STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

IFN-gamma-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-alpha

The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of eotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-alpha. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner.     

Regulation of murine lymphokine production in vivo. Ultraviolet radiation exposure depresses IL-2 and enhances IL-4 production by T cells through an IL-1-dependent mechanism

The exposure of experimental animals to the inflammatory effects of ultraviolet radiation (UVR) is known to cause depressions in their ability to initiate and effectuate various types of cellular immune responses. Contact-type and delayed-type hypersensitivity, plus the ability to generate protective forms of anti-viral and anti-tumor immunity, are all affected by the prior exposure of normal animals to the effects of this physical agent. Presently, the cellular and molecular mechanism(s) responsible for mediating the changes in immune function observed in UVR-exposed animals is not fully understood. Herein we report that one reproducible consequence of exposing normal mice to low doses of UVR is a dramatic change in the pattern of lymphokines secreted by their activated T cells. Lymphocytes isolated from UVR-exposed donors produce/secrete greatly reduced levels of the T cell lymphokines IL-2 and IFN-gamma activation in vitro with protein Ag of the polyclonal T cell stimulant anti-CD3. The secretion of IL-4 by these lymphocyte cultures, however, is consistently elevated in comparison to normal controls. Further studies determined that a similar change in lymphokine production was induced when mice were treated with either bacterial LPS or rIL-1 beta, a cytokine known to be elevated in vivo after UVR or LPS exposure. The ability of IL-1 to facilitate a change in the capacity of T lymphocytes to produce/secrete lymphokines after in vitro activation does not appear to represent a direct effect of this cytokine on lymphocyte or accessory cell targets because addition of IL-1 beta to cultures of Ag-primed lymphocytes obtained from normal donors was incapable of altering the pattern of lymphokine production. Collectively, our present results add further support to the hypothesis that UVR-induced elevations in endogenous IL-1 are, in part, responsible for the immunomodulatory effects of UVR. These findings provide compelling evidence that UVR, plus other agents capable of endogenously stimulating the production of IL-1, may function to alter the expression of different effector mechanisms in vivo. This could be facilitated through selective reductions in lymphokines produced by Th-1-type cells (IL-2 and IFN-gamma) and a simultaneous augmentation in a lymphokine produced by Th-2-type cells (IL-4).     

Th2 Cytokines Augment IL-31/IL-31RA Interactions via STAT6-dependent IL-31RA Expression

Interleukin 31 receptor α (IL-31RA) is a novel Type I cytokine receptor that pairs with oncostatin M receptor to mediate IL-31 signaling. Binding of IL-31 to its receptor results in the phosphorylation and activation of STATs, MAPK, and JNK signaling pathways. IL-31 plays a pathogenic role in tissue inflammation, particularly in allergic diseases. Recent studies demonstrate IL-31RA expression and signaling in non-hematopoietic cells, but this receptor is poorly studied in immune cells. Macrophages are key immune-effector cells that play a critical role in Th2-cytokine-mediated allergic diseases. Here, we demonstrate that Th2 cytokines IL-4 and IL-13 are capable of up-regulating IL-31RA expression on both peritoneal and bone marrow-derived macrophages from mice. Our data also demonstrate that IL-4Rα-driven IL-31RA expression is STAT6 dependent in macrophages. Notably, the inflammation-associated genes Fizz1 and serum amyloid A (SAA) are significantly up-regulated in M2 macrophages stimulated with IL-31, but not in IL-4 receptor-deficient macrophages. Furthermore, the absence of Type II IL-4 receptor signaling is sufficient to attenuate the expression of IL-31RA in vivo during allergic asthma induced by soluble egg antigen, which may suggest a role for IL-31 signaling in Th2 cytokine-driven inflammation and allergic responses. Our study reveals an important counter-regulatory role between Th2 cytokine and IL-31 signaling involved in allergic diseases.     

IFN-gamma production and cytotoxicity of IL-2-activated murine NK cells are differentially regulated by MHC class I molecules

Activation of NK cells by target cells leads to cytotoxicity as well as production of various cytokines including IFN-gamma. MHC class I molecules on target cells regulate NK cytotoxicity. However, little is known about the regulation of IFN-gamma production by NK cells. We examined the production of IFN-gamma in individual murine NK cells stimulated with tumor cell lines by flow cytometric analysis of intracellular IFN-gamma. Among several tumor lines tested, the rat basophilic leukemia line RBL-1 induced particularly high level of IFN-gamma production in IL-2-activated NK cells, whereas other lines, including the prototypic NK target YAC-1, induced very low or no IFN-gamma production. Transfection of murine classical MHC class I molecules into RBL-1 cells substantially inhibited IFN-gamma production. This inhibition of IFN-gamma production by MHC class I was independent of Ly-49 or CD94/NKG2A expression on NK cells. These results indicate that some target cells directly stimulate IL-2-activated NK cells and induce IFN-gamma production, but the requirements for the induction of IFN-gamma production seem different from those for NK cytotoxicity. Furthermore, similar to NK cytotoxicity, induction of IFN-gamma production is inhibited by MHC class I on stimulating cells. However, the MHC class I-specific receptors inhibiting IFN-gamma production are different from those for NK cytotoxicity.     

Cutting edge: IL-5 primes Th2 cytokine-producing capacity in eosinophils through a STAT5-dependent mechanism

Both type-2 CD4(+) Th cells (CD4(+)Th2) and type-2 innate effector cells play critical roles in generating type-2 immunity that can either be protective against parasitic infection or cause tissue damage in allergy and asthma. How innate effector cells acquire the capacity to produce Th2 cytokines is not entirely known. We previously showed that IL-4 induced differentiation of Th2 cytokine-producing eosinophils. To determine whether other Th2 cytokines can also induce Th2 cytokine-producing capacity in innate effector cells, we cultured bone marrow progenitor cells in the presence of various Th2 cytokines. IL-5, but not IL-13 or IL-25, primed bone marrow progenitor cells to differentiate into robust IL-4-producing cells. The majority of IL-4-producing cells induced by IL-5 were eosinophils. Importantly, IL-5 completely depended on STAT5 to promote IL-4-producing capacity in eosinophils. Thus, our study demonstrates that IL-5 functions as a potent factor that drives bone marrow progenitor cells into IL-4-producing eosinophils.     

人胎脾细胞IL—2和IL—6的产生及其与NK功能发育的关系

The production of IL-2, IL-6 by fetal splenic mononuclear cells (FSMC) and relationship between them and the ontogenetic development of natural killer cell function were studied in human fetal spleens. As a results, before 20 weeks of gestation, both IL-2 and NK cell activities were not measured, but IL-6 was done. It was found that IL-2, IL-6 and NK cell activities were increased with the gestational age, and shown that there were linear positive correlation between the activities of three ones above (r > 0.86). Before the birth, the induced IL-2 activity was the same as adult levels (p > 0.05), although both IL-6 production and NK activity in fetal spleens were significantly lower than that in adults (p < 0.01). Lastly, the production of IL-2, IL-6 in relation to the functional development of NK cells during the embryonic development was discussed.     

IL-21 induces the functional maturation of murine NK cells

IL-21 is a recently identified cytokine that stimulates mouse NK cell effector functions in vitro. In this study we demonstrate that IL-21 achieves its stimulatory effect by inducing the development of mature NK cells into a large granular lymphocyte phenotype with heightened effector function. IL-21 treatment results in increased cell size and granularity and a corresponding decrease in cell viability and proliferative potential. These cells up-regulate the expression of the inhibitory CD94-NKG2A receptor complex and the activation markers CD154 and killer cell, lectin-like-receptor G1. Surprisingly, IL-21 treatment also results in down-regulation of the pan-NK marker, NK1.1. Coinciding with these cellular changes IL-21 enhances cytolytic capacity across a spectrum of target sensitivities and induces IL-10 and IFN-gamma production. In vivo treatment with IL-21 results in a very similar activation and phenotypic maturation of NK cells as well as a potent increase in NK cell-mediated anti-tumor immunity that is perforin dependent. These developmental changes suggested that IL-21 functions to induce the terminal differentiation of mouse NK cells, resulting in heightened NK cell-mediated cytotoxicity and immune surveillance.     

IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130(F/F) knock-in mutant mice displaying hyperactivated IL-6-dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130(F/F) mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130(F/F) mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130(F/F):Stat3(+/-) mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130(F/F) mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.     

IL-4-dependent up-regulation of IL-4 receptor expression in murine T and B cells

This study examined mRNA levels and cell surface expression of IL-4 receptor (IL-4R) in murine T and B cells after incubation with IL-4. Northern blot analysis of mRNA levels of T cells isolated from mesenteric lymph nodes and spleen revealed that IL-4 induced a transient augmentation of IL-4R mRNA in a dose-dependent manner. Maximal levels of mRNA were detected as early as 5 h after initiation of culture. These data were complemented by studies examining the cell surface expression of IL-4R using an anti-IL-4R mAb. Resting T and B lymphocytes express IL-4R (T greater than B) and incubation of these cells with exogenous IL-4 increased IL-4R expression to a maximum after 24 h. This effect was abolished after addition of anti-IL-4 antibody. Continuous incubation of T cells in the presence of high concentrations of IL-4 resulted in a down-regulation of IL-4R expression. Addition of the protein synthesis inhibitor cycloheximide blocked the induced increases in IL-4R expression, indicating the requirement for de novo protein synthesis. Both the levels of mRNA and cell surface expression of IL-4R were not affected by addition of exogenous IL-2, and IL-4 regulation of IL-4R expression was not influenced by the immunosuppressive drug cyclosporin A. These data demonstrate that in T and B cells, IL-4 induces a transient up-regulation of IL-4 mRNA levels that is subsequently reflected in increased numbers of IL-4R displayed on the cell surface. This regulation of IL-4R expression by IL-4 provides an important mechanism for amplification of IL-4-dependent activation pathways.