Cellular and Molecular Biology of Inflammation and Repair

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This is a proposal to continue the study of the cellular and molecular biology of cells participating in the early inflammatory response to injury. The hypothesis to be tested is that neutrophils (PMN) act as instructive cells in sterile inflammation by regulating macrophage phenotypic expression and directing macrophages along the alternative or anti-inflammatory activation pathway. The hypothesis predicts that the suppression of macrophage inflammatory phenotype by PMN is evident locally at sites of tissue injury, and systemically during the response to stimuli such as hemorrhage or long bone fracture. Recent progress on this project provides support for the hypothesis. In this regard, wounds in neutropenic mice contain more TNF-a and IL-6 than in normal controls, and macrophages isolated from neutropenic wounds contain and release excess TNF-a and IL-6. Evidence garnered in vitro demonstrated PMN release soluble factor(s) less than 3000 Da in molecular mass that are not adenosine, NO, or products of COX, that induce the expression of an anti- inflammatory phenotype consisting of alterations in cytokine/chemokine mRNA and protein expression, and the suppression of superoxide production in macrophages. Added relevance to these observations is given by the finding that human PMN culture supernatants suppress TNF-a release from LPS-stimulated human monocyte-derived macrophages. Systemically, injection of LPS into neutropenic animals results in serum TNF-a concentrations 15-fold higher than those in controls, and in increased hepatic and splenic TNF-a mRNA content. In order to test the hypothesis, the proposal is structured in four non-overlapping Specific Aims designed to: I) A. Characterize the wound PMN phenotype. B. Define the molecular identity of the factor(s) released by PMN that suppress macrophage activation. II) Establish the mechanism of suppression of macrophage TNF-a production by PMN factor(s). Ill)A. Define the phenotype of the wound macrophage. B) Investigate the impact of PMN. and PMN inhibitory products on macrophage phenotype and gene expression in acute sterile inflammation, and IV) Determine the role of PMN and PMN secretory products in the systemic response to injury. Completion of the proposed studies will characterize the anti-inflammatory activity of PMN at the molecular, cellular, tissue, and systemic levels, and expand current understanding of a previously unrecognized role of PMN in determining the phenotype of macrophages in inflammation.