Recruitment of various inflammatory cells in response to humoral or cellular immune responses is central in tissue damage in human disease; however, the events involved remain incompletely understood. Recent advances in molecular cloning of cellular chemoattractants called chemokines have opened a new avenue to determine the means by which the body controls the influx of cells. The chemokine are a family of small heparin-binding proteins divided into three subfamilies depending on the presence of conserved cysteines (C-X-C, C-C, and C) which are neutrophil, monocyte, and lymphocyte chemoattractants, respectively. Although numerous studies suggest that chemokine are major attractants for inflammatory cells in vitro, generation of modulators is required to assess their actual pathogenic role in vivo. Ligand-based mutagenesis or receptor antagonists (Ras) is a novel approach to generate modulators. Preliminary studies with recombinant MCP-1 RA generated in E. coli. dramatically decreased the influx on monocyte/macrophages in experimental tubulointerstitial nephritis (TIN) in rats. The potency of Ras in blocking macrophage chemotaxis in vivo implies a new manipulative approach for chemokine with therapeutic value in human disease. This proposal will focus on developing RA for the recently cloned lymphocyte chemoattractant, lymphotactin (Ltn), and MIP-1alpha. MIP-1 RA will target the """"""""shared"""""""" C-C chemokine MIP-1/RANTES receptor involved in lymphocyte/monocyte attraction. MCP-1 RA altered the biology of the inflammatory process in TIN by delaying resolution of the initial neutrophil infiltrate, presumably by altering macrophage uptake of neutrophils. The implied new role of MCP-1 in homeostasis by the modulation of macrophage-mediated phagocytosis of apoptotic neutrophils will be studied in vitro and in vivo. The role of these chemokine will be examined in renal immune disease models in which monocytes and/or lymphocytes are major effector cells. The in vivo contribution will be determined via the use of neutralizing antibodies and/or Ras. Molecular chaperones, GroESL, will be coexpressed and these chemokine analogues in E. coli. to attain biologically active recombinant peptides in vivo. Therefore specific aim one now is to examine the contribution of two different C-C chemokines MCP-1 and MIP-1 in the mononuclear cell infiltration and the TIN model using either neutralizing antibodies or the receptor antagonist. In addition, it is to examine the role of macrophage mediated phagocytosis of apoptotic neutrophils.
Specific Aim 2 is to characterize the role of rat lymphotactin as well as MIP1 using specific receptor antagonists in both the model of tubulointerstitital nephritis and the anti-GBM model in order to determine the role of chemokines in the T-lymphocyte infiltration in these models. The planned studies will lead to better understanding of the structure/activity of chemokine, the biology of renal inflammation, the mechanisms of monocyte/macrophage and lymphocyte influx, the role of C-C and C chemokine in immune inflammatory injury, and will provide valuable information on the potential use of chemokine Ras in humans.
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