Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-γ, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-γ was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-γ synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.

Eosinophilic inflammation is the characteristic feature of bronchial asthma (1, 2, 3). Activated T cells and T cell cytokines including IL-5 are crucially involved in the local infiltration and activation of eosinophils (4, 5). IL-5 is a potent growth factor, differentiation factor, activating factor, and chemotactic factor for human eosinophils (6, 7). The number of CD4+ T cells expressing IL-5 mRNA is increased in the bronchial mucosa of symptomatic asthmatics (8), is correlated with the number of activated eosinophils, and is further increased upon allergen inhalation challenge (9, 10). IL-5, but not IL-4, mRNA expression in the bronchial mucosa is correlated with the severity of asthma (11). Bronchoalveolar lavage fluid obtained from atopic and nonatopic asthmatics showed an increased concentration of IL-5 (12). IL-5 is the predominant eosinophil-active cytokine present in bronchoalveolar lavage fluid obtained from asthmatics (13). Serum IL-5 concentration was elevated in symptomatic asthmatics and decreased after oral prednisolone therapy (14). The degree of IL-5 mRNA expression correlated well with the degree of eosinophilic infiltration, whereas that of IL-3 and GM-CSF did not (15). Several experimental asthma models revealed that administration of anti-IL-5-neutralizing Ab inhibited the recruitment of eosinophils into the lung and abrogated late phase bronchoconstriction (16, 17, 18, 19), clearly indicating the essential role of IL-5 in the late phase asthmatic response. In addition, we have reported that IL-5 production by CD4+ T cells of asthmatic patients is significantly enhanced compared with that of healthy controls (20). T cells producing IL-3, GM-CSF, and IL-5 were also demonstrated in the late phase cutaneous reaction of atopic dermatitis patients upon Ag challenge (21). Accumulating evidence collectively indicates that IL-5 is the key cytokine involved in the allergic disorders associated with eosinophilic inflammation, and the regulation of T cell IL-5 synthesis seems to be an effective way to manage eosinophilic disorders including bronchial asthma and atopic dermatitis.

Glucocorticoids (GC)3 have been the most effective treatment for chronic asthma (22). The efficacy of GC is ascribed to their multiple pharmacological actions, one of which is the suppression of inflammatory cytokine production (23, 24). We and others have reported that IL-5 synthesis by peripheral Th cells is inhibited by GC in vitro (20, 25, 26, 27). GC, however, possess a wide range of pharmacological actions not only on the immune system, but also on various tissues and organs to cause multiple side effects including hypertension, diabetes mellitus, osteoporosis, etc., which often limit their clinical efficacy. The immunosuppressants FK506 and cyclosporin A were recently found to be highly effective for severe atopic dermatitis (28, 29). These agents have a more restricted target specificity compared with GC, but still interfere with multiple T cell functions, thereby causing generalized immune suppression. An agent capable of selectively regulating IL-5 synthesis with little effect on other major T cell cytokines such as IL-2 and IL-4 would provide an ideal treatment for eosinophilic inflammation without severe side effects including general immune suppression.

To elucidate the molecular requirements for T cell IL-5 synthesis, the possible involvement of p38 mitogen-activated protein (MAP) kinase in IL-5 synthesis was examined in the present study. p38 MAP kinase, which is also termed cytokine-suppressive anti-inflammatory drug-binding protein (CSBP) (30) or RK (31), is one of the three groups of MAP kinases that mediate numerous biological signals on cell proliferation, differentiation, and death. p38 MAP kinase is activated by treatment of cells with LPS, cytokines, and stress (32, 33). MAPKAP kinase-2 was first identified as a p38 MAP kinase substrate, which in turn phosphorylates HSP-25/27 (31, 34). Several transcription factors, including ATF-2 (35), CHOP/GADD153 (36), MAX (37), myocyte enhancer factor 2C (38), and ternary complex factor (39, 40, 41), have been found to be activated by p38 MAP kinase. In addition to the original p38 (also termed p38α, CSBP2, or SAPK2A), the p38 subgroup of MAP kinases now consists of CSBP1 (17), Mxi2 (37), p38β (also known as SAPK2B), p38-2 (also known as p38β2) (42, 43), p38γ (also known as ERK6 or SAPK3) (44, 45), and p38δ (also known as SAPK4) (43, 46, 47, 48). SB203580, a pyridinylimidazole compound, is a highly specific inhibitor of p38α, p38β, and p38-2 MAP kinases (42, 49). It has been reported that SB203580 inhibits the production of TNF-α and IL-1 by LPS-induced human monocytes at the translational level (30, 50, 51, 52), and IL-6 production by TNF-α-stimulated murine fibrosarcoma cells at the transcriptional level (53, 54). Induction of p38 MAP kinase in murine T cells has recently been reported, although its functional significance remains unclear (55). In the present study, we demonstrated the role of p38 MAP kinase in T cell cytokine production, and indicated that human IL-5 synthesis is regulated by a mechanism distinct from those regulating other major T cell cytokines, IL-2, IL-4, and IFN-γ.

PMA was purchased from Sigma (St. Louis, MO). Anti-CD3 (Leu4), anti-CD4 (Leu2), anti-CD8 (Leu3), and anti-CD28 (L293) mAbs were purchased from Becton Dickinson (San Jose, CA). Anti-CD3 mAb (OKT3) was from Ortho (Raritan, NJ). Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) assay kit and PhosphoPlus p38 MAP kinase (Tyr182) Ab kit were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal Ab against p38 MAP kinase (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). MAPKAP kinase-2 immunoprecipitation kinase assay kit and MAP kinase immunoprecipitation kinase assay kit were obtained from Upstate Biotechnology (Lake Placid, NY). Human rIL-2 was provided by Shionogi Pharmaceutical (Osaka, Japan). rDer f 2 was kindly provided by Dr. Y. Okumura (Asahi Brewery, Tokyo, Japan) (56). AIM-V medium (Life Technologies, Gaithersburg, MD) was used for culturing T cell clones.

Der f 2-specific human T cell clones were derived from PBMC of atopic asthmatic donors by Ag stimulation, followed by the limiting dilution method, as described previously (57). Briefly, PBMC (2 × 106/ml) were cultured with rDer f 2 protein (1 μg/ml) for 10 days in 24-well culture plates, and nonadherent cells were recovered. Then 102–104 live cells were cultured in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark) with Ag and 2500 rad-irradiated autologous PBMC (5 × 104 cells). Fresh medium containing 10 U/ml rIL-2 was added once per week. When fewer than 1 of 10 wells contained proliferating cells, the resulting cell lines were considered to have originated from a single clone. To ensure their clonality, these T cell clones were further subcloned by limiting dilution using irradiated autologous PBMC and Ag. After 10 to 14 days, expanding cultures were transferred to 24-well culture plates (Becton Dickinson). T cell clones were maintained by antigenic stimulation with irradiated autologous PBMC (2 × 106/well) and rDer f 2 protein every 2–3 wk.

T cells were harvested at least 10 days after the last antigenic stimulation, layered onto Ficoll-Paque, and centrifuged. The interface was recovered, washed twice, and resuspended in fresh medium. The resulting preparation usually consisted of more than 98% CD3-positive cells, as determined by flow cytometry. Cells (105/well) were cultured in triplicate with various stimuli in 96-well round-bottom culture plates for 24 h, and then supernatants were harvested and kept frozen at −70°C until use. In some cultures, wells were preincubated with 10 μg/ml anti-CD3 mAb (OKT3) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight. To obtain cytoplasmic RNA, T cells (4 × 106/well) were cultured in 24-well culture plates for the designated time periods. For proliferation analysis, cells were cultured for 72 h. [3H]Thymidine (0.5 μCi/well) was pulsed for the last 16 h.

IL-5 was measured by a sandwich ELISA using monoclonal anti-human IL-5 (D138) as the capture Ab and biotinylated purified rabbit anti-human IL-5 as the second Ab, as described previously (58). The linear portion of the standard curve was between 3.9 and 500 pg/ml. IL-2, IL-4, IFN-γ, and TNF-α were measured by specific ELISA (DuoSet ELISA Development System; Genzyme, Cambridge, MA), according to the manufacturer’s directions.

RNA was extracted from the pelleted cells essentially following the one-step acid guanidinium isothiocyanate/phenol-chloroform extraction method of Chomczynski and Sacchi (59) using Isogene (Nippongene, Tokyo, Japan). RNA (10 μg/lane) was denatured at 55°C for 15 min in electrophoresis buffer (40 mM MOPS, pH 7, 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50% formamide), electrophoresed through 1% agarose gel containing 2.2 M formaldehyde, and then transferred to nylon membrane filters (Hybond-N; Amersham, Arlington Heights, IL). To confirm the equal distribution of RNA in each lane, the gels were stained with ethidium bromide and visualized under UV light. The blots were prehybridized and hybridized at 42°C in 50% formamide, 5× SSC (1× SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7), 5× Denhardt’s solution (1× Denhardt’s = 200 μg/ml Ficoll, 200 μg/ml polyvinyl pyrrolidone, and 200 μg/ml BSA fraction V), 20 mM phosphate buffer (pH 6.5), and 100 μg/ml denatured salmon sperm DNA. Hybridization was conducted for 16 h after adding [32P]dCTP-labeled probes prepared using a Multiprime DNA labeling kit (Amersham). After washing to a final stringency of 0.1× SSC, 0.1% SDS at 65°C, the hybridized blots were exposed to RX film (Fuji Photo Film, Tokyo, Japan) at −70°C using a screen intensifier. The RNA blots were later stripped in 0.1% SDS at 100°C to remove traces of radioactive hybridized probes. The blots were then hybridized with other nonlymphokine probes to ensure that approximately equal amounts of RNA were present in each lane. The cDNA probes used were as follows: human IL-5 cDNA was a 0.8-kbp BamHI fragment (60), and mouse α-tubulin cDNA was a 0.6-kbp EcoRI/HindIII fragment (61). Autoradiography of the resulting Northern blots was quantified by scanning densitometry.

Activity of p38 MAP kinase and its substrate, MAPKAP kinase-2, was analyzed by in vitro kinase assay, as previously described (62). Briefly, cells were lysed on ice in 1 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X, 50 mM NaF, 2 mM Na3VO4, 30 mM Na4P2O7, 1 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF). Insoluble nuclear material was pelleted by centrifugation at 14,000 × g for 10 min at 4°C, and the supernatant (50 μg total protein) was precleared with 30 μl protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°C. Then 2 μl rabbit polyclonal anti-p38 MAP kinase Ab (Santa Cruz) was added to the precleared lysates, and each mixture was incubated for 2 h at 4°C. The mixtures were further incubated for 1 h at 4°C after the addition of 15 μl protein A-Sepharose beads. The immunoprecipitates were washed twice with lysis buffer and then twice with kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 10 mM MgCl2, 2 mM DTT, and 0.1 mM Na3VO4), and resuspended in 30 μl kinase assay buffer containing 1 μg GST-ATF-2 fusion protein (Santa Cruz), 50 μM ATP, and 10 μCi [γ-32P]ATP. The reaction mixtures were incubated at 30°C for 30 min, terminated by the addition of 30 μl of 2× SDS sample buffer containing 20 mM DTT, and then boiled for 5 min. Phosphorylation of the substrate proteins was examined by SDS-PAGE (12% gel), followed by autoradiography.

For determination of the effect of SB203580 on the in vivo activity of p38 MAP kinase, MAPKAP kinase-2 activity in cell lysates was measured by means of a MAPKAP kinase-2 immunoprecipitation assay kit, according to the manufacturer’s protocol. Briefly, cells were incubated with or without SB203580 for 30 min, stimulated, and then lysed in cold lysis buffer. After centrifugation and normalization of the protein content, extracts (100 μg protein) were mixed with 2 μg sheep anti-MAPKAP kinase-2 antiserum (Upstate Biotechnology) for 2 h at 4°C. The MAPKAP kinase-2 and anti-MAPKAP kinase-2 immune complexes were precipitated with protein G-Sepharose beads (Pharmacia Biotech). The immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer, and then resuspended in 30 μl kinase assay buffer containing 100 μM substrate peptide KKLNRTLSVA (31), 50 μM ATP, and 10 μCi [γ-32P]ATP. The reactions were incubated at 30°C for 30 min, and blotted onto p81 phosphocellulose paper. The papers were washed twice in 1% acetic acid and twice in water, and then measured with a scintillation counter.

Activity of p44/42 MAP kinase was analyzed by means of a MAP kinase immunoprecipitation assay kit (Upstate Biotechnology), according to the manufacturer’s protocol. Briefly, p44/42 MAP kinase was immunoprecipitated from cell lysates using anti-p44/42 MAP kinase Ab. In vitro kinase assay was performed using myelin basic protein as a substrate. The reactions were incubated at 30°C for 30 min, and blotted onto p81 phosphocellulose paper, which was measured with a scintillation counter. To analyze the in vitro effect of SB203580 on p44/42 MAP kinase, the precipitated enzyme obtained from the stimulated T cell clones was aliquoted, and in vitro kinase assay was performed in the presence or absence of SB203580.

Activity of JNK was analyzed by means of a SAPK/JNK assay kit (New England Biolabs). Cell lysates were incubated with GST-c-Jun fusion protein-coated beads overnight at 4°C. The resulting pellet was washed twice with lysis buffer, twice with the kinase buffer devoid of ATP, suspended in the same kinase buffer without ATP, and aliquoted. In vitro kinase assay was performed after the addition of 50 μM ATP and 10 μCi [γ-32P]ATP in the presence or absence of various concentrations of SB203580. The reactions were incubated at 30°C for 30 min, blotted onto p81 phosphocellulose paper, and measured with a scintillation counter.

Statistical analysis was performed using Student’s t test. A value of p < 0.05 was considered statistically significant. Values are presented as mean ± SEM.

The first experiment was performed to determine whether human IL-5 synthesis could be suppressed by SB203580, a selective inhibitor of p38 MAP kinase. T cell clones were used for experiments at least 10 days after the last antigenic stimulation. As described in Materials and Methods, T cells obtained from the interface of the Ficoll-Paque density gradient consisted of more than 98% pure CD3+CD4+ cells. They were washed three times, resuspended in fresh medium, and stimulated by either immobilized OKT3 mAb, immobilized OKT3 mAb plus soluble anti-CD28 mAb, PMA plus soluble anti-CD28 mAb, or rIL-2 (100 U/ml). OKT3 mAb, a stimulating anti-CD3 mAb, activates T cells by cross-linking TCR-CD3 complexes that physiologically transduce activating signals into the cytoplasm (63). As shown in Fig. 1 A, IL-5 production was clearly induced upon activation, and suppressed by SB203580 in a dose-dependent manner, although even at the highest concentration (3 μM), a partial response (10–40%) remained. Essentially the same dose responses in the effects of SB203580 on IL-5 production were observed for all four stimulation protocols. The viability of the cells was determined by the trypan blue dye exclusion test and was not changed significantly by the addition of SB203580 after 24 h, excluding nonspecific toxicity of the agent at these concentrations (data not shown).

FIGURE 1.

SB203580 inhibited IL-5 synthesis of human Th cells activated by various stimuli.A, Human T cell clones (105 per well) were stimulated for 24 h by either immobilized OKT3 mAb (□), immobilized OKT3 mAb plus soluble anti-CD28 mAb (▵), PMA plus soluble anti-CD28 mAb (▪), or rIL-2 (○, 100 U/ml) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. Supernatants were harvested after 24 h and assayed for IL-5 by a specific ELISA. Data are expressed as the mean of triplicate cultures ± SEM. IL-5 production in unstimulated cultures was always below the detection limit of the ELISA system (<1 pg/ml). B, T cell clones (105; □, HK5; ▵, YA5; ▪, HK2; ○, YA4) were cultured in 96-well round-bottom culture plates pretreated with OKT3 mAb (10 μg/ml).

FIGURE 1.

SB203580 inhibited IL-5 synthesis of human Th cells activated by various stimuli.A, Human T cell clones (105 per well) were stimulated for 24 h by either immobilized OKT3 mAb (□), immobilized OKT3 mAb plus soluble anti-CD28 mAb (▵), PMA plus soluble anti-CD28 mAb (▪), or rIL-2 (○, 100 U/ml) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. Supernatants were harvested after 24 h and assayed for IL-5 by a specific ELISA. Data are expressed as the mean of triplicate cultures ± SEM. IL-5 production in unstimulated cultures was always below the detection limit of the ELISA system (<1 pg/ml). B, T cell clones (105; □, HK5; ▵, YA5; ▪, HK2; ○, YA4) were cultured in 96-well round-bottom culture plates pretreated with OKT3 mAb (10 μg/ml).

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The effect of SB203580 on IL-5 synthesis was confirmed using other T cell clones (Fig. 1 B). SB203580 was effective in suppressing IL-5 synthesis by all four T cell clones stimulated with immobilized OKT3 mAb in a dose-dependent manner. Essentially the same results were obtained using 10 other T cell clones (data not shown).

The effect of SB203580 on the proliferation of activated T cell clones was examined. Significant proliferation of T cells was induced by all of the four stimulation protocols, and was proliferation not affected by SB203580 (Fig. 2). Essentially the same results were obtained using 10 other T cell clones, indicating that the observation was not restricted to this particular clone (data not shown). The result further confirmed that the effect of SB203580 on IL-5 production was not due to nonspecific toxicity of the agent.

FIGURE 2.

SB203580 did not inhibit the proliferation of activated T cells. T cell clones (105 per well) were stimulated for 72 h by either immobilized OKT3 mAb (□), immobilized OKT3 mAb plus soluble anti-CD28 mAb (▵), PMA plus soluble anti-CD28 mAb (▪), or rIL-2 (○, 100 U/ml) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. [3H]Thymidine (0.5 μCi/well) was pulsed for the last 16 h. Data are expressed as the mean of triplicate cultures ± SEM. [3H]Thymidine incorporation by unstimulated cells was 840 ± 266.

FIGURE 2.

SB203580 did not inhibit the proliferation of activated T cells. T cell clones (105 per well) were stimulated for 72 h by either immobilized OKT3 mAb (□), immobilized OKT3 mAb plus soluble anti-CD28 mAb (▵), PMA plus soluble anti-CD28 mAb (▪), or rIL-2 (○, 100 U/ml) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. [3H]Thymidine (0.5 μCi/well) was pulsed for the last 16 h. Data are expressed as the mean of triplicate cultures ± SEM. [3H]Thymidine incorporation by unstimulated cells was 840 ± 266.

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The effect of SB203580 on the production of other major T cell cytokines such as IL-2, IL-4, and IFN-γ was next examined. As shown in Fig. 3,A, detectable amounts of IL-4, IL-5, and IFN-γ were produced upon stimulation with immobilized OKT3 mAb. Addition of SB203580 suppressed IL-5 production in a dose-dependent manner, but did not affect IL-4 or IFN-γ production at any concentrations tested. Upon stimulation with immobilized OKT3 mAb plus soluble anti-CD28 mAb, IL-2, IL-4, IL-5, and IFN-γ were produced (Fig. 3 B). Again, IL-5, but not IL-2, IL-4, or IFN-γ production was suppressed by the addition of SB203580, clearly excluding nonspecific toxicity of the agent and indicating that the effect of p38 MAP kinase inhibitor on T cell cytokine production is restricted to IL-5 synthesis. The absence of an effect of SB203580 on IL-2, IL-4, and IFN-γ synthesis was confirmed using 10 other Th clones (data not shown).

FIGURE 3.

SB203580 did not inhibit IL-2, IL-4, or IFN-γ production. T cell clones (105 per well) were stimulated for 72 h by either immobilized OKT3 mAb (A) or immobilized OKT3 mAb plus soluble anti-CD28 mAb (B) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. Supernatants were harvested after 24 h and assayed for IL-2, IL-4, IL-5, and IFN-γ by specific ELISAs. Data are expressed as the mean of triplicate cultures ± SEM. The minimum detectable concentrations of ELISA systems were 6 pg/ml for IL-2, 3 pg/ml for IL-4, 1 pg/ml for IL-5, and 10 pg/ml for IFN-γ. Production of cytokines in the unstimulated cultures was always below the detection limit of the ELISA system.

FIGURE 3.

SB203580 did not inhibit IL-2, IL-4, or IFN-γ production. T cell clones (105 per well) were stimulated for 72 h by either immobilized OKT3 mAb (A) or immobilized OKT3 mAb plus soluble anti-CD28 mAb (B) in 96-well round-bottom culture plates. Designated concentrations of SB203580 were included from the start of some cultures. Supernatants were harvested after 24 h and assayed for IL-2, IL-4, IL-5, and IFN-γ by specific ELISAs. Data are expressed as the mean of triplicate cultures ± SEM. The minimum detectable concentrations of ELISA systems were 6 pg/ml for IL-2, 3 pg/ml for IL-4, 1 pg/ml for IL-5, and 10 pg/ml for IFN-γ. Production of cytokines in the unstimulated cultures was always below the detection limit of the ELISA system.

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p38 MAP kinase is activated upon stimulation of cells by the dual specificity kinases that phosphorylate the threonine and tyrosine residues in its TGY motifs (30). To verify that p38 MAP kinase activity was induced under the conditions used in the present study, p38 MAP kinase was immunoprecipitated from the cellular extracts with anti-p38 MAP kinase Ab, and in vitro kinase assays were performed using GST-ATF-2 fusion protein as a substrate. As shown in Fig. 4, phosphorylation of the GST-ATF-2 fusion protein was induced by the treatment of human Th clones with immobilized OKT3 mAb in a time-dependent manner, clearly indicating that p38 MAP kinase was induced upon activation of human Th cells. Western blotting of each immunoprecipitation sample was performed, as described previously (62), and demonstrated that equivalent amount of p38 MAP kinase protein was present in each sample (data not shown).

FIGURE 4.

In vivo activation of p38 MAP kinase activity in human Th clones. Human Th clones were stimulated with immobilized OKT3 mAb for the indicated time periods, and then cell lysates were prepared. Specific polyclonal Abs were used to immunoprecipitate p38 MAP kinase from the cell lysates, and in vitro kinase assays were performed using 10 μCi [γ-32P]ATP and 1 μg GST-ATF-2 fusion protein as the substrate. The result is the representative of three experiments that showed essentially the same results.

FIGURE 4.

In vivo activation of p38 MAP kinase activity in human Th clones. Human Th clones were stimulated with immobilized OKT3 mAb for the indicated time periods, and then cell lysates were prepared. Specific polyclonal Abs were used to immunoprecipitate p38 MAP kinase from the cell lysates, and in vitro kinase assays were performed using 10 μCi [γ-32P]ATP and 1 μg GST-ATF-2 fusion protein as the substrate. The result is the representative of three experiments that showed essentially the same results.

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MAPKAP kinase-2 is a substrate of p38 MAP kinase in several cell types (34). To determine whether MAPKAP kinase-2 is activated in human Th clones stimulated with immobilized OKT3 mAb and down-regulated by SB203580, MAPKAP kinase-2 was immunoprecipitated from the cell lysates obtained from unstimulated and stimulated Th clones, and then in vitro kinase assays were performed using the peptide KKLNRTLSVA as a substrate (31). As shown in Fig. 5,A, MAPKAP kinase-2 activity was induced upon stimulation, and was significantly inhibited by SB203580 in a dose-dependent manner, clearly indicating that p38 MAP kinase was activated in vivo and inhibited by SB203580. The result is representative of three separate experiments with different batches of cells. The concentration response relationship observed in the suppression of p38 MAP kinase activity was quite similar to that observed in the suppression of IL-5 synthesis (Fig. 1).

FIGURE 5.

In vivo activation of MAPKAP kinase-2 by activated human Th clones.A, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the absence or presence of SB203580 (0.1, 1, and 10 μM). Specific polyclonal Abs were used to immunoprecipitate MAPKAP kinase-2 from the cell lysates, and in vitro kinase assays were performed using 10 μCi [γ-32P]ATP and the substrate peptide KKLNRTLSVA. Data are expressed as the mean ± SEM of triplicate cultures. ∗, p < 0.01 compared with the value for the stimulated culture without SB203580. B, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min. Specific polyclonal Abs were used to immunoprecipitate p44/42 MAP kinase from the cell lysates, and in vitro kinase assays were performed in the presence and absence of SB203580 using myelin basic protein as a substrate. Data are expressed as the mean ± SEM of triplicate experiments. C, GST-c-Jun fusion protein-coated beads were used to pull down JNKs from the activated T cell clones, and in vitro kinase assays were performed after the addition of 50 μM ATP and 10 μCi [γ-32P]ATP in the presence and absence of SB203580. Data are expressed as the mean ± SEM of triplicate experiments. D, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the presence or absence of SB203580 (0.1, 1, and 10 μM). Specific polyclonal Abs were used to immunoprecipitate p44/42 MAP kinase from the cell lysates, and in vitro kinase assays were performed using myelin basic protein as a substrate. Data are expressed as the mean ± SEM of triplicate experiments. E, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the presence or absence of SB203580 (0.1, 1, and 10 μM). GST-c-Jun fusion protein-coated beads were used to pull down JNKs, and in vitro kinase assays were performed after the addition of 50 μM ATP and 10 μCi [γ-32P]ATP. Data are expressed as the mean ± SEM of triplicate experiments.

FIGURE 5.

In vivo activation of MAPKAP kinase-2 by activated human Th clones.A, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the absence or presence of SB203580 (0.1, 1, and 10 μM). Specific polyclonal Abs were used to immunoprecipitate MAPKAP kinase-2 from the cell lysates, and in vitro kinase assays were performed using 10 μCi [γ-32P]ATP and the substrate peptide KKLNRTLSVA. Data are expressed as the mean ± SEM of triplicate cultures. ∗, p < 0.01 compared with the value for the stimulated culture without SB203580. B, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min. Specific polyclonal Abs were used to immunoprecipitate p44/42 MAP kinase from the cell lysates, and in vitro kinase assays were performed in the presence and absence of SB203580 using myelin basic protein as a substrate. Data are expressed as the mean ± SEM of triplicate experiments. C, GST-c-Jun fusion protein-coated beads were used to pull down JNKs from the activated T cell clones, and in vitro kinase assays were performed after the addition of 50 μM ATP and 10 μCi [γ-32P]ATP in the presence and absence of SB203580. Data are expressed as the mean ± SEM of triplicate experiments. D, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the presence or absence of SB203580 (0.1, 1, and 10 μM). Specific polyclonal Abs were used to immunoprecipitate p44/42 MAP kinase from the cell lysates, and in vitro kinase assays were performed using myelin basic protein as a substrate. Data are expressed as the mean ± SEM of triplicate experiments. E, Human Th clones were stimulated with immobilized OKT3 mAb for 40 min in the presence or absence of SB203580 (0.1, 1, and 10 μM). GST-c-Jun fusion protein-coated beads were used to pull down JNKs, and in vitro kinase assays were performed after the addition of 50 μM ATP and 10 μCi [γ-32P]ATP. Data are expressed as the mean ± SEM of triplicate experiments.

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It has been reported recently that SB203580 suppressed several JNK isoforms (41, 61). Possible effect of SB203580 on other groups of MAP kinases was next examined. p44/42 MAP kinase was immunoprecipitated from the stimulated T cell clones. JNKs were also precipitated from the same T cell clones using GST-c-Jun fusion protein-coated beads. The effect of SB203580 was analyzed by in vitro kinase assay. As shown in Fig. 5, B and C, SB203580 at the concentrations employed in the present experiments did not significantly affect the activity of p44/42 MAP kinase (B) and JNKs (C). Essentially the same results were obtained using p44/42 MAP kinase and JNKs precipitated from two other T cell clones (data not shown). Lack of SB203580 effect on p44/42 MAP kinase and JNKs in vivo was also confirmed (Fig. 5, D and E). Incubation of the cells during in vivo stimulation with SB203580 (0.1, 1, 10 μM) did not significantly affect the activity of p44/42 MAP kinase (D) and JNKs (E).

The effect of SB203580 on IL-5 mRNA expression was determined by Northern blot analysis. It was shown by preliminary experiments that IL-5 mRNA was detectable at 4 h following stimulation, peaked at 8 h, and then declined (data not shown). RNA was prepared from the T cell clones stimulated in the presence of various concentrations of SB203580 (0.3, 1, and 3 μM). The result shown in Fig. 6 A is representative of three separate experiments employing different T cell clones. IL-5 mRNA expression induced by immobilized anti-CD3 stimulation was inhibited by SB203580 in a dose-dependent manner. Essentially the same results were obtained at 4 and 12 h following stimulation (data not shown).

FIGURE 6.

SB203580 suppressed Il-5 mRNA expression of human Th cells. A, Human T cell clones (2 × 106 per well) were stimulated for 8 h by immobilized OKT3 mAb in 24-well culture plates. SB203580 (0, 0.3, 1, and 3 μM) was included from the start of some cultures (lanes 1–3). RNA was extracted, electrophoresed (10 μg/each lane), blotted, and hybridized with IL-5 cDNA probe and α-tubulin cDNA probe. B, Relative levels of IL-5 mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the α-tubulin signal. Data are expressed as the mean ± SEM of three experiments.

FIGURE 6.

SB203580 suppressed Il-5 mRNA expression of human Th cells. A, Human T cell clones (2 × 106 per well) were stimulated for 8 h by immobilized OKT3 mAb in 24-well culture plates. SB203580 (0, 0.3, 1, and 3 μM) was included from the start of some cultures (lanes 1–3). RNA was extracted, electrophoresed (10 μg/each lane), blotted, and hybridized with IL-5 cDNA probe and α-tubulin cDNA probe. B, Relative levels of IL-5 mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the α-tubulin signal. Data are expressed as the mean ± SEM of three experiments.

Close modal

Our present findings clearly demonstrated that p38 MAP kinase regulates IL-5 synthesis by human Th cells. p38 MAP kinase was activated upon stimulation of human Th clones (Fig. 4), and inhibited by SB203580 (Fig. 5,A). SB203580 suppressed IL-5 production, but not IL-2, IL-4, or IFN-γ production (Fig. 3, A and B). Proliferation of Th clones was not affected by SB203580 (Fig. 2). Selective involvement of p38 MAP kinase in IL-5 synthesis indicates a differential regulation of the two Th2 cytokines, IL-4 and IL-5.

The involvement of Th cells in the pathogenesis of bronchial asthma has been well documented (4, 5, 14). Th2-type deviation of immune response has been implicated in eosinophilic inflammation, as the local production of IL-3, IL-4, and IL-5 is up-regulated (10, 65). Among T cell cytokines, IL-5 is a prerequisite for the development of tissue eosinophilia (13, 18, 66). Nakajima et al. reported that T cells and IL-5 are essential for eosinophilic infiltration into the lung (67). We have reported that IL-5 synthesis by Th cells obtained from atopic and nonatopic asthmatics is significantly enhanced compared with that in normal subjects (20, 68). Although GC, FK506, and cyclosporin A are effective in treating eosinophilic inflammation, a more specific inhibitor of IL-5 synthesis would be desirable, considering the broad side effects of these immunosuppressive agents.

SB203580, a pyridinyl imidazole derivative, has been shown to be a highly selective inhibitor of p38 MAP kinase, as it had no action on a wide range of other kinases, including p42 and p54 MAP kinase, and phosphatases (30, 69, 70). We also confirmed its lack of effect on the activation of p44/42 MAP kinase and JNKs in our experimental condition (Fig. 5, B and C). Recent studies revealed that p38 MAP kinase is involved in a variety of biological responses, including cell growth and stress responses (32, 33). The involvement of p38 MAP kinase in cytokine synthesis by monocytes and fibroblasts has been relatively well investigated (53, 54), whereas its involvement in T cell responses remains largely unclear. Crawley et al. recently reported that p38 MAP kinase is activated in IL-2-stimulated murine T cell lines and is involved in the proliferation of T cells (70). We have for the first time demonstrated that p38 MAP kinase is involved in IL-5 synthesis by human Th cells. The effect of SB203580 on IL-5 synthesis is a direct action on T cells, as the Th cell preparations employed in this study consisted of more than 98% CD3+CD4+ cells.

The most outstanding finding of our present study is its selective effect on IL-5 synthesis. Besides IL-5, Th clones stimulated by anti-CD3 Ab released IL-2, IL-4, and IFN-γ into the culture supernatants (Fig. 3, A and B). The production of IL-5 alone was inhibited by SB203580, while synthesis of other cytokines was unaffected. The result was confirmed using more than 10 Th clones to show that the finding was not confined to a specific clone (data not shown). This finding supports our previous reports indicating that the synthesis of IL-5 is regulated by a distinct mechanism from that of other major T cell cytokines such as IL-2, IL-4, and IFN-γ (25, 71, 72), in which it was shown that the transcriptional activity of the IL-5 gene is induced in Th cells stimulated with IL-2, whereas the IL-4 gene, another Th2 cytokine gene, is not induced. The IL-2R signal did not induce NF-AT-, AP-1-, or NF-κB-binding activity, while it induced equivalent IL-5 gene transcription compared with the TCR signal, by which NF-AT-, AP-1-, and NF-κB-binding activity was significantly induced (72).

IL-5 is produced by Th cells in response to various activation signals. Not only the activating signals mediated through the TCR, but also those mediated through the IL-2R and CD28 induced cytokine synthesis and cell proliferation (Figs. 1 and 2). IL-5 synthesis induced by all four stimulation protocols was suppressed by SB203580 at similar concentrations. These results suggest that p38 MAP kinase is involved in IL-5 synthesis in general, but is not involved in specific signal transduction such as the TCR signal, IL-2R signal, and PMA plus anti-CD28 signal. SB203580 did not suppress IL-5 synthesis completely, but partial responses always remained unsuppressed, suggesting the involvement of several pathways in IL-5 synthesis that are not sensitive to SB203580. The precise molecules involved in the transduction of IL-2 signal and PMA plus anti-CD28 signal leading to IL-5 synthesis warrant further investigation, although we have reported that unique yet-undefined transcription factor is involved in IL-5 gene transcription, which is sensitive to FK506 (72).

SB203580 did not affect the proliferative response of Th clones stimulated by the same experimental conditions as the cytokine production (Fig. 2). It has been reported that SB203580 inhibited the proliferation of fibroblasts and mature T cells (70). Although the reason for the apparent discrepancy between their results and ours is not clear at this moment, the absence of an effect of SB203580 on proliferation was confirmed using more than 10 independent Th clones (data not shown).

The activation of p38 MAP kinase under our experimental condition was confirmed by immunoprecipitation, followed by in vitro kinase assay (Fig. 4), consistent with the report by Salmon et al. (55). SB203580 inhibited the activity of MAPKAP kinase-2 (Fig. 5,A), indicating that MAPKAP kinase-2 was induced mainly by p38 MAP kinase, rather than p42 MAP kinase in the human Th clones. These results confirmed the fact that p38 MAP kinase is induced upon activation and down-regulated by SB203580 in human Th cells. Moreover, SB203580 inhibited the activation of MAPKAP kinase-2 with a similar concentration response relationship to the suppression of IL-5 synthesis (Fig. 1), consistent with the notion that IL-5 synthesis of human Th cells is dependent on p38 MAP kinase activity. It has been reported recently that SB203580 suppressed several JNK isoforms at relatively high concentrations (10–100 μM) (41, 64). To show the specificity of the agent for the inhibition of IL-5 synthesis, possible effect on p44/42 MAP kinase and JNKs was examined. SB203580 significantly suppressed IL-5 synthesis at 1 μM (IC50 of 0.3–1 μM), while it did not significantly affect the activity of either p44/42 MAP kinase or JNKs at the same concentration (Fig. 5, B and C). Lack of the effect on p44/42 MAP kinase was consistent with the reports by others (30, 41). Lack of the effect on JNK was consistent with Dean et al. reporting that the agent had little effect on monocyte JNKs up to 2 μM (64). Whitmarsh et al. reported that SB203580 suppressed JNK2β1 and JNK2β2 activity at the concentration of 10 μM and higher, but did not affect JNK1 or JNK2α isoforms up to 10 μM (41), consistent with our observation that SB203580 only slightly reduced the activity of JNKs only at 10 μM concentration (Fig. 5,C). Absence of SB203580 effect on p44/42 MAP kinase and JNKs in vivo was also confirmed (Fig. 5, D and E).

Rincón et al. investigated the regulation of p38 MAP kinase in the murine T cell populations that had been developed into Th1 and Th2 phenotypes under the influence of IL-12 and IL-4, respectively (73). They found that p38 MAP kinase activity was induced by Th1- but not Th2-developing cells upon stimulation, and p38 MAP kinase was selectively involved in IFN-γ synthesis. The discrepancy between their observation and ours might be explained by the species difference, or more likely by the fact that murine Th cell populations studied in their experiments were cultured under a strong influence of the priming cytokines (IL-4 for Th2 and IL-12 for Th1) to extremely polarize into either phenotype (74), whereas human Th clones derived from the peripheral blood exhibited mostly Th0 phenotype (57, 71). The lack of SB203580 effect on human T cell IFN-γ synthesis was confirmed using more than 10 Th clones and primary PBMC (data not shown). Matsuda et al. reported that p38 MAP kinase was involved in IL-2 gene transcription by a transformed human T cell line, Jurkat cell (75), although Rincón et al. and we have confirmed the absence of SB203580 effect on IL-2 synthesis using nontransformed Th cells. As T cell clones employed in the present study seemed to be designated as Th2 phenotype and produced very low amount of TNF-α (less than 10 pg/ml), we could not confirm the findings recently reported by Schafer et al. that SB203580 suppressed the production of TNF-α through the inhibition of p38 MAP kinase, employing an influenza hemagglutinin-specific human T cell clone (76).

In regard to the mode of SB203580 action on cytokine synthesis, IL-5 mRNA expression was significantly inhibited by the agent in a dose-dependent manner (Fig. 6 A). The finding is consistent with the reports made by several investigators using cells of non-T lymphoid lineage that SB203580 acts at the level of transcription (53, 54). Our present finding does not exclude posttranscriptional and/or translational regulation indicated by several reports (30, 50, 51, 52, 62). The target transcription factor(s) for IL-5 gene regulation by p38 MAP kinase warrants further investigation employing nuclear run-on and gel-shift assays.

In conclusion, our present study clearly indicates that IL-5 synthesis by human Th cells is regulated by p38 MAP kinase. As p38 MAP kinase is not essential for the synthesis of other major T cell cytokines, thorough elucidation of the molecules involved in human IL-5 synthesis will facilitate the future development of a specific IL-5 synthesis inhibitor as a novel treatment for allergic diseases.

We thank Dr. Y. Okumura for providing rDer f 2 protein, Ms. Yoko Nakagawa and Ms. Yumiko Asakura for technical assistance, and Dr. Wendy A. Gray for reviewing this manuscript.

1

This work was supported in part by a grant to A.M. from the Suzuken Memorial Foundation.

3

Abbreviations used in this paper: GC, glucocorticoid; ATF, activating transcription factor; CSBP, cytokine-suppressive anti-inflammatory drug-binding protein; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MAPKAP, MAP kinase activated protein; SAPK, stress-activated protein kinase.

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