Neutrophil serine proteases exert proteolytic activity on endothelial cells

Uwe Jerke, Daniel Perez Hernandez, Patrick Beaudette, Brice Korkmaz, Gunnar Dittmar, Ralph Kettritz*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

41 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)764-775
Number of pages12
JournalKidney International
Volume88
Issue number4
DOIs
Publication statusPublished - 3 Oct 2015
Externally publishedYes

Keywords

  • ANCA
  • endothelium
  • inflammation
  • proteomic analysis
  • vasculitis

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