106 Human airway smooth muscle cell responses to TLR-ligands are enhanced by oncostatin M

Introduction: Airway smooth muscle cells (ASMC) are involved in lung inflammatory responses through alterations in secretion of chemokines and growth factors as well as proliferation or contractile functions. Thus ASMC contribute to levels of chemokines such as MCP-1 and eotaxins, that can enhance cellular infiltration, and modulators of extracellular matrix such as VEGF. Since ASMC express TLRs, and the lung mucosa is exposed to TLR ligands through environmental/infectious agents, we assessed in vitro responses of human (H) ASMC to TLR-ligands alone or in context of gp130 cytokines that are elevated during lung inflammation. Methods: HASMC were cultured and stimulated with ligands for TLR-3 (poly IC) and TLR-4 (LPS) alone and in combination with gp130 cytokine family members including Oncostatin M. Supernatants were saved for ELISA analysis and cell extracts were prepared for RNA analysis and cell signaling protein analysis by immunoblots. Results: TLR-3 ligand induced MCP-1, IP-10, IL-6 and eotaxin-3 responses by HASMC in dose dependent fashion, however reduced VEGF levels (protein and mRNA). TLR-4 ligand showed lower activity. OSM induced IL-6, MCP-1 and VEGF expression in vitro, whereas other members of the gp130 cytokine family (LIF, IL-31, IL-6) did not. Combination experiments showed that HASMC treated with 0.1ng/ml OSM responded to poly IC or LPS at 10-fold less TLR-ligand concentration indicating enhanced sensitivity to TLR-3 and −4 ligands. Immunoblots showed that OSM stimulation in combination with either poly IC or LPS activated the NFkappa B pathway (IkB alpha degradation) to a greater extent than OSM or TLR ligand stimulus alone. Furthermore, OSM induced an increase in TLR-3 mRNA and CD14 mRNA. Conclusion: OSM increases the responses of HASMC to TLR-3 and TLR4 ligands, including NFkappa B activation, elevation of receptor chains for TLRs, and chemokine release, and thus is potentially integral to exacerbation of lung inflammatory diseases. This work was supported by the Canadian Institutes for Health Research.