Stat5a inhibits IL-12-induced Th1 cell differentiation through the induction of suppressor of cytokine signaling 3 expression

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a(-/-)) CD4(+) T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-gamma production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a(-/-) CD4(+) T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a(-/-) CD4(+) T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a(-/-) CD4(+) T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4(+) T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a(-/-) CD4(+) T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.     

Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis

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.     

IL-4 induces in vivo production of IFN-gamma by NK and NKT cells

Although IL-4 and IFN-gamma often have opposite effects and suppress each other's production by T cells, IL-4 can stimulate IFN-gamma production. To characterize this, we injected mice with IL-4 and quantified IFN-gamma production with the in vivo cytokine capture assay. IL-4 induced Stat6-dependent IFN-gamma production by NK and, to a lesser extent, NKT cells, but not conventional T cells, in 2-4 h. Increased IFN-gamma production persisted at a constant rate for >24 h, but eventually declined, even with continuing IL-4 stimulation. This eventual decline in IFN-gamma production was accompanied by a decrease in NK and T cell numbers. Consistent with a dominant role for NK cells in IL-4-stimulated IFN-gamma secretion, IL-4 induction of IFN-gamma was B and T cell-independent; suppressed by an anti-IL-2Rbeta mAb that eliminates most NK and NKT cells; reduced in Stat4-deficient mice, which have decreased numbers of NK cells; and absent in Rag2/gamma(c)-double-deficient mice, which lack T, B, and NK cells. IL-4-induced IFN-gamma production was not affected by neutralizing IL-12p40 and was increased by neutralizing IL-2. IL-13, which signals through the type 2 IL-4R and mimics many IL-4 effects, failed to stimulate IFN-gamma production and, in most experiments, suppressed basal IFN-gamma production. Thus, IL-4, acting through the type 1 IL-4R, induces Stat6-dependent IFN-gamma secretion by NK and NKT cells. This explains how IL-4 can contribute to Th1 cytokine-associated immune effector functions and suggests how IL-13 can have stronger proallergic effects than IL-4.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

A heritable defect in IL-12 signaling in B10.Q/J mice. I. In vitro analysis

B10.Q mice are normally susceptible to the induction of collagen-induced arthritis. We noted that one subline of B10.Q mice, B10.Q/J, was completely resistant to disease induction when immunized with collagen in CFA. B10.Q/J mice have a global defect in the generation of Th1 responses, and Ag-specific T cells derived from this strain failed to produce IFN-gamma. Because T cells from these mice could produce normal amounts of IFN-gamma when activated by IL-12/IL-18-independent stimuli, the defect appeared to be a failure to respond to IL-12. This defect extended to NK cells, which also failed to produce IFN-gamma when stimulated by IL-12. The capacity of NK cells, but not activated T cells, to produce IFN-gamma in response to IL-12 could be partially restored by IL-18. The expression of the IL-12R beta1- and beta2-chains on T cells and NK cells from B10.Q/J mice was normal. However, activated T cells from B10.Q/J mice did not signal normally through the IL-12R and manifested a defect in their capacity to phosphorylate Stat4. This defect was partial in that it could be overcome by increasing both the concentration of IL-12 and the incubation times in the Stat4 phosphorylation assays. Because Stat4 function is apparently intact in B10.Q/J mice, the defect in IL-12 signaling can be localized between the IL-12R complex and Stat4. This subtle abnormality in IL-12 responsiveness results in a profound defect in the generation of Th1 cells and the development of autoimmune disease.     

Cytokine production by Vgamma(+)-T-cell subsets is an important factor determining CD4(+)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis

Two coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vgamma4 Vdelta4 cells, which are strongly positive for gamma interferon (IFN-gamma), whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vgamma1(+) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4(+)-, IFN-gamma(+)-cell responses in both H3- and H310A1-infected mice yet decreased the CD4(+)-, IL-4(+)-cell response. Depleting Vgamma4(+) cells suppressed myocarditis and reduced CD4(+) IFN-gamma(+) cells but increased CD4(+) IL-4(+) T cells. The role of cytokine production by Vgamma1(+) and Vgamma4(+) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-gamma expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vgamma4 and Vgamma1(+) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma, whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4. Stat4ko Vgamma1(+) cells (IL-4(+)) suppress myocarditis. Stat6ko Vgamma1(+) cells (IFN-gamma(+)) were not inhibitory. Stat6ko Vgamma4(+) cells (IFN-gamma(+)) significantly enhanced myocarditis. Stat4ko Vgamma4(+) cells (IL-4(+)) neither inhibited nor enhanced disease. These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4(+)-Th-cell response. The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4(+)-Th-cell phenotype.     

Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.     

Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile

The concept that IL-4 is the primary signal for Th2 lymphocyte differentiation has recently been put in doubt by studies in which the production of Th2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell analysis that CD4+ lymphocytes with a classical Th2 phenotype (IL-4+, IL-5+, IFN-gamma-, IL-2-) develop in significant numbers in helminth-infected mice deficient in either IL-4R alpha-chain or Stat6. While an expanded population of Th1 (IL-4-, IL-5-, IFN-gamma+, IL-2+) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokine pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected by in vitro neutralization of endogenous IL-4 or IFN-gamma. Nevertheless, while addition of exogenous rIL-12 resulted in transitory IFN-gamma production by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 synthesis was preserved in the former, but temporarily ablated in the latter cells. Importantly, IL-4+ IFN-gamma- and IL-4- IFN-gamma+ populations similar to those arising in helminth-infected Stat6-deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4+CD62Lhigh lymphocytes from uninfected mice of the same strain. Together, the results of these single cell analysis experiments demonstrate that IL-4R/Stat6 signaling, while influencing the final frequency of Th2 lymphocytes, is not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once they have emerged.     

Suppressive effect of IL-4 on IL-13-induced genes in mouse lung

Although IL-4 signals through two receptors, IL-4R alpha/common gamma-chain (gamma(c)) and IL-4R alpha/IL-13R alpha1, and only the latter is also activated by IL-13, IL-13 contributes more than IL-4 to goblet cell hyperplasia and airway hyperresponsiveness in murine asthma. To determine whether unique gene induction by IL-13 might contribute to its greater proasthmatic effects, mice were inoculated intratracheally with IL-4 or IL-13, and pulmonary gene induction was compared by gene microarray and real-time PCR. Only the collagen alpha2 type VI (Ca2T6) gene and three small proline-rich protein (SPRR) genes were reproducibly induced > 4-fold more by IL-13 than by IL-4. Preferential IL-13 gene induction was not attributable to B cells, T cells, or differences in cytokine potency. IL-4 signaling through IL-4R alpha/gamma(c) suppresses Ca2T6 and SPRR gene expression in normal mice and induces these genes in RAG2/gamma(c)-deficient mice. Although IL-4, but not IL-13, induces IL-12 and IFN-gamma, which suppress many effects of IL-4, IL-12 suppresses only the Ca2T6 gene, and IL-4-induced IFN-gamma production does not suppress the Ca2T6 or SPRR genes. Thus, IL-4 induces genes in addition to IL-12 that suppress STAT6-mediated SPRR gene induction. These results provide a potential explanation for the dominant role of IL-13 in induction of goblet cell hyperplasia and airway hyperresponsiveness in asthma.     

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.     

Augmentation of antigen-presenting and Th1-promoting functions of dendritic cells by WSX-1(IL-27R) deficiency

WSX-1 is the alpha subunit of the IL-27R complex expressed by T, B, NK/NKT cells, as well as macrophages and dendritic cells (DCs). Although it has been shown that IL-27 has both stimulatory and inhibitory effects on T cells, little is known on the role of IL-27/WSX-1 on DCs. LPS stimulation of splenic DCs in vivo resulted in prolonged CD80/CD86 expression on WSX-1-deficient DCs over wild-type DCs. Upon LPS stimulation in vitro, WSX-1-deficient DCs expressed Th1-promoting molecules higher than wild-type DCs. In an allogeneic MLR assay, WSX-1-deficient DCs were more potent than wild-type DCs in the induction of proliferation of and IFN-gamma production by responder cell proliferation. When cocultured with purified NK cells, WSX-1-deficient DCs induced higher IFN-gamma production and killing activity of NK cells than wild-type DCs. As such, Ag-pulsed WSX-1-deficient DCs induced Th1-biased strong immune responses over wild-type DCs when transferred in vivo. WSX-1-deficient DCs were hyperreactive to LPS stimulation as compared with wild-type DCs by cytokine production. IL-27 suppressed LPS-induced CD80/86 expression and cytokine production by DCs in vitro. Thus, our study demonstrated that IL-27/WSX-1 signaling potently down-regulates APC function and Th1-promoting function of DCs to modulate overall immune responses.     

Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

Stat6 and IRS-2 are two important signaling proteins that associate with the cytoplasmic tail of the interleukin 4 (IL-4) receptor. Data from numerous in vitro experiments have led to a model for IL-4 signal transduction in which the Stat6 signaling pathway is responsible for the IL-4 induced changes in gene expression and differentiation events, while the IRS-2 signaling pathway provides mitogenic and antiapoptotic signals. In order to determine the relative contributions of these signaling molecules in primary lymphocytes, we have examined IL-4 responses in T cells from mice deficient for either Stat6 or IRS-2 as well as from mice doubly deficient for both genes. Both IRS-2 and, especially, Stat6 are shown to be critically involved in IL-4-induced proliferation of T cells, presumably through the cooperative regulation of the Cdk inhibitor p27kip1. Like Stat6-deficient Th cells, IRS-2-deficient cells are also compromised in their ability to secrete Th2 cytokines, revealing a previously unrecognized role for IRS-2 in Th2 cell development. Although Stat6 and/or IRS-2 expression is required for IL-4-induced proliferative and differentiative responses, both signaling proteins are dispensable for the antiapoptotic effect of IL-4. However, treatment of lymphocytes with a protein tyrosine phosphatase inhibitor is able to block the antiapoptotic effect of IL-4 specifically in Stat6- or IRS-2-deficient cells and not in wild-type cells. Our results suggest that Stat6 and IRS-2 cooperate in promoting both IL-4-induced proliferative and differentiating responses, while an additional signaling mediator that depends on protein tyrosine phosphatase activity contributes to the antiapoptotic activities of IL-4 in primary T cells.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.