IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression. Using EMSA gel shift assay and an antibody to Stat6, we phenotyped two breast cancer cell lines, ZR-75-1 being active Stat6(high) phenotype and BT-20 being defective Stat6(null) phenotype, respectively. Breast cancer cells carrying Stat6(null) phenotype exhibited increased spontaneous apoptosis compared with those carrying Stat6(high) phenotype. Expression microarray analyses demonstrated that IL-4 upregulated CCL26, SOCS1, CISH, EGLN3, and SIDT1, and downregulated DUSP1, FOS, and FOSB, respectively, in these breast cancer cells. Among those genes, CCL26 and SOCS1 were known genes regulated by IL-4/Stat6 pathway, but CISH, EGLN3, SIDT1, DUSP1, FOS, and FOSB were novel genes demonstrated to be IL-4 responsive for the first time. IL-4 also upregulated 38 genes unique to Stat6(null) BT-20 cells and 23 genes unique to Stat6(high) ZR-75-1 cells, respectively. Furthermore, Stat6(high) and Stat6(null) cells showed very different profiles of constitutively expressed genes relevant to apoptosis and metastasis among others, which serve as a valuable expression database and warrant for detailed studies of IL-4/Stat6 pathway in breast cancer.     

Increased expression and activation of IL-12-induced Stat4 signaling in the mucosa of ulcerative colitis patients

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases with unsolved pathogenesis. Imbalanced Th1/Th2 may play a role in the sustained inflammation of IBD. In China, CD is rare but the incidence of UC has been rising steadily in the last two decades. We investigated the expression of IL-12 (p40) and IFN-γ, and the activational state of Stat4 signaling in mucosal tissues at the site of disease from 30 active UC patients in comparison with 30 healthy controls. RT-PCR analyses revealed increased mRNA expression of IL-12 (p40) but not IFN-γ in UC patients. Western blot analyses discovered, for the first time, increased levels of constitutive Stat4 in the cytoplasm and phosphorylated Stat4 in the nucleus of mucosal cells from UC patients. We conclude that a heightened, perhaps persistent, activational state of IL-12/Stat4, and/or IL-23/Stat4 signaling may be present in active Chinese UC patients, and possibly involved in chronic inflammation in UC.     

Long-term suppression of tumor growth by TNF requires a Stat1- and IFN regulatory factor 1-dependent IFN-gamma pathway but not IL-12 or IL-18

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-gamma pathway consisting minimally of IFN-gamma, IFN-gamma receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-gamma-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-gamma nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-gamma was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-gamma pathway that without measurable IFN-gamma production or activity affects the ability of TNF to suppress tumor development.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

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.     

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.     

Regulation of macrophage activation markers by IL-4 and IFN-gamma is subpopulation-specific

We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.