Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145-1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, beta-casein, and kappa-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MMPs were assessed by Northern blot. Rat alveolar macrophages were found to produce a low constitutive level of MMP-2 (72-kD gelatinase A) that was only modestly upregulated following stimulation with phorbol myristate acetate, bacterial lipopolysaccharide, or immunoglobulin A-containing immune complexes. Although control cells were found to produce little or no MMP-9 (92-kD gelatinase B) or MMP-12 (metalloelastase), both enzymes were markedly upregulated upon stimulation. In the same stimulated macrophages there was little activity against type I collagen (associated with MMP-13 [collagenase-3] on the basis of Western blotting), no activity suggestive of stromelysin or matrilysin, and no measurable secretion of the serine proteinases, elastase and cathepsin G. These data demonstrate the ability of rat alveolar macrophages to elaborate certain MMPs under proinflammatory conditions, consistent with their possible involvement in the progression of acute inflammation.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.

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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...

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.