Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells.
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Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine. (+info)
TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs.
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The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta. (+info)
Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype.
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We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency. (+info)
The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway.
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Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon gamma (IFN-gamma) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-gamma production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-gamma production in Stat4-deficient T cells, but augmented IFN-gamma expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-gamma reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting. (+info)
Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells.
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CD4(+) and CD8(+) T cells exhibit important differences in their major effector functions. CD8(+) T cells provide protection against pathogens through cytolytic activity, whereas CD4(+) T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-gamma, which can contribute to protective immunity. Here we show that CD4(+) and CD8(+) T cells differ in their regulation of IFN-gamma production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-gamma induced by interleukin (IL)-12/IL-18 signaling, but only CD4(+) T cells require Stat4 for IFN-gamma induction via the TCR pathway. In response to antigen, CD8(+) T cells can produce IFN-gamma independently of IL-12, whereas CD4(+) T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-gamma production based on differences in TCR signaling between CD4(+) and CD8(+) T cells. (+info)
Orally induced peripheral nonresponsiveness is maintained in the absence of functional Th1 or Th2 cells.
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Intragastric administration of soluble protein Ags results in peripheral tolerance to the fed Ag. To examine the role of cytokine regulation in the induction of oral tolerance, we fed OVA to mice deficient in Th1 (Stat 4-/-) and Th2 (Stat 6-/-) cells and compared their response to that of normal BALB/c controls. We found that, in spite of these deficiencies, OVA-specific peripheral cell-mediated and humoral nonresponsiveness was maintained in both Stat 4-/- and Stat 6-/- mice. In the mucosa, both Peyer's patch T cell proliferative responses and OVA-specific fecal IgA were reduced in Stat 4-/- and Stat 6-/- mice fed OVA but not in normal BALB/c controls. Mucosal, but not peripheral, nonresponsiveness was abrogated by the inclusion of a neutralizing Ab to TGF-beta in the culture medium. Our results show that, in the periphery, tolerance to oral Ag can be induced in both a Th1- or Th2-deficient environment. In the mucosa, however, the absence of Th1 and Th2 cytokines can markedly affect this response, perhaps by regulation of TGF-beta-secreting cells. (+info)
Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis.
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IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-gamma production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-gamma production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo. (+info)
Requirement for type 2 NO synthase for IL-12 signaling in innate immunity.
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Interleukin-12 (IL-12) and type 2 NO synthase (NOS2) are crucial for defense against bacterial and parasitic pathogens, but their relationship in innate immunity is unknown. In the absence of NOS2 activity, IL-12 was unable to prevent spreading of Leishmania parasites, did not stimulate natural killer (NK) cells for cytotoxicity or interferon-gamma (IFN-gamma) release, and failed to activate Tyk2 kinase and to tyrosine phosphorylate Stat4 (the central signal transducer of IL-12) in NK cells. Activation of Tyk2 in NK cells by IFN-alpha/beta also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine signaling and function in innate immunity. (+info)