FUNCTIONAL ANALYSIS OF A PDGF-INDUCIBLE CYTOKINE

点击翻译按钮获取中文摘要

Monocyte chemoattractant protein-1 (MCP-1) is a member of a recently defined family of chemotactic factors called "chemokines," which are notable for their ability to attract specific leukocyte subsets. MCP-1, originally cloned as the PDGF-inducible JE gene, attracts monocytes and memory T lymphocytes to the exclusion of other leukocytes, such as neutrophils. Because of its attractant properties and its patterns of expression, MCP-1 has been hypothesized to play a pathogenetic role in a variety of diseases having a monocyte inflammatory component, such as atherosclerosis. So far, however, there have been no direct demonstrations of MCP-1's causal participation in disease. Under the auspices of this grant, structure/activity analyses defined regions of MCP-1 required for its chemoattractant activity, demonstrated that MCP-1 acts as a dimer, and generated a dominant suppressor mutant. In addition, transgenic mice overexpressing MCP-1 provided evidence that MCP-1 is involved in resistance to intracellular pathogens such as L. monocytogenes and M. tuberculosis. This grant renewal seeks: (1) to extend the structure/activity analyses to test regions of MCP-1 and its receptor for sites of interaction; and (2) to create MCP-1-deficient mice to test hypotheses about MCP-1's involvement in disease. The following specific aims are designed to accomplish these goals: SPECIFIC AIM 1: Regions of MCP-1 previously defined as critical for activity will be analyzed by scanning mutagenesis to identify specific amino acids involved in receptor interaction. Previously unexamined areas of MCP-1 will also be analyzed. Regions of MCP-1 receptor B will also be tested by substituting extracellular domains of IL-8 receptor 1 and by site-directed mutagenesis. Peptides based on these findings will be tested for their inhibitory or activating properties. SPECIFIC AIM 2: My laboratory has recently developed MCP-1-deficient mice by targeted gene disruption. These mice will be characterized for their baseline phenotype and their responses to immunologic and infectious challenges. MCP-1's role in atherosclerosis will be investigated by subjecting these mice to atherogenic diets and by breeding the disrupted alleles into an atherosclerosis-prone genetic background. SPECIFIC AIM 3: Many chemokines have overlapping properties, suggesting that redundancy might prevent the appearance of an abnormal phenotype in MCP-1-deficient mice. Since chemokine genes cluster, multiple simultaneous "knockouts" would be technically difficult to construct by breeding. Instead, transgenic mice expressing dominant suppressor chemokine variants will be constructed. Their mating will produce mice multiple simultaneous disruption of several chemokines.