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In Vivo Suppression of Immune Complex-Induced Alveolitis by Secretory Leukoproteinase Inhibitor and Tissue Inhibitor of Metalloproteinases 2 MICHAEL S

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In Vivo Suppression of Immune Complex-Induced Alveolitis by Secretory Leukoproteinase Inhibitor and Tissue Inhibitor of Metalloproteinases 2 MICHAEL S Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11523-11527, December 1993 Medical Sciences In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2 MICHAEL S. MULLIGAN*, PAUL E. DESROCHERSt, ARUL M. CHINNAIYANt, DOUGLAS F. GIBBS*, JAMES VARANI*, KENT J. JOHNSON*, AND STEPHEN J. WEISSt* Departments of *Pathology and tlnternal Medicine, University of Michigan, Ann Arbor, MI 48109 Communicated by Hilary Koprowski, August 12, 1993 ABSTRACT The pulmonary tree is exposed to neutrophil- cellular matrix (4, 5). Because rat and human neutrophil derived serine proteinases and matrix metalloproteinases in proteinases display considerable homology (6), we reasoned inflaMmatory lung diseases, but the degree to which these that the rat model might afford the opportunity to assess the enzymes participate in tissue injury remains undefined, as does role of leukocyte-derived proteolytic enzymes in lung dam- the therapeutic utility of antiproteinase-based interventions. age in vivo and evaluate the therapeutic efficacy of antipro- To address these issues, an in vivo rat model was examined in teinases relevant to human intervention. Herein, we demon- which the intrapulmonary deposition of immune complexes strate that the serine proteinase inhibitor, secretory leuko- initiates a neutrophil-mediated acute alveolitis. In vitro studies proteinase inhibitor (SLPI; refs. 7 and 8), and the matrix demonstrated that rat neutrophils can release neutrophil metalloproteinase inhibitor, tissue inhibitor of metallopro- elastase and cathepsin G as weDl as a neutrophil progelatinase, teinases 2 (TIMP-2; refs. 9 and 10), are able to specifically which was subsequentiy activated by either chlorinated oxi- regulate homologous rat proteinases in vitro and can, when dants or serine proteinases. Based on structural homologies administered in vivo, potently suppress immune complex- that exist between rat and human neutrophil proteinases, rat induced alveolitis. neutrophil elastase and cathepsin G activities could be specif- icaily regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activ- METHODS ity proved sensitive to inhibition by recombinant human tissue Neutrophil Preparation. Neutrophils were isolated from inhibitor of metailoproteinases 2. When either of the recom- glycogen-induced peritoneal exudates in Long-Evans male binant antiproteinases were instilled intratracheally, in vivo rats (Charles River Breeding Laboratories; 300-350 g) 4 hr lung damage as assessed by increased permeability or hemor- after the i.p. injection ofsterile 1% glycogen (4, 5). Harvested rhage was signcany reduced. Furthermore, the coadmin- neutrophils were suspended in Hanks' balanced salt solution istration of the serine and matrix metailoproteinase inhibitors (pH 7.4; GIBCO). almost completely prevented pulmonary damage while effect- Reaction Conditions. Neutrophils were stimulated either ing only a modest decrease in neutrophil influx. These data with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), sur- support a critical role for neutrophil-derived proteinases in face-bound immune complexes [prepared with bovine serum acute lung damage in vivo and identify recombinant human albumin (BSA; Boehringer Mannheim), and rabbit anti-BSA secretory leukoproteinase and recombinant human tissue in- polyclonal antisera (Cappel) as described (11)] or with hibitor of metalloproteinases 2 as potentially efficacious inter- N-formylmethionylleucylphenylalanine (fMet-Leu-Phe; 1 ventions in inflammatory disease states. ,uM) in the absence or presence ofcytochalasin B (10 .g/ml), catalase (20 jig/ml), azide (1 mM), L-methionine (5 mM), Neutrophils play a critical role in the development of inflam- recombinant (human) [r(h)] SLPI (Synergen, Boulder, CO) or matory lung diseases by infiltrating the pulmonary tree and r(h)TIMP-2 (Amgen). Hypochlorous acid (HOCl) and releasing tissue-destructive mediators (1-3). Although the N-chloramine generation was quantiated as described (12). primary species used by neutrophils to damage host tissues Proteinase Assays. Neutrophils (1 x 106 cells per ml) were in vivo remain undefined, the lung is exposed to an increased stimulated for 90 min at 37°C as described above and the burden of leukocyte-derived proteolytic enzymes in disease cell-free releasates were assayed for rat neutrophil elastase states ranging from respiratory distress syndromes to cystic (RNE) and cathepsin G activity with methoxysuccinyl- fibrosis (1-3). In vitro studies have demonstrated that neu- alanylalanylprolylvalyl-p-nitroanilide (1.0 mM; Calbiochem) trophil serine and metalloproteinases can degrade the extra- and succinyl-alanylalanylprolylphenyl-p-nitroanilide (1.0 cellular matrix, hydrolyze serum proteins associated with the mM; Calbiochem), respectively, as described (13). Results clotting and complement cascades, and mediate cytotoxic are expressed as nmol of substrate cleaved per hr at 25°C. effects (1, 2). However, despite their destructive potential in For matrix metalloproteinase assays, neutrophils (20 x 106 vitro, the degree to which neutrophil proteinases participate cells per ml) were stimulated as described above and the in pulmonary damage in vivo remains the subject of conjec- cell-free releasates were treated with a1-proteinase inhibitor ture. (25 pg/ml; Calbiochem) and phenylmethylsulfonyl fluoride (1 In a rat model of acute alveolitis, the intrapulmonary mM) to inhibit serine proteinase activity (13). Latent matrix deposition of immune complexes initiates a proinflammatory metalloproteinases were activated with 4-aminophenylmer- cascade wherein infiltrating neutrophils compromise endo- curic acetate (APMA; 0.5 mM) for 0.5-3 hr at 37(C (14). Type thelial and epithelial permeability barriers while simultane- ously disrupting structural barriers established by the extra- Abbreviations: APMA, 4-aminophenylmercuric acetate; BSA, bo- vine serum albumin; PMA, phorbol 12-myristate 13-acetate; r(h), recombinant (human); RNE, rat neutrophil elastase; SLPI, secretory The publication costs ofthis article were defrayed in part by page charge leukoproteinase inhibitor; TIMP-2, tissue inhibitor of metallopro- payment. This article must therefore be hereby marked "advertisement" teinases 2. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 11523 Downloaded by guest on September 30, 2021 11524 Medical Sciences: Mulligan et al. Proc. Natl. Acad Sci. USA 90 (1993) I collagenolytic and gelatinolytic activities were determined trophils in the presence of methionine (5 mM), the RNE and by incubating releasates with rat tail type I collagen (1 ,uM) cathepsin G activities in the releasates increased 2.5 ± or gelatin (1 uM) for 18 hr at 25°C and 3 hr at 37°C, 0.9-fold (n = 4) and 3.6 ± 0.9-fold (n = 5), respectively. respectively. Substrate hydrolysis was determined by SDS/ Human neutrophils degrade collagenous substrates by PAGE as described (14). Zymography was performed under releasing and activating the metalloproteinase zymogens, nonreducing conditions in SDS/8.5% polyacrylamide gels neutrophil procollagenase and neutrophil progelatinase (1, impregnated with either ,B-casein (1 mg/ml; Sigma) or gelatin 14). To determine the ability of rat neutrophils to release (1 mg/ml; Sigma) (14). collagenase, supematants were analyzed initially by P-casein Acute Alveolitis Model. Lung injury was induced in rats by zymography (14). A single faint band of proteolytic activity the i.v. injection of 10 mg of BSA and the intratracheal was detected at =100 kDa (Fig. 2A, lane 1), but the organo- instillation of 2.5 mg of rabbit anti-BSA polyclonal antisera mercurial activator, APMA, failed to induce a shift in the (4, 5). Tissue injury was assessed 4 hr after injection as molecular mass ofthe proteinase (lane 2) and the caseinolytic changes in vascular permeability or hemorrhage (4, 5). Per- activity was not inhibited by EDTA (lane 3). Furthermore, meability values and hemorrhage were defined as the ratio of when cell-free releasates were incubated with native type I 1251-labeled BSA or 51Cr-labeled erythrocytes in lungs, re- collagen in the absence or presence of APMA, no colla- spectively, to the amount present in 1.0 ml of blood (4, 5). genolytic activity could be detected (Fig. 2B, lane 2). Addi- Where indicated, proteinase inhibitors were coinstilled in- tional studies revealed that the caseinolytic activity could be tratracheally with antisera. Lungs were extracted for myelo- attributed to a serine proteinase-proteinase inhibitor com- peroxidase as an indicator ofneutrophil content as described plex (unpublished observation) while attempts to detect (5). collagenase activity in peripheral blood rat neutrophils were Statistical Analysis. All values are expressed as mean ± SD similarly unsuccessful. unless indicated otherwise. Data were analyzed by analysis Despite the absence of collagenase activity in neutrophil of variance and a comparison of means was determined by releasates, gelatin zymography revealed two major bands of the least significant difference test. Statistical significance proteolytic activity at =97 and =230 kDa (Fig. 2A, lane 4). was defined at P < 0.05. When cell-free supematants were incubated with APMA RESULTS prior to zymography (Fig. 2A, lane 5), these bands decreased in intensity and a major new band at =75 kDa appeared in a Characterization of Serine and Metafloproteinase Release manner
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