Biochemical mechanisms that interact with membrane-associated IL-1 RII (60-kDa decoy) receptors in populations of adherent macrophages and vascular endothelium

associated IL-1 RII (decoy) receptor complexes expressed by populations of adherent macrophages and vascular endothelium. The initial research strateg y utilized to achieve this objective involved delineating the ability of ma crophage activation or exposure of macrophages and vascular endothelium to a spectrum of enzyme proteases to influence the expression of membrane-asso ciated IL-1 RE (decoy) or generate soluble fragments of this receptor compl ex. Results from these investigations revealed that stimulated macrophages displayed proportional increases in both the expression of membrane-associa ted IL-1 RH (decoy) and release of soluble receptor fragments. Exposure of macrophages and vascular endothelium to the reference proteases discovered the ability of cathepsin-D to biochemically deplete membrane-associated IL- 1 RII (decoy) in addition to generating soluble fragments of this receptor complex. Complementary investigations isolated a carboxyl/aspartate proteas e from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated IL-1 RII (decoy) and gener ation of soluble receptor fragments, Evaluation of pepstatin-A binding prot eins by SDS-PAGE identified a primary protein fraction with a molecular mas s of 47-52 kDa that closely correlates with the known molecular size of leu kocyte cathepsin-D fractions. Macrophage pepstatin-A binding protein fracti ons evaluated by nondenaturing haemoglobin-substrate PAGE (Hb-PAGE) analysi s detected a lucent proteolytic band at 47-52 kDa. Macrophage pepstatin-A b inding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated IL-1 RI I (60-kDa decoy) receptor complexes expressed by macrophages and vascular e ndothelium. (C) 2001 Elsevier Science Inc. All rights reserved.