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Published on: May 31, 2018
Biochemical alteration of membrane-associated IL-6 RI (80-kDa) in adherent macrophages and vascular endothelium
C P Coyne1, T Howell, J Baravick
1Veterinary Research Program, Veterinary Pharmacology Research Laboratory, College of Veterinary Medicine, Mississippi State University, Mississippi, USA. coyne@cvm.msstate.edu
Abstract:
The potential biochemical mechanisms that mediate the 'shedding' of soluble IL-6 RI (80-kDa) receptor fragments in populations of adherent macrophages were explored. Stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-6 RI (80-kDa) and the release of soluble receptor fragments. The use of protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases in this process. Cathepsin-D depleted membrane-associated IL-6 RI (80-kDa) complexes and generated soluble receptor fragments. A carboxyl/aspartate protease from activated macrophages isolated utilizing pepstatin-A affinity chromatography, was also found to affect membrane-associated IL-6 RI (80-kDa) complexes and generate soluble receptor fragments. Most likely, this fraction corresponded to cathepsin-D based upon its origin, pepstatin-A binding avidity, Hb-PAGE zymography, and hydrolysis of an enzyme-specific substrate. We conclude that cathepsin-D can generate soluble fragments of IL-6 RI (80-kDa) expressed by both macrophages and vascular endothelium.
Insights
This study reveals how macrophages release soluble Interleukin-6 Receptor (IL-6 RI) fragments. Cathepsin-D, a protease, is identified as a key enzyme responsible for generating these soluble IL-6 RI fragments.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Interleukin-6 receptor (IL-6 RI) shedding produces soluble fragments.
- Understanding the mechanisms of IL-6 RI shedding is crucial for immune response modulation.
Purpose of the Study:
- To investigate the biochemical pathways involved in the release of soluble IL-6 RI (80-kDa) fragments from macrophages.
- To identify the specific proteases responsible for IL-6 RI shedding.
Main Methods:
- Macrophage stimulation and analysis of membrane-bound and soluble IL-6 RI.
- Use of protease inhibitors to identify enzyme classes involved.
- Enzyme isolation via pepstatin-A affinity chromatography and characterization (zymography, substrate hydrolysis).
Main Results:
- Stimulated macrophages showed increased membrane-associated IL-6 RI (80-kDa) and soluble fragment release.
- Protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases.
- Cathepsin-D was identified as a key enzyme, depleting membrane IL-6 RI and generating soluble fragments.
Conclusions:
- Cathepsin-D plays a significant role in generating soluble IL-6 RI (80-kDa) fragments.
- This mechanism is relevant for macrophages and vascular endothelium.
- The findings provide insights into the regulation of IL-6 signaling.
