In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2.

Abstract

The pulmonary tree is exposed to neutrophilderived serine proteinases and matrix metalloproteinases in inflaMmatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. cellular matrix (4, 5). Because rat and human neutrophil proteinases display considerable homology (6), we reasoned that the rat model might afford the opportunity to assess the role of leukocyte-derived proteolytic enzymes in lung damage in vivo and evaluate the therapeutic efficacy of antiproteinases relevant to human intervention. Herein, we demonstrate that the serine proteinase inhibitor, secretory leukoproteinase inhibitor (SLPI; refs. 7 and 8), and the matrix metalloproteinase inhibitor, tissue inhibitor of metalloproteinases 2 (TIMP-2; refs. 9 and 10), are able to specifically regulate homologous rat proteinases in vitro and can, when administered in vivo, potently suppress immune complexinduced alveolitis. METHODSNeutrophil Preparation. Neutrophils were isolated from glycogen-induced peritoneal exudates in Long-Evans male rats (Charles River Breeding Laboratories; 300-350 g) 4 hr after the i.p. injection of sterile 1% glycogen (4,5). Harvested neutrophils were suspended in Hanks' balanced salt solution (pH 7.4; GIBCO).Reaction Conditions. Neutrophils were stimulated either with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), surface-bound immune complexes [prepared with bovine serum albumin (BSA; Boehringer Mannheim), and rabbit anti-BSA polyclonal antisera (Cappel) as described (11)] or with N-formylmethionylleucylphenylalanine (fMet-Leu-Phe; 1 ,uM) in the absence or presence ofcytochalasin B (10 .g/ml), catalase (20 jig/ml), azide (1 mM), L-methionine (5 mM), recombinant (human) [r(h)] SLPI (Synergen, Boulder, CO) or r(h)TIMP-2 (Amgen). Hypochlorous acid (HOCl) and N-chloramine generation was quantiated as described (12).Proteinase Assays. Neutrophils (1 x 106 cells per ml) were stimulated for 90 min at 37°C as described above and the cell-free releasates were assayed for rat neutrophil elastase (RNE) and cathepsin G activity with methoxysuccinylalanylalanylprolylvalyl-p-nitroanilide (1.0 mM; Calbiochem) and succinyl-alanylalanylprolylphenyl-p-nitroanilide (1.0 mM; Calbiochem), respectively, as described (13). Results are expressed as nmol of substrate cleaved per hr at 25°C.For matrix metalloproteinase assays, neutrophils (20 x 106 cells per ml) were stimulated as described above and the cell-free releasates were treated with a1-proteinase inhibitor (25 pg/ml; Calbiochem) and phenylmethylsulfonyl fluoride (1 mM) to inhibit serine proteinase activity (13). Latent matrix metalloproteinases were activated with 4-aminophenylmercuric acetate (APMA; 0.5 mM) for 0.5-3 hr at 37(C (14). Type Abbreviations: APMA, 4-aminophenylmercuric acetate; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; r(h), recombinant (human); RNE, rat neutrophil elastase; SLPI, secretory leukoproteinase inhibitor; TIMP-2, tiss...

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