Selective induction of p38 mitogen-activated protein kinase activity following A6H co-stimulation in primary human CD4+ T cells
Tord Labuda,
Anette Sundstedt and
Mikael Dohlsten
BMC Immunobiology, Department of Tumor Immunology, University of Lund, Sölvegatan 21, 223 62 Lund, Sweden
Correspondence to:
T. Labuda
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Abstract
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We have recently described the novel A6H antigen expressed on human peripheral blood T cells and on renal cell carcinoma cells. Cross-linking of the A6H antigen results in co-stimulation of human CD4+ T cells, characterized by induction of the transcription factor activator protein-1 (AP-1), proliferation and prominent IFN-
production, but low levels of IL-2. The proximal signaling events associated with A6H ligation include protein tyrosine kinase phosphorylation and association of p56 Lck, ZAP-70 and the TCR
chain. In this study we show that A6H co-stimulation selectively induced activation of the p38 mitogen-activated protein kinase (MAPK) pathway, whereas no significant c-Jun N-terminal kinases (JNK) activity was observed. In contrast, CD28 co-stimulation resulted in both p38 and JNK MAPK activities. Human CD4+ T cells co-stimulated with A6H up-regulated AP-1 binding proteins reactive with a proximal AP-1 binding site in the human IFN-
promoter and a consensus AP-1 binding site. Moreover, preincubation of the T cells with the specific p38 MAPK inhibitor SB203580 resulted in decreased AP-1 binding following A6H or CD28 co-stimulation. This suggests that the p38 MAPK pathway is required for induction of full AP-1 binding activity in human CD4+ T cells co-stimulated with A6H or CD28. Blocking the p38 MAPK pathway by SB203580 completely inhibited IFN-
production from A6H co-stimulated T cells and radically reduced IFN-
production from T cells co-stimulated with anti-CD28. In contrast, no significant inhibition of IL-2 production was seen after blocking of the p38 MAPK in either A6H or CD28 co-stimulated T cells. Since the p38 MAPK recently has been shown to be critically involved in regulation of IFN-
production from Th1 cells, we propose that A6H co-stimulation induces a specific pathway, mediated via p38 and AP-1 activation, for induction of a Th1 profile in human CD4+ T cells.
Keywords: co-stimulatory molecules, human, signal transduction, T lymphocytes, transcription factors
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Introduction
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T cell activation to proliferation and cytokine production requires at least two signals. The first signal is generated by interaction between the TCR and the MHCantigen and a second signal provided by co-stimulatory molecules on an antigen-presenting cell (1). Several co-stimulatory molecules have been described, including B7, LFA-3 and the ICAMs binding to their counter receptors CD28, CD2 and LFA-1 respectively, on the T cell (24). We have recently described the existence of a new co-stimulatory molecule on human T cells (5,6). This molecule, 120140 kDa, is recognized by the mAb A6H at similar densities on a subpopulation of both CD4+ and CD8+ T cells but is only co-stimulatory for the CD4+ T cells. Co-ligation of the A6H antigen and the CD3 complex on CD4+ T cells induces activation characterized by proliferation and cytokine production, including high levels of IFN-
and tumor necrosis factor (TNF) but low levels of IL-2 (5,6).
The production of cytokines in T cells is critically regulated at the transcriptional level and several transcription factors including Oct-1, AP-1, NF-
B and nuclear factor of activated T cells (NF-AT) have been shown to be involved in regulation of both the IL-2 (7,8) and IFN-
gene promoters (912). Activator protein-1 (AP-1) proteins seem to play a central role in the regulation of the IL-2 promoter by binding to the functionally important AP-1 site in the IL-2 promoter (13) as well as participating in the formation of transcriptionally active NF-AT and NF-IL-2 (14,15). Moreover, recent studies implicate a critical role for AP-1 in regulation of IFN-
promoter activity in T cells stimulated with mitogens or IL-18 (16).
We have previously shown that A6H co-stimulation of primary CD4+ human T cells results in enhanced expression of AP-1 binding proteins, whereas no increase in NF-
B or Oct proteins could be detected compared to anti-CD3 stimulation alone (6). The AP-1 transcription factor is composed of different members of the Fos and Jun family of proteins (17). Homodimers of Jun but not Fos do bind to an AP-1 containing oligonucleotide but heterodimers formed between the different Jun and Fos proteins have an enhanced binding activity compared to JunJun homodimers (18). AP-1 is regulated both at the level of jun and fos gene transcription, and by post-translational modification of their gene products. Synthesis and trans-activating capacities of c-Jun and c-Fos have been shown to be regulated by mitogen-activated protein kinases (MAPK). The MAPK constitute a group of signaling molecules characterized by phosphorylation of tyrosine and threonine residues by specific MAPK kinases. The MAPK described in mammalian cells include the extracellular regulated kinases (ERK) (19,20), The c-Jun N-terminal kinases (JNK, also known as SAPK) (2123), Fos regulating kinase (FRK) (24) and p38 (2527). ERK activation occurs through the TCRCD3 complex via activation of p21ras and subsequent activation of the RafMEK kinase cascade (2830). The downstream effect of ERK on AP-1 is mediated by phosphorylation of the transcription factor Elk-1, a protein involved in up-regulation of the c-fos gene (31,32). JNK activation is also mediated through p21ras but requires an additional co-stimulatory signal mediated by the CD28 receptor (22). Downstream, JNK has been shown to phosphorylate the transcription factor c-Jun in its N-terminal activating domain (3335). FRK is a 88 kDa MAPK kinase that has been implicated in post-translational modification of c-Fos by phosphorylation of a threonine residue within its activation domain (24). No information on the regulation of FRK in T cells is presently available. p38 is activated by CD3 cross-linking (36,37). However the participation of co-stimulatory signals to p38 activation in T cells remains obscure. Salmon et al. have recently shown a synergistic effect of CD3 and CD28 cross-linking on p38 activity, although CD28 cross-linking alone was unable to activate the p38 MAPK (37). In contrast, DeSilva et al. demonstrated that p38 activity is fully induced by anti-CD3 alone and CD28 co-stimulation did not enhance the effect even at low anti-CD3 concentrations (36). The downstream effect of p38 includes activation of the transcription factors ATF-2 and Elk-1 (38). Elk-1 have been implicated in the up-regulation of the c-fos gene and the transcription factor ATF-2 can form heterodimers with Jun subunits suggesting that p38 may be involved in regulation of AP-1 binding activity.
In the present study, we have evaluated the relative contribution of A6H co-stimulation on p38 and JNK MAPK activity in CD4+ human T cells.
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Methods
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Reagents
Polyclonal rabbit anti-p38, anti-JNK-1 and GSTATF-2 (a substrate for p38 kinase assay) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A GSTc-Jun179 expression plasmid was a gift from Dr M. Karin (UCSD, La Jolla, CA). Production and purification of GSTc-Jun protein were performed as previously described (21). The mAb A6H (5,39) was a gift from Dr R. Vessella (University of Washington, Seattle, WA). The mAb anti-C215 (reactive with a human colon carcinoma antigen; used as IgG control mAb) has been described earlier (40), the mAb anti-CD28 was obtained from Immunotech (Westbrook, ME), and the anti-c-Jun mAb and goat anti-mouse Ighorseradish peroxidase conjugate were from Transduction Laboratories (Lexington, KY). The p38-specific inhibitor SB203580 was purchased from Calbiochem (La Jolla, CA). T4 polynucleotide kinase was purchased from Promega (Madison, WI). Poly(dIdC)2, Ficoll-Paque and Percoll were obtained from Amersham-Pharmacia Biotech (Uppsala, Sweden).
Media used
Complete medium: RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10 mM HEPES, 4 mM L-Glutamine, 1 mM pyruvate, 0.1% NaHCO3 and 10% FCS.
Cell separation and culture
Human mononuclear cells were isolated from buffy coats by centrifugation on Ficoll-Paque and Percoll gradients, and depleted of monocytes by the use of a gelatin column as previously described (41). CD4+ T cell subsets were obtained by using the MACS cell sorting system (Miltenyi Biotec, Sunnyvale, CA) according to the manufacturer's recommendations. The purity of the T cells was regularly >98% as determined by FACS analysis.
mAb linking to Dynabeads
Combinations of mAb OKT-3/C215, OKT-3/A6H and OKT-3/CD28 at a ratio of 1:1 (w/w) or A6H, C215 and OKT-3 alone were linked to Dynabeads precoated with sheep anti-mouse IgG, as earlier described (5,42).
Thymidine incorporation and cytokine assays
CD4+ T cells (105) and mAb-coated beads (1.5x105) were added to flat-bottomed 96-well microtiter plates and incubated at 37°C. For p38 inhibition, cells were preincubated in the presence of various concentrations of SB203580 or the solvent DMSO (0.05%) for 2030 min at 37°C. During the last 4 h of a 72120 h culture period, cells were incubated with 0.5 µCi of [3H]thymidine, harvested onto filter paper and the thymidine incorporation was measured in a ß-scintillation counter.
The IFN-
activity in culture supernatants was assessed in an anti-viral cytopathic assay using Wish cells and MTT as previously described (5,43). IL-2 activity in the culture supernatants was assessed utilizing the IL-2-dependent murine T cell line CTLL-2 as described (5,43).
Protein extraction, immunoprecipitation and immunocomplex kinase assay
Cells (20x106 per experiment) for immunoprecipitation were incubated with beads at a ratio of 1:1.5 at 37°C or phorbol myristate acetate 25 ng/ml and ionomycin 1.5 µg/ml. For p38 inhibition, cells were preincubated for 30 min at 37° C with various concentrations of SB203580 or the solvent DMSO. After stimulation, the cells were rapidly pelleted and the reaction stopped by lysing the cells in ice-cold JNK lysis buffer (20 mM TrisHCl, pH 7.7, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP-40, 1 mM p-nitrophenylphosphate, 10 mM ß-glycerophosphate, 0.1 mM Na3VO4, 1 mM PMSF and a cocktail of protease inhibitors Complete; Boehringer Mannheim, Mannheim, Germany) or p38 lysis buffer (20 mM TrisHCl, pH 7.5, 10% glycerol, 1% Triton X-100, 137 mM NaCl, 25 mM ß-glycerophosphate, 2 mM EDTA, 0.5 mM DTT, 1 mM Na3VO4, 2 mM Na-pyrophosphate, 10 µg/ml leupetin and 1 mM PMSF). Polyclonal rabbit antisera (2 µg/ml) was added to precleared lysates before incubation with Protein ASepharose beads. The immunoprecipitates were pelleted, washed 3 times in lysis buffer and 2 times in JNK kinase buffer (20 mM HEPES, pH 7.6, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 2 mM DTT, 1 mM p-nitrophenylphosphate, 10 mM ß-glycerophosphate and 0.1 mM Na3VO4) or p38 kinase buffer (25 mM HEPES, pH 7.4, 25 mM ß-glycerophosphate, 25 mM MgCl2, 0.5 mM DTT and 0.1 mM Na3VO4). The pellets were resuspended in 30 µl of kinase buffer containing 1 µg of GSTc-Jun or GSTATF-2, 1 µM ATP and 1 µCi of [
-32P]ATP. Incubations were conducted for 20 min at 30°C, then stopped by addition of 10 µl of 4xSDS sample buffer. Phosphorylation of the substrates was analyzed by 10% SDSPAGE followed by autoradiography of the dried gel. The optical density of each band was measured using a Agfa Duo Scanner (Agfa-Gevaert, Mortsel, Belgium) and a densitometry program, Gel Pro (Media Cybernetics, Silver Spring, MD).
Nuclear extracts and mobility shift assay
Freshly isolated resting human CD4+ T cells at a concentration of 10x106/well were stimulated with OKT-3/C215, OKT-3/A6H or OKT-3/anti-CD28 mAb linked to Dynabeads. Nuclear extracts were prepared as previously described (6). The protein concentrations of all extracts were measured using the BioRad protein assay kit (BioRad, Hercules, CA). Binding reactions were carried out with the same amount of protein in each reaction (0.51 µg of nuclear protein) in binding buffer (AP-1 consensus: 10 mM TrisHCl, pH 7.5, 25 mM NaCl, 1 mM EDTA, 1 mM DTT and 5% glycerol), (AP-1 human IFN-
promoter: 25 mM HEPES, 150 mM KCl, 5mM DTT and 10% glycerol), 2 µg poly(dIdC)2 as a non-specific competitor and 30x103 c.p.m. of 32P end-labeled double-stranded oligonucleotide as described (6,44). The double-stranded oligonucleotides used in the binding assay had the following sequences: AP-1 consensus (5'-CTA GTG ATG AGT CAG CCG GAT C-3') or the proximal AP-1 site in the human IFN-
promoter (16) (5'-ATG GGT CTG TCT CAT CGT CAA AGG A-3').
Western blot analysis
Western blot analysis was performed as previously described (45). Briefly, protein extracts were separated on 10% SDSPAGE and electroblotted onto nitrocellulose membranes. The membranes were probed with specific c-Jun mAb (Transduction) or specific c-Fos mAb (Santa Cruz Biotechnology). The immuncomplexes were detected by a goat anti-mouse Ighorseradish peroxidase conjugate and were visualized using an enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech).
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Results
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A6H co-stimulation induces proliferation and cytokine production in CD4+ T cells.
CD4+ T cells were cultured in the presence of beads coupled with OKT3 (against the CD3
chain) and A6H, anti-CD28 or C215 (isotype control) as previously described (5,6). The CD4+ T cells proliferated strongly when activated with OKT3/A6H or OKT3/anti-CD28 (Fig. 1A
) and (6). In contrast, no or marginal proliferation was seen after stimulation with OKT3 alone or the OKT3/C215 combination. The proliferative response to A6H showed a slower kinetics compared to anti-CD28 co-stimulation and reached peak levels after 5 days of stimulation (Fig. 1A
) (5). The cytokine production induced by A6H co-stimulation resulted in high levels of IFN-
but low levels of IL-2 (Figs 1B,C
) (5). In contrast, co-ligation of CD28 induced high levels of both IFN-
and IL-2. Interestingly, the IL-2/IFN-
ratio was ~1:1 in CD28 co-stimulated T cell cultures, whereas A6H co-stimulation resulted in a IL-2/IFN-
ratio of 1:100. No cytokine production was detected from T cells stimulated with OKT3 alone or the OKT3/C215 control beads.
A6H co-stimulation selectively utilizes the p38 MAPK pathway
The ERK, JNK and p38 MAPK pathways have previously been shown to be activated in T cells upon stimulation. ERK requires only anti-CD3 or phorbol myristate acetate (PMA) for maximal induction, whereas JNK requires either PMA and Ca2+ ionophore or antibodies to TCR and CD28 (22). Involvement of the p38 pathway in T cell co-stimulation remain controversial (36,37). However, a recent study has suggested an important role of p38 in the induction of IFN-
production in Th1 T cells (46). Since A6H has been shown to be a strong inducer of IFN-
, we investigated the role of A6H co-stimulation on p38 and JNK-1 activities in human CD4+ T cells. We cultured T cells in the presence of mAb-coated beads for 15, 30, 45 and 60 min, and analyzed the ability of immunoprecipitated p38 and JNK-1 to phosphorylate ATF-2 or GSTc-Jun respectively. Interestingly, a strong synergistic effect on p38 activity was observed by, anti-CD3 (OKT3) and A6H after 15 min of stimulation; the effect was still strong after 30 min of activation (Fig. 2A
) and declined thereafter (data not shown). In contrast, A6H induced only a small, transient increase in JNK-1 activity over T cells stimulated with the control OKT3/C215 (Fig 2B
). CD28 co-stimulation induced substantial activity of both p38 and JNK-1 (Fig. 2A and B
). Optimal stimulation with PMA and ionomycin induced potent activation of both p38 and JNK-1. Moreover, nuclear extracts from unstimulated T cells or T cells stimulated with the control OKT3/C215 contained small amounts of c-Jun and A6H co-stimulation did not increase the levels of c-Jun detected. In contrast CD28 co-stimulation induced a substantial increase in the amount of c-Jun as judged by Western blot (Fig. 2C
). Furthermore, nuclear extracts from A6H as well as CD28 co-stimulated CD4+ T cells contained increased levels of c-Fos protein as compared to nuclear extracts from CD4+ T cells stimulated with the control OKT3/C215 (data not shown).
The p38 inhibitor SB203580 reduces AP-1 binding activity in co-stimulated CD4+ T cells.
We have previously shown that co-ligation of the A6H antigen and the CD3 complex on CD4+ T cells induced expression of Jun and Fos proteins binding to a AP-1 consensus motif, whereas no increase in NF-
B and Oct binding proteins was seen compared to T cells stimulated with anti-CD3 alone (6). In order to investigate whether A6H induced AP-1 binding proteins reactive with a proximal AP-1 site from the human IFN-
promoter, we cultured resting CD4+ T cells in the presence of beads as described for 20 h. Resting T cells or T cells stimulated with OKT3/C215 contained no or minimal amounts of AP-1 binding proteins, whereas A6H or CD28 co-stimulation resulted in weak expression of AP-1 binding proteins reactive with the AP-1 site from the human IFN-
promoter (Fig. 3A
) as well as strong binding to a consensus AP-1 (Fig. 3B
). To verify that the binding to AP-1 oligonucleotides was specific, we performed competition experiments with unlabeled oligonucleotides. AP-1 binding activity in nuclear extracts to consensus AP-1 or AP-1 from the human IFN-
promoter was completely blocked by a 50-fold molar excess of unlabeled oligo but was not competed by excess of unlabeled consensus NF-
B (data not shown).
To evaluate whether inhibition of the p38 pathway could affect AP-1 binding activity in A6H co-stimulated T cells, we preincubated CD4+ T cells with or without the specific p38 inhibitor SB203580 prior to stimulation and subsequent electrophoretic mobility shift assay (EMSA) on consensus AP-1. Interestingly, SB203580 (5 µM) could significantly decrease the AP-1 binding activity in T cells co-stimulated with A6H or CD28 (Fig. 3B
), suggesting that maximal induction of AP-1 binding proteins requires the p38 MAPK pathway.
p38 is required for IFN-
production from CD4+ T cells co-stimulated with A6H
The p38 pathway has been implicated in IFN-
production from Th1 T cells (46). Moreover, our results suggest that A6H co-stimulation utilizes the p38 pathway, at least in part, for induction of AP-1 binding proteins in human CD4+ T cells. We therefore analyzed the importance of the p38 pathway on proliferation and cytokine production from CD4+ T cells co-stimulated with A6H. CD4+ T cells preincubated in normal media or with various concentrations of SB203580 were incubated with beads for 35 days as described, and thereafter analyzed for proliferation and production of IFN-
and IL-2. The proliferative response in T cells co-stimulated with A6H could be reduced by ~50% using the highest concentration (10 µM) of the inhibitor but lower concentrations had no significant effects (Fig. 4B
). The proliferative response of CD28 co-stimulated cells was also decreased using the highest concentration (10 µM) of SB203580, but to a lesser extent (Fig. 4A
). The production of IFN-
from CD28 or A6H co-stimulated T cells was radically affected by the highest concentration of the inhibitor (Fig. 4C and D
). Lower concentrations of the inhibitor (5 and 1 µM respectively) had no significant effect on IFN-
production from the CD28 co-stimulated T cells. In contrast, the levels of IFN-
from A6H co-stimulated T cells were significantly reduced even at 5 µM. IL-2 production of A6H or CD28 co-stimulated T cells was not significantly affected even by the highest concentration of the p38 inhibitor (Fig. 4E and F
).
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Discussion
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We have previously shown that A6H co-stimulation of CD4+ human T cells is sufficient for some of the early events in T cell activation. A6H ligation alone was sufficient for tyrosine phosphorylation of p56 Lck, whereas phosphorylation and co-ligation of the TCR
chain and ZAP-70 with Lck required cross-linking of both A6H and the CD3 complex (47). We now show that A6H co-stimulation activates the p38 MAPK, whereas no or little JNK activity could be detected in the same T cells.
Several distinct MAPK signal transduction pathways have been identified in mammalian cells, leading to the activation of the MAPK ERK, JNK, FRK and p38 respectively (24,4850). One of the targets for MAPK activation in mammalian cells is the transcription factor AP-1. AP-1 is composed of members of the Fos and Jun family of DNA binding proteins (17,51) which bind to the TPA-response element (TRE) with the consensus sequence TGACTCA (51). However, related atypical TRE also bind AP-1 complexes and the binding capacity to a specific TRE is dependent on the composition of the AP-1 complex (51,52). The MAPK signaling pathways regulate AP-1 activity both by increasing the abundance of the AP-1 components and by directly stimulating their activity (51). ERK family members (53,54) as well as JNK (55) and p38 (38) have been described to be involved in phosphorylation of several sites within the C-terminal end of the transcription factor Elk-1. The transcription factor Elk-1 has thus been suggested to integrate multiple MAPK signal transduction pathways at the c-fos serum response element to increase c-fos transcription (56). The transcriptional response of the c-jun promoter is primarily mediated by two TRE, which are constitutively occupied and preferentially bind heterodimers of c-Jun and ATF-2 (56). Both c-Jun and ATF-2 are phosphorylated in their activation domain by JNK, resulting in increased c-jun transcription (57,58). In contrast, p38 can phosphorylate and activate ATF-2 but not c-Jun (38,59).
The p38 MAPK-specific inhibitor SB203580 is a pyridinyl imidiazole compound that can inhibit p38 activity by binding in the ATP binding pocket (60). Neither JNK nor ERK is inhibited by SB203580 in the same concentrations (61,62). Our results show that A6H co-stimulation is sufficient for AP-1 induction and that preincubating the T cells with the p38 inhibitor can significantly reduce the AP-1 binding activity. Similarly, the p38 inhibitor reduced AP-1 binding activity in CD28 co-stimulated T cells. These results suggest that full induction of AP-1 binding activity in T cell co-stimulation requires participation of the p38 MAPK pathway. A6H co-stimulation was able to induce p38 but minimal JNK activity compared to anti-CD3 stimulation alone, whereas CD28 co-stimulation induced p38 as well as JNK activities. Furthermore, A6H co-stimulation did not elevate the levels of c-Jun compared to T cells stimulated with anti-CD3 alone, whereas a substantial increase of c-Jun was detected in extracts from CD28 co-stimulated T cells. These results suggest that A6H co-stimulation affects the AP-1 binding activity in human T cells primarily at the level of c-fos transcription, whereas CD28 co-stimulation regulates the transcription of both c-fos and c-jun. Indeed nuclear extracts from A6H co-stimulated T cells contained elevated levels of c-Fos proteins compared to T cells stimulated with anti-CD3 alone (data not shown).
AP-1 has been implicated in the regulation of expression of number of cytokines produced by T cells including the IL-2, TNF and IFN-
genes. The IFN-
and TNF genes are regulated by AP-1 protein in complex with other transcription factors, and ATF-2/c-Jun have been shown to cooperate with NF-ATp and bind to a CRE site resulting in TNF-
production in calcium-stimulated T cells (63). The IFN-
gene has also recently been shown to be critically regulated by AP-1 in primary human T cells stimulated with anti-CD3/anti-CD28 or stimulated with IL-18 (12,16). In addition at least three more binding sites in the 160 bp proximal region of the IFN-
promoter (NF-AT/AP-1, GATA/AP-1 and ATF-2/AP-1) have been shown to bind AP-1 components (9,10,64). In our study, T cells co-stimulated with A6H or CD28 induced similar, but weak, AP-1 binding to the 190 proximal AP-1 (12) site of the human IFN-
promoter.
Rincon et al. have recently shown that the p38 inhibitor SB203580 can block the production of IFN-
from murine CD4+ Th1 clones (46). We now show that inhibition of the p38 pathway is sufficient for blocking IFN-
production from CD4+ human T cells co-stimulated by A6H, suggesting that IFN-
production in CD4+ T cells co-stimulated by A6H is critically regulated by the p38 MAPK. CD28 co-stimulated T cell production of IFN-
was also drastically reduced following p38 inhibition, although to a lesser extent, implicating that CD28 co-stimulation utilizes additional signal transduction pathway(s) for induction of IFN-
production. Reduced IFN-
reporter gene activation by a dn-p38 expression plasmid has been demonstrated by Rincon et al. following CD28 co-stimulation but no inhibition of IFN-
production using a dn-JNK expression plasmid was seen in this study (46). However, these studies were done in Jurkat T cells and not primary T cells.
The effect of p38 inhibition on proliferation was also more profound in T cells co-stimulated with A6H than T cells co-stimulated with CD28. This might reflect that CD28 signaling is mediated via both p38 and JNK, whereas A6H ligation has no significant effect on JNK activation. IL-2 production from T cells co-stimulated with CD28 or A6H was not affected significantly by inhibition of the p38 pathway, which suggests that IL-2 production in CD4+ human T cells is independent of the p38 MAPK pathway. The levels of IL-2 in supernatants from T cells co-stimulated with A6H were extremely low compared to CD28 co-stimulated T cells. Recent studies have shown that the key regulatory elements in the human IL-2 promoter are the proximal AP-1 and NF-
B sites (65). We have previously shown that A6H co-stimulation does not increase NF-
B binding to a consensus NF-
B oligonucleotide compared to anti-CD3 stimulation alone (6). Since A6H co-stimulation is able to induce IFN-
production but only low levels of IL-2, this suggests a less prominent role for NF-
B binding proteins in the IFN-
promoter compared to the IL-2 promoter.
Taken together we have, for the first time, demonstrated that A6H co-stimulation results in selective activation of the p38 MAPK. Furthermore, inhibition of the p38 pathway by SB203580 reduced A6H-induced AP-1 binding activity and blocked IFN-
production in primary human CD4+ T cells. Previous studies have shown that blockade of the p38 MAPK results in inhibition of IFN-
production from mouse Th1 clones, whereas no effect of p38 inhibition could be seen on production of IL-4 from Th2 clones (46). Thus, a reasonable interpretation of our data is that A6H co-stimulation induces AP-1 binding activity, mediated primarily by the p38 pathway, which results in production of IFN-
, thereby skewing the T cells towards a Th1 type of response. Indeed, no IL-4 production could be seen in supernatants from A6H co-stimulated T cells (data not shown). In addition, the low levels of IL-2 (IL-2/IFN-
ratio of 1:100) seen following A6H co-stimulation suggests that the A6H pathway primarily is involved in sustaining a Th1 profile and contributes less to clonal expansion of naive T cells.
Moreover, our results further add to the signal transduction pathway that is utilized by A6H co-stimulation of primary T cells. However, it remains to be investigated how the proximal signaling events induced by A6H co-stimulation, characterized by tyrosine phosphorylation and association between the protein tyrosine kinases Lck, Zap-70 and the TCR
chain, are linked to activation of the p38 MAPK.
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Acknowledgments
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We thank Drs R. Vessella (University of Washington Seattle, WA) and M. Karin (UCSD, La Jolla, CA) for the generous gift of the A6H mAb and GSTc-Jun expression plasmid respectively. The Swedish Cancer Society supported this work.
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Abbreviations
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AP-1 activator protein-1 |
EMSA electrophoretic mobility shift assay |
ERK extracellular regulated kinase |
FRK Fos regulated kinase |
JNK c-Jun N-terminal kinase |
MAPK mitogen-activated protein kinase |
NF-AT nuclear factor of activated T cells |
PMA phorbol myristate acetate |
TNF tumor necrosis factor |
TRE TPA-response element |
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Notes
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Transmitting editor: H. Wigzell
Received 2 July 1999,
accepted 2 November 1999.
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