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*
Department of Medicine, Hospital for Special Surgery, Cornell University Medical College,
Cornell/Rockefeller/Sloan-Kettering Tri-Institutional M.D.-Ph.D. Program, and
Graduate Program in Immunology, Cornell University Graduate School of Medical Sciences, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor subunit, IL-2, IL-7, and IL-15, was suppressed by
preculture in IL-4. Activation of the Jak1 and Jak3 kinases that are
proximal to Stat5 in the IL-2-Jak-STAT signaling pathway was
suppressed, and this correlated with inhibition of IL-2Rß subunit
expression. In contrast to suppression of Stat5, proliferative
responses to IL-2 were augmented in IL-4-cultured cells, and activation
of proliferative pathways leading to activation of mitogen activated
protein kinases, induction of expression of Myc, Fos, Pim-1, and cyclin
D3, and decreased levels of the cyclin-dependent kinase inhibitor p27
were intact. These results identify molecular mechanisms underlying
interactions between IL-4 and IL-2 in T cells and demonstrate that one
mechanism of regulation of IL-2 activity is selective and differential
modulation of signaling pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and a common -chain
(c)4, the
latter used by other cytokine receptors including the IL-4, IL-7, IL-9,
and IL-15R (reviewed in 4 . Signaling by IL-2 occurs through high
or intermediate affinity receptors containing /ß/c,
or ß/c chains, respectively. The IL-2R subunit
primarily increases the affinity of ligand binding and is not known to
contain a signaling domain, whereas the ß and c
subunits participate in both ligand binding and signal transduction.
Signaling by the ß and c proteins is mediated by
kinases that are associated with or recruited to the cytoplasmic
domains of these molecules. Ligation of the IL-2R triggers several
signal transduction pathways, including the Jak-STAT pathway (4, 5, 6, 7, 8, 9, 10, 11)
and distinct pathways leading to activation of Myc (12, 13), Bcl-2
(14), signal transducing adaptor molecule (15) and Ras-mitogen
activated protein kinase (MAPK) pathways (16, 17, 18, 19, 20). Genetic approaches
using receptor mutations in transfected cell lines have demonstrated
that these pathways are independent and have linked specific pathways
with effects on cell phenotype. For example, the Ras pathway is
involved in proliferation (17), whereas the Jak-STAT pathway is
involved in gene activation (18, 21). Triggering of individual or
subgroups of signaling pathways by IL-2, resulting in selective
activation of distinct cellular responses, has not to our knowledge
been previously described.
The protein tyrosine kinases Jak1 and Jak3 that are associated with the
IL-2Rß and c subunits, respectively, are activated
shortly after binding of IL-2 to its receptor (22, 23, 24). Subsequently,
specific tyrosine residues in the cytoplasmic domains of the IL-2Rß
and c subunits become phosphorylated. The ß-chain
phosphotyrosine motifs provide docking sites that are recognized by the
SH2 domain of Stat5, a member of the STAT family of transcription
factors (6, 9, 11). After recruitment to the receptor complex, Stat5
becomes phosphorylated, dimerizes, translocates to the nucleus, and
activates transcription. Although the role of the Jak kinases, Myc, and
Bcl-2 in proliferation by IL-2 is well established (12, 13, 14, 15, 23, 25),
the role of Stat5 is not clear, because reports differ in their
conclusions (7, 19, 20, 26, 27), and Stat5 may promote proliferation
through an indirect mechanism (28).
Differentiation of T cells is determined, in great part, by regulatory
cytokines present during activation. IL-12 and IL-4 are major
regulatory cytokines involved in differentiation of Th1 and Th2 cells,
respectively (reviewed in 29 . One important mechanism of
regulation of T cell differentiation is blocking of signaling pathways
that are triggered by antagonistic cytokines. For example, recent
studies have demonstrated that cells cultured in IL-4 lose
responsiveness to IL-12 by down-regulation of the IL-12Rß2 subunit
and consequently, Stat4 activation (30, 31). Similarly, studies have
shown that IFN- can down-regulate expression of its own receptor
ß-chain in Th1 cells, thereby allowing Th1 cells to escape the
antiproliferative effects of IFN- (32, 33, 34). IL-2 plays a role in T
cell differentiation by synergizing with IL-12 in driving proliferation
and differentiation toward a Th1 effector phenotype (35).
Interestingly, experiments using both in vitro and in vivo systems have
demonstrated that IL-2 is also necessary for IL-4-mediated
differentiation of Th2 cells (36, 37, 38, 39). Because the induction of both
Th1 and Th2 cells requires IL-2, we investigated whether IL-4 can
modulate IL-2R expression or signaling, and thus modify effects of this
cytokine on T cell phenotype. Our results suggest that the Jak-STAT and
proliferative pathways emanating from the IL-2R can be selectively and
differentially regulated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whole blood or buffy coats (New York Blood Center, New York, NY) were obtained from disease-free donors, and mononuclear cells (MNC) were obtained by density gradient centrifugation using Ficoll metrizoate (Lymphoprep; Life Technologies, Gaithersburg, MD). MNC were depleted of CD56+ NK cells using the MACS separation system (Miltenyi Biotech, San Jose, CA) and CD56-specific paramagnetic beads. Cells were cultured at 37°C, 5% CO2, in a humidified atmosphere in complete media (CM) containing RPMI 1640 (Life Technologies) supplemented with 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were for cultured 1–4 days in CM containing 1–2.5 µg/ml PHA (Sigma, St. Louis, MO) and cytokines as noted in figure legends. For acute stimulation of PHA-activated T cells, cells were harvested, washed, and incubated with the indicated cytokines for 10 min in CM. In pilot experiments, cells were evaluated on a daily basis throughout the 4 days of culture by obtaining cell counts, assessing viability using trypan blue exclusion and propidium iodide (PI) staining, determining entry into cell cycle using flow cytometry and [3H]thymidine incorporation, and determining cell surface phenotype using flow cytometry. There was no proliferation during the culture period, consistent with previous reports demonstrating that, in the absence of exogenous IL-2, human T cells do not divide during the 3–4 days after mitogen stimulation (40, 41). No differences in cell numbers and viability were detected between control and IL-4-treated cultures, and no outgrowth or selection of any T cell populations was detected.
Flow cytometry
Cultures were analyzed using flow cytometry as previously
described (42) using the following Abs: T cell and activation markers,
4B5 (anti-CD3), 3G10 (anti-CD25), CF1 (anti-CD122), AG184
(anti-c); NK cell marker, 3G8 (anti-CD16); and
monocyte marker, IV.3 (anti-CD32 monocyte-specific epitope). AG184
was purchased from PharMingen (San Diego, CA), 4B5, 3G10, and 3G8 from
Boehringer Mannheim (Indianapolis, IN), CF1 from Immunotech (Westbrook,
ME) and IV.3 from Medarex (New Lebanon, NH). Analyses were done using a
FACScan flow cytometer with CellQuest software (Becton Dickinson, San
Jose, CA).
Cell lysis and electrophoretic mobility shift assays (EMSA)
EMSA were performed as previously described (43). Briefly, cell extracts were obtained by lysing cells in hypertonic buffer (for EMSA) or buffer containing 0.1 mM EDTA, 200 mM NaCl, 50 mM Tris-Cl (pH 7.5), 0.5 mM DTT, 200 µM PMSF, 4 mM NaF, 0.5% Nonidet P-40, 2 mM Na3VO4, and 10% glycerol (for Jak immunoprecipitates or EMSA), as described (42). Protein concentration was determined by the Bradford method, and 5 µg of cell extract were incubated with 0.5 ng of 32P-labeled double-stranded oligonucleotide containing a STAT site derived from the IFN response factor (IRF)-1 gene promoter. For supershift experiments, 1 µl of affinity purified anti-Stat5 or PU.1 (irrelevant control) Abs (Santa Cruz Biotechnology, Santa Cruz, CA) was added to cell extracts before incubation with oligonucleotide probe. Samples were resolved on 4.5% polyacrylamide gels in 0.25Xx TBE at 9V/cm at room temperature. Gels were dried and visualized by autoradiography.
Immunoblotting and immunoprecipitation
Whole cell lysates or immunoprecipitated proteins were fractionated on 7.5 or 10% polyacrylamide gels using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). mAbs against Jak1, Jak3, Stat5, and extracellular stimulus-regulated kinase (ERK2) were obtained from Transduction Laboratories (Lexington, KY), polyclonal Abs against Jak1, Jak3, Stat5, ERK2, Myc, Bcl2, cyclin D3, and p27 were obtained from Santa Cruz Biotechnology, and antiphosphotyrosine Ab (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).
Immunoprecipitates were performed using polyclonal Abs as described (42) by adjusting cell extract volume to 0.5 ml and incubating with 2 µg of specific Ab for 4 h at 4°C. Immunoprecipitates were collected using protein A and protein G agarose beads (Pierce Chemical, Rockford, IL), washed twice in lysis buffer, once in PBS, resolved by SDS-PAGE, and analyzed by immunoblotting with mAbs.
Analysis of mRNA levels
Total cellular RNA was isolated using RNAzol (Cinna Scientific,
Friendswood, TX) according to the instructions of the manufacturer. For
Northern hybridization analysis, 5 µg of RNA were fractionated on
1.2% formaldehyde agarose gels, transferred to Hybond-N membranes, and
hybridized with random primer labeled (Boehringer Mannheim) DNA probes
using standard techniques as described (42). cDNA probes were provided
by K. Smith (44) and M. Kaplan (45). Filters were hybridized overnight
at 42°C in 50% (v/v) formamide, 10% Dextran, 10 mM Tris-Cl (pH
7.5), 5Xx SSC, 1x Denhardt’s solution (34), 0.1% SDS, and 100
µg/ml sheared herring sperm DNA, and were washed in 2Xx SSC for 30
min at room temperature, 42°C and 55°C. For RT-PCR, RNA was treated
with RNase-free DNase, and cDNA was obtained using Maloney murine
leukemia virus reverse transcriptase (Life Technologies). A total of
2.5% of each cDNA was subjected to 25 cycles of PCR using conditions
that result in a single specific amplification product of the correct
size (Ref. 44 and data not shown): 30-s denaturation at 94°C, 1-min
annealing at 55°C, and 30-s extension at 72°C in a GeneAmp 9600
thermal cycler (Perkin-Elmer, Norwalk, CT). dNTPs were used at
100 µM and 1 µCi of [-32P]dATP was added to each
reaction. No amplification products were obtained when reverse
transcriptase was omitted, indicating the absence of contaminating
genomic DNA. Amplification was empirically determined to be in the
linear range. Oligonucleotide primers for the cytokine responsive (CR)
genes were provided by Smith and co-workers (44), and primer sequences
for glyceraldehyde phosphate dehydrogenase (GAPDH), Myc, and Fos
(written 5'
3') are as follows: GAPDH, GTG AAG GTC GGA GTC AAC
and TGG AAT TTG CCA TGG GTG; Myc, GGC GGC CGG CTA GGG TGG AAG A and CCT
GGC AGC GGC GGG GAA GTG T; and Fos, CCG AGA TTG CCA ACC TGC TGA A and
CAC TGG GCC TGG ATG ATG C.
Proliferation analysis
Cells were seeded in 96-well, flat-bottom, tissue culture cluster plates at a density of 5 x 105 cells/well and stimulated with human rIL-2 (Boehringer Mannheim) for 24 h. After 18 h of culture, 20 µl of a 50 µCi/ml [3H]thymidine solution was added to the culture media and culture continued for an additional 6 h. Subsequently, cells were washed, lysed, and harvested on an automated cell harvester (Harvester 96; Tomtec, Orange, CT) and extracts blotted on filter pads. Filters were placed in scintillation fluid and [3H]thymidine incorporation quantitated using a Wallac Microbeta Trilux scintillation counter (Wallac, Gaithersburg, MD).
PI DNA staining
DNA content was measured by staining with PI (Sigma). Cells were harvested and adjusted to a density of 5 x 105 cells/ml and fixed in 75% ethanol/PBS supplemented with 1% FBS. Fixed cells were stained with 20 µg/ml of PI, treated with 10 µg/ml DNase-free RNase (Boehringer Mannheim) for 2 h, and analyzed using a FACScan flow cytometer and CellQuest software.
Analysis of ERK2 activity
ERK2 immunoprecipitates were washed twice in immunoprecipitate
buffer (42), once in PBS, once in kinase buffer containing 25 mM HEPES,
10 mM MnCl2, 0.5 mM DTT, and 5 µM ATP, and resuspended in
50 µl of kinase buffer containing 10 µCi of
[-32P]dATP (NEN; Life Sciences Products, Boston, MA),
and 5 µg of the substrate myelin basic protein (Life Technologies).
After incubation for 45 min at room temperature, the reaction was
terminated by the addition of gel loading buffer and the products were
fractionated on 12% SDS polyacrylamide gels and visualized by
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of Stat5 is a major signaling event triggered by IL-2
and therefore, the effect of IL-4 on IL-2 induction of Stat5
DNA-binding activity was analyzed. Freshly isolated MNC from normal
donors were activated with PHA and cultured for a period of 3–4 days
without exogenous cytokines or in the presence of exogenous IL-4 or
IL-12. Subsequently, cells (>90% T cells) were washed and stimulated
for 10 min with IL-2, and cell extracts were assayed for STAT
DNA-binding activity by EMSA using a radiolabeled IRF-1 oligonucleotide
probe that binds Stat1, Stat5, and Stat6. Treatment with IL-2 resulted
in the rapid induction of a DNA-protein complex that likely corresponds
to Stat5 (see below) in control and IL-12-treated cells (Fig. 1
A, lanes 4
and 6). In contrast, activation of Stat5 was suppressed in
IL-4-treated cells (Fig. 1A, lane 5 and Fig.
1B, lane 2). Suppression of Stat5 activation in
IL-4-cultured cells was consistently detected in over 20 independent
experiments using different blood donors and a broad range of IL-2
doses (between 5 and 100 U/ml; data not shown). Immunoblot analysis
demonstrated comparable levels of Stat5 in all extracts (Fig.
1A, lower panel and data not shown),
indicating that IL-4 suppressed activation of DNA binding and did not
decrease Stat5 protein levels. Consistent with previous results from
several laboratories, including ours (43, 46, 47), DNA protein
complexes were detected in activated T cells before stimulation with
IL-2 (Fig. 1A, lanes 1–3).
Supershift experiments showed that the faint lower complex present
before and after IL-2 stimulation corresponded to Stat1 (data not
shown; see 43 . Stat1 activity was suppressed in cells cultured in
IL-4 (most apparent in Fig. 1B, lanes 1 and
2) and increased in cells cultured in IL-12 (Fig.
1A, lane 3). Stat1 activity was not increased by
IL-2 stimulation (Fig. 1A, lanes
4–6).
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B). Incubation of cell
extracts with Abs against Stat5 before the addition of radiolabeled IRF
oligonucleotide, resulted in disruption of the IL-2-induced complex
(but not the Stat1 complex) and the appearance of more slowly migrating
DNA-protein complexes (Fig. 1B, lanes 3 and
4) compared with extracts incubated with matched control
(anti-PU.1) Abs (lanes 1 and 2).
Incubation with anti-Stat1 Ab resulted in disruption of the Stat1,
but not Stat5, complex (data not shown). Taken together with published
data from other investigators showing that IL-2 activates Stat5 in T
cells (6, 8, 9, 48), these results confirm that the IL-2-induced
complex contains Stat5. To determine whether IL-4 may nonspecifically
suppress Jak-STAT signaling, the activation of Stat1 by IFN- in
IL-4-treated cultures was assayed. Levels of IFN--induced Stat1
complexes were similar in cells that were cultured in the presence or
absence of IL-4 (Fig. 1B, lanes 5 and
6). These data suggest that IL-4 specifically blocks
activation of Stat5 by IL-2 in PHA-activated T cells.
Tyrosine phosphorylation of Stat5 is required for DNA binding. The
effect of IL-4 on IL-2-induced tyrosine phosphorylation was examined by
immunoprecipitation of Stat5 followed by phosphotyrosine immunoblotting
(Fig. 1C). IL-2-induced tyrosine phosphorylation of Stat5
(lane 2) was suppressed by IL-4 (lane
4) to an extent comparable with suppression of DNA binding. These
results confirm the DNA-binding results (Fig. 1, A and
B) and suggest that culture in IL-4 suppressed the
activation of Stat5.
Activation of Stat5 by IL-2 depends on membrane proximal events
including the phosphorylation and activation of Jak1 and Jak3 kinases,
upstream events occurring shortly after ligand engagement of the IL-2R
(9, 18, 20, 24, 25, 48). Jak1 and Jak3 levels were comparable in both
control and IL-4-treated cells (data not shown). Tyrosine
phosphorylation of Jak1 and Jak3 were analyzed using
immunoprecipitation and phosphotyrosine immunoblotting (Fig. 2). IL-2 induced tyrosine phosphorylation
of Jak1 in control cells but not in cells cultured with IL-4 (Fig. 2, top panel). Subsequent immunoblot analysis of the same
membrane using Jak1 Abs demonstrated comparable levels of Jak1 in the
immunoprecipitates, showing that the decrease in phosphotyrosine levels
was not due to variable levels or degradation of the Jak1 protein (Fig. 2). Similarly, the IL-2-induced increase in tyrosine phosphorylation of
Jak3 was suppressed in cells cultured with IL-4 (Fig. 2, third
panel). In contrast to Jak1, IL-2-induced Jak3
phosphorylation was not completely blocked in IL-4-treated cells
(lane 4) and detection of this residual
phosphorylation was reproducible. Immunoblotting of the same filter
with anti-Jak3 Ab revealed that protein levels of Jak3 were
comparable in all immunoprecipitates (Fig. 2). These results show that
culture in IL-4 suppressed the activation of Jak-STAT signaling by IL-2
in T cells.
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-chain) in T cells by a mechanism involving Stat5 (49, 50), we
investigated the regulation of CD25 cell surface expression in
IL-4-treated cultures. Baseline expression of CD25 in cells cultured in
IL-4 was similar to expression in control cells immediately after
culture (Fig. 3, top
panel). As expected, stimulation with IL-2 for 24 h
induced an increase in CD25 expression in cells previously cultured in
the presence of PHA alone (Fig. 3, lower panel). The
response to IL-2 was suppressed by 
50% in IL-4-treated cells (mean
fluorescence values were 2838 in control cells and 1571 in IL-4-treated
cells in the representative experiment shown in Fig. 3). This result
was reproduced in more than five independent experiments. Thus,
induction of CD25, a Stat5 target gene (49, 50), by IL-2 was suppressed
by IL-4 in parallel with suppression of Stat5 activation.
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A).
Surprisingly, IL-2 induced Myc mRNA expression in IL-4-treated cells
(Fig. 4A, lanes 2 and 4), and
the level of induction appeared comparable to IL-2-stimulated control
cells, when signal intensities are corrected for variable loading of
lanes using GAPDH levels (lanes 1 and
3). This result suggested that IL-4 may selectively suppress
expression of IL-2 genes that are highly dependent on Stat5, but not
genes activated through other signaling pathways. This idea was tested
by using RT-PCR to screen the induction of eight IL-2-inducible genes,
including genes that were originally termed the CR genes (44). IL-4
suppressed IL-2 induction of CIS, but not Myc (Fig. 4B, panels
1 and 5); these results are comparable with those
obtained using Northern hybridization (Fig. 4A) and further
validate the use of RT-PCR to monitor changes in mRNA levels, as
described in Materials and Methods. The induction of Myc was
further confirmed at the protein level (see Fig. 9 below). In addition
to suppression of CIS, IL-4 suppressed the IL-2 induction of CR1, the
PGE2 receptor (CR3), and CR8, but did not suppress
induction of Fos, SATB1 (CR4), and Pim-1 (CR-7) (Fig. 4B).
Interestingly, some of the genes not suppressed by IL-4, such as Fos,
Myc, and Pim-1, are believed to play an important role in cell
proliferation, whereas genes inhibited in IL-4-treated cells such as
CIS and the PGE2 receptor, have been implicated in
inhibition of cytokine signaling or inhibition of growth (51, 52, 53, 54).
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A major feature of IL-2 stimulation of T cells is induction of
proliferation (1) and IL-2-induced proliferation was measured in cells
cultured in IL-4. After 4 days, cells were removed from culture and
incubated in medium containing IL-2 for an additional 24 h and
proliferation assayed by measuring incorporation of
[3H]thymidine. IL-4-treated cells incorporated a greater
amount of [3H]thymidine over a broad range of IL-2
concentrations, as compared with control cultures (Fig. 5A). The levels of IL-2R
expression and differences in ß-chain expression between control and
IL-4-treated cells (see below) did not change during the culture period
(data not shown). Consistent with these results, the increase in cell
number in IL-4-treated cultures was greater than that in control
cultures after incubation with IL-2 (data not shown).
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B). DNA content in resting
cells exhibits a major fluorescence peak (2N) using flow cytometry when
stained with PI and fluorescence beyond that peak (>2N) indicates DNA
synthesis and entry into the cell cycle. IL-2 induced fluorescence
beyond the 2N peak in 3% of control cells and in 11% of cells
cultured in IL-4 (Fig. 5B). Detection of subgenomic DNA
levels (measured to the left of the 2N peak) indicates DNA degradation
(<2N) usually accompanying apoptosis. The percentage of cells
containing subgenomic DNA levels after IL-2 stimulation was comparable
in cells cultured with or without IL-4 (Fig. 5B). Similar to
results of the [3H]thymidine incorporation experiments,
these results were consistent in multiple experiments using different
blood donors. These data suggest that proliferative pathways are fully
functional despite diminished activation of the Jak-STAT signaling
pathway. IL-4 regulation of IL-2R ß-chain expression
Studies by other laboratories have demonstrated that
phosphotyrosine motifs representing Stat5 docking sites are
contained in the IL-2R ß-chain (21) that is associated with Jak1.
Decreased Jak1 and Stat5 activation after culture with IL-4 could be
explained by inhibition of the Jak kinases or by decreased expression
of IL-2Rß. Therefore, cell surface expression of IL-2R subunits and
other cell surface Ags was determined using flow cytometry (Fig. 6). After 4 days of culture, >90% of
cells were CD3+ (Fig. 6A), indicating a
predominant and comparable population of T cells in both control and
IL-4-treated cultures. Further analysis verified the absence of NK
cells (bright CD16+) and monocytes (express IV.3 epitope of
CD32) in the cultures (Fig. 6, B and E). The
slight reduction in CD25 expression in IL-4-treated cells (without any
additional IL-2 stimulation) seen in this experiment (Fig. 6C) was not consistently detected. Interestingly, decreased
expression of the c chain was observed after culture
with IL-4 (Fig. 6D). The most plausible explanation for
lower c expression is ligation of the c
chain by IL-4, and subsequent endocytosis (55). Expression of the
IL-2Rß subunit (CD122) was determined over 4 days of culture (Fig. 7). Constitutive, low level expression of
ß-chain was observed in freshly isolated MNC before culture,
consistent with previous reports (56). Over a period of 4 days, a
gradual increase in CD122 expression was observed in cells cultured
with PHA in the absence of exogenous IL-4. In contrast, cells incubated
with PHA in the presence of IL-4 did not show an increase in ß-chain
expression. The difference in ß-chain expression between control and
IL-4-treated cells was maximal at 3–4 days and persisted for at least
5 days (data not shown). Addition of exogenous IL-2 or neutralization
of endogenous IL-2 in the cultures did not affect ß-chain expression
or the difference in ß-chain levels between control and IL-4-treated
cells, indicating that IL-4 did not regulate IL-2Rß expression by
modulating endogenous IL-2 production (data not shown). A highly
reproducible suppression of induction of IL-2Rß and c
expression was observed in multiple cultures using cells from over 20
different blood donors. These results on cell surface expression of
IL-2R components are consistent with a previous study demonstrating
that IL-4 preculture reduced the number of IL-2 binding sites on
lymphocytes (57). The decrease in IL-2Rß expression suggests that one
mechanism for decreased Stat5 activation is decreased availability of
phosphotyrosine motifs that correspond to Stat5 docking sites that are
present in the cytoplasmic domain of the ß-chain, although additional
mechanisms may also play a role.
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c. In this
case, one would predict that culture in IL-4 should suppress Stat5
activation by other cytokines that utilize c, such as
IL-7, IL-9, and IL-15, and that culture in IL-2 should suppress
activation of Stat6 by IL-4. Indeed, activation of Stat5 by IL-7 and
IL-15, which also shares the ß subunit with IL-2, was suppressed by
IL-4 (Fig. 8A); IL-9 did not
activate Stat5 in control cells and thus could not be assessed (Fig. 8A). Culture of cells in IL-2 resulted in a suppression of
Stat6 activation by IL-4 (Fig. 8B, lanes 3
and 6). These results suggest that competition for common
receptor subunits is another mechanism by which IL-4 suppresses IL-2
signaling.
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The proliferation and flow cytometry data (
Figs. 5–7) suggest
that signals activated by a low number of high affinity IL-2R can
effectively drive proliferation in T cells cultured in IL-4. We
investigated the molecular mechanism underlying proliferative
responsiveness in these cells. T cell proliferation in response to IL-2
has been linked to activation of the ERK kinases, members of the MAPK
family of protein kinases that are downstream effectors in the Ras
pathway (19, 20, 58). Although IL-2 can activate the Ras-MAPK pathway,
IL-4 does not (59). In vitro kinase assays were used to determine
whether activation of ERKs might contribute to the proliferation seen
in the IL-4-treated cultures stimulated with IL-2. Baseline ERK2 kinase
activity was detected in control (PHA-activated) cells and there was no
further increase after IL-2 stimulation (Fig. 9A, lanes 1
and 2). Baseline ERK2 activity in PHA-activated cells is
consistent with previously reported results, was suppressed using
PD98059, a specific inhibitor of MEK1 (the kinase directly upstream of
ERK2) (Fig. 9A, right panel), and was low
in resting cells (L.B.I., unpublished data). Lower baseline ERK2 kinase
activity was detected in cells cultured in IL-4 and increased
significantly after a 10 min incubation with IL-2 (Fig. 9A,
lanes 3 and 4). Immunoblot analysis of a
fraction of the ERK2 immunoprecipitates showed comparable levels of
ERK2 protein, indicating that the differential kinase activity was not
due to the absence or degradation of the ERK2 protein (Fig. 9A, lower panel).
The product of the c-myc proto-oncogene has been well
characterized for its participation in cell proliferation (12, 13, 60).
Induction of Myc protein levels by IL-2 was measured in cells cultured
in the presence or absence of IL-4. Myc levels were undetectable before
IL-2 stimulation and were induced to comparable levels after an 18-h
stimulation with IL-2 in both control cells and those cultured in IL-4
(Fig. 9B, top panel); these results are
consistent with the analysis of Myc mRNA levels (Fig. 4). Entry into
the cell cycle in response to IL-2 is associated with the induction of
expression of cyclin D3, and decreased levels of the p27 cyclin
dependent kinase inhibitor, and these results were reproduced in our
system (Fig. 9B, second and third
panels, lanes 1–3). IL-4
treatment alone had no effect on cyclin D3 levels, but preculture in
IL-4 resulted in a more rapid and slightly greater induction of cyclin
D3 expression by IL-2 (Fig. 9B, second
panel). Culture in IL-4 alone resulted in decreased p27
levels (third panel), and levels of the
anti-apoptotic protein Bcl-2 were comparable in all lanes,
demonstrating comparable protein loading (Fig. 9B,
bottom panel). These data, together with Fig. 4, show
that IL-2 can activate several different signaling pathways important
in proliferation, those mediated by MAPKs, Myc, Fos, Pim-1, and cyclin
D3 in IL-4-cultured cells to a level comparable to activation levels in
control cells. Decreased p27 expression in IL-4-treated cells suggests
that IL-4 may prime cells to enter the cell cycle in response to an
IL-2-triggered proliferative signal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differential regulation of IL-2 signaling pathways has important
functional implications for the expansion of different cell populations
by IL-2. IL-2 and IL-12 synergize in generating Th1 cells, and IL-2 has
been proposed to function as a Th1-promoting cytokine. However, IL-2 is
also necessary for the generation of Th2 cells (29, 37, 38, 39) and thus
must be able to drive proliferation of these cells without changing
their phenotype toward that of a Th1 cell. Our data show that IL-4, the
major Th2-promoting cytokine, suppresses signals that may be important
for development of the Th1 phenotype, and others have shown that IL-4
can block signaling by the Th1-promoting cytokine IL-12 (30, 31).
Extinction of IL-12 signaling is one mechanism by which cells may
commit to a Th2 phenotype, and modulation of IL-2 signaling corresponds
to a mechanism by which IL-2 can expand Th2 precursors without strong
activation of IL-2-inducible Stat5 target genes important for Th1
development. Of note, IFN- signaling was not suppressed in cells
cultured in IL-4 (Fig. 1), consistent with the dominance of IFN-
over IL-4 in regulation of T cell phenotype (31). Our data do not
resolve the issue of the role of Stat5 in T cell proliferation because
Stat5 activation was not completely blocked, and sufficient Stat5 may
be activated by IL-2 in IL-4-treated cells to contribute to
proliferative responses.
Expression of high affinity IL-2R containing , ß, and
c subunits was not completely blocked because expression
of the -chain was not significantly affected (Fig. 6), expression of
the ß-chain was detected albeit at low levels (Fig. 7), and cells
responded to low doses of IL-2, as assessed by proliferation, cell
counts, and gene activation (
Figs. 3–5 and 9; A.C., unpublished data).
Because the ß-chain contains docking sites for Stat5, decreased
ß-chain expression provides one plausible mechanism for suppression
of Stat5 activation, which was also not suppressed completely (Fig. 1).
Indeed, experiments with multiple donors have demonstrated a
correlation between the level of inhibition of ß-chain expression and
the level of inhibition of Stat5 (A.C., unpublished data). Decreased
IL-2Rß expression also provides a plausible mechanism for the
decreased activation of Jak1 and Jak3 kinases. It is unlikely that the
recently described SOCS/JAB/SSI Jak kinase inhibitors (61, 62, 63) play a
major role in inhibition, because these molecules would be expected to
also inhibit Jak1 and Jak2 associated with the IFN- receptor, and
inhibition of IFN- signaling was not detected (Fig. 1). We have not
formally excluded the possibility that novel proteins which bind to
tyrosine phosphorylated STATs (64) may contribute to the mechanism of
inhibition. Such putative inhibitors, which to date have only been
described for Stat3, would not affect Jak tyrosine phosphorylation and
thus are unlikely to play an important role in the inhibition of Stat5
in our system.
The preservation of an intact and augmented proliferative response to
IL-2, despite lower numbers of IL-2R, demonstrates synergy between IL-2
and IL-4 in driving cellular proliferation. One molecular mechanism
underlying this synergy is the effective activation by IL-2 in
IL-4-cultured cells of several molecules that play an important role in
promoting proliferation, including ERK and induction of expression of
Myc, Fos, Pim-1, and cyclin D3. Previous work suggests that activation
of IL-2 signaling pathways leading to Fos and Myc expression is
sufficient for proliferation, regardless of the activation state of
other IL-2 signaling pathways (13). Effective induction of Myc, Fos,
Pim-1, cyclin D3, and MAPK pathways suggests that IL-4 augments or
amplifies a weak proximal IL-2-triggered proliferative signal, such
that weak activation of Jak3 (Fig. 2) results in stronger activation or
induction of downstream molecules that drive proliferation (Figs. 4 and 9). In contrast to Myc, Fos, Pim-1, and cyclin D3, IL-4 suppressed IL-2
induction of genes that play a role in inhibition of signaling and
proliferation, such as CIS and the PGE2 receptor (51, 52, 53, 54).
Furthermore, IL-4 itself activates several proliferative pathways and
regulates components of the cell cycle machinery (45, 65), including
triggering decreases in levels of the p27 cell cycle inhibitor (Fig. 9B), and we propose that this primes cells for a response to
an IL-2-triggered proliferative signal. Thus, the increased
proliferation detected in the IL-4-cultured cells after IL-2
stimulation is likely secondary to a combination of amplification of an
IL-2 signal leading to the expression of genes important in
proliferation, and priming of cells to respond to this signal through
regulation of components of the cell cycle machinery. These results are
consistent with previous work demonstrating an important role for Jaks
in proliferation because: 1) proliferative responses to IL-2 in the
absence of Jak1 have been described in a cell line (66), and are
attenuated but present in lymphocytes from Jak1 knockout mice (67); and
2) activation of Jak3 in our experiments was not completely suppressed
(Fig. 2), in contrast to complete absence of Jak3 activity in patients
with Jak3 deficiency or c mutations, or in Jak3 knockout
mice (23, 68, 69, 70, 71). Moreover, it is not clear that Jak3 plays a critical
nonredundant role in all lymphocyte proliferation, because circulating
lymphocytes in older Jak3 or c knockout mice exhibit a
hyperactivated, proliferating phenotype (72, 73).
An important feature of cytokine action is pleiotropy, the triggering of different responses in different cell types. The molecular basis of pleiotropy is not completely understood, but has been attributed to cell-specific expression of genes and effector molecules. Our results demonstrate that the response of a cell to a cytokine can be modified by extracellular stimuli that differentially suppress or augment different signaling pathways that emanate from the cytokine receptor. In the case of IL-2, the molecular mechanism underlying the interaction with IL-4 is the differential regulation of Stat5 activation and of other proliferative pathways. This finding represents a novel mechanism for regulating the action of IL-2 and for increasing the pleiotropic effects of this cytokine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lionel B. Ivashkiv, Department of Medicine, Hospital for Special Surgery, Cornell University Medical College, 535 East 70th Street, New York, NY 10021. E-mail address:
3 Present address: Department of Neurosurgery, University of Texas, Houston, TX 77030.
4 Abbreviations used in this paper: c, common -chain; MAPK, mitogen activated protein kinase; GAPDH, glyceraldehyde phosphate dehydrogenase; CR, cytokine responsive; EMSA, electromobility shift assay; IRF, IFN response factor; ERK, extracellular stimulus-regulated kinase; MNC, mononuclear cells; PI, propidium iodide.
Received for publication February 26, 1998. Accepted for publication October 9, 1998.
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