FUNCTIONAL ANALYSIS OF A PDGF INDUCIBLE CYTOKINE

点击翻译按钮获取中文摘要

DESCRIPTION (investigator's abstract): Chemokines are small proteins that control normal leukocyte trafficking and are responsible for leukocyte recruitment in inflammatory disorders. The physiology of this protein family is made extraordinarily complex by the existence of over 50 chemokine ligands and 20 receptors. In order to understand chemokine biology and to discover methods for therapeutically modulating their activity, we have studied one chemokine in depth, namely monocyte chemoattractant protein-1 (MCP- 1). MCP-1, a CC chemokine that was first cloned as a PDGF-inducible mRNA from mouse fibroblasts, attracts monocytes, NK cells, and memory T cells in vitro. Under the auspices of this grant, we used genetically modified mice to demonstrate that: MCP-1 is a potent monocyte-specific chemoattractant in vivo; it is uniquely required for monocyte recruitment in inflammation despite the existence of other monocyte chemoattractants that activate MCP-1's receptor, CCR2; and, its absence protects mice from atherosclerosis. Recently, we have shown that MCP-1 is required for the induction of Th2 polarized immune responses. This finding is surprising because MCP-l's primary in vivo functions were thought to involve only innate immunity, and because CCR2-deficient mice have a Th1 defect. The work in this application is designed to elucidate details of MCP-1's mechanisms of action in vivo by addressing two hypotheses: (1) MCP-1 expression by cells in secondary lymphoid organs directly stimulates Th2 polarization through a pathway that may be IL-4-independent; and (2) This effect is specific for MCP-1 but may work through a receptor other than CCR2. To test these hypotheses, I propose the following specific aims: Specific Aim 1: Determine the physiological basis for MCP-1's influence on Th2 polarization. This will be approached by reconstituting polarized immune responses in vitro using cells from MCP-1 -I- and MCP-1 +1+ mice and testing inferences drawn from these experiments by in vivo adoptive transfer experiments. Specific Aim 2: Examine the molecular basis for MCP-1's influence on T helper cell polarization. We will test whether IL-4 is required for MCP-1-induced Th2 polarization, whether MCP-1 activates the same downstream targets as IL-4, and whether BCL-6 (a transcriptional repressor that suppresses Th2 polarization) is in the MCP- 1 pathway. Specific Aim 3: Determine whether MCP-1-mediated Th2 polarization depends specifically on MCP-1 and CCR2. Other receptors will be tested for their ability to effect Th2 polarization by MCP- 1, and another CCR2 ligand, MCP-3, will be knocked into the MCP-1 locus to test whether MCP-1 is specifically required.