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Pathophysiology. 2003 Jan;9(2):115-125.

Biochemical entities that influence membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complex expression and receptor fragment production in adherent vascular endothelium.

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1
The Veterinary Pharmacology Research Laboratory, Department of Basic Science, Veterinary Research Program, College of Veterinary Medicine, Wise Center Drawer V, Mississippi State University, 39762, Mississippi, USA

Abstract

The research aim of the present investigation was to identify leukocyte enzyme-proteases that have the capacity to biochemically recruit the passive participation of vascular endothelium in cytokine receptor 'shedding' phenomenon involving membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Achieving this research objective involved the design of a laboratory approach that delineated to what extent enzyme-proteases released by activated macrophages directly interact with, and liberate soluble fragments of membrane-associated cytokine receptor complexes. Results from this segment of the investigation revealed that cathepsin-D, a leukocyte carboxyl/aspartate protease, altered the integrity and generated soluble fragments of TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. Furthermore, laboratory findings also suggested that cathepsin-D possessed the ability to variably deplete biologically functional membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Complementary investigations isolated a carboxyl/aspartate protease 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 TNF RII (80-kDa) and IL-1 RI (80-kDa) in addition to the generation of soluble receptor fragments. Analysis of macrophage pepstatin-A binding proteins by SDS-PAGE identified a primary fraction with a molecular mass of 47-52-kDa that closely correlated with the known molecular mass of leukocyte cathepsin-D. Evaluation of macrophage pepstatin-A binding-protein fractions by non-denaturing Hb-PAGE detected a lucent proteolytic band at 47-52-kDa compatible with the known molecular mass of leukocyte cathepsin-D. Macrophage pepstatin-A binding 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 TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. The relevance of this concept is in part based on investigations that have discovered that genetic 'knock-out' mice incapable of expressing IL-1 RI (80-kDa) or TNF RI (55-kDa) receptor complexes are highly resistant to developing the pathophysiological alterations classically associated with conditions of endotoxic-shock.

PMID:
14567944

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