functional variant in the CD209 promoter is
associated with DQ2-negative celiac disease in the Spanish population. World J Gastroenterol,
2006 Jul 21;12(27):4397-400.
3.- Puig-Kröger A, Domínguez-Soto A, Martínez-Muñoz L, Serrano-Gómez D, Lopez-Bravo M,
Sierra-Filardi E, Fernández-Ruiz E, Ruiz-Velasco N, Ardavín C, Groner Y, Tandon N, Corbí AL,
Vega MA. RUNX3 negatively regulates CD36 expression in myeloid cell lines. J Immunol, 2006 Aug
15;177(4):2107-14.
4.- Gómez LM, Anaya JM, Sierra-Filardi E, Cadena J, Corbí A, Martín J. Analysis of DC-SIGN
(CD209) functional variants in patients with tuberculosis. Hum Immunol, 2006 Oct;67(10):808-11.
5.- Serrano-Gómez D, Martínez-Nuñez RT, Sierra-Filardi E, Izquierdo N, Colmenares M, Pla J,
Rivas L, Martinez-Picado J, Jimenez-Barbero J, Alonso-Lebrero JL, González S, Corbí AL. AM3
modulates dendritic cell pathogen recognition capabilities by targeting DC-SIGN. Antimicrob
Agents Chemother, 2007 Jul;51(7):2313-23.
6.- Sierra-Filardi E, Serrano-Gómez D, Martínez-Nuñez RT, Caparrós E, Delgado R, MuñozFernández MA, Abad MA, Jimenez-Barbero J, Leal M, Corbí AL. Structural requirements for
multimerization of the pathogen receptor DC-SIGN (CD209) on the cell surface. J Biol Chem, 2008
Feb 15;283(7):3889-903.
7.- Fernández de Palencia P, Werning ML, Sierra-Filardi E, Dueñas MT, Irastorza A, Corbí AL,
López P. Probiotic properties of the 2-substituted (1,3)-β-D-glucan producing Pedioccus parvulus 2.6.
Appl Environ Microbiol, 2009 Jul;75(14):4887-91.
8.- Sierra-Filardi E, Puig-Kröger A, Domínguez-Soto A, Samaniego R, Corcuera MT, LópezAguado F, Ratnam M, Sánchez-Mateos P, Corbí AL. Folate receptor beta is expressed by tumorassociated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages.
Cancer Res, 2009 Dec 15;69(24):9395-403.
159
Anexo
9.- Sierra-Filardi E, Estecha A, Samaniego R, Fernández-Ruiz E, Colmenares M, SánchezMateos P, Steinman RM, Granelli-Piperno A, Corbí AL. Epitope mapping on the dendritic cell-specific
ICAM3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor. Mol Immunol, 2010
Jan;47(4):840-848.
10.- Martin-Gayo E, Sierra-Filardi E, Corbí AL, Toribio ML. Plasmacytoid dendritic cells resident
in human thymus drive natural Treg cell development. Blood, en prensa, 2010.
11.- Sierra-Filardi E, Vega MA, Sánchez-Mateos P, Puig-Kröger A, Corbí AL. Heme
Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10
release. Immunobiology, en prensa, 2010.
12.- Garai-Ibabe G, Dueñas MT, Irastorza A, Sierra-Filardi E, Werning ML, López P, Corbí AL,
Fernández de Palencia P. Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus
suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods.
Bioresour Technol, en prensa, 2010.
13.- Domínguez-Soto A, Sierra-Filardi E, Puig-Kröger A, Blanca Pérez-Maceda B, GómezAguado F, Corcuera MT, Sánchez-Mateos P, Corbí AL. M-CSF promotes DC-SIGN expression in
M2-polarized and Tumor-Associated Macrophages. J Immunol, en revisión, 2010.
14.- Sierra-Filardi E, Puig-Kröger A, Sánchez-Mateos P, Vega MA, Corbí AL. Activin prevents
the acquisition of M2/anti-inflamatoy markers and skews the macrophage cytikine profile.
Manuscrito en preparación.
160