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Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
and promote delayed-type hypersensitivity responses (2).
In contrast, Th cells that are activated in the presence of IL-4
differentiate into Th2 cells, produce mainly IL-4 and IL-5, and promote
humoral and allergic responses (2). Mutually exclusive use of the IL-12 or IL-4 signal transduction pathway by Th1 and Th2 cells, respectively, has spurred significant interest in understanding how regulation of these pathways are coupled to the differentiation process. IL-12 or IL-4 signaling is mediated by STAT4 and STAT6, respectively (3). Because transduction of cytokine signals through STAT proteins generally results in transcriptional activation of STAT-inducible genes, it is tacitly assumed that Th cells differentiate into Th1 or Th2 phenotype because of differential activation of genes that drives them to their respective developmental state. However, recent studies showing that cytokine signaling is under negative feedback regulation by a multimember family of proteins called suppressors of cytokine signaling (SOCS)2 have raised the possibility that differentiation toward the Th1 or Th2 pathway may be mediated in part by the selective repression of IL-12/STAT4 or IL-4/STAT6-signaling pathways, respectively (4).
The SOCS family of proteins is at present composed of eight members
characterized by the presence of a Src homology 2 domain and a
C-terminal conserved domain called the SOCS box (5, 6).
Evidence to date suggests that mRNA transcripts encoding SOCS are
selectively up-regulated in response to several cytokines including
IFN-, IL-2, IL-3, IL-4, IL-6, IL-12, IL-13, leukemia-inhibitory
factor, stem cell factor, GM-CSF, growth factor, leptin, and
erythropoietin (7). The SOCS proteins translated from the
corresponding mRNA transcripts inhibit requisite cytokine-induced
signaling pathways by classical feedback circuits (5, 6, 7).
The inhibitory effects derive from direct interaction of SOCS Src
homology 2 domains with cytokine receptors and/or Janus kinases (JAK)
leading to the recruitment of SOCS proteins to the signaling complex,
their inhibition of the binding of STATs to tyrosine-phosphorylated
cytokine receptors, and suppression of the catalytic activities of JAKs
(8, 9, 10, 11). Gene targeting has been used to generate
SOCS1-/-, SOCS2-/-, and
SOCS3-/- mice (12, 13, 14, 15, 16). Deletion
of SOCS1 or SOCS3 is lethal in mice; SOCS3 null mice die in utero and
mice lacking SOCS1 die within 3 wk after birth. Analysis of the
physiological functions of these SOCS proteins reveals requirement of
SOCS3 in fetal liver erythropoiesis and placental development, whereas
SOCS2 is important in postnatal growth. In contrast, SOCS1 is crucial
in regulation of IFN- pathways. SOCS1-/-
mice have defective thymocyte development, and overexpression of SOCS1
impairs pre-TCR-induced thymocyte proliferation, suggesting that
inhibition of cytokine signaling has important influence on T cell
differentiation (17, 18).
In this study, we have characterized the repertoires of SOCS mRNAs and proteins expressed in naive and differentiated Th cells to examine the possibility that mutually exclusive patterns of cytokine expression by Th1 and Th2 cells derive from differential expression of SOCS proteins. We show here that Th1 and Th2 cells preferentially express distinct SOCS proteins that may play a role in the selective inhibition of STAT4/IL-12- and STAT6/IL-4-signaling pathways of Th cells. We discuss the implications of our findings on Th cell lineage commitment and/or maintenance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes expressing HEL-specific TCR were isolated from
spleens and lymph nodes of HEL-specific TCR-transgenic (Tg) mice
designated "3A9" (a generous gift from M. Davis, Stanford
University, Stanford, CA) as described (19). Briefly,
CD4+ cells were isolated and purified (>97%) Th
cells were cultured at 2.5 x 105/ml in RPMI
1640 supplemented with 50 µM 2-ME, antibiotics, and 10% FCS
(complete medium) with 10x irradiated syngeneic splenocytes (as APC)
in the presence of 2 µg/ml HEL (Sigma-Aldrich, St. Louis, MO), 10
ng/ml IL-12 (Sigma-Aldrich), and 10 µg/ml anti-IL-4 Ab (BD
PharMingen, San Diego, CA) for Th1 or 0.2 µg/ml HEL, 10 ng/ml IL-4
(BD PharMingen), 10 µg/ml anti-IFN- Ab (BD PharMingen), and 10
µg/ml anti-IL-12 Ab (BD PharMingen) for Th2. After 4 days,
cultured cells were expanded with 40 IU/ml IL-2 (Chiron, Emeryville,
CA) for 4 days, and these cells are designated as resting Th1 or Th2
cells. Some cells were restimulated at 2.5 x
105/ml with 10x irradiated syngeneic APC in the
presence of either 2 µg/ml HEL, 40 IU/ml IL-2, and 10 ng/ml IL-12 for
Th1 or 0.2 µg/ml HEL, 40 IU/ml IL-2, and 10 ng/ml IL-4 for Th2. Three
days later, cells were harvested, washed, resuspended in RPMI- 1640,
and designated as activated Th1 or Th2 cells. The cells designated as
"Th" were obtained by incubating purified
CD4+ cells, with HEL in the absence of the
polarizing cytokines or their Abs.
For signal transduction studies, Th1 and Th2 cell lines were established and maintained in IL-2 (40 IU/ml) under polarization conditions described above. However, the HEL protein and APC were substituted by anti-CD3 (0.1 µg/ml) and anti-CD28 (0.5 µg/ml) Abs (BD PharMingen). Activation of the STAT4 or STAT6 signal transduction pathway was analyzed in Th1 and Th2 cells cultured for 2 h under starvation conditions (1% BSA, RPMI 1640). The cells were then treated with IL-12 (10 ng/ml) or IL-4 (10 ng/ml) for 15 or 30 min.
Isolation of naive CD4+ T cells and FACS analysis
CD4+ T lymphocytes from spleens and lymph nodes of 3A9 mice were purified as described above. The cells were stained with PE-Cy5-CD4 or FITC-CD62 ligand (CD62L) Abs (BD PharMingen), gated, and sorted out in FACSear Plus-SE cell sorter (BD Biosciences, San Jose, CA). For FACS analysis, purified CD4+ T cells (3 x 105) in FACS buffer (1x HBSS, 1% FCS, 10 mM HEPES, 0.2% NaN3) were mixed with staining Ab and incubated at room temperature for 15 min. The cells were then washed twice with FACS buffer, fixed in 1% formaldehyde, and analyzed. mAbs used for staining are anti-CD4, CD62L, CD25, and their corresponding isotype control Abs (BD PharMingen). Analysis was performed on the BD Biosciences FACSCalibur.
Cytokine measurements
For measurement of cytokines produced by the polarized cells, Th1 or Th2 cells were cultured as described above. Supernatants were collected after the second cycle of activation and assayed for cytokine secretion by ELISA, using kits obtained from Endogen (Woburn, MA).
RNase protection assay
RNA (10 µg) was hybridized overnight with
[
-32P]UTP-radiolabeled RNA probes
transcribed in vitro from cDNA templates indicated in Fig. 1
A. Overlapping ssRNA on
hybridized dsRNAs was digested, and protected dsRNA duplexes were
fractionated on denaturing-urea gels and processed for
autoradiography.
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Northern blot analysis was performed with 20 µg RNA as
described (20). The integrity and comparability of RNA
preparations used for analysis were verified by agarose-formaldehyde
gel electrophoresis; comparable amounts of 18S and 28S rRNAs were
detected for all RNA preparations. Mouse -actin, SOCS1, SOCS2, and
SOCS3 cDNAs were used as hybridization probes. SOCS-specific cDNAs were
kindly provided by D. Hilton (Walter and Eliza Hall Institute,
Melbourne, Australia) and H. Young (National Institutes of Health,
Bethesda, MD).
Western blotting and immunoprecipitation analyses
Preparation of whole cell lysates, immunoprecipitation, and
immunodetection were performed as described (21). Briefly,
samples (40 µg/lane) were fractionated on 4–20% gradient SDS-PAGE,
and SOCS1, SOCS2, SOCS3, or -actin-specific (Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-pSTAT4- or
anti-pSTAT6-specific (Zymed Laboratories, San Francisco, CA)
polyclonal Abs were used as probes. For immunoprecipitation, 0.2 mg
whole cell extract was incubated with protein G-agarose (Pharmacia
Biotech, Piscataway, NJ), and anti-SOCS2 Ab for 1 h at 4°C
and immunoprecipitates were washed four times in lysis buffer before
electrophoresis. Preimmune serum was used in parallel as controls and
signals were detected with HRP-conjugated secondary
F(ab')2 Ab (Zymed Laboratories) using the ECL
system (Amersham, Arlington Heights, IL).
RT-PCR analysis
RNA (5 µg), SuperScript II Reverse Transcriptase (Life
Technologies, Gaithersburg, MD), and
oligo(dT)12–16 were used for first-strand
synthesis as previously described (21). Samples were
subjected to hot-start PCR in a reagent mix containing
digoxigenin-11-dUTP (Roche, Indianapolis, IN) and AmpliTaq Gold DNA
polymerase (Applied Biosystems, Foster City, CA). Primers used for PCR
amplification are as follows. For SOCS1, 5'-CTCGAGTAGGATGGTAGCACGCAA-3'
and 5'-CATCTTCACGCTGAGCGCGAAGAA-3'; SOCS2,
5'-GACCAGCTGTCTGGGACGTGTTGA-3' and
5'-GAGAGAGAAATACTTATACCTGGAAT-3'; SOCS3',
5'-TGCGCCATGGTCACCCACAGCAAGTTT-3' and
5'-GCTCCTTAAAGTGGAGCATCATACTGA-3'. For T-bet,
5'-TGCCTGCAGTGCTTCTAACA-3' and 5'-TGCCCCGCTTCCTCTCCAACCAA-3'.
For c-maf, 5'-GTGCAGCAGAGACACGTCCT-3' and
5'-CAACTAGCAAGCCCACTC-3'. For GATA-3,
GAAGGCATCCAGACCCGAAAC-3' and 5'-ACCCATGGCGGTGACCATGC-3'. For
-actin, 5'-GTGGGCCGCTCTAGGCACCAA-3' and
5'-TCTTTGCCAATAGTGATGACTTGGC-3'. Amplification was conducted for 35
cycles of 30 s each at 95°C, 60°C, and 72°C, followed by a
final 10-min extension at 72°C. Under this condition, amplification
was within the linear portion of the Taq amplification
curve. First-strand synthesis containing each mRNA sample but no
reverse transcriptase was performed to control for possible DNA
contamination of mRNAs used as target for PCR amplification; failure to
obtain RT-PCR products with any of the PCR amplimers confirmed the
absence of contaminating DNA templates. PCR-amplified fragments were
fractionated on 1.5% agarose gels. PCR products from naive cells were
transferred onto nylon membranes, and signal detection was by the
nonradioactive method as recommended for the ECL detection system
(Amersham).
Quantitative RT-PCR analysis
First-strand synthesis was performed as described above. A negative control reaction without reverse transcriptase was performed for each RNA sample. RNA samples were normalized to 18S rRNA using the Taqman Ribosomal RNA Control Reagents kit (Applied Biosystems). Real-time PCR was performed on an ABI 7700 (Applied Biosystems) or ICycler iQ Real Time PCR (Bio-Rad, Hercules, CA) Sequence Detection System with the following primers: for SOCS1, 5'-ACCTTCTTGGTGCGCGAC-3' and 5'-AAGCCATCTTCACGCTGAGC-3'; for SOCS2, 5'-GGTTGCCGGAGGAACAGTC-3' and 5'-GAGCCTCTTTTAATTTCTCTTTGGC-3'; for SOCS3, 5'-CCTTCAGCTCCAAAAGCGAG-3' and 5'-GCTCTCCTGCAGCTTGCG-3'. Hybridization probes used are: for SOCS1 (6-carboxyfluoresein (6FAM)-TCGCCAACGGAACTGCTTCTTCG-6-carboxytetramethylrhodamine (TAMRA); for SOCS2 (6FAM-CGCGTCTGGCGAAAGCCCTG-TAMRA); for SOCS3 (6FAM-CCAGCTGGTGGTGAACGCCGT-TAMRA). PCR primers and probes for GAPDH are described in the TaqMan Rodent GAPDH Control Reagents kit (Applied Biosystems). PCR parameters are as recommended for the TaqMan Universal PCR master mix kit (Applied Biosystems). Triplicate samples of 10-fold serial dilutions of SOCS1, SOCS2, or SOCS3 plasmid cDNA were assayed and used to construct the standard curves.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have shown that SOCS proteins inhibit IL-4
signaling and the cross-talk between IFN- and IL-4 signaling
pathways in hemopoietic cells (22, 23). Because these
pathways have been implicated in Th cell differentiation and lineage
commitment, we hypothesized that SOCS genes might be
differentially expressed in Th cells and may contribute to
establishment of stable Th1 or Th2 phenotype. To examine these
possibilities, we generated HEL-specific Th1 and Th2 cells from the
spleen and lymph nodes of HEL-specific TCR Tg mice and characterized
them by RPA and ELISA cytokine assays. As shown in Fig. 1
, the Th1
cells express relatively high levels of IFN- but undetectable
amounts of IL-4 or IL-5 mRNAs, whereas the Th2 cells contain relatively
high levels of IL-4 and IL-5 mRNAs. Nonpolarized Th cells produced
lower levels of all three cytokines. Adoptive transfer of the Th1 cells
into Tg mice expressing the HEL protein in the ocular lens produced
delayed-type hypersensitivity-like ocular inflammatory disease, whereas
large numbers of the Th2 cells induced eosinophilic inflammation only
in immunodeficient recipients (19), further underscoring
the fact that polarized T cells used in this study have the requisite
phenotype expected of Th1 or Th2 lineage.
SOCS1, SOCS2, and SOCS3 genes are differentially expressed in Th1 and Th2 cells
We then examined the repertoire of SOCS family members expressed
in HEL-stimulated nonpolarized (Th), Th1, and Th2 cells by Northern
blot analysis. We found significantly higher levels of SOCS1 and SOCS2
mRNA transcripts in Th1 cells than in Th2 cells (Fig. 2A). In contrast, we detected
a considerably higher level of the SOCS3 mRNA transcript in Th2 cells,
with very little SOCS3 expression in Th1 cells. Nonpolarized Th cells,
consisting of both Th1 and Th2 cells, produced low levels of all three
SOCS transcripts. These results were obtained in three separate
experiments that used independently derived Th, Th1, and Th2 cells.
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B). Conversely, we detected
SOCS3 protein in Th2 but not in Th1 cells. Although we could not detect
SOCS2 protein in either Th1 or Th2 cells by Western blot analysis (not
shown), it was detected in both cell types by immunoprecipitation,
albeit with higher amounts in Th1 cells (Fig. 2B). The low
SOCS2 protein level is surprising considering that Th1 cells express
high levels of SOCS2 mRNA transcripts. This may suggest that the SOCS2
protein is either unstable in Th1 cells or that its steady-state
concentration is regulated at the level of translation. Together, our
protein and mRNA data provide the first evidence that SOCS are
differently expressed in Th1 and Th2 cells. Quantitative PCR analysis of SOCS1, SOCS2, and SOCS3 expression in Th1 and Th2 cells
We determined the abundance of SOCS1, SOCS2, and SOCS3 in Th1 and
Th2 cells by real-time quantitative PCR assay. In Fig. 3, we show real-time PCR amplification
profiles of mouse SOCS1, SOCS2, and SOCS3 cDNAs from resting Th, Th1,
or Th2 cells. Standard curves generated from the SOCS cDNA dilution
series showed excellent linearity indicating precise, quantitative
relationship between cDNA copy number, and fluorescence signal
intensity within the dynamic range of the assay (data not shown).
Assuming that a typical mammalian cell contains 10 pg RNA, our
real-time PCR data indicate that Th1 cells contain 
1276 copies of
SOCS1 transcripts per cell, as compared with only 246 copies in a Th2
cell. A similar analysis revealed a value of 1225 and 320 copies of
SOCS2 transcripts in the Th1 and Th2 cell, respectively. In contrast,
1297 copies of SOCS3 transcripts are detected per Th2 cell and only 56
copies in Th1 cell. The relatively high levels of SOCS expression in
Th1 and Th2 cells may be sufficient to influence phenotype-specific
cytokine signaling and maintenance of the corresponding differentiated
state.
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Given the differential patterns of SOCS expression in Th1 and Th2
cells, we predicted that IL-4 signaling would be inhibited in Th1 cells
by the relatively high levels of SOCS1 and SOCS2 expression in these
cells. Conversely, we expected marked inhibition of IL-12 signaling in
Th2 cells by high constitutive expression of SOCS3. To test this
hypothesis, we established long term Th1 and Th2 cell lines as
described above. Before use in our signaling studies, we verified that
these cells do indeed retain their respective phenotypes by examining
expression patterns of transcription factors that characterize Th1 or
Th2 lineage. Consistent with published reports (2, 3), Th1
cells expressed high levels of T-bet, whereas Th2 cells expressed
relatively higher levels of GATA-3 and c-maf than did Th1
cells (Fig. 4A). Analysis of
these cells by real-time PCR verified that they retain their expected
patterns of SOCS expression, with Th1 cells expressing relatively
higher levels of SOCS1 and SOCS2, whereas Th2 cells contain higher
levels of SOCS3 (Fig. 4B). We then examined activation of
STAT6 and STAT4 after treatment of the cells with either IL-4 or IL-12
by Western blotting. In Th2 cells, IL-4 induced strong tyrosine
phosphorylation of STAT6, whereas activation of STAT6 in Th1 cells is
barely detected (Fig. 4C). We also detected activated STAT6
in untreated Th2 but not in Th1 cells, suggesting that constitutive and
inducible IL-4/STAT6 signals in Th1 cells may be inhibited in part by
high SOCS1 and SOCS2 expression. In contrast, IL-12 induced high levels
of tyrosine-phosphorylated STAT4 in Th1 but not Th2 cells, suggesting
that the high constitutive SOCS3 expression may inhibit IL-12/STAT4
pathway in these cells (Fig. 4D). Although these results
clearly show that expression of specific SOCS correlates with
activation of specific STAT signaling pathways, this does not
necessarily constitute proof that the specific SOCS members mediate the
relevant pathways in vivo.
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To determine whether differential regulation of the
SOCS is intrinsic to Th cells or derives from effects of
cytokines that polarize Th cells to the Th1 or Th2 phenotype, we
analyzed SOCS expression in naive nonpolarized
CD4+ T cells. Fresh CD4+
cells from the spleen and lymph nodes were isolated on a cell sorter as
described in Materials and Methods. More than 99% of the
sorted cells express cell surface CD4. FACS analysis of the cells
further reveal that they express high levels of CD62L and low levels of
CD25, features associated with a naive phenotype (Fig. 5). Analysis of cDNAs from these cells
for the expression of SOCS1, SOCS2, or SOCS3 gene
by quantitative RT-PCR reveals constitutive expression of SOCS1, SOCS2,
and SOCS3 mRNA transcripts (Fig. 6).
However, in contrast to significant amounts of mRNAs expressed in Th,
Th1, and Th2 cells that allow their detection by the relatively
insensitive Northern blot assay, the levels of SOCS transcripts in
naive Th cells are very low; their detection required at least 35
cycles of PCR amplification (Fig. 6A). Of particular note is
the relatively low abundance of SOCS1 transcripts detected by real-time
PCR in the naive nonpolarized cells (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and signaling molecules (JAKs,
STATs, Tec, p95vav). In this study, we found that three members of the SOCS family are differentially expressed in differentiated Th cells. Th1 cells predominantly express SOCS1 and SOCS2 but synthesize minute amounts of SOCS3 mRNAs or protein. Conversely, Th2 cells express very high levels of SOCS3 but low levels of SOCS1 protein and mRNA. The relatively high levels of SOCS mRNAs and proteins and their restricted patterns of expression in Th1 and Th2 cells underscore the importance of particular SOCS members in Th cell subtypes.
Furthermore, our data suggest that IL-4/STAT6 signaling is repressed in
Th1 cells that constitutively express high levels of SOCS1 but not in
Th2 cells that contain relatively low amounts of SOCS1. This
observation is consistent with another report showing that SOCS1
inhibits IL-4 signaling in M12 B cell line (22).
Constitutive expression of SOCS1 in Th1 cells is therefore consistent
with a Th cell differentiation model in which SOCS1 promotes
differentiation toward the Th1 lineage by stably inhibiting IL-4
signaling, thereby repressing Th2 differentiation. Because IFN-
signaling is dependent on activated STAT1 and tyrosine phosphorylation
of STAT1 is under feedback-inhibition by SOCS1 (5, 6, 24),
constitutive expression of SOCS1 may also lead to abrogation of IFN-
signaling and autocrine stimulation of Th1 cells by IFN-. In fact,
the inability to respond to IFN- is a hallmark feature of Th1 cells
as Th2 cells do respond to IFN- signaling, and this has been
attributed to the absence of IFN-R2 receptors in Th1 cells
(25). We suggest that in addition to this explanation, it
may well be that constitutive expression of SOCS1 provides a fail-safe
mechanism that ensures absence of IFN- signaling in Th1 cells. Thus,
similar to T-bet, SOCS1 is a Th1 lineage marker and presumably promotes
commitment to the Th1 phenotype.
Although the roles of either SOCS2 or SOCS3 in lymphoid cell
development have not been established, the preferential use of SOCS3 in
Th2 but not Th1 cells is suggestive of its role in the maintenance of
the Th2 cell lineage. Therefore IL-12/STAT4 signaling is repressed in
Th2 cells with high constitutive expression of SOCS3 but not in Th1
cells that contain relatively low amounts of this SOCS member (Fig. 4).
Although there is no direct evidence implicating SOCS3 in the
regulation of IL-12, the very high endogenous levels of SOCS3 in Th2
but not in Th1 cells suggest that SOCS3 may constitutively repress
IL-12/STAT4 signaling in Th2 cells. Furthermore, in contrast to the low
levels of SOCS1 mRNA, SOCS3 mRNA is expressed at relatively high levels
in nonpolarized Th cells, suggesting that this SOCS member may also
contribute to the initial phase of the differentiation process.
Constitutive expression of SOCS2 in naive and differentiated Th cells
is particularly intriguing in that this SOCS member functions primarily
to regulate growth through its inhibitory effects on growth
hormone-induced STAT5-dependent gene transcription. Given that IL-2
effects on Th cells are mediated by STAT5-dependent signaling pathways,
overriding the negative regulation of these pathways by SOCS2 and
possibly other SOCS proteins may be a necessary requirement for
Ag-dependent proliferation and activation of Th cells. Ongoing studies
are examining the levels of other SOCS family members such as SOCS4–7,
cytokine-induced Src homology 2-containing protein (CIS), proteins
containing SOCS box and WD-40 repeats (WSB), proteins containing SOCS
box and ankyrin repeats (ASB), proteins containing SOCS box and SPRY
domains (SSB), and proteins containing SOCS box and a GTPase domain
(RAR) in Th cells and investigating their possible involvement in Th
differentiation process and/or maintenance of the polarized patterns of
cytokine expression in Th1 and Th2 cells.
A major issue that must be resolved relates to the relative
contributions of cytokine and TCR signaling in differential regulation
of SOCS expression in Th1 and Th2. The fact that SOCS expression is
relatively low in naive Th cells and is up-regulated in response to
activation or cytokine signaling suggests that the Th cell
differentiation process and expression of SOCS proteins are
coordinately regulated. On one hand, the up-regulation of SOCS
expression by stimulation with the HEL protein in nonpolarized Th cells
suggests that TCR signaling contributes to SOCS induction. In contrast,
the preferential up-regulation of SOCS1 expression in Th1 but not Th2
cells after IL-12 stimulation argues for a role of cytokine signaling
in SOCS regulation. Our data further suggest that high constitutive
SOCS3 expression may be the default pattern of SOCS expression in
CD4+ cells as naive Th cells, nonpolarized
activated Th cells, and Th2 cells express relatively high levels of
SOCS3 and very low levels of SOCS1. The functional consequences of
elevated expression of SOCS3 may be to drive the majority of
CD4+ toward a Th2 pattern of cytokine expression.
In view of the significant increase of SOCS3 expression in response to
Ag stimulation, it is tempting to speculate that a major impact of TCR
signaling is transcriptional activation of the SOCS3 gene of
naive CD4+. However, cytokines can override the
effects of SOCS3 during Th differentiation. In fact, a major
effect of IL-12 signaling in nonpolarized Th cells is to down-regulate
SOCS3 expression in cells committed to the Th1 lineage. In
contrast to Th2, Th, and naive CD4+ cells, Th1
differentiation requires IL-12/STAT4 signaling and induction of
SOCS1. Selective induction of SOCS1 in
differentiating Th1 cells may inhibit IFN--induced phosphorylation
of STAT1, thereby preventing autoregulation of Th1 cells by IFN-.
Thus, on activation in a target tissue during the course of an
inflammatory response, Th1 cells produce copious amounts of IFN-
that act on cells in their microenvironment, but they are refractile to
the effects of this cytokine on gene transcription and cell cycle
regulation.
In summary, our data show that SOCS mRNAs and proteins are differentially expressed in Th1 and Th2 cells. The polarized expression of SOCS proteins in differentiated Th lymphocytes suggests that IL-12/STAT4- and IL-4/STAT6-signaling pathways may be repressed, at least in part, by a SOCS3- or SOCS1-mediated mechanism, respectively. Thus, Th cells that constitutively repress STAT6 signals become Th1 cells, and those that inhibit STAT4 activities develop into the Th2 phenotype. Our findings provide impetus for further investigations into the roles of negative regulatory factors in Th cell differentiation and, given the critical roles that SOCS proteins play in the termination of immune or cytokine responses, it is likely that other members of the SOCS family may also regulate Th cell lineage commitment and maintenance. These findings are of interest not merely in the context of Th differentiation but also because SOCS1 and SOCS3 proteins can serve as potential therapeutic targets for immune modulation therapy to skew the immune response toward a desirable outcome.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; HEL, hen egg lysozyme; Tg, transgenic; JAK, Janus kinase; TAMRA, 6-carboxytetramethylrhodamine; CD62L, CD62 ligand; 6FAM, 6-carboxyfluoresein.
Received for publication December 12, 2001. Accepted for publication January 24, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98:597.[Medline]
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J. M. Zimmerer, G. B. Lesinski, S. V. Kondadasula, V. I. Karpa, A. Lehman, A. RayChaudhury, B. Becknell, and W. E. Carson III IFN-{alpha}-Induced Signal Transduction, Gene Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of Cytokine Signaling Proteins J. Immunol., April 15, 2007; 178(8): 4832 - 4845. [Abstract] [Full Text] [PDF]
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K. Numata, M. Kubo, H. Watanabe, K. Takagi, H. Mizuta, S. Okada, S. L. Kunkel, T. Ito, and A. Matsukawa Overexpression of Suppressor of Cytokine Signaling-3 in T Cells Exacerbates Acetaminophen-Induced Hepatotoxicity J. Immunol., March 15, 2007; 178(6): 3777 - 3785. [Abstract] [Full Text] [PDF]
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S. J. Orr, N. M. Morgan, J. Elliott, J. F. Burrows, C. J. Scott, D. W. McVicar, and J. A. Johnston CD33 responses are blocked by SOCS3 through accelerated proteasomal-mediated turnover Blood, February 1, 2007; 109(3): 1061 - 1068. [Abstract] [Full Text] [PDF]
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C. Fujimoto, C.-R. Yu, G. Shi, B. P. Vistica, E. F. Wawrousek, D. M. Klinman, C.-C. Chan, C. E. Egwuagu, and I. Gery Pertussis Toxin Is Superior to TLR Ligands in Enhancing Pathogenic Autoimmunity, Targeted at a Neo-Self Antigen, by Triggering Robust Expansion of Th1 Cells and Their Cytokine Production J. Immunol., November 15, 2006; 177(10): 6896 - 6903. [Abstract] [Full Text] [PDF]
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 N. M. Moutsopoulos, N. Vazquez, T. Greenwell-Wild, I. Ecevit, J. Horn, J. Orenstein, and S. M. Wahl Regulation of the tonsil cytokine milieu favors HIV susceptibility J. Leukoc. Biol., November 1, 2006; 80(5): 1145 - 1155. [Abstract] [Full Text] [PDF]
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Y. Li, N. Chu, A. Rostami, and G.-X. Zhang Dendritic Cells Transduced with SOCS-3 Exhibit a Tolerogenic/DC2 Phenotype That Directs Type 2 Th Cell Differentiation In Vitro and In Vivo J. Immunol., August 1, 2006; 177(3): 1679 - 1688. [Abstract] [Full Text] [PDF]
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 I. Kinjyo, H. Inoue, S. Hamano, S. Fukuyama, T. Yoshimura, K. Koga, H. Takaki, K. Himeno, G. Takaesu, T. Kobayashi, et al. Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-{beta}1 J. Exp. Med., April 17, 2006; 203(4): 1021 - 1031. [Abstract] [Full Text] [PDF]
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 C. Tortorella, I. Stella, G. Piazzolla, V. Cappiello, O. Simone, A. Pisconti, and S. Antonaci Impaired Interleukin-12-Dependent T-Cell Functions During Aging: Role of Signal Transducer and Activator of Transcription 4 (STAT4) and Suppressor of Cytokine Signaling 3 (SOCS3). J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2006; 61(2): 125 - 135. [Abstract] [Full Text] [PDF]
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 J. L. Stark and A. H. Cross Differential expression of suppressors of cytokine signaling-1 and -3 and related cytokines in central nervous system during remitting versus non-remitting forms of experimental autoimmune encephalomyelitis Int. Immunol., February 1, 2006; 18(2): 347 - 353. [Abstract] [Full Text] [PDF]
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N. Kozma, M. Halasz, B. Polgar, T. G. Poehlmann, U. R. Markert, T. Palkovics, M. Keszei, G. Par, K. Kiss, J. Szeberenyi, et al. Progesterone-Induced Blocking Factor Activates STAT6 via Binding to a Novel IL-4 Receptor J. Immunol., January 15, 2006; 176(2): 819 - 826. [Abstract] [Full Text] [PDF]
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 S. N. Georas, J. Guo, U. De Fanis, and V. Casolaro T-helper cell type-2 regulation in allergic disease Eur. Respir. J., December 1, 2005; 26(6): 1119 - 1137. [Abstract] [Full Text] [PDF]
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A. Suto, H. Nakajima, N. Tokumasa, H. Takatori, S.-i. Kagami, K. Suzuki, and I. Iwamoto Murine Plasmacytoid Dendritic Cells Produce IFN-{gamma} upon IL-4 Stimulation J. Immunol., November 1, 2005; 175(9): 5681 - 5689. [Abstract] [Full Text] [PDF]
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 R. A. Tripp, C. Oshansky, and R. Alvarez Cytokines and Respiratory Syncytial Virus Infection Proceedings of the ATS, August 1, 2005; 2(2): 147 - 149. [Abstract] [Full Text] [PDF]
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 S. Babu, V. Kumaraswami, and T. B. Nutman Transcriptional Control of Impaired Th1 Responses in Patent Lymphatic Filariasis by T-Box Expressed in T Cells and Suppressor of Cytokine Signaling Genes Infect. Immun., June 1, 2005; 73(6): 3394 - 3401. [Abstract] [Full Text] [PDF]
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H. Takatori, H. Nakajima, S.-i. Kagami, K. Hirose, A. Suto, K. Suzuki, M. Kubo, A. Yoshimura, Y. Saito, and I. Iwamoto Stat5a Inhibits IL-12-Induced Th1 Cell Differentiation through the Induction of Suppressor of Cytokine Signaling 3 Expression J. Immunol., April 1, 2005; 174(7): 4105 - 4112. [Abstract] [Full Text] [PDF]
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S. Canfield, Y. Lee, A. Schroder, and P. Rothman Cutting Edge: IL-4 Induces Suppressor of Cytokine Signaling-3 Expression in B Cells by a Mechanism Dependent on Activation of p38 MAPK J. Immunol., March 1, 2005; 174(5): 2494 - 2498. [Abstract] [Full Text] [PDF]
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M. Watanabe, S. Watanabe, Y. Hara, Y. Harada, M. Kubo, K. Tanabe, H. Toma, and R. Abe ICOS-Mediated Costimulation on Th2 Differentiation Is Achieved by the Enhancement of IL-4 Receptor-Mediated Signaling J. Immunol., February 15, 2005; 174(4): 1989 - 1996. [Abstract] [Full Text] [PDF]
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N. Kienzle, S. Olver, K. Buttigieg, P. Groves, M. L. Janas, A. Baz, and A. Kelso Progressive Differentiation and Commitment of CD8+ T Cells to a Poorly Cytolytic CD8low Phenotype in the Presence of IL-4 J. Immunol., February 15, 2005; 174(4): 2021 - 2029. [Abstract] [Full Text] [PDF]
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H. Yang, M. Kala, B. G. Scott, E. Goluszko, H. A. Chapman, and P. Christadoss Cathepsin S Is Required for Murine Autoimmune Myasthenia Gravis Pathogenesis J. Immunol., February 1, 2005; 174(3): 1729 - 1737. [Abstract] [Full Text] [PDF]
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W.-M. Yu, S. Wang, A. D. Keegan, M. S. Williams, and C.-K. Qu Abnormal Th1 Cell Differentiation and IFN-{gamma} Production in T Lymphocytes from Motheaten Viable Mice Mutant for Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-1 J. Immunol., January 15, 2005; 174(2): 1013 - 1019. [Abstract] [Full Text] [PDF]
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 G. J. Vanasse, R. K. Winn, S. Rodov, A. W. Zieske, J. T. Li, J. C. Tupper, J. Tang, E. W. Raines, M. A. Peters, K. Y. Yeung, et al. Bcl-2 Overexpression Leads to Increases in Suppressor of Cytokine Signaling-3 Expression in B Cells and De novo Follicular Lymphoma Mol. Cancer Res., November 1, 2004; 2(11): 620 - 631. [Abstract] [Full Text] [PDF]
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D. Artis, A. Villarino, M. Silverman, W. He, E. M. Thornton, S. Mu, S. Summer, T. M. Covey, E. Huang, H. Yoshida, et al. The IL-27 Receptor (WSX-1) Is an Inhibitor of Innate and Adaptive Elements of Type 2 Immunity J. Immunol., November 1, 2004; 173(9): 5626 - 5634. [Abstract] [Full Text] [PDF]
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C.-R. Yu, R. M. Mahdi, S. Ebong, B. P. Vistica, J. Chen, Y. Guo, I. Gery, and C. E. Egwuagu Cell Proliferation and STAT6 Pathways Are Negatively Regulated in T Cells by STAT1 and Suppressors of Cytokine Signaling J. Immunol., July 15, 2004; 173(2): 737 - 746. [Abstract] [Full Text] [PDF]
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 C. Brender, R. Columbus, D. Metcalf, E. Handman, R. Starr, N. Huntington, D. Tarlinton, N. Odum, S. E. Nicholson, N. A. Nicola, et al. SOCS5 Is Expressed in Primary B and T Lymphoid Cells but Is Dispensable for Lymphocyte Production and Function Mol. Cell. Biol., July 1, 2004; 24(13): 6094 - 6103. [Abstract] [Full Text] [PDF]
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J. A. Johnston Are SOCS suppressors, regulators, and degraders? J. Leukoc. Biol., May 1, 2004; 75(5): 743 - 748. [Abstract] [Full Text] [PDF]
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S. H. Jackson, C.-R. Yu, R. M. Mahdi, S. Ebong, and C. E. Egwuagu Dendritic Cell Maturation Requires STAT1 and Is under Feedback Regulation by Suppressors of Cytokine Signaling J. Immunol., February 15, 2004; 172(4): 2307 - 2315. [Abstract] [Full Text] [PDF]
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V. Athie-Morales, H. H. Smits, D. A. Cantrell, and C. M. U. Hilkens Sustained IL-12 Signaling Is Required for Th1 Development J. Immunol., January 1, 2004; 172(1): 61 - 69. [Abstract] [Full Text] [PDF]
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R. Lund, T. Aittokallio, O. Nevalainen, and R. Lahesmaa Identification of Novel Genes Regulated by IL-12, IL-4, or TGF-{beta} during the Early Polarization of CD4+ Lymphocytes J. Immunol., November 15, 2003; 171(10): 5328 - 5336. [Abstract] [Full Text] [PDF]
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P. Anderson, A. Sundstedt, L. Li, E. J. O'Neill, S. Li, D. C. Wraith, and P. Wang Differential activation of signal transducer and activator of transcription (STAT)3 and STAT5 and induction of suppressors of cytokine signalling in Th1 and Th2 cells Int. Immunol., November 1, 2003; 15(11): 1309 - 1317. [Abstract] [Full Text] [PDF]
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P. S. Grutkoski, Y. Chen, C. S. Chung, and A. Ayala Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes J. Leukoc. Biol., November 1, 2003; 74(5): 916 - 922. [Abstract] [Full Text] [PDF]
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 C.-R. Yu, R. M. Mahdi, S. Ebong, B. P. Vistica, I. Gery, and C. E. Egwuagu Suppressor of Cytokine Signaling 3 Regulates Proliferation and Activation of T-helper Cells J. Biol. Chem., August 8, 2003; 278(32): 29752 - 29759. [Abstract] [Full Text] [PDF]
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 T. WERNER, S. FESSELE, H. MAIER, and P. J. NELSON Computer modeling of promoter organization as a tool to study transcriptional coregulation FASEB J, July 1, 2003; 17(10): 1228 - 1237. [Abstract] [Full Text] [PDF]
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S. Thomas, R. Kumar, A. Preda-Pais, S. Casares, and T.-D. Brumeanu A Model for Antigen-Specific T-Cell Anergy: Displacement of CD4-p56lck Signalosome from the Lipid Rafts by a Soluble, Dimeric Peptide-MHC Class II Chimera J. Immunol., June 15, 2003; 170(12): 5981 - 5992. [Abstract] [Full Text] [PDF]
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J. R. Mead, T. R. Hughes, S. A. Irvine, N. N. Singh, and D. P. Ramji Interferon-gamma Stimulates the Expression of the Inducible cAMP Early Repressor in Macrophages through the Activation of Casein Kinase 2. A POTENTIALLY NOVEL PATHWAY FOR INTERFERON-gamma -MEDIATED INHIBITION OF GENE TRANSCRIPTION J. Biol. Chem., May 9, 2003; 278(20): 17741 - 17751. [Abstract] [Full Text] [PDF]
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M. Zhang, M. S. Vacchio, B. P. Vistica, S. Lesage, C. E. Egwuagu, C.-R. Yu, M. P. Gelderman, M. C. Kennedy, E. F. Wawrousek, and I. Gery T Cell Tolerance to a Neo-Self Antigen Expressed by Thymic Epithelial Cells: The Soluble Form Is More Effective Than the Membrane-Bound Form J. Immunol., April 15, 2003; 170(8): 3954 - 3962. [Abstract] [Full Text] [PDF]
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D. V. R. Bullen, T. M. Baldwin, J. M. Curtis, W. S. Alexander, and E. Handman Persistence of Lesions in Suppressor of Cytokine Signaling-1-Deficient Mice Infected with Leishmania major J. Immunol., April 15, 2003; 170(8): 4267 - 4272. [Abstract] [Full Text] [PDF]
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A. Matsumoto, Y.-i. Seki, R. Watanabe, K. Hayashi, J. A. Johnston, Y. Harada, R. Abe, A. Yoshimura, and M. Kubo A Role of Suppressor of Cytokine Signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated Interleukin 2 Production J. Exp. Med., February 17, 2003; 197(4): 425 - 436. [Abstract] [Full Text] [PDF]
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 Y.-i. Seki, K. Hayashi, A. Matsumoto, N. Seki, J. Tsukada, J. Ransom, T. Naka, T. Kishimoto, A. Yoshimura, and M. Kubo Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation PNAS, October 1, 2002; 99(20): 13003 - 13008. [Abstract] [Full Text] [PDF]
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S. F. Soriano, P. Hernanz-Falcon, J. M. Rodriguez-Frade, A. M. de Ana, R. Garzon, C. Carvalho-Pinto, A. J. Vila-Coro, A. Zaballos, D. Balomenos, C. Martinez-A., et al. Functional Inactivation of CXC Chemokine Receptor 4-mediated Responses through SOCS3 Up-regulation J. Exp. Med., August 5, 2002; 196(3): 311 - 321. [Abstract] [Full Text] [PDF]
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