Neutrophil serine proteases exert proteolytic activity on endothelial cells

Abstract

Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases.

Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease

“…Likewise, cleavage of annexin 1 by PR3 in the context of neutrophil activation reduces the anti-inflammatory activity of annexin 1 and points to a proinflammatory role of PR3. Transfer of PR3 into the cytosol of endothelial cells during neutrophil-endothelial cell interactions has also been inferred as a trigger of endothelial cell apoptosis, which appears to contribute to small vessel damage at sites of inflammation (Pendergraft et al, 2004;Jerke et al, 2015).…”

“…Identification of PR3 as the third elastase-like protease Baggiolini et al, 1978Baggiolini et al, 1988 Elastolytic activity of PR3 showed in vivo Kao et al, 1988Kao et al, 1989 Role of PR3 in cell differentiation attributed Bories et al, 1989Bories et al, 1990 First synthetic inhibitors of PR3 reported Groutas et al, 1990 cDNA cloning of PR3 performed Campanelli et al, 1990 PR3 identified as the target antigen for C-ANCA Jenne et al, 1990 PR3 detected on the neutrophil cell surface Csernok et al, 1990Csernok et al, 1991 First biochemical and enzymatic characterization of PR3 performed Rao et al, 1991 Inhibition of PR3 by elafin investigated Wiedow et al, 1991Wiedow et al, 1992 Gene mapping of PR3 performed Sturrock et al, 1992 Substrate binding site of PR3 mapped using synthetic substrates and inhibitors Brubaker et al Kam et al, 1992aKam et al, ,b 1993 PR3 gene (PRTN3) localized on 19p13.3 Sturrock et al, 1993 Interaction of PR3 with SLPI investigated Rao et al, 1993Rao et al, 1995 Bimodal distribution of PR3 on resting neutrophil surface observed Halbwachs-Mecarelli et al, 1995 Recombinant production of PR3 in human mast cell line-1 cells as active enzyme performed Specks et al, 1996 Crystal structure of PR3 identified Fujinaga et al, 1996 Allosteric modulation of PR3 activity by C-ANCA reported Hinkofer et al, 2015 Endothelial cytoskeletal target substrates of PR3 identified Jerke et al, 2015 Inhibitors and Antibodies Targeting PR3 605 than HNE (Korkmaz et al, 2005a(Korkmaz et al, , 2013aSinden et al, 2015). Moreover, PR3 m was shown to inhibit the phagocytosis of apoptotic neutrophils by binding to calreticulin on apoptotic neutrophils (Gabillet et al, 2012).…”

“…Crosstalk between neutrophils and endothelial cells at sites of inflammation affects both cytokine networks and cell viability. Jerke et al (2015) recently identified 82 novel endothelial proteins cleaved by PR3 and other NSPs and generated extended cleavage signatures for NSPs (Table 1). Most of the identified proteins are cytoskeletal proteins, and inhibition of NSPs activity in vitro preserved the endothelial cytoskeletal structure.…”

“…A large number of studies have provided significant evidence that PR3 contributes to inflammatory tissue damage at different levels (Box 1) and can be regarded as a promising therapeutic target (Korkmaz et al, , 2013bJerke et al, 2015). In this review, we first provide an overview of PR3, its functional biochemistry, and protein/chemical inhibitors.…”

“…The natural substitutions found in the gibbon PR3 homolog, which are not equally distributed but are clustered on one side of the surface, allowed us to identify the conformational epitopes of the group 1 and group (Jerke et al, 2015) and FRET substrates derived from P21 (Korkmaz et al, 2007), NFkB, PML-RAR-a, pro-IL8 (Korkmaz et al, 2013a), protein C inhibitor (Korkmaz et al, 2013a), and PAI-1 (Korkmaz et al, 2002) that are selectively cleaved by PR3 and elastase. IL, interleukin; NFkB, nuclear factor kB; PAI-1, plasminogen activator inhibitor; PML-RAR-a, promyelocytic leukemia-retinoic acid receptor-a; TIM-3, T-cell Ig mucin domain-containing molecule 3.…”