Interleukin 1 (IL-1) is an important mediator in immune and inflammatory responses. IL-1 activity is expressed by both secreted and membrane associated molecules. Secreted IL-1 has been extensively characterized. However, the biochemical nature and the function of membrane IL-1 are largely unknown. This proposal addresses basic questions about the biology and biochemistry of membrane IL-1 using human endothelial cells (HEC) as the prototypical membrane IL-1 producing cells. 1) The conditions for induction of membrane IL-1: membrane IL- 1 expression will be examined on human umbilical vein endothelial cells incubated with a panel of immune and inflammatory stimulants in vitro. The treated cells will be either fixed with paraformaldehyde or fractionated by hypotonic lysis and ultracentrifugation to isolate membranes. IL-1 activity will be assayed using the D10.G4.1 IL-1 indicator line. 2) The relationship of membrane and secreted IL-1 will be examined by biochemical, immunochemical, and molecular approaches. The possible precursor-product relationship of membrane and secreted IL-1 will be examined by kinetic and pulse-labeling studies of IL-1 secretion and membrane expression. The antigenic and biochemical relationship of membrane and secreted IL-1 will be examine by anti-IL-1 inhibition of membrane IL-1 function and immunoprecipitation of metabolic or surface labeled cells. Transfection of BW5147 or Cos 7 cells with cDNA encoding known IL-1 alpha or IL-1 beta proteins or endothelial cell-derived IL-1 proteins will be used to establish if membrane and secreted IL-1 are encoded by the same genes and whether expression of membrane versus secreted IL-1 is controlled by transcriptional, post-transcriptional, or post-translational events. 3) The association of IL-1 with the cell membrane will be examined by attempting to extract IL-1 activity from membranes with chaotropic ions, detergents, and proteolytic or lipolytic enzymes. These studies will indicate if membrane IL-1 is an integral membrane protein or associated with an integral membrane protein or membrane lipid. 4) The function of membrane IL-1 will be assessed by comparing membrane and secreted IL-1 as i) co-factors in mitogen induced T cell activation (a prototypical immune activity), ii) stimulants of fibroblast proliferation, and iii) inducers of endothelial cell- leukocyte adhesion (examples of inflammatory responses). The specificity of these responses will be determined by blocking studies with anti-IL-1 antibodies.
| Leung, D Y; Cotran, R S; Kurt-Jones, E et al. (1989) Endothelial cell activation and high interleukin-1 secretion in the pathogenesis of acute Kawasaki disease. Lancet 2:1298-302 |
| Leung, D Y; Cotran, R S; Kurt-Jones, E et al. (1989) Endothelial activation in the pathogenesis of Kawasaki disease. Trans Assoc Am Physicians 102:131-8 |
