Mononuclear phagocytic cells (MPCs) play a pivotal role in the initiation and resolution of inflammatory lung diseases. MPC recruitment and activation are mediated by monocyte chemoattractant produced at the inflammatory focus. Recently, rat monocyte chemoattractant protein 1 (MCP-1), a peptide specific for monocyte/macrophage chemotaxis and activation, has been sequenced and cloned, facilitating investigation of the role of MCP-1 in inflammatory diseases using animal models. Using two well characterized rat models of inflammatory lung injury, we have identified rat alveolar MPCs as a cellular source of MCP-1 during both the acute and chronic inflammatory response in the lung. We hypothesize that lung MPCs are both a source and effector cell of MCP-1 during inflammatory lung injury, dependent on their state of differentiation.
The specific aims of this proposal are to define 1) the relative contribution of alveolar MPC derived MCP-1 to total MCP-1 in the lung 2) the critical cytokine networks which regulate the genetic expression and synthesis of MCP-1 in the lung 3) the role of mCD14 expression and specific signal transduction events (e.g. phospholipase D [PLD] activity, calcium flux, protein tyrosine phosphorylation events) in LPS induced expression, synthesis and secretion of MCP-1 receptor expression and specific signal transduction event (PLD activity, protein tyrosine phosphorylation events) in MCP-1 induced activation of human monocytes and the relationship to MPC differentiation. Methods: Expression, synthesis and secretion of MCP-1 and specific cytokines which regulate MCP-1 will be determined by Northern blot analysis, in situ hybridization analysis and immunohistochemistry, functional assays and cytokine specific neutralization assays. Receptor expression (CD14, MCP-1) will be determined by binding assays using I labeled substrates and/or by flow cytometry. PLD activity will be determined in labeled cells by accumulation of 14C phosphatidic acid and, in the presence of ethanol, 14C phosphatidyl ethanol. Ca2+ will be determined using Fura-2 loaded cells. Protein tyrosine phosphorylation events will be identified by effects of tyrosine kinase and tyrosine phosphatase inhibitors and by analysis of tyrosine phosphorylated proteins. It is anticipated that results of these studies will further define the role of MCP-1 in inflammatory lung injury and identify specific signal transduction events involved in MCP-1 synthesis and/or MCP-1 induced activation in MPCs, dependent on their state of differentiation.
