Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-gamma production in mice - PubMed (original) (raw)

Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-gamma production in mice

Rajkumar Noubade et al. J Clin Invest. 2007 Nov.

Abstract

Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell-mediated models of organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-gamma production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-gamma expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-gamma production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.

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Figures

Figure 1. H1R is required for IFN-γ production by CD4+ T cells.

Purified CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs either (A) in the presence of IL-12 (4 ng/ml) and anti–IL-4 mAb (10 μg/ml), (B) in the presence of IL-4 (30 ng/ml) and anti–IFN-γ mAb (10 μg/ml), or (C) in the presence of TGF-β (1 ng/ml), IL-6 (30 ng/ml), and anti–IFN-γ (10 μg/ml) and anti-IL4 mAbs (10 μg/ml). After 4 days, the cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h. Production of (A) IFN-γ, (B) IL-4, and (C) IL-17 was determined by ELISA in triplicate. *P < 0.05, Student’s t test. (D) CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs. After 4 days, cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h, and IFN-γ production was determined by ELISA. **P = 0.002, Student’s t test. (E and F) WT and H1RKO CD4+ T cells were stimulated as in D for the indicated periods of time. Supernatants were analyzed for (E) IFN-γ (F = 168.8, P < 0.0001, 2-way ANOVA; **P < 0.01, ***P < 0.001, Bonferroni corrected post-hoc comparison) and (F) IL-2 by ELISA. (G) CD4+ T cells from WT and H1RKO mice were stimulated as in E, and 18 h [3H]-thymidine incorporation was measured in total 72 h culture. Data are representative of at least 2 independent experiments.

Figure 2. Expression and function of HA-H1R in HEK293T cells.

(A) HEK293T cells were transfected with empty pEGZ (control) and pEGZ-HA-H1R plasmids, and the expression of HA-H1R was determined by Western blot using an anti-HA mAb. Data are representative of at least 3 independent experiments. (B) HEK293T cells were transfected as in A, fixed, permeabilized, and stained with an anti-HA mAb (red) and Topro-nuclear dye (blue). EGFP expression (green) represents transfected cells. Cells were visualized by confocal microscopy. Data are representative of at least 3 independent experiments. (C) HEK293 cells were transfected with pHA-H1R-Gα11 fusion construct, and membrane fractions were isolated and used in [35S]GTPγS binding assays in the absence (basal) or presence of 10–4 M histamine. Samples were then used in immunoprecipitation using Gα11 antiserum, and the bound [35S]GTPγS was measured by liquid-scintillation spectrometry. ***P < 0.001, Student’s t test.

Figure 3. Hrh1 is downregulated upon activation in CD4+ T cells.

(A) CD4+ T cells from WT and H1RKO mice were stimulated in the presence of anti-CD3 and anti-CD28 mAbs for 16 h and then retrovirally transduced with pEGZ-HA-H1R or with empty pEGZ control plasmids. Transduced, sorted EGFP+ cells were then restimulated with anti-CD3 mAb, and 24 h later, the supernatants were harvested for determination of IFN-γ by ELISA in triplicate. Data are representative of 2 independent experiments. ***P < 0.001, Student’s t test. (B) Freshly isolated CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs. After 24 h, IFN-γ production was determined by ELISA. Data are representative of at least 3 independent experiments. ***P < 0.001, Student’s t test. (C and D) CD4+ T cells were isolated from WT mice and stimulated with anti-CD3 and anti-CD28 mAbs. Cells were harvested at the indicated time points; total RNA was isolated and used to examine Hrh1 expression by conventional RT-PCR with Hprt1 as the endogenous control (C) and by quantitative real-time RT-PCR relative to B2m as the endogenous control (D). Data (representative of at least 3 independent experiments) are presented as expression relative to the unstimulated CD4+ T cells.

Figure 4. Transgenic expression of H1R in H1RKO CD4+ T cells complements IFN-γ production.

(A) Hrh1 transgene expression was analyzed by RT-PCR in CD4+ T cells from WT and H1RKO mice and the 2 independent lines of H1R transgenic mice crossed with H1RKO mice, HIRKO-Tg1 and HIRKO-Tg3. (B) CD4+ T cells were stained with anti-HA mAb (red) and visualized by confocal microscopy. Nuclear stain Topro (blue) is shown. (C) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs for 72 h, and IFN-γ was determined by ELISA. Data are expressed as IFN-γ production relative to that by WT cells (set as 100%). (D) CD4+ T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated as in C for the indicated periods of time, and IFN-γ was determined by ELISA. F = 55.1, P < 0.0001, 2-way ANOVA. **P < 0.01, ***P < 0.001, Bonferroni corrected post-hoc comparison. (E) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of IL-12 (4 ng/ml) and anti-IL-4 mAb (10 μg/ml). After 4 days, cells were restimulated and IFN-γ production was determined. F = 25.4, P < 0.001; 1-way ANOVA. **P < 0.01, Bonferroni corrected post-hoc comparison. (F) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs. After 4 days, cells were restimulated with anti-CD3 mAb for 24 h, and IFN-γ production was determined by ELISA. F = 288.0, P < 0.0001; 1-way ANOVA. ***P < 0.001, Bonferroni corrected post-hoc comparison. Data are representative of at least 3 independent experiments.

Figure 5. Activation of p38 MAPK by TCR ligation requires H1R signals.

(A) Purified CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and nuclear extracts were prepared and analyzed for NF-κB DNA binding by EMSA. (B) CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-STAT1 (P-STAT1) and total STAT1 by Western blot analysis. Actin was used as loading control. (C) CD4+ T cells from WT and H1RKO mice were treated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-p38 MAPK and total p38 by Western blot analysis. (D) CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-ERK and total ERK by Western blot analysis. (E) CD4+ T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for phospho-p38, total p38, and actin by Western blotting. Data are representative of at least 2 independent experiments.

Figure 6. Activation of p38 MAPK by TCR ligation is mediated by histamine/H1R binding.

(A) CD4+ T cells from WT and H1RKO mice were treated with histamine (10–7 M) for the indicated periods of time in the histamine-free medium. Whole-cell extracts were used to analyze phospho-p38, total p38, and actin by Western blotting. (B) CD4+ T cells were isolated from WT and H1RKO mice and stimulated with anti-CD3 and anti-CD28 mAbs in the histamine free-medium for the indicated periods of time. CD4+ T cells stimulated in medium containing 10–7 M histamine (Hist) are shown as positive control for p38 MAPK activation. Phospho-p38, total p38, and actin are shown. (C) CD4+ T cells from WT and H1RKO mice were incubated with anti-CD3 and anti-CD28 mAbs, 10–7 M histamine, or both in the histamine-free medium for 30 minutes, and whole-cell lysates were analyzed for phospho-p38, total p-38, and actin by Western blotting. (D) CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for T-bet expression by Western blot. Actin is shown as loading control. (E) Purified CD4+ T cells from WT, H1RKO, and H1RKO-MKK6Glu Tg mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and supernatants were analyzed for IFN-γ production by ELISA in triplicate. F = 21.7, P < 0.0001, 2-way ANOVA. ***P < 0.001, Bonferroni corrected post-hoc comparison. Data are representative of at least 2 independent experiments.

Figure 7. H1R signaling directly in CD4+ T cells regulates encephalitogenic Th1 effector responses.

Clinical EAE course (A and B), severity of CNS pathology (C and D), and ex vivo cytokine responses (E and F) of WT, H1RKO, and H1RKO-Tg mice were compared following immunization with MOG35–55-CFA plus PTX (A, C, and E) or 2× MOG35–55 and CFA (B, D, and F). Cytokine production was assessed by stimulating splenocytes with MOG35–55 on day 10 after injection, and supernatants were collected and quantified by ELISA in triplicate. The significance of differences in the course of clinical disease, CNS pathology indices, and cytokine responses were assessed by regression analysis (63), χ2 test, or ANOVA followed by Bonferroni corrected post-hoc comparisons. With the exception of TNF-α and IL-17 production, significant differences among the strains were detected for all parameters at P < 0.0001 — WT, H1RKO-Tg1, and H1RKO-Tg3 groups were equivalent and all significantly different from the H1RKO group.

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Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-gamma production in mice - PubMed (original) (raw)