By
From the * Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut
06520-8011; Section of Rheumatology, Department of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut 06520-9031; and § Howard Hughes Medical Institute, Yale
University School of Medicine, New Haven, Connecticut 06520-8011
Interleukin (IL)-4 is the most potent factor that causes naive CD4+ T cells to differentiate to
the T helper cell (Th) 2 phenotype, while IL-12 and interferon trigger the differentiation of
Th1 cells. However, the source of the initial polarizing IL-4 remains unclear. Here, we show
that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells
to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells. These results
show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting
cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.
In response to pathogens, naive CD4+ T cells differentiate into effector Th1 and Th2 cells. Th1 cells produce
IL-2, IFN- If cytokines are indeed the driving force behind CD4+ T
cell differentiation, where does the initial polarizing cytokine come from? Several findings suggest that during the
initiation of a Th1 response, IL-12 is produced particularly
by macrophages in response to certain microbial antigens,
while NK cells are the main source of IFN- In an attempt to find potential cytokines that, like IL-12,
could be produced by classical APCs and could trigger the
initial IL-4 production by CD4+ T cells, we analyzed the
modulatory effects of IL-6, a cytokine involved in different
aspects of the immune response and acute phase response
(for review see references 14 and 15). Here we show that
IL-6 is able to initiate the polarization of naive CD4+ T cells
to effector Th2 cells by inducing the production of endogenous IL-4. In addition, IL-6 also antagonizes the IL-12-
mediated differentiation of Th1 cells. We postulate that IL-6
is a key factor in the choice between a Th1 or Th2 immune response.
Cell Preparation and Reagents.
Total CD4+ T cells were isolated from spleen and lymph nodes from either wild-type B10.BR,
wild-type C57BL/6J, or C57BL/6J-backcrossed IL-6-deficient
mice by negative selection as previously described (16). The naive
population (CD4+CD45RBhighCD44low) was purified from total
CD4+ T cells obtained from cytochrome (Cyt)1 c TCR transgenic mice by staining with a red613-conjugated anti-CD4 mAb, a
biotinylated anti-CD44 mAb, FITC-conjugated anti-CD45RB
mAb, and PE-conjugated streptavidin (PharMingen, San Diego,
CA) and cell sorting by using a FACSPLUS®. Mitomycin C-treated
(50 µg/ml) syngeneic splenocytes were used as source of APCs.
, and TNF-
, which are involved in cellmediated inflammatory reactions. Th2 cells secrete mainly
IL-4, IL-5, IL-6, IL-10, and IL-13, which mediate B cell
activation, antibody production, and the regulation of Th1
responses (for review see reference 1). In general, a Th1 response helps eradicate intracellular microorganisms, whereas a Th2 response can control extracellular pathogens. Development of an inappropriate response can lead to ineffective
immunity and may even be pathogenic. Thus, the factors
that regulate the polarization to either a Th1 or Th2 immune response are critical, but remain unclear. The dose of
antigen and the type of APC, and/or the co-stimulatory
pathways (2), have been postulated to be some of the
polarizing factors. However, the most effective inducer of
CD4+ T cell differentiation appears to be the local cytokine
environment. It is clear that the cytokine IL-12 directs differentiation to a Th1 phenotype (7, 8), while IL-4 can drive
differentiation to a Th2 phenotype (9, 10).
in response to
IL-12 (7, 11). In the case of a Th2 response, the initial
source of IL-4 is less clear, since none of the classical APCs
make IL-4. Some non-APCs, such as mast cells and basophils, can produce IL-4, but these cells are not abundant in
the lymphoid organs where T cell priming occurs (12).
Recently, it has been shown that IL-4 is also produced by a
minor subpopulation of T cells, the CD3+CD4+NK1.1+
cells, which may therefore have a role (13). However, the
production of IL-4 by mast cells and basophils is a late
event and it is not yet clear how the CD4+NK1.1+ cells
become activated. An alternative possibility, however, is
that other cytokines may induce the initial production of
IL-4 by the CD4+ T cells; after this initial stimulus, the secreted IL-4 could act in an autocrine fashion, upregulating
IL-4 production and inhibiting IFN-
production, thereby
polarizing the differentiation of Th2 cells.
Cell Surface Staining.
Expression of IL-6R was analyzed by
FACS® by double staining with a FITC-conjugated anti-CD4
mAb and a biotinylated anti-IL-6R
mAb (PharMingen) in
combination with PE-conjugated streptavidin.
Competitive Reverse Transcriptase-PCR.
Total RNA was extracted as described (17) from 2 × 105 cells mixed with 104 Raji
cells which were added at harvest as an internal control, and the
amount of human MHC class II HLA-DR cDNA from Raji cells was used as an internal standard. In brief, after dilution (1/2) of reverse transcription reaction mixture, 5 µl was assayed for levels of
DR cDNA by PCR using DR-specific primers in the presence of
DR competitor construct (50 pg) to confirm the efficiency of RNA
extraction and reverse transcriptase (RT)-PCR procedure in each
group. IL-4 transcript levels in the 5 µl of diluted (1/2) reverse transcription reaction mixture was semi-quantitated using the competitor
(167 fg) in the presence of specific primers as described (18). DR
primers used were (5-CGAGTTCTATACTGAATCCTG, and
3
-GTTCTGCTGCATTGCTTTTGC). Competitor amounts
shown (competition cDNA, fg or pg) are corrected to represent the
amount of IL-4 or DR gene and not as the total plasmid amount. A
multiple cytokine-containing competitor construct was a gift from
R.M. Locksley (University of California, San Francisco, CA).
Cytokine Production.
ELISA assays were performed using purified anti-IL-4, anti-IL-12, and anti-IL-6 mAbs (3 µg/ml) as
primary antibodies, the corresponding biotinylated anti-IL-4,
anti-IL-12 and anti-IL-6 mAbs (1 µg/ml; PharMingen), and
horseradish peroxidase-conjugated avidin D (2.5 µg/ml) (Vector
Labs., Inc., Burlingame, CA), the TMB microwell peroxidase
substrate and stop solution (Kirkegaard & Perry Labs., Inc., Gaithersburg, MD), using the recommended protocol (PharMingen). Recombinant IL-4 (DNAX) and IFN- (GIBCO BRL, Gaithersburg, MD) were used as standards. The specific activity of the IL-4
and the IFN-
that were used as standards for the ELISA assays
were 108 U/mg for IL-4 and 107 U/mg for IFN-
.
IL-6 is produced by a wide spectrum of cells including
fibroblasts, endothelial cells, neuronal cells, macrophages,
mast cells, tumor cell lines, and CD4+ Th2 cells, but from
an immunological point of view, APCs represent the major
source of IL-6 (14, 19). To determine the potential role of
IL-6 in differentiation of naive CD4+ T cells into effector
Th1 and Th2 cells, we first analyzed the effect of exogenous IL-6. Total mouse CD4+ T cells were isolated (16)
and stimulated to differentiate with Con A with or without
IL-4 (Th2) or IL-12 (Th1) in the presence or absence of
exogenous IL-6. After 4 d, the effector Th1 or Th2 cells
were exhaustively washed, counted, and equal number of
cells were restimulated with Con A for 24 h before harvesting the supernatant which was then analyzed for cytokine
production. Interestingly, even in the absence of any polarizing cytokine, IL-6 directed the differentiation of the
CD4+ cells to a Th2 phenotype, since the cells produce
high amounts of IL-4, but not IFN- (Fig. 1 A). IL-6 did
not modify the differentiation of the Th2 cells directed by
IL-4. However, differentiation into Th1 cells by IL-12,
was impaired in the presence of IL-6. Thus, Th1 cells differentiated with Con A and IL-12 in the presence of IL-6,
produced less IFN-
, and more IL-4 (Fig. 1 A). IL-6, like
IL-2, has been described to be a growth factor for a number of cells (14, 15). However, only IL-6, but not IL-2, was
able to modify the polarization of the CD4+ cells to Th2
phenotype (Fig. 1 A), indicating that IL-6 is involved in
differentiation rather than growth of T cells. To eliminate the possibility that IL-6 could favor the expansion or IL-4
secretion of the CD4+ memory subpopulation, which has
been described to display a Th2 phenotype (20), we analyzed the role of IL-6 in the differentiation of purified naive CD4+ T cells. Thus, we used CD4+ T cells from TCR
transgenic mice (21), which express the
and
chain of
the TCR that recognizes a pigeon Cyt c peptide; before
further purification, 90-95% of the CD4+ T cells express
the naive phenotype. Total CD4+ T cells were stained with
anti-CD44 and anti-CD45RB mAbs and the naive CD4+
CD44lowCD45RBhigh population was isolated by cell sorting and activated with the same polyclonal stimulus, Con A,
in the presence or absence of different cytokines. We observed that IL-6, in the absence of any other cytokine, was
able to promote the differentiation of naive CD4+ cells to
IL-4-producing cells (Fig. 1 B). In addition, we also analyzed the effect of IL-6 in the differentiation of naive
CD4+ T cells stimulated by specific antigen. The presence
of IL-6 during the activation with Cyt c peptide drove differentiation of naive CD4+ T cells into IL-4-producing effector Th2 cells as well or better than IL-4 (Fig. 1 C).
Therefore, the modulatory effect of IL-6 in T cell differentiation is not a consequence of an expansion of the memory
subpopulation.
Other cytokines have also been described to indirectly
modulate the polarization towards Th1 and Th2. IL-10, a
Th2 cytokine, promotes Th2 and inhibits Th1 cells, their
cytokines, and related immune phenomena. Although the
mechanism is not clear yet, several lines of evidence indicate that IL-10 reduces the Th1 response by an inhibitory
effect on the IL-12 expression by APCs such as macrophages
(22). In contrast, the immunoregulatory role of IL-6 in
the polarization of Th1 and Th2 is not an indirect effect on
the APCs. Thus, IL-6 inhibited IFN- production and
stimulated IL-4 synthesis by Th1 cells that have been differentiated in the presence of exogenous IL-12 (Fig. 1, A
and B), eliminating the possibility of inhibiting the production of IL-12 by APCs. In addition, IL-6 did not affect the
expression on the APCs of co-stimulatory molecules such
as B7.1 and B7.1, which have also been involved in the differentiation of Th1 and Th2 cells (5, 6; data not shown). To further prove that the effect of IL-6 was on T cells directly, rather than APCs, we differentiated CD4+ T cells
with immobilized anti-CD3 mAb plus anti-CD28 mAb in
the complete absence of APCs, and then restimulated these
cells with anti-CD3 mAb alone. The presence of IL-6 (or
IL-4 as a control) during the first culture resulted in an increase of IL-4 production and a dramatic reduction of IFN-
production (Fig. 1 D) by the cells that were elicited, showing that IL-6 directly favors the polarization of naive CD4+
T cells to Th2 cells via the T cell.
IL-4 is the most effective differentiation factor for Th2
cells; it acts by promoting the secretion of more IL-4 and
inhibiting the production of IFN- by T cells (9, 10). It
was therefore possible that IL-6 could directly upregulate
the synthesis of IL-4 by T cells and, consequently, that the
IL-6 effect was mediated through IL-4, which would be
responsible for the suppression of Th1 differentiation, while
favoring a Th2 response. To address this hypothesis, CD4+
T cells were differentiated with Con A and IL-6 in the
presence or absence of neutralizing anti-IL-4 mAb, and after 4 d, the cells were washed and restimulated with Con A. The ability of IL-6 to polarize CD4+ T cells toward the
Th2 phenotype was blocked by anti-IL-4 mAbs, since the
cells were unable to produce IL-4 upon restimulation (Fig.
1 E). These results indicated that the differentiation of Th2
cells by IL-6 is dependent on the endogenous production
of IL-4. It was therefore possible that IL-6 may trigger the
Th2 pathway by inducing small amounts of IL-4, which in
turn would act as an autocrine differentiation factor for the
Th2 cells.
As mentioned above, APCs represent the major source
of IL-6 early in the immune response. To examine the
physiological role of IL-6 in T cell differentiation, we first
measured the production of IL-6 during the differentiation
of Th1 and Th2 cells. CD4+ T cells were stimulated with
Con A and IL-4 or IL-12 in the presence of APCs, and supernatants were harvested after different periods of time to
measure IL-6 secretion. After 2 d of culture, identical levels
of IL-6 were detected in the IL-4 and IL-12 cultures (Fig. 2 A). However, while the IL-6 level in the presence of
IL-12 was sustained during the course of T cell differentiation, it decayed dramatically during the differentiation of
Th2 cells in the presence of IL-4. No IL-6 was detected after restimulation of either Th1 or Th2 cells with Con A in
the absence of APCs (data not shown), suggesting that the
IL-6 that we detected during the first stimulation was secreted mainly by the APCs. The difference in the kinetics of IL-6 synthesis during the differentiation of Th1 and Th2
could, therefore, be due either to upregulation of IL-6 on
the APCs in the presence of IL-12, or greater IL-6 consumption by Th2 cells than by Th1 cells. To test this, we
examined IL-6 production during the differentiation of
Th1 and Th2 in the presence of an anti-IL-6 receptor (IL-6R)
mAb, to block IL-6 consumption. In the presence of anti-
IL-6R mAb IL-6 did not diminish during the differentiation of Th2 cells (Fig. 2 B). These data indicate that the IL-6
produced by APCs was consumed during the differentiation of Th2 cells, but not during differentiation of Th1
cells, supporting the idea that IL-6 plays a role during Th2
polarization by acting directly on T cells. IL-6R is a heterodimer of the signal transducer gp130 (which is also a
component of the IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, and onconstatin M receptors) and the
specific IL-6R chain (25). IL-6R has been found in both unstimulated CD4+ and CD8+ T cell subsets and its
expression is downregulated upon activation (31). Differential expression of the IL-6R
chain during Th1 or Th2
differentiation could explain the higher IL-6 consumption by the Th2 cells. We also analyzed, therefore, the expression of IL-6R
during the differentiation of CD4+ T cells
in effector Th1 or Th2 cells. However, the expression of
cell surface IL-6R
was regulated similarly during the differentiation of Th1 or Th2 cells in the presence of either
IL-4 or -12 (Fig. 2 C). Low levels of IL-6R
were present
on unstimulated CD4+ T cells, and downmodulation occurred after the first day of stimulation, remaining at almost
undetectable levels during the differentiation of both Th1
and Th2 cells. These results indicated that the effects of IL-6
in the differentiation of Th2 cells were not due to a differential distribution of the IL-6R
.
If IL-6 was required for Th2 cell differentiation in vitro,
it would follow that T cells from IL-6/
mice should be
incapable of developing Th2 effector cells. The experiments described below show that this appears to be true. In
correlation with the previous characterization of IL-6
/
mice (32, 33), analysis of cellular populations in the spleen and thymus did not show any differences between wildtype (WT) and IL-6
/
mice (data not shown), and IL-6
/
mice developed normally. We therefore used splenocytes
from IL-6
/
mice as APCs to further establish that the
production of endogenous IL-6 by the APC plays a critical
role in the polarization of the CD4+ T cells to effector Th2
cells. We purified CD4+ T cells from WT mice and stimulated them for 4 d with Con A in the presence of APCs
from WT mice or IL-6
/
mice in the absence of any
added exogenous cytokines. Restimulation with Con A resulted in significantly less IL-4 production in cells differentiated in the presence of IL-6
/
than in WT APCs (Fig.
3 A). Although the production of IFN-
under these conditions (absence of exogenous IL-12) was low, the levels of
IFN-
produced by CD4 cells differentiated in the presence of IL-6
/
APCs were higher than those produced
by cells stimulated in the presence of WT APCs (data not
shown).
In vivo experiments have showed an impaired immune
response in IL-6/
mice. These mice fail to control infection with Listeria monocytogenes and have a diminished T cell-
dependent body response against vesicular stomatitis virus
(32). In addition, CD4+ cells from WT mice infected
with Candida albicans express IL-4, but reduced IgE and IL-4
mRNA was observed in IL-6
/
mice (35). Nevertheless,
due to the numerous effects of IL-6 in vivo, it is difficult to
study by which mechanism IL-6 affects the immunopathology of infection and whether this is mediated by a modification in T cell differentiation. We examined, therefore, the in vitro differentiation of CD4+ T cells from IL-6-deficient mice. IL-6
/
CD4+ T cells stimulated with Con A
in the presence of WT APCs, which can provide the IL-6
required, produced IL-4, but this IL-4 production was impaired when the IL-6 pathway was blocked by the addition of anti-IL-6 mAbs (Fig. 3 B). Most significantly, however,
was that in the complete absence of IL-6, when IL-6
/
CD4+ T cells were stimulated in the presence of IL-6
/
APCs, no in vitro Th2 response was obtained (Fig. 3 B).
The difference between complete absence of IL-4 production when IL-6
/
APCs were used with IL-6
/
T cells
compared with IL-6
/
APCs and WT T cells may have
been the result of either IL-6 production from contaminating WT APCs in the T cells or a contribution of IL-6 from
the WT T cells. Moreover, high IL-4 production was restored in cells that were differentiated in the presence of
IL-6
/
APCs together with an exogenous source of IL-6
(Fig. 3 B). Similar levels of IL-4 were detected when
CD4+ T cells were differentiated in the presence of IL-4,
and no significant additional increase was observed when
both IL-4 and IL-6 were present during the differentiation,
indicating again that both IL-4 and IL-6 were using the
same pathway.
To demonstrate whether the upregulation of IL-4 production by IL-6 was due to an induction of IL-4 gene expression rather than a posttranslational mechanism, e.g., increasing IL-4 secretion, we examined the levels of IL-4
mRNA. Naive CD4+ T cells were isolated from Cyt c
TCR transgenic mice (see above) and stimulated with specific Cyt c peptide in the absence or presence of IL-6 or
IL-4. After 4 d, cells were washed and restimulated with
Cyt c peptide, and total RNA was isolated 20 h later. The expression of the IL-4 gene was determined by competitive
RT-PCR of IL-4-specific transcripts (18). The presence of
exogenous IL-6 or IL-4 during the differentiation (Th2
cells) resulted in the induction of high levels of IL-4
mRNA upon antigen restimulation (Fig. 3 C). Quantitation of IL-4 transcripts in cells differentiated in the presence
of IL-6 or IL-4 were 100-fold higher than in the absence of
cytokines (data not shown). Together, these data indicate that IL-6 causes the differentiation of Th2 cells by upregulating IL-4 gene expression. The transcriptional regulation
of the IL-4 gene has not yet been characterized as extensively as the regulation of the IL-2 gene. Several positive
and negative regulatory elements in the 5 flanking region of
the IL-4 gene have been described (36). Among five
different nuclear factor of activated T cells (NFAT) binding
elements, one located between
90 and
60 is important for the activation of the IL-4 gene expression. Like the distal NFAT site in the IL-2 promoter (40), this element
binds NFAT (either NFATp or NFATc) in association
with AP-1 components (37, 43). In addition, recently three
functional binding sites for the C/EBP family of transcriptional factors have been identified in the IL-4 promoter (44),
which might be regulated by IL-6 since members of this
family (NF-IL6) have already been described as having been
activated by IL-6 in other systems (45). Whether IL-6
acts through these sites will be the subject of future studies.
Both in vivo and in vitro experiments support the idea
that, although other factors can play immunomodulatory
roles, IL-4 and IL-12 are both necessary and sufficient for
the polarization of Th1 and Th2 during the response to different pathogens. Considerable interest in the initial source
of the IL-4 that drives Th2 cell differentiation has been expressed and recent attention has focused on mast cells or
NK1.1+ CD4 T cells. We propose here that IL-6 is one of
the stimuli in the initial production of IL-4 which provides
a new mechanism to initiate Th2 CD4+ T cell differentiation. The levels of IL-6 that we used in these experiments
are relatively high, just as the levels of IL-4 that also must
be used to direct Th2 differentiation in vitro are (500- 1,000 U/ml IL-4). Presumably, for both IL-4 and IL-6
produced endogenously, lower levels produced in a local
microenvironment suffice. Consistent with this idea, although the IL-6 effect is mediated through IL-4 (since it is
blocked by anti-IL-4), the levels of IL-4 that can be detected are very low, presumably because of local consumption, but are sufficient to direct Th2 cell differentiation. Our data suggest a model (Fig. 4) for Th2 differentiation
that is strikingly similar to the mechanism used for Th1 differentiation. In both cases, the source of the polarizing cytokine is the APC, IL-12 in the case of Th1 cells and IL-6
in the case of Th2 cells. Although APCs are the major
source of IL-6 in vitro, other cell types that produce IL-6,
such as fibroblasts, endothelial cells, keratinocytes or others,
could also contribute to the overall in vivo IL-6 production. In our in vitro model, activation of the APC is proposed to result in the secretion of IL-6 which, in combination with the antigen, will participate in the induction of
IL-4 gene expression in naive CD4+ T cells. In the second
step, the low levels of IL-4 secreted by the T cells is sufficient to induce upregulation of the IL-4 and IL-4 receptor
genes in an autocrine manner, while it inhibits the expression of the IFN- gene. Cells are therefore subsequently polarized to a Th2 phenotype using their own source of IL-4.
Address correspondence to Dr. Richard A. Flavell, Section of Immunobiology, Yale University School of Medicine, PO Box 208011, New Haven, CT 06520-8011. M. Rincón's present address is the University of Vermont, Department of Medicine, Given Medical Bldg., Burlington, VT.
Received for publication 30 October 1996
This work was supported by National Institutes of Health grants CA65861 and 1-P01-AI30548. R.A. Flavell is an investigator and T. Nakamura was an associate of the Howard Hughes Medical Institute. E. Fikrig is a Pew Scholar.We thank S. Samanta for technical assistance and F. Manzo for assistance with manuscript preparation. We also thank Genetics Institute for providing the IL-12 and DNAX Institute for a gift of IL-4.
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