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. 2009 Nov 6;284(45):30815-24.
doi: 10.1074/jbc.M109.052472. Epub 2009 Sep 10.

MKP-1 is necessary for T cell activation and function (V体育安卓版)

Yongliang Zhang  1 , Joseph M Reynolds, Seon Hee Chang, Natalia Martin-Orozco, Yeonseok Chung, Roza I Nurieva, Chen Dong
Affiliations

MKP-1 is necessary for T cell activation and function

Yongliang Zhang et al. J Biol Chem. .

Abstract

MAPKs are evolutionarily conserved immune regulators VSports手机版. MAPK phosphatases (MKPs) that negatively regulate MAPK activities have recently emerged as critical players in both innate and adaptive immune responses. MKP-1, also known as DUSP1, was previously shown to negatively regulate innate immunity by inhibiting pro-inflammatory cytokine production. Here, we found that MKP-1 is necessary in T cell activation and function. MKP-1 deficiency in T cells impaired the activation, proliferation, and function of T cells in vitro, associated with enhanced activation of JNK and reduced NFATc1 translocation into the nucleus. Consistently, MKP-1(-/-) mice were defective in anti-influenza immunity in vivo and resistant to experimental autoimmune encephalomyelitis. Our results thus demonstrate that MKP-1 is a critical positive regulator of T cell activation and function and may be targeted in treatment of autoimmune diseases. .

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"V体育安卓版" Figures

FIGURE 1.
FIGURE 1.
Regulation of MAPK activation by MKP-1 in T cells. A, naïve CD4+ T cells were activated with or without anti-CD3 antibody. In vitro differentiated Th1, Th2, or Th17 cells were activated with anti-CD3 antibody. MKP-1 gene expression was determined by real-time reverse transcription-PCR. B, naïve CD4+ T cells were activated with PMA and ionomycin for the indicated times. Cells were lysed, and cell extracts were probed with antibodies to phospho (p)-p46 and phospho-p54 isoforms of JNK, total JNK, phospho-ERK, ERK, phospho-p38, and p38. Relative MAPK activation was determined by the levels of phospho-MAPKs normalized to their total protein expression. The data are representative of four separate experiments.
FIGURE 2.
FIGURE 2.
MKP-1 regulates T cell activation and effector function in vitro. A, naïve CD4+ T cells were activated with different concentrations of anti-CD3 antibody in the presence or absence of anti-CD28 antibody, and their IL-2 production and [3H]thymidine uptake were measured. B, IL-2 production and [3H]thymidine incorporation were measured in WT and KO CD8+ T cells in response to different concentrations of anti-CD3 antibody in the presence or absence of anti-CD28 antibody. C, nuclear fractions were prepared from WT and KO naïve CD4+ T cells. NFATc1 expression was examined by Western blot analysis. D–F, naïve CD4+ T cells from WT and KO mice were differentiated into Th1 (D), Th2 (E), and Th17 (F) cells, followed by anti-CD3 stimulation before ELISA assay and intracellular cytokine staining. *, p < 0.05; **, p < 0.005. G, purified naïve CD8+ T cells were activated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 5 days. Cells were harvested and washed three times with RPMI 1640 complete medium, followed by restimulation with plate-bound anti-CD3 antibody for 24 h. Cytokine concentrations in the culture supernatant were measured by ELISA. The data are representative of three separate experiments.
FIGURE 3.
FIGURE 3.
MKP-1 is required for antigen-specific T cell responses in vivo. WT and KO mice (n = 5) were immunized with OVA in CFA. On day 8, splenocytes were isolated and stimulated with peptide as indicated. A, IL-2 production and cell proliferation were measured after 1 and 3 days of stimulation, respectively. B, cytokine production was determined by ELISA after 3 days of stimulation. C, anti-OVA antibodies of different isotypes in sera from immunized mice were measured by ELISA. The data are representative of two independent experiments with similar results.
FIGURE 4.
FIGURE 4.
MKP-1-deficient mice are defective in anti-influenza responses. WT and KO mice were infected with 13 hemagglutinating units of PR8 influenza virus. A, changes in body weight of WT and KO mice after infection were monitored daily. B, mice were killed 7 days after infection. Hemagglutinin (HA) and neuraminidase (NA) gene expression in the lungs of day 7 influenza-infected WT and KO mice was analyzed by real-time reverse transcription-PCR. C, virus-specific CD8+ T cells in BAL were determined by staining with DbNP366 tetramer and anti-CD8 antibody. D, IFNγ-producing CD8+ cells in BAL from day 7 influenza-infected WT and KO mice in response to PA224–233 peptide stimulation were analyzed by intracellular cytokine staining. E, IFNγ-producing CD4+ cells in BAL from day 7 influenza-infected WT and KO mice in response to NP311–325 peptide stimulation were analyzed by intracellular cytokine staining. F, IFNγ concentrations in BAL from day 7 influenza-infected WT and KO mice were measured by ELISA. G, IFNγ concentrations in culture supernatants of lung-infiltrated leukocytes from day 7 influenza-infected WT and KO mice in response to PA224–233 (MHCI) and NP311–325 (MHCII) peptide stimulation were determined by ELISA. The data are representative of four independent experiments with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.005.
FIGURE 5.
FIGURE 5.
MKP-1-deficient mice are resistant to EAE disease. A, WT and MKP-1 KO mice were immunized with MOG35–55 peptide to induce EAE. EAE disease was scored. B, mononuclear cells in the central nervous system were stained with anti-CD4 and anti-CD11b antibodies. C, central nervous system-infiltrated leukocytes were activated with PMA and ionomycin in the presence of GolgiPlug. IFNγ and IL-17 production by CD4+ cells was examined by intracellular cytokine staining. Total cytokine-producing cells were calculated and averaged. D and E, splenocytes from MOG-immunized WT and KO mice were restimulated with MOG35–55 peptide. IL-2 production and [3H]thymidine incorporation were determined (D). IFNγ and IL-17 concentrations in the culture supernatant were measured by ELISA (E). The data are representative of two independent experiments with similar results.
FIGURE 6.
FIGURE 6.
MKP-1 deficiency in CD4+ T cells causes resistance to EAE disease. CD4+ T cells were purified from WT and MKP-1 KO mice. 5 × 106 WT or KO cells were injected into Rag1−/− mice through the tail vein. 24 h after T cell transfer, Rag1−/− recipients (five mice in each group) were immunized with MOG35–55 peptide to elicit EAE. A, EAE disease was scored. B, mononuclear cells in the central nervous system were stained with anti-CD4 and anti-CD11b antibodies. C, central nervous system-infiltrated leukocytes were activated with PMA and ionomycin in the presence of GolgiPlug. IFNγ and IL-17 production by CD4+ cells was examined by intracellular cytokine staining. Total cytokine-producing cells were calculated and averaged. The data are representative of two independent experiments with similar results.
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