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The Journal of Immunology, 1999, 162: 659-668.
Copyright © 1999 by The American Association of Immunologists

T Cell Activation Signals Up-Regulate p38 Mitogen-Activated Protein Kinase Activity and Induce TNF-{alpha} Production in a Manner Distinct from LPS Activation of Monocytes

Peter H. Schafer, Liwen Wang, Scott A. Wadsworth, Janet E. Davis and John J. Siekierka1

Drug Discovery Research, R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, 08869


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
p38 mitogen-activated protein kinase (MAPK) (p38) is involved in various cellular responses, including LPS stimulation of monocytes, resulting in production of proinflammatory cytokines such as TNF-{alpha}. However, the function of p38 during antigenic stimulation of T cells is largely unknown. Stimulation of the human Th cell clone HA-1.70 with either the superantigen staphylococcal enterotoxin B (SEB) or with a specific antigenic peptide resulted in p38 activation and the release of TNF-{alpha}. MAPK-activated protein kinase-2 (MAPKAPK-2), an in vivo substrate for p38, was also activated by T cell signaling. SB 203580, a selective inhibitor of p38, blocked p38 and MAPKAPK-2 activation in the T cell clone but did not completely inhibit TNF-{alpha} release. PD 098059, a selective inhibitor of MAPK kinase 1 (MEK1), blocked activation of extracellular signal-regulated kinase (ERK) and partially blocked TNF-{alpha} production by the clone. In human peripheral T cells, p38 was not activated by SEB, but rather by CD28 cross-linking, whereas in the human leukemic T cell line Jurkat, p38 was activated by CD3 and CD28 cross-linking in an additive fashion. TNF-{alpha} production by peripheral T cells in response to SEB and anti-CD28 mAb correlated more closely with ERK activity than with p38 activity. Therefore, various forms of T cell stimulation can activate the p38 pathway depending on the cells examined. Furthermore, unlike LPS-stimulated monocytes, TNF-{alpha} production by T cells is only partially p38-dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mitogen-activated protein kinases (MAPKs)2 are serine/threonine protein kinases that transduce signals originating from extracellular events, such as cell surface receptor engagement, culminating in the regulation of a cellular response, such as growth or differentiation (reviewed in Refs. 1, 2). The MAPKs include the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinase/stress activated protein kinases (JNK/SAPK), and the p38 MAPKs (p38s). These three families of MAPKs form three parallel signaling cascades activated by distinct and sometimes overlapping sets of stimuli. In general, ERKs are activated by mitogenic factors, while the JNKs and p38s are activated by stress-inducing agents or proinflammatory cytokines (2).

p38 was originally identified as a MAPK activated by LPS stimulation of monocytes (3) and was later shown to regulate LPS-induced IL-1ß and TNF-{alpha} release in these cells (4). Members of the p38 family, including p38{alpha} (CSBP2/RK/SAPK2a) (4), p38ß (SAPK2), p38ß2, p38{gamma} (ERK6/SAPK3) (5), p38{delta}(SAPK4) (6, 7), and Mxi2 (8), all bear significant homology to HOG1, the MAPK responsive to hyperosmolarity in Saccharomyces cerevisiae (9). Like the yeast homologue, mammalian p38s are activated by osmotic stress as well as by other forms of environmental stress including UV light, arsenite, heat shock (10, 11, 12), and the proinflammatory cytokines IL-1ß and TNF-{alpha} (10, 13, 14, 15).

p38 activation has been shown to be regulated by the small GTP-binding proteins Rac and Cdc42 and by p21-activated kinase-1 (Pak1) (16). Apoptotic signals can activate p38 via the Ser/Thr protein kinase ASK1 (a MAP kinase kinase kinase) and the dual specificity (Ser/Thr and Tyr-phosphorylating) MAP kinase kinases immediately upstream of p38, MKK3 and MKK6 (17). MAPK-activated protein kinase-2 (MAPKAPK-2) is an in vivo substrate for p38, which in turn phosphorylates the small heat-shock protein Hsp27 (14, 18). Other substrates for p38 include MAPKAPK-3 (19) and the transcription factors ATF2 (20, 21, 22, 23, 24), CHOP/GADD153 (25), MEF2C (26), Elk1 (12), and Max (8).

In addition to regulating IL-1 and TNF-{alpha} synthesis by monocytes, p38 also controls several other cellular responses. For example, p38 activity is essential for the production of IL-10 and prostaglandin H synthase-2 (PGHS-2) by monocytes (27, 28), and the production of PGHS-2, metalloproteinases, and IL-6 from fibroblasts and endothelial cells (15, 29). Functional roles for p38 in lymphocytes have also been described. p38 is constitutively active in mouse thymocytes, suggesting a role in T cell survival and/or differentiation (30). In mouse lymph node or splenic T cells and in mouse T cell lines, anti-CD3 mAbs have recently been shown to activate p38 (31, 32). Moreover, IL-2 or IL-7 can induce an increase in p38 activity in T cell lines (33). Ag receptor or Fas-mediated apoptosis of T and B cells is accompanied by p38 activation, although inhibition of p38 activity alone does not prevent cell death (31, 34, 35, 36). Thus, it is becoming increasingly clear that p38 may participate in a variety of T cell responses.

Many of the studies on p38 function have been facilitated by the availability of pyridinyl imidazoles such as SB 203580 that specifically inhibit p38, but not other MAPKs, by occupying the p38 ATP-binding site (37, 38). Pyridinyl imidazoles have been shown to inhibit IL-1 and TNF-{alpha} biosynthesis in human monocytes (4, 39, 40) by a mechanism involving inhibition of mRNA translation (41, 42, 43) and to have therapeutic activity in inflammatory disease models (44).

TNF-{alpha} is released not only by monocytes and macrophages in response to LPS (endotoxin) produced by Gram-negative bacteria, but also by T cells in response to superantigen (exotoxin) produced by certain Gram-positive bacteria (45). For example, the lethal shock in mice triggered by the superantigen staphylococcal enterotoxin B (SEB) is mediated by T cell TNF-{alpha} production (46). Although the function of p38 in TNF-{alpha} production by monocytes has been well established, its role in TNF-{alpha} production by T cells has not yet been examined.

Here we report that in the Ag-specific human Th cell clone HA-1.70, TCR-mediated signaling via the superantigen SEB, or the specific Ag, influenza hemagglutinin (HA) peptide, up-regulated p38 activity. Antigenic stimulation also induced the activation of the p38 substrate MAPKAPK-2 and the release of TNF-{alpha}. However, TNF-{alpha} release was not completely blocked by the p38-specific inhibitor SB 203580, despite inhibition of p38 enzymatic activity. Both p38 activation and TNF-{alpha} release were inhibited by cyclosporin A (CsA), indicating that these events were dependent upon TCR-mediated signal transduction. In human peripheral blood T cells, p38 was activated differently than in the clone, being induced by CD28 cross-linking rather than SEB binding to the TCR. This CD28-mediated p38 activation was not blocked by CsA. However, as in the T cell clone, TNF-{alpha} release by peripheral T cells was not completely blocked by SB 203580, but was more thoroughly blocked by the MAPK kinase 1/ERK kinase (MEK1) inhibitor PD 098059. These data indicate that various routes of T cell stimulation can lead to p38 activation and TNF-{alpha} release depending on the cells examined. Furthermore, in contrast to monocytes, TNF-{alpha} release by T cells is only partially p38-dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, Abs, and inhibitors

HA-1.70 is an HA-specific, HLA-DR1-restricted human T lymphocyte clone from Dr. David Eckels (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). Methods for its derivation, cloning, and screening have been described elsewhere (47). The clone was restimulated weekly using gamma-irradiated (3000 rads) PBMC from a DR1-positive individual along with 2 µM antigenic peptide corresponding to HA residues 306–320 in RPMI 1640/15% human serum/15% IL-2-conditioned supernatant (Collaborative Biomedical Products, Bedford, MA). Routine testing indicated no evidence of mycoplasma contamination. Before use, cells were passed over Ficoll-Hypaque gradients to remove residual stimulator cells and washed extensively in medium without IL-2.

Jurkat, EHM (DR1+ EBV line from the 10th International Histocompatibility Workshop) and ARENT (DR6+ EBV line from NIGMS Human Genetic Mutant Cell Repository, Camden, NJ) were maintained in RPMI 1640/10% FBS (HyClone Laboratories, Logan, UT).

The anti-human CD28 mAb CD28.2 and the mouse IgG1 isotype control mAb were obtained from PharMingen (San Diego, CA). Anti-CD3{epsilon} mAb OKT3 was produced by Ortho Pharmaceutical (Raritan, NJ). Mouse {gamma}-globulin and the F(ab')2 fragment of goat anti-mouse IgG (H+L) were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Anti-p38 C-20 and anti-ERK2 C-14 rabbit polyclonal Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPKAPK-2 sheep polyclonal Ab was obtained from Upstate Biotechnology (Lake Placid, NY). SB 203580 and CsA were purchased from CalBiochem (La Jolla, CA). PD 098059 was purchased from Research Biochemicals International (Natick, MA). Rapamycin and FK520 were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). CTLA4-Ig fusion protein was purchased from Ancell (Batport, MN). Anti-B7-1-FITC (anti-CD80 mAb BB1), anti-B7-2-FITC (anti-CD86 mAb IT2.2), and the isotype control mouse mAbs IgM-FITC and IgG2b-FITC were obtained from PharMingen. Anti-CD28-phycoerythrin (PE) and the isotype control mouse mAb IgG1-PE were purchased from Becton Dickinson (San Jose, CA).

Flow cytometry

One million cells per sample were stained with 1 µg anti-CD28-PE, anti-B7-1-FITC, anti-B7-2-FITC, or isotype control mAbs in PBS/5% FCS/0.02% NaN3 on ice in a volume of 50 µl. After 30 min, the cells were washed once, fixed in 1% formaldehyde, and analyzed using a FACSsort (Becton Dickinson).

Purification of peripheral T cells and adherent monocytes

Whole blood from healthy donors was obtained by venipuncture into heparinized vacuum tubes and centrifuged at 600 x g for 10 min at room temperature. Buffy coats were harvested, underlaid with Accu-Paque (Accurate Chemical and Scientific, Westbury, NY), and centrifuged at 1100 x g for 20 min at room temperature. PBMC were harvested, washed twice with HBSS (Life Technologies), resuspended in RPMI 1640/10% FBS/1x penicillin-streptomycin-glutamine at 107 PBMC/ml, and placed in tissue culture petri dishes for 1 h at 37°C, 5% CO2 to allow monocytes to adhere. Adherent monocytes were cultured in RPMI 1640/10% FBS/1x penicillin-streptomycin-glutamine. Nonadherent cells were removed by gentle pipetting and combined with sheep anti-mouse IgG Dynabeads M-450 prebound with anti-CD33, CD16, and CD56 mAbs (PharMingen, San Diego, CA), and anti-CD19, MHC class II, and CD14 mAb-coated paramagnetic beads (Dynabeads M-450, Dynal, Lake Success, NY) according to manufacturer’s instructions to deplete non-T cells. The purified T cell population was 98% CD3+CD2+ by flow cytometry.

Immune complex kinase assay

Adherent monocytes (4 x 106 per sample), purified HA-1.70 T cell clone or Jurkat cells (5 x 106 per sample), and peripheral blood T cells (6 x 106 per sample) were stimulated in 1 ml RPMI 1640 with LPS (Sigma, St. Louis, MO) at 10 ng/ml, SEB (Sigma) at 0.1 µg/ml, and anisomycin (Sigma) at 5 µg/ml for 15 min at 37°C. OKT3 mAb and CD28.2 mAb were prebound to cells on ice at 10 µg/ml, cells were spun to remove unbound mAb, then bound mAbs were cross-linked with F(ab')2 goat anti-mouse IgG at 30 µg/ml for 15 min at 37°C. For stimulation with specific Ag, HA 306–320 peptide was used at a final concentration of 2.0 µg/ml and preincubated with the EHM cell line (2 x 106 cells/ml) for 30 min at 37°C. After adding the T cell clone, the cell suspension was centrifuged at 300 x g for 5 min, then incubated at 37°C for 10 min. Cells were then diluted in four volumes of cold RPMI 1640, centrifuged at 600 x g for 5 min at 4°C, and lysed in 300 µl NP40 lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P 40, 1 mM Na3VO4) containing 1x EDTA-free complete protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN). Lysates were spun at 16,000 x g for 10 min and precleared once with 50 µl of a 50% Protein A (Sigma) slurry for 30 min at 4°C. Kinases were immunoprecipitated with 2 µg Ab and 50 µl of a 50% of Protein A slurry for 2 h at 4°C. Immunoprecipitates were washed twice with NP40 lysis buffer and once with kinase reaction buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 20 mM ß-glycerophosphate, and 1x EDTA-free complete protease inhibitor mixture). Immune complex kinase reactions were performed in kinase reaction buffer containing 50 µM ATP with 0.5 µg of kinase-inactive glutathione S-transferase (GST)-MAPKAPK-2 (Upstate Biotechnology) as substrate for p38, 2 µg Hsp27 (StressGen, Victoria, BC, Canada) as MAPKAPK-2 substrate, or 2 µg myelin basic protein (MBP) (Life Technologies) as ERK substrate and 5 µCi {gamma}-32P-ATP (3000 Ci/mmol, Amersham Life Science, Arlington Heights, IL) per sample. After 20 min at 30°C, reactions were stopped by adding 2x SDS sample buffer (Novex, San Diego, CA) containing 10% 2-ME and boiling for 5 min. Samples were electrophoresed in 10% Tris-Glycine polyacrylamide gels (Novex) and transferred to nitrocellulose or poly(vinylidene difluoride) membranes (Novex). Membranes were exposed to Hyperfilm-MP (Amersham) with intensifying screens. Individual bands were quantified on a Storm 840 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).

Western blotting

Nitrocellulose membranes were briefly soaked in ddH2O, then blocked in Tris-buffered saline (Bio-Rad Laboratories, Hercules, CA)/0.05% Tween 20 (TTBS) containing 5% BSA for 1 h at room temperature or overnight at 4°C. Membranes were probed with anti-p38 or anti-ERK2 at 0.1 µg/ml in TTBS for 1 h, washed four times with TTBS, then incubated with goat-anti-rabbit IgG conjugated to either horseradish peroxidase or alkaline phosphatase (Amersham) at 1:10,000 in TTBS/5% goat serum. Membranes were washed four times with TTBS and developed using either enhanced chemiluminescence (ECL) reagents (Amersham or NEN Life Science) and Hyperfilm-ECL (Amersham) for the horseradish peroxidase-conjugated secondary Ab or Vistra ECF substrate (Amersham) and the Storm 840 PhosphorImager system for the alkaline phosphatase-conjugated secondary Ab.

TNF-{alpha} production and cell proliferation assays

Adherent monocytes (4 x 105 cells/ml), clone HA-1.70 (2 x 106 cells/ml), or peripheral blood T cells (2–3.75 x 106 cells/ml) were cultured in 96-well tissue culture plates in RPMI 1640/1% FBS (for monocytes) or 10% FBS (for T cells) in a total volume of 0.2 ml per well in duplicate. Cells were pretreated with inhibitors for 1 h at 37°C, then stimulated as described for immune complex kinase assays. Supernatants were harvested 16 h after stimulation and assayed for TNF-{alpha} using a human TNF-{alpha} ELISA kit (Genzyme, Cambridge, MA) according to the manufacturer’s instructions. Proliferation was measured by incorporation of [3H]thymidine (1 µCi/well; 25 Ci/mM sp. act.) (Amersham) during the 24 h following harvesting of supernatants from duplicate cultures unless otherwise stated, harvesting onto filter mats, and counting in a 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS stimulates p38-dependent TNF-{alpha} release from human monocytes

To examine the p38 dependence of TNF-{alpha} release in monocytes, human monocytes were purified by plastic adherence from freshly isolated PBMCs, pretreated with various concentrations of the specific p38 inhibitor SB 203580, stimulated with LPS, and in vitro p38 kinase assays were performed. LPS stimulation increased p38 activity 3.2-fold, as measured by phosphorylation of MAPKAPK-2, a known in vivo substrate of p38 (Fig. 1GoA). Pretreatment with SB 203580 before stimulation effectively reduced the level of p38 activity, with 65% inhibition at 0.5 µM, 82% inhibition at 1 µM, and complete inhibition at 5 µM. Western blotting of p38 verified that similar levels of the enzyme were present in all samples.



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  		FIGURE 1. p38-dependent TNF-{alpha} release from human monocytes. A, In vitro immune complex kinase assay for p38 activity. Adherent monocytes (4 x 106 cells/sample) were pretreated with the indicated concentrations of SB 203580 for 1 h, then stimulated with LPS for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions performed using kinase-inactive GST-MAPKAPK-2 as substrate. Proteins were separated by SDS-PAGE and analyzed by autoradiography. The fold increase in p38 activity is shown underneath each lane. Anti-p38 immunoprecipitates were analyzed by immunoblotting with anti-p38 (C-20) Ab as a loading control. B, In vitro immune complex kinase assay for MAPKAPK-2 activity. The same monocyte lysates from A were immunoprecipitated with anti-MAPKAPK-2 Ab, and in vitro kinase reactions were performed using Hsp27 as substrate. Proteins were separated by SDS-PAGE and analyzed by autoradiography. C, Adherent monocytes (4 x 105 cells/ml) were pretreated with the indicated concentrations of SB 203580 diluted in 0.1% DMSO or PD 098059 diluted in 0.5% DMSO for 1 h, then stimulated with LPS overnight at 37°C. Supernatants were assayed for TNF-{alpha} by ELISA. TNF-{alpha} levels from cells treated with SB 203580 or PD 098059 are expressed as a percentage compared with control cells stimulated in the presence of 0.1% DMSO or 0.5% DMSO, respectively. Maximum TNF-{alpha} levels ranged from 1 to 5 ng/ml.

 
MAPKAPK-2 is one of the immediate downstream substrates for p38 in vivo (14, 18). Therefore, activation of p38 within cells can be assessed indirectly by quantifying the ability of MAPKAPK-2 to phosphorylate its substrate Hsp27 in vitro. This is advantageous because it may reflect the activation of other p38 family members capable of phosphorylating it, namely p38ß and p38ß2 (48), which would otherwise not be detected by the direct p38{alpha} immune complex kinase assay. Also, SB 203580, which binds the ATP-binding site of p38 (49) and does not inhibit MAPKAPK-2 (L. Wang, unpublished observations, and 37 , could be partially washed away during immunoprecipitation, thereby leading to artificially high p38 activity in immune complex kinase assays. Accordingly, MAPKAPK-2 activity was increased over twofold in LPS-stimulated monocytes, and this activation was more sensitive to inhibition by SB 203580, with complete inhibition at 0.5 µM (Fig. 1GoB).

In Fig. 1GoC, monocytes were pretreated with SB 203580, stimulated with LPS, and the levels of TNF-{alpha} in 16-h culture supernatants were measured by ELISA. SB 203580 inhibited TNF-{alpha} release with a 50% inhibitory concentration (IC50) of ~0.15 µM (Fig. 1GoC), which is in agreement with previous reports (40, 41, 44). These results confirmed the correlation between p38 activation and TNF-{alpha} release from LPS-stimulated monocytes. To examine the dependence of TNF-{alpha} production on the ERK pathway, PD 098059, an inhibitor of MEK1, an upstream activator of ERK1 and ERK2 (50, 51), was also used. PD 098059 blocked TNF-{alpha} production by adherent monocytes in response to LPS, with an IC50 of ~3 µM (Fig. 1GoC). These results are consistent with the reported activity of PD 098059, which has been shown to block MEK1 activity in vitro with an IC50 of 2–7 µM (51). PD 098059 has also been shown to block TNF-{alpha} release from IL-17-stimulated human macrophages (52). Thus, LPS-stimulated TNF-{alpha} production in adherent monocytes is dependent on both the p38 and the ERK pathways.

SEB and specific Ag activate p38 in human T cell clone, but TNF-{alpha} release is partially p38-independent

T cells are another source of TNF-{alpha} produced during the course of an immune response. Unlike monocytes, T cells do not respond to LPS because they lack CD14, the LPS receptor. Rather, TNF-{alpha} production from T cells requires signaling through the TCR/CD3 complex as well as a costimulatory molecule, usually CD28 (reviewed in Refs. 53 and 54). This activation can be accomplished using either a superantigen or a specific MHC class II/peptide complex in the presence of proper costimulation. Clone HA-1.70 is an HA-specific, DR1-restricted human T cell clone, representative of a memory-type T cell (47). HA-1.70 can be activated by SEB, which binds to the Vß3 chain of the TCR, in the absence of APC.

SEB stimulation of HA-1.70 resulted in a threefold increase of p38 activity (Fig. 2GoA, lane 6). By comparison, the translational inhibitor anisomycin, which is known to activate p38 (55), caused a sevenfold increase in p38 activity (Fig. 2GoA, lane 2). Pretreatment of clone HA-1.70 with SB 203580 before stimulation inhibited SEB-induced p38 activation 75% at 0.5 µM and 80% at 5 µM. Anisomycin-induced activity was inhibited 72% at 0.5 µM and 93% at 5 µM. MAPKAPK-2 was also activated by SEB and anisomycin, as shown by phosphorylation of its substrate, Hsp27, and this activation was inhibitable by pretreatment of the cells with SB 203580 (Fig. 2GoB), confirming the activation of p38 within this clone.



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  		FIGURE 2. SEB-induced p38 activation and TNF-{alpha} release from the human T cell clone HA-1.70. A, In vitro immune complex kinase assay for p38 activity: T cell clone HA-1.70 (5 x 106 cells/sample) was pretreated with indicated concentrations of SB 203580 for 1 h, then stimulated with anisomycin and SEB for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions performed using GST-MAPKAPK-2 as substrate. Proteins were separated by SDS-PAGE and analyzed by autoradiography. The fold increase in p38 activity is shown underneath each lane. Anti-p38 immunoprecipitates were analyzed by immunoblotting with anti-p38 (C-20) Ab as a loading control. B, In vitro immune complex kinase assay for MAPKAPK-2 activity. The same T cell lysates from B were immunoprecipitated with anti-MAPKAPK-2 Ab, and in vitro kinase reactions were performed using Hsp27 as substrate. The proteins were separated by SDS-PAGE and analyzed by autoradiography. C, Human T cell clone HA-1.70 (2 x 106 cells/ml) was pretreated with the indicated concentrations of SB 203580 diluted in 0.1% DMSO for 1 h, then stimulated with SEB (0.1 µg/ml) for 16 h at 37°C, and supernatants were harvested for TNF-{alpha} assay. Cell proliferation over the next 24 h was measured by [3H]TdR incorporation. TNF-{alpha} production and cell proliferation are expressed as a percentage compared with control cells stimulated in the presence of 0.1% DMSO. Control cultures typically produced TNF-{alpha} levels in the range of 275–600 pg/ml.

 
To examine the dependence of TNF-{alpha} production on p38 activity in T cells, HA-1.70 was pretreated with various concentrations of SB 203580, stimulated with SEB, and levels of TNF-{alpha} in 16-h culture supernatants were measured by ELISA. Unlike monocytes, T cell production of TNF-{alpha} was not completely inhibited by SB 203580, with no more than 70% inhibition at concentrations as high as 10 µM (Fig. 2GoC). At high concentrations, SB 203580 began to inhibit cell proliferation, thereby reducing the number of T cells available to produce TNF-{alpha}. Thus, in an SEB-stimulated T cell clone, TNF-{alpha} release was only partially inhibitable by SB 203580.

The effects of specific Ag, HA peptide 306–320, on p38 activation and TNF-{alpha} release from clone HA-1.70 were also examined by treatment with various concentrations of SB 203580 before the addition of irradiated APC and HA peptide. p38 was activated 3.1-fold by presentation of antigenic HA peptide by the EHM B cell line, which expresses the MHC class II allele appropriate for stimulation of HA-1.70 (HLA-DR1), but not by presentation of HA peptide by the ARENT cell line, which expresses HLA-DR6 (Fig. 3GoA). Activation was 80–95% inhibited by SB 203580 at 0.5–5 µM (Fig. 3GoA), similar to the inhibition of SEB-induced activation.



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  		FIGURE 3. Specific Ag (HA peptide)-induced p38 activation and TNF-{alpha} release from the human T cell clone HA-1.70. A, In vitro immune complex kinase assay for p38 activity. T cell clone HA-1.70 (5 x 106 cells/sample) was pretreated with indicated concentrations of SB 203580 for 1 h, then stimulated with HA peptide and stimulator cells (2 x 106 cells/sample) for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions performed using GST-MAPKAPK-2 as substrate. Proteins were separated by SDS-PAGE and analyzed by autoradiography. The fold increase in p38 activity is shown underneath each lane. Anti-p38 immunoprecipitates were analyzed by immunoblotting with anti-p38 (C-20) Ab as a loading control. B, Human T cell clone HA-1.70 (2 x 106 cells/ml) was pretreated with indicated concentrations of SB 203580 diluted in 0.1% DMSO for 1 h, then stimulated with HA peptide at 2.0 µg/ml presented by DR1+ EHM stimulator cells (8 x 105 cells/ml) for 16 h at 37°C, and supernatants were harvested for TNF-{alpha} assay. Cell proliferation over the next 24 h was measured by the [3H]TdR incorporation. TNF-{alpha} production and cell proliferation are expressed as a percentage compared with control cells stimulated in the presence of 0.1% DMSO. Control TNF-{alpha} production was 1000 pg/ml.

 
HA-1.70 also produced TNF-{alpha} in response to HA peptide presented by EHM cells (Fig. 3GoB), but not when presented by ARENT cells (data not shown). SB 203580 inhibited TNF-{alpha} production no more than 40%, even at concentrations as high as 5.0 µM (Fig. 3GoB). At this concentration, SB 203580 affected TNF-{alpha} production and cell proliferation to the same extent, inhibiting ~25% (Fig. 3GoB). Therefore, TNF-{alpha} production elicited by specific peptide Ag presented by APC was only moderately p38-dependent.

ERK-dependence of TNF-{alpha} release from human T cell clone

To further examine the mechanism by which TCR signals mediate TNF-{alpha} production, another MAPK pathway was investigated. TNF-{alpha} production by human T cells activated with anti-CD3 mAb and phorbol ester or with anti-CD3 and anti-CD28 mAbs has been blocked using the MEK1 inhibitor PD 098059 (56). Therefore, we surmised that stimulation of the HA-1.70 with SEB might also result in ERK-dependent TNF-{alpha} production. To test this, the clone was stimulated with SEB in the presence of PD 098059 and assayed for ERK2 activity and TNF-{alpha} production. SEB induced a 17.8-fold activation of ERK2 (Fig. 4GoA), compared with a 6.1-fold increase in p38 activity in the same sample (Fig. 4GoB). PD 098059 (10 µM) inhibited ERK2 activity by 67% while actually causing a slight increase in p38 activity, demonstrating the specificity of PD 098059 for the MEK1-ERK pathway. This inhibitor blocked TNF-{alpha} production by 45% at 15 µM (Fig. 4GoC). PD 098059 has previously been shown to block TNF-{alpha} production from human peripheral T cells by 80% at 12 µM (56). This compound had no effect on cell proliferation at concentrations up to 15 µM and inhibited proliferation by <20% at 50 µM (Fig. 4GoC). Therefore, TNF-{alpha} production in clone HA-1.70 stimulated with SEB appears to be partially ERK-dependent.



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  		FIGURE 4. The effects of the MEK1 inhibitor and CTLA4-Ig on ERK activity, p38 activity, and TNF-{alpha} production by SEB-stimulated T cell clone HA-1.70. A, In vitro immune complex kinase assay for ERK activity: T cell clone HA-1.70 (5 x 106 cells/sample) was pretreated with indicated concentrations of the MEK1 inhibitor PD 098059 (10 µM), the p38 inhibitor SB 203580 (1 µM), or CTLA4-Ig (10 µg/ml) for 1 h, then stimulated with SEB for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-ERK2 (C-14) Ab, and in vitro kinase reactions performed using MBP as substrate. Anti-ERK2 immunoprecipitates were analyzed by immunoblotting with anti-ERK2 (C-14) Ab as a loading control. B, The same cell lysates as in A were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions performed using GST-MAPKAPK-2 as substrate. C, Human T cell clone HA-1.70 (2 x 106 cells/ml) was pretreated with the indicated concentrations of PD 098059 diluted in 0.5% DMSO for 1 h, then stimulated with SEB (0.1 µg/ml). After 16 h, supernatants were harvested for TNF-{alpha} ELISA. TNF-{alpha} levels are expressed as a percentage compared with control cells stimulated in the presence of 0.5% DMSO, typically 275–600 pg/ml. D, Clone HA-1.70 (2 x 106 cells/ml) was pretreated with the indicated concentrations of CTLA4-Ig for 1 h, then stimulated with SEB (0.1 µg/ml). After 16 h, supernatants were harvested for TNF-{alpha} ELISA. TNF-{alpha} levels ranged from 240 to 350 pg/ml.

 
SEB-induced p38 activation in T cell clone is CD28-independent

The requirement of costimulatory signals for T cell activation with superantigens has been controversial. It has been shown that a CD28-deficient mouse strain could not produce serum TNF-{alpha} in response to toxic shock syndrome toxin-1 (57), but a different CD28-deficient mouse strain could produce serum TNF-{alpha} in response to a primary SEB injection (58). Anti-B7 mAbs blocked SEB-induced proliferation of mouse spleen cells (59), but CTLA4-Ig did not prevent proliferation or IL-2 production by human CD4+ T cells activated by SEA in the presence of APC (60). Thus, superantigen-induced T cell activation may display different requirements for B7-CD28-mediated costimulation, depending on the nature of the particular system.

Flow cytometric analysis of HA-1.70 showed that this clone expresses CD28 as well as its ligands B7-1 and B7-2 (Fig. 5Go). Thus, the clone has the potential to provide itself with B7 and CD28-mediated costimulatory signals. To determine whether the p38 activation observed in clone HA-1.70 upon stimulation with SEB was dependent upon endogenous B7-mediated costimulation, cells were pretreated with CTLA4-Ig to prevent B7-1 and B7-2 binding to CD28. Neither p38 nor ERK activation was inhibited by the addition of CTLA4-Ig (10 µg/ml) (Fig. 4Go, A and B). Rather, p38 and ERK2 activity were moderately increased. CTLA4-Ig also did not inhibit TNF-{alpha} production by the clone, although it did completely block IL-2 production by Jurkat cells stimulated with SEE in the presence of Raji B cells (data not shown). The addition of blocking mAb against B7-1 or B7-2 also failed to inhibit p38 activation or proliferation of the clone (data not shown). Hence, the activation of p38 by SEB in clone HA-1.70 appears to be independent of costimulation mediated by B7. This finding is similar to the reported inability of CTLA4-Ig to block SEA plus APC-induced proliferation and IL-2 production by human CD4+ T cells (60). However, we cannot rule out the possibility that other self-costimulatory interactions in HA-1.70 may have contributed to the observed activation of p38 by superantigen.



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  		FIGURE 5. CD28, B7-1, and B7-2 expression patterns in human T cell clone HA-1.70, human peripheral blood T cells, and Jurkat cells. HA-1.70 (white shading), peripheral blood T cells (gray shading), and Jurkat cells (black shading) were stained with: A, anti-CD28-PE (solid lines) or the isotype control mouse IgG1-PE (dotted lines); B, anti-B7-1-FITC (solid lines) or the isotype control mouse IgM-FITC (dotted lines); C, anti-B7-2-FITC (solid lines) or the isotype control mouse IgG2b-FITC (dotted lines).

 
p38 activation in peripheral blood T cells by CD28 cross-linking

To analyze p38 activation through the TCR and CD28 in a system that is dependent upon CD28 costimulation, T cells were purified from human peripheral blood by magnetic immunodepletion of non-T cells from PBMC. Peripheral blood T cells have been shown to proliferate in response to SEB in the presence of anti-CD28 mAb (61). Purified T cells expressed CD28, but little or no B7-1 or B7-2 (Fig. 5Go). The T cells were stimulated with SEB and anti-CD28 for both the TNF-{alpha} production assay and p38 in vitro kinase assay. Unlike the clone, the addition of SEB to purified normal T cells resulted neither in TNF-{alpha} production (Fig. 6GoA) nor in p38 activation (Fig. 6GoB). Anti-CD28 mAb alone did not induce TNF-{alpha} production, but did result in activation of p38. The combination of SEB and anti-CD28 mAb was required for TNF-{alpha} induction (Fig. 6GoA), which was accompanied by a modest increase in p38 activity (Fig. 6GoB). Pretreatment of the T cells with SB 203580 (1 µM) caused only a modest (<20%) reduction in TNF-{alpha} production, but completely inhibited p38 activity. Thus, as with clone HA-1.70, TNF-{alpha} production in normal primary T cells was not entirely p38-dependent.



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  		FIGURE 6. p38 activation in human peripheral blood T cells and Jurkat cells. A, CD3+ T cells were purified from peripheral blood by negative selection and stimulated (3.75 x 106 cells/ml) with SEB and/or anti-CD28 mAb plus F(ab')2 goat anti-mouse IgG either alone or in combination, with or without 1 µM SB 203580. After 16 h, culture supernatants were harvested for TNF-{alpha} assay. The average of two separate experiments is shown. Control TNF-{alpha} production in the presence of 0.1% DMSO was 200–600 pg/ml. B, Peripheral blood T cells (6 x 106 cells/sample) purified and stimulated as in A were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions performed using GST-MAPKAPK-2 as substrate. C, Jurkat T cells (5 x 106/sample) were stimulated with anti-CD3 and/or anti-CD28 mAb plus F(ab')2 goat anti-mouse IgG for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions were performed using GST-MAPKAPK-2 as substrate.

 
The Jurkat leukemic human T cell line was also analyzed for p38 activation via the TCR and CD28. Jurkat cells express high levels of CD28 (Fig. 5GoA) and B7-1 (Fig. 5GoB), but little or no B7-2 (Fig. 5GoC). Thus, like clone HA-1.70, Jurkat cells have the potential to constitutively provide costimulatory interactions. Because Jurkat cells express the Vß8 TCR chain rather than the Vß3 chain required for SEB binding, the anti-CD3{epsilon} mAb OKT3 was used to signal through the TCR/CD3 complex. Anti-CD3 mAb caused a 2.3-fold activation of p38 in Jurkat cells (Fig. 6GoC). Anti-CD28 mAb CD28.2 activated p38 a modest 1.7-fold, while the simultaneous cross-linking of both CD3 and CD28 resulted in a 3.7-fold activation (Fig. 6GoC). Thus, both TCR/CD3 and CD28 signals activated p38 in Jurkat cells, and the combined signals were approximately additive.

ERK-dependent TNF-{alpha} release by peripheral blood T cells

Because TNF-{alpha} production in clone HA-1.70 was found to involve the MEK1/ERK pathway, peripheral blood T cells were also treated with the MEK1 inhibitor PD 098059 and monitored for ERK activity and TNF-{alpha} production. PD 098059 inhibited ERK activity and TNF-{alpha} similarly, with nearly identical IC50 values of 4 and 8 µM, respectively, while cell proliferation was relatively unaffected (<30% inhibition) (Fig. 7GoA). By comparison, SB 203580 inhibited p38 activity with an apparent IC50 of 0.2 µM, yet inhibited TNF-{alpha} production no more than 40% (Fig. 7GoB). TNF-{alpha} production in the presence of SB 203580 appeared to correlate with cell proliferation, suggesting that the decrease in cytokine production might have been caused by a decrease in cell number (Fig. 7GoB). Overall, TNF-{alpha} production in peripheral T cells correlated better with ERK activity than with p38 activity.



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  		FIGURE 7. ERK-dependent TNF-{alpha} production by human peripheral blood T cells. CD3+ T cells purified from peripheral blood were pretreated with the indicated concentrations of PD 098059 diluted in 0.5% DMSO (A) or SB 203580 diluted in 0.1% DMSO (B) and stimulated with SEB and anti-CD28 plus F(ab')2 goat anti-mouse IgG. For ERK and p38 in vitro kinase assays, cells (6 x 106/sample) were lysed 15 min poststimulation. For TNF-{alpha} production, cells (2 x 106/ml) were cultured for 48 h, and cell proliferation was measured by [3H]TdR incorporation during the 24 h following supernatant harvest. TNF-{alpha} production and proliferation of cells treated with SB 203580 or PD 098059 are expressed as a percentage of control cultures stimulated in the presence of 0.1% DMSO or 0.5% DMSO, respectively. Control TNF-{alpha} levels were 200–700 pg/ml.

 
SEB-induced p38 activation is calcineurin-dependent, but CD28-mediated p38 activation is not

The Ca2+/calmodulin-regulated serine/threonine phosphatase calcineurin is essential for TCR-mediated signal transduction. The immunosuppressants CsA and FK506 inhibit calcineurin activity by forming complexes with the immunophilins cyclophilin and FK506-binding protein 12 (FKBP12), respectively, which subsequently bind to calcineurin and inhibit activation of transcription factors such as NFAT (62, 63). Unlike TCR/CD3 signaling, CD28 signaling is largely insensitive to CsA and FK506 (64). Therefore, these compounds were used to distinguish between TCR/CD3- and CD28-mediated signaling in clone HA-1.70. Rapamycin, like FK506, binds with high affinity to FKBP12, but the complex fails to inhibit calcineurin activity (63). Therefore, rapamycin can be used to competitively reverse inhibition by FK506. When added to HA-1.70 before SEB stimulation, both CsA and FK520 (a less potent analogue of FK506) inhibited TNF-{alpha} production (Table IGo). Rapamycin alone had no effect, but prevented the inhibition of TNF-{alpha} production by FK520, as expected.


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  		Table I. TNF-{alpha} production is calcineurin-dependent1

 
SEB-mediated activation of p38 was blocked by either CsA or FK520, but not rapamycin (Fig. 8GoA). Rapamycin prevented the inhibition of p38 by FK520, verifying the specificity of FK520 in this system (Fig. 8GoA). In contrast, the activation of p38 in response to anisomycin was not inhibited by CsA (Fig. 8GoB), indicating that anisomycin activates p38 in a manner distinct from p38 activation by TCR/CD3 signaling. In purified peripheral blood T cells, CsA and FK520 were unable to block CD28-mediated activation of p38 by more than 25 or 30% (Fig. 8GoC). These results indicate that the activation of p38 by SEB stimulation in clone HA-1.70 is calcineurin-dependent and, therefore, predominantly TCR/CD3-mediated. In contrast, in peripheral T cells, TCR signaling did not appear to be involved in p38 activation because p38 was not inhibited by CsA, and SEB plus anti-CD28 did not increase p38 activation above that seen with anti-CD28 alone.



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  		FIGURE 8. SEB-induced p38 activation in clone HA-1.70 is calcineurin-dependent, but CD28-mediated p38 activation in peripheral blood T cells is not. A, Clone HA-1.70 (5 x 106 cells/sample) was pretreated with 2 µM rapamycin for 45 min or 100 ng/ml CsA and/or 25 nM FK520 for 15 min, then stimulated with SEB for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions were performed using GST-MAPKAPK-2 as substrate. B, Clone HA-1.70 (5 x 106 cell/sample) was pretreated with 1.0 µM SB 203580 for 45 min or 100 ng/ml CsA for 15 min, then stimulated with anisomycin for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reaction were performed using GST-MAPKAPK-2 as substrate. C, Peripheral blood T cells (6 x 106 cells/sample) were pretreated with 2 µM rapamycin for 45 min or 100 ng/ml CsA and/or 25 nM FK520 for 15 min, then stimulated with anti-CD28 mAb plus F(ab')2 goat anti-mouse IgG, with or without SEB, for 15 min at 37°C. Total cell lysates were immunoprecipitated with anti-p38 (C-20) Ab, and in vitro kinase reactions were performed using GST-MAPKAPK-2 as substrate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data documenting activation of p38 kinase in the human T cell clone HA-1.70 by the superantigen SEB or specific Ag, HA peptide, is the first demonstration that physiological T cell stimuli can activate the p38 MAPK pathway. Activation required TCR-mediated signals, as evidenced by the inhibition of p38 activation by the calcineurin inhibitor CsA, a potent inhibitor of TCR-mediated signaling that does not inhibit the CD28 costimulatory signaling pathway (64). The inability to inhibit p38 activation or TNF-{alpha} production with CTLA4-Ig or anti-B7 mAbs suggests that activation of this clone was CD28-independent. Taken together, these results are similar to that of a previous study using a mouse Th1 clone, which showed that p38 was activated by anti-CD3 mAb alone in a manner not augmented by anti-CD28 mAb (32). However, the potential activation of p38 in clone HA-1.70 via costimulatory signals other than those induced by B7-CD28 interactions could not be ruled out.

To directly address the role of CD28 signaling in p38 activation, we used peripheral blood T cells and the T cell line Jurkat, both of which express higher levels of CD28 than the HA-1.70 clone. We found that in Jurkat cells, p38 was activated by both anti-CD3 and anti-CD28 mAbs in an additive fashion. This finding is similar to those of previous studies using Jurkat cells, which have shown that p38 can be activated by anti-CD3 mAb alone (65) and synergistically using both anti-CD3 and anti-CD28 mAbs (66). Surprisingly, we found that in peripheral blood T cells, p38 was activated by CD28 cross-linking alone, but not by SEB stimulation alone, and p38 activity in cells treated with SEB and anti-CD28 mAb was not inhibited by CsA. Also, anti-CD3 mAb alone did not induce p38 activation in peripheral blood T cells (data not shown). The biological relevance and downstream effects of this CD28-induced p38 activation will be discussed in detail elsewhere.3 These results are somewhat different from those reported for IL-2-treated mouse lymph node T cells, where CD3 and CD28 mAbs have been shown to activate p38 synergistically (31). The ability of anti-CD3 mAb to activate p38 in IL-2-treated mouse lymph node T cells, but not in resting human peripheral blood T cells, may be the result of a difference in the activation state of the cells or a difference in the stimulating ability of the mAbs used.

Interestingly, although activation of the T cell clone by antigen resulted in p38 activation and TNF-{alpha} release, the p38-specific inhibitor SB 203580 did not completely inhibit TNF-{alpha} release. TNF-{alpha} production stimulated by APC presentation of antigenic peptide was less inhibitable than TNF-{alpha} production stimulated by SEB. This may be due to additional or stronger costimulatory signals provided by the APC. In peripheral blood T cells, inhibition of TNF-{alpha} production by SB 203580 was also very weak, and correlated with inhibition of cell proliferation. In monocytes, p38 inhibitors have been shown to prevent LPS-induced TNF-{alpha} mRNA translation at the initiation step (41, 42), via a mechanism involving an AUUUA repeat motif in the 3'-untranslated region (43). In contrast, our data indicate the existence of an additional, p38-independent, pathway for TNF-{alpha} production in T cells. This pathway may involve ERK activity, as suggested by the inhibition of TNF-{alpha} production by the MEK1 inhibitor PD 098059 in clone HA-1.70 (Fig. 4Go), and in peripheral T cells (Fig. 7Go). The degree of inhibition of TNF-{alpha} production by PD 098059 in peripheral T cells stimulated with SEB and anti-CD28 mAb (Fig. 7Go) was very similar to that previously reported using stimulation with anti-CD3 and anti-CD28 mAbs (56). Thus, TNF-{alpha} production in monocytes and T cells is differentially dependent on p38.

SB 203580 also did not affect Ag-driven proliferation of clone HA-1.70. Because this clone is IL-2-dependent, the lack of inhibition of proliferation suggests that p38 is not required for IL-2R expression or IL-2 signaling in these cells. This is in contrast to a recent study showing that IL-2 could activate p38 in a mouse T cell line, and that SB 203580 could inhibit proliferation of human PBMC stimulated with anti-CD3 plus IL-2 (33). Inhibition in this case was observed with concentrations of SB 203580 in the micromolar range (IC50 = 3 µM), doses at which we saw no more than a 25% decrease in proliferation (correlating with decreased cell viability). Furthermore, the IC50 for SB 203580 inhibition of p38 enzymatic activity or the activity of its in vivo substrate MAPKAPK-2 was <=0.5 µM, suggesting that the higher concentrations used to inhibit proliferation in the previous study may have inhibited other pathways. For example, SB 203580 can inhibit p56lck at high concentrations (IC50 ~5 µM) (J. Davis, unpublished observations).

Recent studies have identified several related p38 family members in addition to the original p38{alpha} (CSBP2, RK) (4). These include p38ß (21), p38ß2, a splice variant of p38ß (48), p38{gamma}/SAPK3 (67), p38{delta}/SAPK4 (7), and Mxi2, an apparent splice variant of p38{alpha} (8). In vitro, SB 203580 inhibits p38{alpha} and p38ß2 and partially inhibits p38ß (48). It does not inhibit p38{gamma} or p38{delta} (48), and its activity against other family members is unknown. Therefore, as with other studies using this inhibitor, our results do not rule out the involvement of p38{gamma} or p38{delta} in the responses measured in these T cells. p38{gamma} does not appear to be expressed in lymphoid tissues (67), while p38{delta} is expressed in lymph node and spleen (7). Further analysis of this issue awaits the availability of anti-p38{delta} Abs. It should also be noted that the anti-p38 Ab used in our studies was raised against the carboxyl-terminal 20 amino acids of p38{alpha} and does not cross-react with p38ß. Therefore, only p38{alpha} was present in the immune complex kinase assays using MAPKAPK-2 as substrate for direct assessment of p38 activity. This was our predominant reason for assaying p38 activity indirectly by precipitating MAPKAPK-2, which is a substrate for p38{alpha}, p38ß, and p38ß2, but not for p38{gamma} and p38{delta} (7). The higher IC50 value for SB 203580 inhibition of SEB-stimulated p38 activity in the direct p38 activity assay vs the indirect MAPKAPK-2 activity assay might be accounted for by the presence of different p38 isoforms that may phosphorylate MAPKAPK-2 in the clone, although SB 203580 is most potent against p38{alpha} in vitro (48). Alternatively, the inhibitor may have been partially washed out during the process of immunoprecipitation, thereby allowing increased kinase activity in the assay. This would not have been a factor in the assay of MAPKAPK-2 activity. Finally, SB 203580 is reportedly p38-specific (37), but it does have detectable activity on at least one other enzyme, p56lck (IC50 ~5 µM), and the possible existence of other kinase or nonkinase targets cannot be absolutely ruled out. Regardless of these considerations, our studies clearly demonstrate that p38{alpha} is activated via various T cell stimulation signals depending on the cell type examined, yet indicate that p38{alpha} and/or p38ß is not required for TNF-{alpha} release from clone HA-1.70 or from normal peripheral blood T cells, in contrast to the critical role of p38 in LPS-induced TNF-{alpha} production in monocytes. It is anticipated that further studies will elucidate the role of p38 kinases in T cell functions and provide valuable insights into the potential of p38 inhibitors as therapeutic agents.


   Acknowledgments
 
We thank B. Fahmy for her excellent assistance with monocyte cell cultures.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. John J. Siekierka, Drug Discovery Research, R. W. Johnson Pharmaceutical Research Institute, Route 202, P.O. Box 300, Raritan, NJ, 08869-0602. E-mail address: Back

2 Abbreviations used in this paper: CsA, cyclosporin A; ERK, extracellular signal-regulated kinase; FKBP12, FK506 binding protein-12; GST, glutathione S-transferase; HA, influenza hemagglutinin; Hsp27; heat shock protein 27; IC50, 50% inhibitory concentration; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; MBP, myelin basic protein; MEK1, MAPK kinase 1/ERK kinase; p38, p38 MAPK; PE, phycoerythrin; PGHS-2, prostaglandin H synthase-2; SAPK, stress-activated protein kinase; SEA, SEB, SEE; staphylococcal enterotoxin A, B, and E. Back

3 Schafer, P. H., S. A. Wadsworth, L. Wang, and J. J. Siekierka. p38{alpha} mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and differentiated TH2 cells. Submitted for publication. Back

Received for publication April 7, 1998. Accepted for publication September 24, 1998.


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 Materials and Methods
 Results
 Discussion
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