Leukocyte-Endothelial Cell Interaction in Aging

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DESCRIPTION (provided by applicant): Chemokine biology has emerged as central to many important immune and inflammatory processes. However, very little is known about the effect of aging on T cell chemokine function. The specific goal of this proposal is to test the hypothesis that aging is associated with increased T cell C-C chemokine receptor expression that is caused by C-C chemokine receptor promoter hypomethylation. Specific Aim 1 will test the first part of the hypothesis that aging is associated with increased T cell C-C chemokine receptor expression. The effects of aging on T cell C-C and C-X-C chemokine receptor expression and function will be defined at 1. The RNA level (screening by microarray GeneChip technology and confirmed by ribonuclease protection assays (RPAs)). 2. The protein level (Western blot). 3. The functional level (in vitro adhesion and chemotactic assays and in vivo murine chemokine-dependent cutaneous inflammation model). In addition, T cell chemokine response following cytokine stimulation will be determined. Specific Aim 2 will test the second half of the hypothesis that promoter hypomethylation is the mechanism responsible for aging-associated increased in T cell C-C chemokine receptor gene expression. Regulation of CCR5 gene at the transcriptional level will be confirmed by RNA degradation studies and In vitro transcription nuclear run-on assay. The role of transcription factors in the regulation of CCR5 gene expression will be excluded by transfection studies using promoter constructs and the luciferase/beta- galactosidase reporter system. Bisulfite sequencing will be done to determine the promoter methylation status of T cells from across the life span. "Patch" methylation of the CCR5 promoter will be done to determine the effect of DNA methylation. Finally, expression of DNA methyltransferases and the related regulatory proteins will be determined by RPAs and Western blots.