Oncostatin M (OSM) primes IL-13- and IL-4-induced eotaxin responses in fibroblasts: Regulation of the type-II IL-4 receptor chains IL-4Rα and IL-13Rα1

Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in regulation of various chronic inflammatory processes. Previous work has shown that OSM induces eosinophil accumulation in mouse lungs in vivo and stimulates the eosinophil-selective chemokine eotaxin-1 synergistically with IL-4 in vitro. To examine the role of receptor regulation by OSM in synergistic eotaxin-1 responses, we here examine the modulation of the type-II IL-4 receptor (IL-4Rα and IL-13Rα1) by OSM and other gp130/IL-6 cytokine family members using NIH3T3 fibroblasts and primary mouse lung fibroblasts. We first show that OSM with either IL-13 or IL-4 synergistically induces eotaxin-1 expression in a dose-dependent fashion. Analysis of IL-4Rα expression at the protein (Western blot and FACS) and RNA (TAQMAN) levels showed that OSM markedly elevates expression by 3 h. OSM enhanced IL-13Rα1 mRNA and induced a smaller but detectable increase in total IL-13Rα1 protein. Priming fibroblasts with OSM for 6 h markedly enhanced subsequent IL-13 and IL-4-induced eotaxin-1 responses and STAT6 tyrosine-641 phosphorylation. Regulation of IL-4Rα by OSM was sensitive to inhibition of the PI3′K pathway by LY294002. These studies provide novel mechanistic insights in OSM role in regulation of synergistic eotaxin-1 responses and IL-4Rα expression in fibroblasts.     

Oncostatin-M up-regulates VCAM-1 and synergizes with IL-4 in eotaxin expression: involvement of STAT6

Oncostatin-M (OSM) is an IL-6/gp130 family member that can stimulate the eosinophil-selective CC chemokine eotaxin-1 in vitro and eosinophil accumulation in mouse lung in vivo. The adhesion molecule VCAM-1 and eotaxin have been implicated in extravasation and accumulation of eosinophils into tissue in animal models of asthma. In this study, we investigated the role of OSM in regulation of VCAM-1 expression, and STAT6 tyrosine 641 phosphorylation in murine fibroblasts. OSM induced VCAM-1 expression in C57BL/6 mouse lung fibroblasts (MLF) and NIH 3T3 fibroblasts at the protein and mRNA level in vitro. OSM also induced STAT6 Y641 phosphorylation in MLF and NIH 3T3 fibroblasts, an activity not observed with other IL-6/gp130 cytokine family members (IL-6, leukemia inhibitory factor, cardiotropin-1, and IL-11) nor in cells derived from STAT6(-/-) mice (STAT6(-/-) MLF). STAT6 was not essential for OSM-induced VCAM-1 or eotaxin-1 as assessed in STAT6(-/-) MLF. Combination of IL-4 and OSM synergistically enhanced eotaxin-1 expression in MLF. IL-4 induction and the IL-4/OSM synergistic induction of eotaxin-1 was abrogated in STAT6(-/-) MLF, however, regulation of IL-6 was similar in -/- or wild-type MLF. Induction of VCAM-1 by OSM was diminished by pharmacological inhibitors of PI3K (LY294002) but not inhibitors of ERK1/2 (PD98059) or p38 MAPK (SB203580). These data support the role of OSM in eosinophil accumulation into lung tissue through eotaxin-1 and VCAM-1 expression and the notion that OSM is able to induce unique signal transduction events through its receptor complex of OSMR beta-chain and gp130.     

Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells

Background

Patients who have esophageal eosinophilia without gastroesophageal reflux disease (GERD) nevertheless can respond to proton pump inhibitors (PPIs), which can have anti-inflammatory actions independent of effects on gastric acid secretion. In esophageal cell cultures, omeprazole has been reported to inhibit Th2 cytokine-stimulated expression of eotaxin-3, an eosinophil chemoattractant contributing to esophageal eosinophilia in eosinophilic esophagitis (EoE). The objective of this study was to elucidate molecular mechanisms underlying PPI inhibition of IL-4-stimulated eotaxin-3 production by esophageal cells.

Methods/Findings

Telomerase-immortalized and primary cultures of esophageal squamous cells from EoE patients were treated with IL-4 in the presence or absence of acid-activated omeprazole or lansoprazole. We measured eotaxin-3 protein secretion by ELISA, mRNA expression by PCR, STAT6 phosphorylation and nuclear translocation by Western blotting, eotaxin-3 promoter activation by an exogenous reporter construct, and STAT6, RNA polymerase II, and trimethylated H3K4 binding to the endogenous eotaxin-3 promoter by ChIP assay. Omeprazole in concentrations ≥5 µM significantly decreased IL-4-stimulated eotaxin-3 protein secretion and mRNA expression. Lansoprazole also blocked eotaxin-3 protein secretion. Omeprazole had no effect on eotaxin-3 mRNA stability or on STAT6 phosphorylation and STAT6 nuclear translocation. Rather, omeprazole blocked binding of IL-4-stimulated STAT6, RNA polymerase II, and trimethylated H3K4 to the eotaxin-3 promoter.

Conclusions/Significance

PPIs, in concentrations achieved in blood with conventional dosing, significantly inhibit IL-4-stimulated eotaxin-3 expression in EoE esophageal cells and block STAT6 binding to the promoter. These findings elucidate molecular mechanisms whereby patients with Th2 cytokine-driven esophageal eosinophilia can respond to PPIs, independent of effects on gastric acid secretion.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.     

A novel human CC chemokine, eotaxin-3, which is expressed in IL-4-stimulated vascular endothelial cells, exhibits potent activity toward eosinophils.

IL-4 has been shown to be involved in the accumulation of leukocytes, especially eosinophils, at sites of inflammation by acting on vascular endothelial cells. To identify novel molecules involved in the IL-4-dependent eosinophil extravasation, cDNA prepared from HUVEC stimulated with IL-4 was subjected to differential display analysis, which revealed a novel CC chemokine designated as eotaxin-3. The human eotaxin-3 gene has been localized to chromosome 7q11.2, unlike most other CC chemokine genes. The predicted mature protein of 71 aa showed 27-42% identity to other human CC chemokines. The recombinant protein induced a transient increase in the cytosolic Ca2+ concentration and in vitro chemotaxis on eosinophils. Furthermore, in cynomolgus monkeys, the accumulation of eosinophils was observed at the sites where the protein was injected. Eotaxin-3 inhibited the binding of 125I-eotaxin, but not 125I-macrophage inflammatory protein-1alpha, to eosinophils and acted on cell lines transfected with CCR-3, suggesting that eotaxin-3 recognized CCR-3. IL-13 as well as IL-4 up-regulated eotaxin-3 mRNA in HUVEC, whereas neither TNF-alpha, IL-1beta, IFN-gamma, nor TNF-alpha plus IFN-gamma did. The expression profile of eotaxin-3 is different from those of eotaxin, RANTES, and monocyte chemoattractant protein-4, which are potent eosinophil-selective chemoattractants and are induced by either TNF-alpha or TNF-alpha plus IFN-gamma. These results suggest that eotaxin-3 may contribute to the eosinophil accumulation in atopic diseases.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.