Cytokines such as tumor necrosis factor (TNF) exert pleiotropic effects on a wide variety of cells and tissues. These alterations are particularly profound in endothelial cells where TNF has been shown to induce adherence for leukocytes and to increase membrane procoagulant activity. Changes of this nature are likely to be central to the pro-inflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function, we utilized differential plaque hybridization to identify TNF immediate early response genes. SEVEN such genes were identified. DNA sequencing revealed that two encoded known cytokine induced genes; endothelial leukocyte adhesion molecule-1 and neutrophil chemotactic factor. One encoded the nuclear protooncogene; c-jun, while another encoded a recently described monocyte specific chemotactic factor NOT previously associated with endothelium. The production of a monocyte chemotaxin by cytokine activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. Significantly, the other three cDNAs are completely novel as judged by data bank searches of partial DNA sequences. Importantly, one of these (A20) contains seven zinc fingers which is characteristic of a certain class of transcriptional factors. To better understand the role of the monocyte chemoattractant and the novel cDNAs in vessel wall disease states, angiogenesis and inflammation we propose the following studies:
Specific Aim 1 : (i) Clone full length cDNAs encoding the novel transcripts and complete their DNA sequencing.
Specific Aim 2 : (i) Make bacterial fusion protein for both the monocyte chemoattractant and the novel cDNAs for rabbit immunization and antibody production.
Specific Aim 3 : Using the monospecific polyclonal antiserum raised in Specific Aim 2, localize both the monocyte chemoattractant and the novel antigens: a) In Vitro, using cytokine treated endothelial and other cells. This will allow subcellular localization of the novel gene products. b) In vivo, in inflammatory states such as wound healing and psoriasis. For the monocyte chemoattractant it will be particularly important to examine atherosclerotic tissue. Further, in situ hybridization studies will be carried out to identify which cells in the tissue under study are actually transcribing the gene of interest.
