Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Hsp60 Proteolysis by Proteinase 3 Induces Neutrophil Degranulation: Implications for Amplification of Injury in Wegener's Granulomatosis.
Sato4, Shinji, Park1, Jin Kyun, Askin3, Frederic, Rosen2, Antony, Levine1, Stuart M.
Extracellular proteolytic processing by proteinase 3 (PR3) released from activated neutrophils has recently been implicated in several important biological processes. Heat shock proteins are induced in response to a variety of inflammatory stimuli, and some, such as Hsp60, can be released into the extracellular environment, where they can exert pro-inflammatory functions. Whether PR3-mediated proteolysis of Hsp60 released in the WG microenvironment contributes to the inflammatory response is unknown.
Human recombinant Hsp60 was incubated for varying times with increasing concentrations of PR3, and cleaved products were detected by SDS-PAGE and immunoblotting. Polymorphonuclear cells (PMNs) were isolated from heparinized peripheral venous blood by density gradient centrifugation. Following co-incubation with intact or PR3-cleaved Hsp60, PMN degranulation was quantified using the b-glucuronidase release assay at baseline and after 90 minutes. Activity units (U) were calculated for each experiment from the observed fluorescent intensities using a standard curve obtained from serial concentrations of a dye standard. The supernatants of TNF-primed PMNs were similarly assessed for Hsp60 proteolytic activity, and the effects of PR3 inhibition using a1-antitrypsin were examined. Hsp60 expression in WG tissue was examined by immunohistochemistry.
Hsp60 was efficiently cleaved by purified PR3 in vitro with a kcat/Km of 7.5 × 104 M -1 S -1. PR3-cleaved HSP60 caused significant b-glucuronidase release from PMNs at 90 minutes compared to intact Hsp60 (73.7 U +/- 22.7 vs. 30.3 U +/- 19.1, p=0.01). Upon PMN priming with TNF-a, small amounts of PR3 released in the supernatants were detected by immunoblotting. Co-incubation of these TNF-primed supernatants with Hsp60 led to Hsp60 processing that was inhibited by the PR3 inhibitor a1-antitrypsin. Subsequent incubation of fresh, non-primed PMNs with this PR3-processed Hsp60 caused significant degranulation compared to those incubated with supernatants from non-TNF-primed PMNs (51.7 U +/- 9.9 vs. 23.5 U +/- 16.7, p=0.005), demonstrating that the PR3 released by primed PMNs can cleave exogenous Hsp60 to further amplify fresh PMN degranulation. Finally, an analysis of WG lung tissues reveals that Hsp60 is highly expressed in the giant cells of WG granulomata.
These studies identify multinucleated giant cells in the WG granuloma as a rich source of Hsp60. TNF-primed PMNs release PR3, and PR3-cleaved Hsp60 induces further degranulation of fresh PMNs, suggesting a novel mechanism whereby PR3 participates in amplification of the local inflammatory response in WG. This reinforcing interaction of target tissue and inflammatory cells in the local WG microenvironment may be useful for disease monitoring and therapy.
To cite this abstract, please use the following information:
Sato, Shinji, Park, Jin Kyun, Askin, Frederic, Rosen, Antony, Levine, Stuart M.; Hsp60 Proteolysis by Proteinase 3 Induces Neutrophil Degranulation: Implications for Amplification of Injury in Wegener's Granulomatosis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :681