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Elastase and metalloproteinase activities regulate soluble complement receptor 1 release
1Department of Research University Hospital Basel, Basel, Switzerland. sadallah@ubaclu.unibas.ch
Abstract:
Complement receptor 1 (CR1) is cleaved from the surface of polymorphonuclear cells (PMN) in the membrane-proximal region to yield a soluble fragment (sCR1) that contains the functional domains. The enzymes involved in this cleavage are produced by the PMN itself, since in vitro stimulation of purified PMN is followed by sCR1 release. Purified human neutrophil elastase (HNE) cleaved CR1 from erythrocytes and urinary vesicles originating from podocytes and enhanced tenfold the cleavage of CR1 from activated PMN. The largest fragment released from PMN by HNE was identical in size to CR1 shed spontaneously. The CR1 fragments cleaved from erythrocytes were functional. The shedding of sCR1 by activated PMN was inhibited by phenylmethylsulfonyl fluoride (80 +/- 10%), alpha1-antiprotease (50 +/- 5%) and elafin (60 +/- 5%). Furthermore the cleavage was blocked by the metalloprotease inhibitor 1,10-phenanthroline (70 +/- 6 %) as well as by a monoclonal antibody against human neutrophil collagenase MMP8 (40 +/- 10%). Maximal inhibition of sCR1 shedding was obtained by a combination of 1,10-phenanthroline with elafin (86 +/- 6%). These inhibitors had no effect on L-selectin shedding, indicating that the cleavage of CR1 was specific. In conclusion, elastase or elastase-like activity may be responsible for the shedding of functional sCR1 in vivo, and this activity is controlled by the local release of PMN metalloproteases and alpha1antiprotease.
Insights
Soluble complement receptor 1 (sCR1) is released from immune cells by enzymes like human neutrophil elastase (HNE). This shedding of functional sCR1 is regulated by metalloproteases and alpha1-antiprotease.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Complement receptor 1 (CR1) is a key immune molecule found on polymorphonuclear cells (PMNs).
- CR1 can be released as a soluble fragment (sCR1), but the mechanisms and enzymes involved are not fully understood.
Purpose of the Study:
- To investigate the enzymes responsible for cleaving CR1 from PMNs and other cells.
- To identify inhibitors that can block the shedding of soluble CR1 (sCR1).
Main Methods:
- In vitro stimulation of purified human PMNs.
- Cleavage assays using purified human neutrophil elastase (HNE) on PMNs, erythrocytes, and urinary vesicles.
- Assessment of sCR1 shedding inhibition using various protease inhibitors (PMSF, alpha1-antiprotease, elafin, 1,10-phenanthroline) and an antibody against MMP8.
- Specificity testing by evaluating the effect of inhibitors on L-selectin shedding.
Main Results:
- Human neutrophil elastase (HNE) cleaves CR1 from erythrocytes and urinary vesicles, and significantly enhances CR1 cleavage from activated PMNs.
- The largest HNE-cleaved fragment from PMNs matches the size of spontaneously shed CR1, and these fragments retain functionality.
- sCR1 shedding is inhibited by serine protease inhibitors (PMSF, alpha1-antiprotease, elafin) and metalloprotease inhibitors (1,10-phenanthroline, anti-MMP8 antibody).
- Combined inhibition with 1,10-phenanthroline and elafin maximally blocked sCR1 shedding, with no effect on L-selectin shedding, indicating specificity.
Conclusions:
- Elastase or elastase-like activity is likely responsible for in vivo shedding of functional sCR1.
- The shedding process is tightly regulated by the local release of PMN-derived metalloproteases and alpha1-antiprotease.