Cleavage of HMGB1 by Proteolytic Enzymes Associated with Inflammatory Conditions

Extracellular HMGB1 acts as an alarmin in multiple autoimmune diseases. While its release and functions have been extensively studied, there is a substantial lack of knowledge regarding HMGB1 regulation at the site of inflammation. Herein we show that enzymes present in arthritis-affected joints process HMGB1 into smaller peptides in vitro. Gel electrophoresis, Western blotting and mass spectrometry analyses indicate cleavage sites for human neutrophil elastase, cathepsin G, and matrix metalloproteinase 3 within the HMGB1 structure. While human neutrophil elastase and matrix metalloproteinase 3 might alter the affinity of HMGB1 to its receptors by cleaving the acidic C-terminal tail, cathepsin G rapidly and completely degraded the alarmin. Contrary to a previous report we demonstrate that HMGB1 is not a substrate for dipeptidyl peptidase IV. We also provide novel information regarding the presence of these proteases in synovial fluid of juvenile idiopathic arthritis patients. Correlation analysis of protease levels and HMGB1 levels in synovial fluid samples did not, however, reveal any direct relationship between the recorded levels. This study provides knowledge of proteolytic processing of HMGB1 relevant for the regulation of HMGB1 during inflammatory disease.

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Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (‘gelatinase’) by human neutrophil elastase and cathepsin G

FEBS Letters ◽

10.1016/0014-5793(89)80657-x ◽

1989 ◽

Vol 249 (2) ◽

pp. 353-356 ◽

Cited By ~ 111

Author(s):

Yasunori Okada ◽

Isao Nakanishi

Keyword(s):

Matrix Metalloproteinase ◽

Neutrophil Elastase ◽

Human Neutrophil ◽

Cathepsin G ◽

Matrix Metalloproteinase 2 ◽

Human Neutrophil Elastase ◽

Matrix Metalloproteinase 3

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Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽

10.1182/blood.v82.7.2188.2188 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2188-2195 ◽

Cited By ~ 45

Author(s):

RC Woodman ◽

PH Reinhardt ◽

S Kanwar ◽

FL Johnston ◽

P Kubes

Keyword(s):

Neutrophil Elastase ◽

Human Neutrophil ◽

Neutrophil Migration ◽

Specific Inhibitor ◽

Neutrophil Infiltration ◽

Cathepsin G ◽

Neutrophil Adhesion ◽

Human Neutrophil Elastase

Abstract The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

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Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽

10.1182/blood.v82.7.2188.bloodjournal8272188 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2188-2195

Author(s):

RC Woodman ◽

PH Reinhardt ◽

S Kanwar ◽

FL Johnston ◽

P Kubes

Keyword(s):

Neutrophil Elastase ◽

Human Neutrophil ◽

Neutrophil Migration ◽

Specific Inhibitor ◽

Neutrophil Infiltration ◽

Cathepsin G ◽

Neutrophil Adhesion ◽

Human Neutrophil Elastase

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

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