IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3+ infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130DeltaSTAT/DeltaSTAT). T cell migration was related to STAT3 activity, because monoallelic deletion of Stat3 in gp130(Y757F/Y757F) mice (gp130Y757F/Y757F:Stat3+/-) corrected the exaggerated responses observed in gp130Y757F/Y757F mice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.     

Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis

We have previously demonstrated that STAT3 hyperactivation via the interleukin 6 (IL-6) cytokine family receptor gp130 in gp130 (Y757F/Y757F) mice leads to numerous hematopoietic and lymphoid pathologies, including neutrophilia, thrombocytosis, splenomegaly, and lymphadenopathy. Because IL-6 and IL-11 both signal via a gp130 homodimer, we report here a genetic approach to dissect their individual roles in these pathologies. Neutrophilia and thrombocytosis were absent in gp130 (Y757F/Y757F) mice lacking either IL-6 (gp130 (Y757F/Y757F): IL-6 (-/-)) or the IL-11 receptor alpha subunit (gp130 (Y757F/Y757F): IL-11Ralpha1 (-/-)), and this was associated with a normalized bone marrow compartment. The elevated myelopoiesis and megakaryopoiesis in bone marrow of gp130 (Y757F/Y757F) mice was attributable to an increase by either IL-6 or IL-11 in the STAT3-driven impairment of transforming growth factor beta (TGF-beta) signaling, which is a suppressor of these lineages. In contrast, the absence of IL-6, but not IL-11 signaling, prevented the splenomegaly, abnormal lymphopoiesis, and STAT3 hyperactivation in lymphoid organs of gp130 (Y757F/Y757F) mice. Furthermore, hyperactivation of STAT3 in lymphoid organs was associated with increased expression of IL-6Ralpha, and IL-6Ralpha expression was reduced in gp130 (Y757F/Y757F): Stat3 (+/-) mice displaying normal levels of STAT3 activity. Collectively, these data genetically define distinct roles of IL-6 and IL-11 in driving pathologic hematopoietic and lymphoid responses mediated by STAT3 hyperactivation.     

Differential regulation of gastric tumor growth by cytokines that signal exclusively through the coreceptor gp130

BACKGROUND & AIMS: We have shown that mice with a mutation in gp130 (gp130(757F/F)), the signal transducing receptor for interleukin (IL)-6 family cytokines, have chronic gastric inflammation and develop distal stomach tumors associated with deregulated phosphorylated STAT3 expression. This model recapitulates many characteristics of intestinal-type gastric cancer in humans. METHODS: To evaluate the role of IL-6 and IL-11 as ligands regulating tumor growth and submucosal invasion, we compared tumor characteristics of gp130(757F/F) mice with gp130(757F/F) mice lacking IL-6 or mature T and B cells. RESULTS: As a result of the gp130(757F/F) mutation, expression of IL-6 and IL-11 was greatly up-regulated concomitant with activation of STAT3 and development of tumors. However, the lack of IL-6 or T and B cells did not impact on tumor growth. While IL-6 did not regulate tumor growth or tumor vascularization, gp130(757F/F)/IL-6(-/-) mice showed approximately 10-20-fold more submucosal tumor invasion, reduced mononuclear inflammatory cell infiltrate, and greater IL-11 and matrix metalloproteinase (MMP)-13 and MMP-9 synthesis than gp130(757F/F) mice. Expression of MMP-13 was largely restricted to tumor-associated stroma, but MMP-9 was also expressed in polymorphonuclear cells and a subset of epithelial cells. In addition, treatment with recombinant IL-11 stimulated expression of MMP-13 and MMP-9 in stomachs of wild-type mice. CONCLUSIONS: Increased submucosal invasion in gp130(757F/F)/IL-6(-/-) mice could not be explained by increased vascularization or reduced immunosurveillance but was most likely facilitated by augmented metalloproteinase activity driven by elevated IL-11 levels.     

Opposing roles of gp130-mediated STAT-3 and ERK-1/ 2 signaling in liver progenitor cell migration and proliferation

Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6(+ve) liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130(deltaSTAT)), hyperactive STAT-3 signaling (gp130(Y757F) and Socs-3(-/deltaAlb)) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6(+ve) LPC numbers were increased with IL-6 treatment in vivo. The gp130(Y757F) mice displayed increased A6(+ve) LPCs numbers compared with wild-type and gp130(deltaSTAT) mice. Numbers of A6(+ve) LPCs were also increased in the livers of CDE treated Socs-3(-/deltaAlb) mice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Conclusion: Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation.     

Suppressor of cytokine signaling 3 regulates CD8 T-cell proliferation by inhibition of interleukins 6 and 27

Suppressor of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine responses. SOCS3 is expressed in peripheral T cells, and recent reports have suggested that overexpression of SOCS3 modulates antigen- and/or costimulation-induced T-cell activation. To study the role of SOCS3 in the regulation of T-cell activation, we used a conditional gene-targeting strategy to generate mice that lack SOCS3 in T/natural killer T cells (Socs3(DeltaLck/DeltaLck) mice). SOCS3-deficient CD8 T cells showed greater proliferation than wild-type cells in response to T-cell receptor (TCR) ligation despite normal activation of signaling pathways downstream from TCR or CD28 receptors. Signaling in response to the gp130 cytokines interleukin (IL)-6 and IL-27 was prolonged in Socs3(DeltaLck/DeltaLck) T cells, and T cells from gp130(Y757F/Y757F) mice, in which the SOCS3-binding site on gp130 is ablated, showed a striking similarity to SOCS3-deficient CD8 T cells. Although the proliferative defect of Socs3(DeltaLck/DeltaLck) T cells was not rescued in the absence of IL-6, suppression of IL-27 signaling was found to substantially reduce anti-CD3-induced proliferation. We conclude that enhanced responses to TCR ligation by SOCS3-deficient CD8 T cells are not caused by aberrant TCR-signaling pathways but, rather, that increased IL-27 signaling drives unregulated proliferation in the absence of SOCS3.     

Lack of gp130 expression in hepatocytes promotes liver injury

BACKGROUND & AIMS: Interleukin 6 (IL-6) contributes via its signal transducer gp130 to the acute phase response (APR) in hepatocytes. Recent studies indicated that IL-6 is involved in the regulation of different pathophysiologic conditions of the liver. To define the IL-6-dependent intracellular pathways more specifically, we generated a hepatocyte-specific gp130 knockout mouse. METHODS: Hepatocyte-specific gp130-deficient mice were generated using the Cre-loxP system. Expression of the Cre recombinase was under the control of a hepatocyte-specific control element. Adult mice were challenged with IL-6, oncostatin M (OSM), and LPS. RESULTS: Cre expression started at day 10.5 postconception, and a complete deletion of gp130 in hepatocytes was found at day 14 during liver development. The adult liver of these mice showed no abnormalities; however, after IL-6 and OSM stimulation, gp130-dependent pathways (STAT3, APR gene expression) were completely blocked in the liver of these animals. Additionally, challenging hepatocyte-specific gp130 knockout animals with lipopolysaccharides (LPS) lead to an onset of acute liver injury with an increase of hepatocyte apoptosis associated with elevated tumor necrosis factor alpha (TNF-alpha) serum levels and reduced nuclear factor kappaB (NF-kappaB) activation in hepatocytes. CONCLUSIONS: Our findings demonstrate that gp130 is of minor relevance for embryonal development of hepatocytes. However, the molecule has an essential role in controlling acute phase gene expression and provides hepatocellular protection after LPS challenge.     

Lack of gp130 expression results in more bacterial infection and higher mortality during chronic cholestasis in mice

Chronic cholestasis is associated with increased bacterial infections and sepsis resulting in higher mortality in humans. In the current study, we investigated the relevance of gp130-dependent pathways after bile duct ligation (BDL). BDL was performed in conditional gp130 knockout (loxP/Cre system) mice and respective controls. Liver injury, regulation of the acute phase response, and the impact on survival and bacterial infections were determined. Acute BDL resulted in increased IL-6 levels, Stat3 activation, and an increase in acute-phase proteins (serum-amyloid-A [SAA]), which was blocked in gp130-deleted animals. In addition, the antimicrobial gene hepcidin was regulated in a gp130-dependent manner after BDL. During chronic cholestasis Stat3 activation was dramatically reduced, while high SAA levels were maintained via gp130-dependent signaling. Inhibition of gp130-dependent pathways resulted in higher mortality and liver damage, which was associated with higher infiltration of immune-activated cells and increased germ number in the liver. In conclusion, during acute and chronic cholestasis, the gp130 system is essential for controlling the acute-phase response. Lack of gp130 expression results in pronounced bacterial growth in bile and liver after BDL, which is associated with higher mortality. Activation of gp130-dependent pathways after BDL is essential and appears to be a therapeutic target during cholestasis.     

Hematopoietic abnormalities in mice deficient in gp130-mediated STAT signaling

OBJECTIVE: Studies on mice lacking the common receptor subunit gp130 reveal that activation of gp130-dependent signaling pathways is essential for normal fetal and adult hematopoiesis. However, the extent to which hematopoiesis is dependent upon activation of a particular gp130 signaling pathway, namely STAT1/3 or SHP2/MAPK, is unknown. This study examined the specific contribution of gp130-mediated STAT1/3 signaling to the regulation of hematopoiesis. MATERIALS AND METHODS: Hematopoiesis was examined at various developmental stages in mice homozygous for a targeted carboxy-terminal truncation mutation in gp130 (gp130(delta)/(delta)) that deletes all STAT1/3 binding sites, thereby abolishing gp130-mediated STAT1/3 activation. RESULTS: Adult gp130(delta)/(delta) mice have increased numbers of immature colony-forming unit spleen progenitor cells in the bone marrow and spleen, elevated numbers of committed myeloid progenitor cells in the spleen and peripheral blood, and leukocytosis. Increased progenitor cell production was observed in gp130(delta)/(delta) fetal livers from 14 days of gestation onward. In contrast, the circulating platelet count was reduced by 30% in gp130(delta)/(delta) mice, without any corresponding decrease in the number of bone marrow and splenic megakaryocytes. In liquid cultures, megakaryocytes from gp130(delta)/(delta) mice are smaller than those from wild-type mice and do not increase in size upon stimulation with interleukin-6 or interleukin-11. Administration of either interleukin-6 or interleukin-11 to gp130(delta)/(delta) mice failed to increase platelet numbers, despite an increase in the production of megakaryocytes. CONCLUSIONS: Collectively, these results reveal that gp130-mediated STAT1/3 activation is required to maintain the normal balance of hematopoietic progenitors during fetal and adult hematopoiesis. Furthermore, they suggest two distinct roles for gp130-mediated STAT1/3 activation in hematopoiesis, one restricting the production of immature hematopoietic progenitor cells and the other promoting the functional maturation of megakaryocytes to produce platelets.     

Contribution of interleukin-6/gp 130 signaling in hepatocytes to the inflammatory response in mice infected with Streptococcus pyogenes

BACKGROUND: Sepsis and septic shock caused by gram-positive bacteria have become increasingly frequent clinical problems. These conditions are accompanied by an overwhelming inflammation in which the liver plays a central role as a source and target of inflammatory mediators. Sepsis is still associated with high mortality rates, and new intervention strategies directed at ameliorating the extent of the inflammatory reaction are strongly needed. Here, we investigated whether blockage of the transducer gp130, a receptor involved in the regulation of the inflammatory response, might be useful in the treatment of experimental gram-positive sepsis. METHODS: An experimental model of gram-positive sepsis was used in which liver-specific gp130-deficient mice (FVB/n alfpCre+ gp130(LoxP/LoxP)) and wild-type mice (FVB/n gp130(LoxP/LoxP)) were intravenously infected with Streptococcus pyogenes. The following parameters were monitored: mortality, bacterial loads in systemic organs, serum inflammatory cytokine levels, and organ damage. RESULTS: We show that infected gp130-deficient mice survived significantly longer, had lower bacterial loads, and developed organ damage more slowly than infected wild-type mice. Furthermore, levels of interferon- gamma , interleukin-6, and the chemokine cytokine-induced neutrophil chemoattractant were significantly lower in gp130-deficient mice than in wild-type mice. Histopathological examination of livers showed lower amounts of neutrophil infiltration, apoptosis, and tissue damage in infected gp130-deficient mice than in wild-type mice. CONCLUSION: Our results demonstrate that the gp130 receptor is involved in the regulation of inflammation during gram-positive sepsis and that blockage of gp130 signaling in hepatocytes could constitute a novel target for adjunctive therapy in patients with sepsis.     

An essential role for gp130 in neointima formation following arterial injury

Interleukin (IL)-6 induced vascular smooth muscle cell (VSMC) motility in a dose-dependent manner. In addition, IL-6 stimulated tyrosine phosphorylation of gp130, resulting in the recruitment and activation of STAT-3. IL-6-induced VSMC motility was found to be dependent on activation of gp130/STAT-3 signaling. IL-6 also induced cyclin D1 expression in a time- and gp130/STAT-3-dependent manner in VSMCs. Suppression of cyclin D1 levels via the use of its small interfering RNA molecules inhibited IL-6-induced VSMC motility. Furthermore, balloon injury induced IL-6 expression both at mRNA and protein levels in rat carotid artery. Balloon injury also caused increased STAT-3 phosphorylation and cyclin D1 expression, leading to smooth muscle cell migration from the media to the intimal region. Blockade of gp130/STAT-3 signaling via adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury-induced STAT-3 phosphorylation and cyclin D1 induction, resulting in reduced smooth muscle cell migration from media to intima and decreased neointima formation. Together, these observations for the first time suggest that IL-6/gp130/STAT-3 signaling plays an important role in vascular wall remodeling particularly in the settings of postangioplasty and thereby in neointima formation.