Inflammatory Response (IR) in the heart, lung, and other vascularized tissues is mediated by the first line of bone marrow-derived cellular responders. These comprise blood phagocytes: PMN leukocytes and monocytes and macrophages, and tissue phagocytes, dendritic cells. In addition, mast cells are important inflammatory responders in tissues. PMN leukocytes and monocytes respond to chemotactic stimuli, emigrate from blood to the sites of tissue injury and produce reactive oxygen and nitrogen intermediates and arachidonic acid metabolites, release lysosomal hydrolases and pro-inflammatory chemokines and cytokines. Some of these key mediators of inflammation are also produced by tissue-based macrophages, dendritic and mast cells. We hypothesize first that insights into the genetic program governing the function of these cells during inflammatory response can be gained by studying differential gene expression second, that disruption or genes encoding mediators or inhibitors of the IR will respectively result in attenuated or heightened IR.
The first aim of this Project, focused on PMNs, monocytes, macrophages, dendritic, and mast cells, is to establish their gene expression profile in resting and stimulated state using probes representing 15,600 genes and ESTs including currently available disrupted genes in a library of ES cell clones. Highly expressed genes in phagocytes and mast cells will be prioritized for transmission to the germline. In the second aim, mutant mice will be phenotypically analyzed for chemotactic response, respiratory burst response during phagocytosis, cell adhesion, chemokine and cytokine expression, and IgE-mediated degranulation (mast cells). The function of specific cell types expressing GFP under the control of the promoter of disrupted gene will be analyzed Mutant mice showing attenuated or heightened response to pro-inflammatory agonist in cell-based assays and in vivo during systematic and localized inflammatory response will be extensively characterized using a combination of biochemical, cell biological and immunological approaches. Cumulatively, we anticipate that advanced phenotypic analysis of mutant mice will expand our knowledge about the genes that control inflammatory stress response in the """"""""first line"""""""" cellular responders and provide potential therapeutic targets and animal models based on """"""""loss of function"""""""" mutations.
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