This application has three specific aims: to establish the mechanism of Interleukin 1 (IL-1) action on T cells; to establish the mechanisms of the temperature sensitivity of T cells; and to purify and characterize an apparently new lymphokine which causes macrophages to secrete IL-1. Current work has suggested that exposure of LBRM IA5 cells to IL- l cause increased phosphorylation of lipocortin, a cell membrane protein which regulates phospholipase A2 (PLA-2) activity. It is proposed to extend this work by repeating it with other cell lines with both IL-1 alpha and beta, and using a variety of different incubation times and adjunct stimuli. The possible involvement of an N protein in the stimulus chain will be investigated using pertussis and cholera toxins. Available evidence strongly suggests that many IL-1 actions are mediated by arachidonic acid metabolites. Since IL-1 action is thought to be deleterious in some circumstances, such as during acute sepsis, knowledge of the pathways of cell activation by IL-1 might allow effective therapeutic interventions. It is already known that IL-1 action is strongly enhanced by small increases in temperature, and current work has shown that one mechanism for this is greatly enhanced secretion of Interleukin 2 by IL-l stimulated T cell lines. lt is proposed now to investigate the effect of temperature on IL-2 receptor expression by T cells, and on the lipocortin phosphorylation and PLA-Z activation discussed above. The most probable cause of an unusually high temperature sensitivity is a lipid phase change, and these membrane events might well depend on the fluidity of the cell membrane, or its inner half. Purification of the lymphokine will be attempted using sizing, ion exchange, isoelectric focusing and reverse phase columns on a pharmacia FPLC system. The molecular properties of the purified activity will be compared to those of known lymphokines, and if it appears to be truly new an attempt will be made to develop monoclonal antibody and use it to identify the cDNA for the gene in a T cell expression library.
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