Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

The src homology 2 domain-containing tyrosine phosphatase 2 regulates primary T-dependent immune responses and Th cell differentiation

The src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) plays an important role in development and in growth factor receptor signaling pathways, yet little is known of its role in the immune system. We generated mice expressing a dominant-negative version of the protein, SHP2(CS), specifically in T cells. In SHP2(CS) mice, T cell development appears normal with regard to both negative and positive selection. However, SHP2(CS) T cells express higher levels of activation markers, and aged mice have elevated serum Abs. This is associated with a marked increase in IL-4, IL-5, and IL-10 secretion by SHP2(CS) T cells in vitro. In addition, primary thymus-dependent B cell responses are deficient in SHP2(CS) mice. We show that whereas TCR-induced linker for activation of T cells phosphorylation is defective, CTLA-4 and programmed death-1 signaling are not affected by SHP2(CS) expression. Our results suggest that a key action of wild-type SHP2 is to suppress differentiation of T cells to the Th2 phenotype.     

Protein Tyrosine Phosphatases,TC-PTP,SHP1, and SHP2, Cooperate in Rapid Dephosphorylation of Stat3 in Keratinocytes Following UVB Irradiation

Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na₃VO₄ desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.     

Stat3 phosphorylation mediates resistance of primary human T cells to regulatory T cell suppression

Human autoimmune diseases are characterized by systemic T cell dysfunction, resulting in chronically activated Th1 and Th17 cells that are inadequately suppressed by regulatory T cells (Tregs). IL-6, which is overexpressed in tissue and serum of patients with autoimmune diseases, inhibits human Treg function. We sought to determine the mechanism for the antitolerogenic properties of IL-6 by examining the signaling pathways downstream of IL-6R in primary human T cells. Inhibition of Stat3 signaling in MLCs containing IL-6 restores Treg-mediated suppression, demonstrating that IL-6-mediated loss of Treg suppression requires phosphorylation of Stat3. Cultures in which either effector T cells (Teffs) or Tregs were pretreated with Stat3 inhibitors indicate that phosphorylated (p)Stat3 is required in both T cell populations for IL-6-mediated reversal of Treg function. IL-21, which signals preferentially through pStat3, also reverses Treg suppression, in contrast to IL-27 and IFN-γ, which signal preferentially through Stat1 and do not inhibit Treg function. Interestingly, both Teffs and Tregs respond to IL-6 stimulation through strong Stat3 phosphorylation with minimal MAPK/Erk activation and moderate Stat1 phosphorylation. Finally, Teffs stimulated strongly through the TCR are also resistant to suppression by Tregs and show concurrent Stat3 phosphorylation. In these cultures, inhibition of pStat3 restores functional suppression by Tregs. Taken together, our findings suggest that an early dominance of Stat3 signaling, prior to subsequent T cell activation, is required for the loss of functional Treg suppression and that kinase-specific inhibitors may hold therapeutic promise in the treatment of autoimmune and chronic inflammatory diseases.     

Monocyte 15-lipoxygenase expression is regulated by a novel cytosolic signaling complex with protein kinase C delta and tyrosine-phosphorylated Stat3

Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)delta. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCdelta with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCdelta and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCdelta association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCdelta and subsequent PKCdelta-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCdelta with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.     

Phosphoinositide 3-kinase-dependent regulation of interleukin-3-induced proliferation: involvement of mitogen-activated protein kinases, SHP2 and Gab2

We have demonstrated previously that class I(A) phosphoinositide 3-kinases play a major role in regulation of interleukin-3 (IL)-3-dependent proliferation. Investigations into the downstream targets involved have identified the MAPK cascade as a target. Expression of Deltap85 and incubation with LY294002 both inhibited IL-3-induced activation of Mek, Erk1, and Erk2. This was most pronounced during the initial phase of Erk activation. The Mek inhibitor, PD98059, blocked IL-3-driven proliferation, an effect enhanced by Deltap85 expression, suggesting that inhibition of Mek and Erks by Deltap85 contributes to the decrease in IL-3-induced proliferation in these cells but that additional pathways may also be involved. To investigate the mechanism leading to decreased activation of Erks, we investigated effects on SHP2 and Gab2, both implicated in IL-3 regulation of Erk activation. Expression of Deltap85 led to a reduction in SHP2 tyrosine phosphorylation and its ability to interact with Grb2 and Gab2 but increased overall tyrosine phosphorylation of Gab2. LY294002 did not perturb SHP2 interactions, potentially related to differences in the effects of these inhibitors on levels of phosphoinositides. These results imply that the regulation of Erks by class I(A) phosphoinositide 3-kinase may contribute to IL-3-driven proliferation and that both SHP2 and Gab2 are possibly involved in this regulation.     

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.     

Identification of novel IL-4/Stat6-regulated genes in T lymphocytes

IL-4, primarily produced by T cells, mast cells, and basophiles, is a cytokine which has pleiotropic effects on the immune system. IL-4 induces T cells to differentiate to Th2 cells and activated B lymphocytes to proliferate and to synthesize IgE and IgG1. IL-4 is particularly important for the development and perpetuation of asthma and allergy. Stat6 is the protein activated by signal transduction through the IL-4R, and studies with knockout mice demonstrate that Stat6 is critical for a number of IL-4-mediated functions including Th2 development and production of IgE. In the present study, novel IL-4- and Stat6-regulated genes were discovered by using Stat6(-/-) mice and Affymetrix oligonucleotide arrays. Genes regulated by IL-4 were identified by comparing the gene expression profile of the wild-type T cells induced to polarize to the Th2 direction (CD3/CD28 activation + IL-4) to gene expression profile of the cells induced to proliferate (CD3/CD28 activation alone). Stat6-regulated genes were identified by comparing the cells isolated from the wild-type and Stat6(-/-) mice; in this experiment the cells were induced to differentiate to the Th2 direction (CD3/CD28 activation + IL-4). Our study demonstrates that a number a novel genes are regulated by IL-4 through Stat6-dependent and -independent pathways. Moreover, elucidation of kinetics of gene expression at early stages of cell differentiation reveals several genes regulated rapidly during the process, suggesting their importance for the differentiation process.     

Stat3 activation in acute lung injury

Stat3 plays diverse roles in biological processes including cell proliferation, survival, apoptosis, and inflammation. Very little is known regarding its activation and function in the lung during acute inflammation. We now show that Stat3 activation was triggered in lungs and in alveolar macrophages after intrapulmonary deposition of IgG immune complexes in rats. Low levels of constitutive Stat3 were observed in normal rat lungs as determined by the EMSA. Stat3 activity in whole lung extracts increased 2 h after initiation of IgG immune complex deposition, reaching maximal levels by 4 h, whereas Stat3 activation was found in alveolar macrophages as early as 30 min after onset of injury. Expression and activation of Stat3 mRNA, protein, and protein phosphorylation was accompanied by increased gene expression of IL-6, IL-10, and suppressor of cytokine signaling-3 in whole lung tissues. Both Tyr(705) and Ser(727) phosphorylation were involved in Stat3 activation as assessed in whole lung extracts. C5a (complement 5, fragment a) per se can induce phosphorylation of Ser(727) of Stat3. In vivo, Stat3 activation was dramatically suppressed by depletion of neutrophils or lung macrophages, resulting in reduced gene expression of IL-6 and IL-10 in whole lung tissues. Using blocking Abs to IL-6, IL-10, and C5a, Stat3 activation induced by IgG immune complexes was markedly diminished. These data suggest in the lung injury model used that activation of Stat3 in lungs is macrophage dependent and neutrophil dependent. IL-6, IL-10, and C5a contribute to Stat3 activation in inflamed rat lung.     

Mammary gland remodeling depends on gp130 signaling through Stat3 and MAPK

The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Delta/Delta). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Delta/Delta mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue.