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Role of Matrix Metalloproteinases in Models of Macrophage-Dependent Acute Lung Injury Evidence for Alveolar Macrophage as Source of Proteinases Douglas F. Gibbs, Thomas P. Shanley, Roscoe L. Warner, Hedwig S. Murphy, James Varani, and Kent J. Johnson Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan

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. 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.

Alveolar macrophages in the lung provide the first line of defense against inhaled organisms and irritants. In addition to this phagocytic clearance role, the alveolar macrophage is known to be a critical modulator of the lung inflammatory response through the production of various proinflammatory and anti-inflammatory cytokines (reviewed in 1–4). What is less well known is the direct role that macrophage products such as oxidants and proteinases play in the development of lung injury. Previous studies from our laboratories have established that neutrophiland macrophage-derived oxidants appear to be important (Received in original form July 13, 1998 and in revised form December 2, 1998) Address correspondence to: Kent J. Johnson, M.D., Dept. of Pathology, The University of Michigan Medical School, M7520 Medical Science Research I, Box 0602, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. E-mail: [email protected] Abbreviations: aminophenyl mercuric acetate, APMA; bronchoalveolar lavage fluid, BALF; bovine serum albumin, BSA; ethylenediamenetetraacetic acid, EDTA; imunoglobulin, Ig; lipopolysaccharide, LPS; Eagle’s minimum essential medium, MEM; matrix metalloproteinase, MMP; permeability index, PI; phorbol myristate acetate, PMA; sodium dodecyl sulfate, SDS; tissue inhibitor of metalloproteinases, TIMP. Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 1145–1154, 1999 Internet address: www.atsjournals.org

in the development of acute lung injury, with hydrogen peroxide, the hydroxyl radical, and nitric oxide species such as the peroxynitrite radical implicated as being phlogistic (5–10). However, inhibitory and time-course studies suggest that these oxidants are not the only species responsible for the lung injury and that their primary effect may be that of modulating the activity of other inflammatory mediators, such as proteinases (11). Recent studies from our laboratories have shown that in the neutrophil-dependent immunoglobulin (Ig)G immune complex–induced lung injury model, proteinases appear critical to the development of injury (12). Lung injury can be partially suppressed by secretory leukoproteinase inhibitor (SLPI), an inhibitor of serine proteinases, and by tissue inhibitor of metalloproteinase (TIMP)-2. Zymographic analysis of the bronchoalveolar lavage fluid (BALF) revealed that both serine proteinases (elastase and cathepsin G) and matrix metalloproteinases (MMPs) including MMP-9 (92-kD gelatinase B) were present and increased over time. By comparison, a model of neutrophil-independent acute lung injury induced by exogenous oxidant generation (glucose/glucose-oxidase) did not have proteinases present in the BALF (13). Corollary in vitro studies with rat neutrophils revealed that these cells had the same spectrum of serine proteinase and MMP secre-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

tion as was observed in vivo (12). Thus, neutrophil-derived serine proteinases and MMPs appear important in the evolution of neutrophil-dependent acute lung injury. The potential role of macrophage-derived proteinases in the development of acute lung injury has not previously been determined. Human macrophages are known to produce several MMPs, including MMP-2 (72-kD gelatinase A), MMP-9, MMP-1 (interstitial collagenase), MMP-3 (stromelysin-1), and MMP-12 (metalloelastase) (14–17). Differentiated alveolar macrophages, as compared with monocytes, do not contain detectable serine proteinase activity. A role for these MMPs has been established in chronic inflammation, where macrophages play a pivotal role in such degenerative inflammatory diseases as arthritis (rheumatoid and osteo) (18–19) and periodontal disease (20–21), where the degradation of extracellular matrix components such as type I and II collagens and proteoglycans is a significant component of disease progression. However, the role of macrophage-derived MMPs in the pathogenesis of acute inflammation is largely unknown. In recent studies utilizing immunohistochemistry, increased expression of MMP-1, MMP-2, and MMP-9 was detected in the lungs of patients with idiopathic pulmonary fibrosis and histocytosis X (22, 23). There are also recent reports of increased MMP-9 expression in the BALF of patients with adult respiratory distress syndrome and asthma (24, 25). Recent experimental studies have described elevated BALF levels of gelatinases and collagenases in animals injured by hyperoxia or with lipopolysaccharide (LPS), but there was no attempt to demonstrate that these activities were derived from macrophages or that they were, in fact, phlogistic (26–28). The studies described herein were undertaken to determine whether MMPs play a role in the progression of macrophage-mediated acute lung injury in the rat. To this end, two models were studied: a macrophage-dependent, neutrophil-independent model of lung injury induced by IgA immune complexes (7, 8, 29), and an LPS-induced injury model known to be both macrophage- and neutrophil-dependent (30). The data from these studies show that both IgAand LPS-induced injury were inhibited by recombinant human TIMP-2; and further, that the component of LPS injury that remained after neutrophil depletion was also inhibitable by TIMP-2. BALFs from injured animals of both models show increased levels of MMPs. Of endothelial cells, fibroblasts, type II epithelial cells, neutrophils, and alveolar macrophages isolated from rat lungs, only the alveolar macrophages showed a spectrum of MMP secretion that matched that seen in BALF. Alveolar macrophages taken from injured lungs in both models were activated in vivo to produce this same spectrum of MMPs. Together the data show that MMPs are produced during macrophage-dependent acute lung injury, that these MMPs play a role in the progression of injury, and that the alveolar macrophage is the likely source of these MMPs.

Materials and Methods Reagents and Materials Recombinant human TIMP-2 was a gift of Dr. Keith Langley of Amgen Pharmaceuticals (Thousand Oaks, CA).

Male Long–Evans rats were purchased from Charles River Breeding Company (Wilmington, MA) and housed under specific pathogen–free conditions in sterile cages under laminar flow. Tissue culture flasks and dishes were purchased from Corning (Corning, NY). Electrophoresis equipment, supplies, and reagents including Coomassie Brilliant Blue 250-R were purchased from Bio-Rad (Hercules, CA). Eagle’s minimal essential medium (MEM), balanced salt solutions, and fetal bovine serum (FBS) were purchased from Irvine Scientific (Irvine, CA). Ketamine was purchased from Fort Dodge Labs (Fort Dodge, IA). Bovine serum albumin (BSA), LPS, phorbol myristate acetate (PMA), gelatin, E64, pepstatin, phenylmethylsulfonyl fluoride (PMSF), b-casein, Triton X-100, ethylenediaminetetraacetic acid (EDTA), aminophenyl mercuric acetate (APMA), dimethyl sulfoxide, and Tris-HCl were purchased from Sigma (St. Louis, MO). Other reagents are noted below in the respective protocols. IgA Immune Complex–Mediated Acute Alveolitis Male (200 to 300 g) Long–Evans rats were used in this study. All experiments were carried out following review and approval by the University Committee on Use and Care of Animals (University of Michigan, Ann Arbor, MI). Rats were subjected to acute experimental alveolitis through the interstitial formation of IgA immune complexes as described previously (7, 8). Briefly, the rats were anesthetized with intraperitoneal Ketamine, after which the trachea was exposed by midline incision, and 300 mg of antidinitrophenyl IgA in sterile normal saline was instilled into the lung in two administrations with a 25-gauge needle. For the animals receiving then, inhibitors were coinstilled with IgA. The total volume was held constant at 300 ml. Following instillation, the incisions were closed with suture. The amount of 3.3 mg of dinitrophenyl (DNP)BSA in 1 ml of sterile normal saline was then instilled intravenously. For those procedures requiring measurement of permeability, this solution was supplemented with 1 mCi 125 I-labeled BSA. At various times following insult, the animals were killed by lethal injection of Ketamine, and the appropriate samples and measurements taken as described later. LPS-Mediated Acute Alveolitis Rats were subjected to acute experimental alveolitis by the intratracheal administration of LPS as described previously (30). Briefly, the rats were anesthetized with intraperitoneal Ketamine, the trachea was exposed, and 50 to 150 mg LPS in sterile normal saline was instilled into the lung in two administrations with a 25-gauge needle. As in the other model, the inhibitors were coinstilled with LPS; the total volume was held constant at 300 ml. For those procedures requiring measurement of permeability, 1 ml of sterile normal saline containing 1 mCi of 125I-labeled BSA was instilled intravenously. At various times following insult, the animals were killed and the appropriate samples and measurements taken as described later. Evaluation of Lung Injury: Permeability Index Animals previously injured and labeled with intravenous 125 I-labeled BSA tracer were assayed for lung permeability

Gibbs, Shanley, Warner, et al.: Matrix Metalloproteinases in Acute Lung Injury

as described elsewhere (4, 8). Briefly, the animals were anesthetized with Ketamine and their abdomens opened surgically. The amount of 1 ml of venous blood was drawn from the vena cava and transferred to counting tubes, and the animals were exsanguinated thereafter. Immediately after exsanguination, the thoracic cavities were opened and the lungs, heart, and pulmonary vasculature removed as a unit. The pulmonary circulation was flushed by injection of 10 ml of saline into the pulmonary artery. The lungs were then separated, rinsed, and transferred to counting vials for determination of radioactive content in a g counter. The permeability index (PI) was then determined by dividing the lung radioactivity by that in 1 ml of blood. Preparation of Histologic Sections of Rat Lungs Animals previously subjected to an injury protocol were killed by lethal injection of Ketamine and exsanguinated, and their thoracic cavities opened to expose the lungs and trachea. A loop of suture was led under the trachea, an incision was made anterior to the loop, and a small luer-lock catheter was fed into the incision and secured with the suture. The lungs, heart, and pulmonary vasculature were removed as a unit. The lungs were then filled with 10 ml of a 50/50 mixture of OCT (Miles, Elkhart, IN) and normal saline from a 10-cc syringe. The lungs were then immediately frozen in liquid nitrogen and kept at 2808C until they could be sectioned. Five-micron sections were prepared with a cryostat microtome and mounted on slides that were then stained with hematoxylin and eosin (H&E). Bronchoalveolar Lavage Animals previously subjected to an injury protocol were lavaged for lung contents. The animals were killed by lethal injection of Ketamine and exsanguinated, and their thoracic cavities opened to expose the lungs and trachea. A loop of suture was led under the trachea, an incision was made anterior to the loop, and a small luer-lock catheter was fed into the incision and secured with the suture. The lungs were then lavaged in situ with 10 ml of sterile normal saline from a 10-cc syringe, which was cycled five times with an average return between 8 and 9 ml. The lavage fluids thereby obtained were kept on ice and immediately centrifuged to remove contaminating cells. In those procedures designed to isolate cells, the lavage step was repeated two more times for a total final volume of 28–29 ml. The fluids were then subjected to various assay techniques as described below. In some experiments (as noted below), BALFs were concentrated 10- to 50-fold with Centricon 10 concentrators. Volume-to-volume control was strictly monitored to maintain sample comparability. In some experiments the BALF cells were put on slides by Cytospin and stained, and differential counts performed. Preparation of Alveolar Macrophages from Bronchoalveolar Lavage Alveolar macrophages were isolated by bronchoalveolar lavage (BAL) as described above. The cells were pelleted by 10 min centrifugation at 400 3 g and plated in MEM supplemented with 0.02% BSA, and penicillin/streptomycin (MEM-BSA). After allowing the cells to adhere to the plates for two hours, nonadherent cells were removed with

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two washes. Morphologic examination and staining for nonspecific esterase or with Griffonia simplicifolia-1 lectin (31) indicated that rat alveolar cells prepared in this manner were routinely . 95% macrophages. Unstimulated control cells and cells stimulated with PMA (100 ng/ml) were then allowed to incubate for various times, after which any cellular debris was removed by centrifugation at 400 3 g for 10 min and the conditioned medium tested as described later. It should be noted that MEM-BSA contains methionine, which is known to prevent oxidative activation of latent MMP by hypochlorous acid (32). In some instances the conditioned medium was concentrated either 10- or 50-fold using Centricon 10 (Amicon, Danvers, MA) concentrators before assay. In these cases, volume-to-volume concentration was strictly controlled. Plasma Preparations Plasma was drawn into heparin-coated tubes from rats 4 h after initiation of injury. Serial 5-fold dilutions were made and analyzed for MMP activity as described later. Culture and Preparation of Rat Type II Epithelial Cells, Fibroblasts, and Endothelial Cells Primary cultures of rat type II alveolar epithelial cells were prepared by the method of Simon and colleagues (31), plated into 24-well dishes, and allowed to grow to confluence. Primary cultures of rat lung fibroblasts were prepared by finely mincing rat lungs with a sterile blade and placing the pieces into a 25-cm2 flask with enough medium to cover the bottom. After allowing the pieces to adhere, the flasks were carefully inverted and placed in an incubator at 378C in 5% CO2 and 100% humidity. After a period of 24 to 48 h, a ring of fibroblasts could be observed colonizing the plastic of the dish around each piece of lung tissue. These were trypsinized and replated into new flasks and kept for use through passage seven. The preparation and characterization of rat pulmonary artery endothelial cells was carried out as described in detail previously (33). The cells were grown in 25-cm2 or in 24-well plates. The growth medium for all cells was MEM containing nonessential amino acids, penicillin/streptomycin, and 10% FBS. Cell growth was at 378C in 5% CO2/95% air and 100% humidity. When cell culture fluid was used for enzyme analysis, the serum in the basal medium was replaced with 0.02% BSA. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Substrate–Embedded Enzymography Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) substrate-embedded enzymography (zymography) was carried out by a modification of the method of Heussen and Dowdle (34). Zymography was used in these studies as the initial approach to identifying and characterizing the MMPs. Briefly, SDS-PAGE gels were prepared for minigels from 30:1 acrylamide/bis according to the recipe that follows, with the added incorporation of either gelatin (1 mg/ml) or b-casein (1 mg/ml) before casting. The gelatin gels were routinely 7.5% acrylamide, whereas the casein gels were routinely cast at 10% acrylamide. The final concentrations of the other com-

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ponents of the gels were: Tris-HCl at pH 8.8 (325 mM), SDS (0.1%), ammonium persulfate (0.05%), and N,N,N9,N9,tetra-methylethelynediamine (TEMED) (0.05%). Various denatured but nonreduced samples and standards were then electrophoresed into the gels at constant voltage of 150 V in an ice bath under nonreducing conditions. When the dye fronts reached a point approximately 0.5 cm from the bottom of the gels, they were removed and subjected to the following washing protocol: twice for 15 min each time in 50 mM Tris buffer (containing 1 mM Ca21 and 0.5 mM Zn21) with 2.5% Triton X-100; once for 5 min in Tris buffer alone; and finally overnight in Tris buffer with 1% Triton X-100. Inhibitors were added as desired to the overnight wash as indicated later. The gels were stained the following morning with Coomassie Brilliant Blue 250-R. Following destaining, zones of enzyme activity were detected as regions of negative staining. Some samples were activated with APMA before electrophoresis to show the band shift that occurs when latent MMPs are activated through cleavage of the N-terminal domain (35).

Results TIMP-2 Inhibition of IgA Immune Complex–Mediated Rat Lung Injury The first studies were undertaken to determine the role of MMPs in macrophage-dependent lung injury. For this, we first characterized the effects of the MMP inhibitor TIMP-2 on IgA immune complex–mediated lung injury. Figure 1A shows the result of studies in which 1 mg TIMP-2 was coinstilled with the injurious anti-DNP IgA intratracheally at time zero. Although the extent of injury varied from animal to animal, when the data were normalized to controls in the calculation of net permeability index (PI) TIMP-2 gave a mean 46 6 12% inhibition, which was statistically significant (P , 0.001). The effect of TIMP-2 was doseresponsive over the range from 0.1 to 1.0 mg, at which its effect was maximized (not shown). Inclusion of an irrelevant protein in place of TIMP-2 had no effect on the injury. The inhibition data provide strong support for the involvement of MMPs in IgA immune complex–induced injury. TIMP-2 Inhibition of LPS-Mediated Rat Lung Injury In similar experiments, TIMP-2 was tested for its ability to inhibit neutrophil- and macrophage-dependent LPS-mediated injury. Figure 1B shows that 1 mg of TIMP-2 instilled intratracheally gave a significant inhibition (25 6 11%, P , 0.05) of the LPS injury. In this model, the inhibitory effect of TIMP-2 was also dose-dependent (data not shown). Thus, as in the IgA model, the partial inhibition of lung injury in the LPS model suggests a role for MMPs in the development of acute lung injury. TIMP-2 Inhibition of LPS-Mediated Rat Lung Injury in Neutrophil-Depleted Rats Our previous studies of the neutrophil-dependent IgG lung injury model showed that TIMP-2 inhibited neutrophil-mediated lung injury (12). However, the data showing that TIMP-2 inhibits the macrophage-mediated IgA injury

suggest that MMPs are also involved in macrophagemediated inflammatory processes. The partial macrophage dependence of the LPS model, combined with the observation that the number of BALF macrophages remains constant during LPS injury (not shown), allowed further testing of the hypothesis that MMPs play a role in macrophage-dependent lung injury independent of neutrophil accumulation. To this end, rats were made greater than 95% neutropenic by the intraperitoneal injection of a rabbit antirat neutrophil antibody 18 h before the start of the experiment. LPS-induced lung injury of these neutrophildepleted rats is shown in Figure 1C, along with data from nondepleted controls and depleted rats in which TIMP-2 was coinstilled with the LPS. Although neutrophil depletion by itself caused a significant reduction in injury (36 6 7% inhibition, P , 0.05), TIMP-2 was still able to provide a further (24 6 6%, P , 0.05) reduction in the remaining injury. These data thus suggest that MMPs play a role in the development of macrophage-dependent lung injury, independent of neutrophils. TIMP-2 Inhibition of Neutrophil Influx in LPS-Mediated Rat Lung Injury In additional studies, lungs from LPS-injured animals and TIMP-2–protected animals were lavaged at the end of the study (e.g., after 6 h) and differential cell counts were performed on the contents. These data are summarized in Figure 2. Whereas the alveolar macrophage counts in the BALF remained the same as in the controls (not shown), a marked increase in BALF neutrophils accompanied LPS injury. The addition of TIMP-2 gave a significant inhibition of this neutrophil influx (49 6 17%, P , 0.05). The reduction in BALF neutrophils supports the hypothesis that MMPs are involved in the process of neutrophil accumulation during lung inflammation. Figure 3 shows histologic sections of lungs from similar experiments, which confirm the BAL results. Histologically, the instillation of LPS induced the accumulation of neutrophils in the lung and this was associated with injury to the alveolar capillaries. The coinstillation of 1 mg TIMP-2 reduced the numbers of neutrophils in the lung, with macrophage numbers appearing unaffected. These data, combined with our previous observations of TIMP-2 inhibition of hemorrhage and vascular permeability in the neutrophil-mediated IgG model (12) as well as in the partially neutrophil-mediated LPS injury models, provide evidence that MMP inhibitors protect, in part, by limiting neutrophil influx into the alveolar space. Zymography of BALF from Injured Rat Lungs The ability of TIMP-2 to attenuate macrophage-dependent lung injury in vivo supports the involvement of MMP activities in the injury process. It was therefore of interest to determine whether increased MMP levels were present in the lung during injury, and if so, to determine the likely source of these MMPs. To this end, BALFs from the IgAinjured and uninjured control animals were collected. After removal of cells and debris, the supernatants were analyzed for evidence of released MMPs by gelatin and casein zymography. These experiments showed that BALF from


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Figure 2. TIMP-2 inhibition of LPS-induced neutrophil migration into the rat lung. Rats were treated with intratracheal injection of either 0.5 mg TIMP-2, 150 mg LPS, or 150 mg LPS plus 0.5 mg TIMP-2 in a total volume of 300 ml. The injury was allowed to progress for 6 h, after which the animals were killed and the number of neutrophils in lung lavage determined as described in MATERIALS AND METHODS. TIMP-2 treatment decreased BALFneutrophils by 49 6 17% (P , 0.05 by ANOVA and Student– Newman–Keuls testing; n 5 9 animals per group for all three groups).

rats injured with IgA immune complexes and killed after 4 h contained increased proteinase activity against gelatin and casein as assessed by zymography (Figure 4). The lavage fluids from negative control animals (receiving antibody but no antigen) showed only a faint gelatinolytic activity at 72 kD and no measurable activity at 92 kD (Figure 4, lane U). The faint 72-kD band was also present in the injured animals (Figure 4, lane 1), but not significantly increased; whereas significant 92-kD activity was present in the BALF from the injured animals (Figure 4, lane 1). In BALF from some of the injured animals, a faint band of gelatinolytic activity could also be identified at 35 kD; often, however, it was too weak to be detected. The 35-kD band appeared more clearly on casein gels (Figure 4, lane 2). All of the activities were inhibited when EDTA was in-

Figure 1. TIMP-2 inhibition of IgA immune complex–induced and LPS-induced acute lung injury. Rats were subjected to injury with IgA immune complexes or with LPS in the presence or absence of 1 mg TIMP-2, injected intratracheally at the time of injury. Values shown (PIs) for control rats were arbitrarily set at 1.0 and the reduction of injury in the presence of TIMP-2 shown as a percentage of the control. In the IgA model ( A), the negative controls were 0.15 6 0.03 PI and the positives were 0.37 6 0.09 PI (n 5 9 animals for both the IgA and IgA 1 TIMP-2 groups, n 5 3 for the negative control group). TIMP-2 gave a mean 46 6 12% inhibition, which was statistically significant ( P , 0.001). In the LPS model (B), the negative controls were 0.14 6 0.05 PI and the

positives were 0.62 6 0.18 PI (n 5 11 animals for both the LPS and LPS 1 TIMP-2 groups, n 5 5 for the negative control group). TIMP-2 gave a mean 25 6 11% inhibition, which was statistically significant (P , 0.05). In C, where indicated, rats were depleted of neutrophils by intraperitoneal injection of rabbit antirat neutrophil antibody 18 h before treatment. This routinely produced greater than 90% neutrophil depletion. Data are normalized to positive and negative controls for comparison. Neutrophil depletion by itself caused a significant reduction in injury (36 6 7% inhibition, P , 0.05 compared with LPS alone). TIMP-2 was able to provide a further 24 6 6% reduction in the remaining injury (P , 0.05 compared with the neutrophil-depleted LPS injury by analysis of variance [ANOVA] and Student–Newman–Keuls testing; n 5 11 per group for all groups except the negative control group, where n 5 5).

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Figure 3. Histology of LPS-injured and TIMP-2–protected rat lungs. Rats were treated with intratracheal injection of either 150 mg LPS or 150 mg LPS plus 0.5 mg TIMP-2 in a total volume of 300 ml. The injury was allowed to progress for 6 h before death, at which time the lungs were snap-frozen in liquid nitrogen. The specimens were then embedded in OCT, and 5-mm sections were prepared and stained as described in M ATERIALS AND METHODS. The LPS-treated animals (A and B) showed a significant number of neutrophils in the lung (filled arrows), which was associated with some hemorrhage and fibrin deposition. Macrophages (open arrows) were also prominent. The addition of TIMP-2 (C and D) caused a significant reduction in the number of neutrophils (filled arrows), whereas numerous macrophages were still present (open arrows) (H&E. Original magnification: A and C, 340; B and D, 3100).

cluded in the overnight buffer (Figure 4, lane 4) but were resistant to PMSF, E64, and pepstatin (not shown), confirming their identity as metalloproteinases. In addition, each of the activities underwent the characteristic band shift to a lower molecular-weight form when subjected to pretreatment with APMA (not shown). These results demonstrate that increased levels of MMPs are expressed in vivo in the lungs during IgA immune complex–induced acute alveolitis. A similar profile is seen in Figure 4, lanes 3 and 5, which shows the data from lavage fluid taken from the lungs of LPS-injured rats. The same gelatinolytic and caseinolytic activities seen in the BALF of IgA-injured animals at 92, 72, and 35 kD were also present in the lungs of LPSinjured animals (Figure 4, lanes 3 and 5). An additional activity of 130 kD was also seen. A similar high molecularweight activity was also previously observed in IgG immune complex–mediated (neutrophil-dependent) injury (12). This band has been shown by others to represent complexes with NGAL, a neutrophil gelatinase–associated lipocalin, which is a component of the tertiary granules of neutrophils (36, 37). Together, these results show that increased levels of MMPs are expressed in vivo in the lungs during LPS-induced lung injury. Gelatin Zymography of Cells Isolated from Rat Lungs To identify potential sources of the MMPs observed in the BALFs from rats in the in vivo injury models, primary cultures of cells endogenous to the rat lung were prepared. The cells were allowed to condition serum-free medium overnight in the presence of PMA. These conditioned me-

dia were then examined by gelatin zymography and compared with the spectrum of MMPs found in the BALF of injured animals. In addition, plasma was obtained from rats and examined in the same assays. Results of these studies are shown in Figure 5. Rat plasma (Figure 5, lane S) showed a strong band of activity at 72 kD with weaker activity at 92 kD. The rat lung fibroblasts (Figure 5, lane RF) produced a strong band of gelatinolytic activity at 72 kD but no other detectable bands. The rat pulmonary artery endothelial cells (Figure 5, lane EC) also demonstrated a 72-kD activity. Rat pulmonary vein and pulmonary microvascular endothelial cells were also tested, with similar results (not shown). The rat type II epithelial cell cultures (Figure 5, lane Epi) demonstrated activities at both 92 and 72 kD, with the 72-kD enzyme being the major activity. As can be seen, the only cell that had a profile of activity (e.g., mostly 92-kD activity with little 72-kD activity) similar to that seen in the BALF of injured rats was the alveolar macrophage (Figure 5, lane M). It should be noted that although PMA was used to stimulate enzyme elaboration by alveolar macrophages in this study, we have also shown that either LPS- or IgA-containing immune complexes induce the same spectrum of enzymes (38). Our previous studies (12) showed that rat neutrophils also secrete a 92-kD gelatinase activity. However, the same studies showed that these cells do not secrete the 72-kD gelatinase activity observed in BALF from IgA- or LPSinjured animals. Further, there are very few neutrophils in the BALF of uninjured animals or of animals injured with IgA immune complexes. These data suggest, therefore, that the alveolar macrophage is the source of much of the MMPs found in the BALF from injured lungs in the mac-


Figure 4. Zymography of BALF from injured rat lungs. Rats were subjected to either IgA immune complex–induced or LPSinduced injury as described in M ATERIALS AND METHODS, after which the animals were killed and BALF collected. Lane U represents gelatin zymography of BALF from uninjured control animals. Lane 1 represents gelatin zymography of BALF taken from the lungs of IgA immune complex–injured rats. Lane 2 represents casein zymography of the same sample. Lane 3 represents gelatin zymography of BALF taken from the lungs of LPS-injured rats, and lane 4 shows inhibition of the gelatinolytic activity by EDTA. The inset in lane 5 shows casein zymography of 50-fold concentrated BALF from LPS-injured lungs.

rophage-dependent IgA model and in the macrophageand neutrophil-dependent LPS model. Production of MMP by Alveolar Macrophages Isolated from IgA-Injured Rat Lungs The next step was to isolate alveolar macrophages from IgA-injured lungs and uninjured controls, allow them to condition the culture media, and screen for MMPs. The results of this experiment are illustrated in Figure 6. As can be seen, cells from the injured lungs (Figure 6, lanes 2 and 5) produce 92-kD gelatinolytic and 35-kD caseinolytic activities without the requirement for additional in vitro stimulation, whereas cells from uninjured lungs have much lower or undetectable activities (Figure 6, lanes 1 and 4). Interestingly, macrophages from these same BALFs also demonstrated a weak band at 22 kD. In the BALFs themselves, the 22-kD activity was seen only after 50-fold concentration (Figure 4, lane 5). Finally, the alveolar macrophages (from either control or injured lungs) demonstrated a faint band of gelatinolytic activity at 72 kD (Figure 6, lanes 1–3). Together these data indicate that alveolar macrophages from injured lungs produce a comparable spec-

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Figure 6. Gelatinolytic activity and caseinolytic activity in 18-h culture fluids from alveolar macrophages taken from uninjured and IgA-injured rats. Macrophages were isolated from rats that had been subjected to IgA immune complex injury as described in MATERIALS AND METHODS section and from antibody only– treated, sham-operated control rats and placed in culture. Lane 1 is a gelatin zymogram of media conditioned by macrophages from the uninjured control animals. Lane 2 is a gelatin zymogram of media conditioned by macrophages taken from the lungs of IgA immune complex–injured rats. Lane 3 is a gelatin zymogram of media conditioned by macrophages taken from the lungs of IgA immune complex–injured rats and stimulated in vitro with 100 ng/ml PMA. Lane 4 is a casein zymogram of media conditioned by macrophages from the uninjured control animals. Lane 5 is a casein zymogram of media conditioned by macrophages from the lungs of IgA immune complex–injured rats. Lane 6 is a duplicate of lane 3, except that the zymogram was treated with TIMP-2 (50 mg/ml) during the overnight development wash.

trum of activities to that found in the BALF of injured rats. Further, the activities appear in the same relative proportions. It is interesting that although macrophages lavaged from control rats rarely demonstrated any 92-kD activity, cells from negative control animals in this experiment showed a weak but detectable 92-kD activity without additional in vitro stimulation (Figure 6, lane 1). It must be remembered, however, that the BALFs were obtained from these animals 4 h after intra-alveolar injection of the antibody. Even in the absence of antigen, it is conceivable that the antibody could elicit a response in the resident macrophages as they attempt to phagocytose or endocytose the instilled protein. As a final step, TIMP-2 was used in an effort to block the activities expressed by stimulated alveolar macrophages. A duplicate of Figure 6, lane 3 (conditioned medium from alveolar macrophages taken from the lungs of IgA-injured rats and stimulated with PMA for 18 h in vitro), was separately developed in zymography buffer containing 50 mg/ml TIMP-2. Figure 6, lane 6, shows that the MMP activities of stimulated rat alveolar macrophages were completely inhibited by TIMP-2.

Discussion Figure 5. MMP activity in rat plasma and in culture fluids from cells endogenous to the rat lung. Rat plasma from LPS-injured rats was diluted 1:50. Primary cultures of rat lung cells were allowed to condition medium as described in M ATERIALS AND METHODS. Lane S is a gelatin zymogram of the plasma. Lanes RF, EC, Epi, and M are gelatin zymograms of conditioned media from PMA-stimulated rat lung fibroblasts, pulmonary artery endothelial cells, rat lung type II epithelial cells, and alveolar macrophages, respectively.

The formation of IgG- or IgA-containing immune complexes in the alveolar wall after intravenous injection of antibody and concomitant intratracheal instillation of antigen results in acute inflammatory lung injury, as does the intratracheal injection of LPS. Past studies have shown that large numbers of neutrophils are recruited to the lung when IgG-containing immune complexes are formed and that the resulting injury is mediated largely by neutrophil

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products (6, 7, 12, 13, 39). In contrast, almost no neutrophils are recruited into lungs with IgA-containing immune complexes, and injury is thought to be due to resident or recruited macrophages (8, 22, 40–42). In LPS-injured lungs, a role for both neutrophils and macrophages has been described (30, 42–44). In spite of these differences, injury in all three models can be largely attenuated by catalase treatment (5, 6, 8, 44). Thus, oxidants appear to play a critical role in all three. However, the effect of antioxidant therapy is overcome with time, suggesting a role for other leukocyte products, such as proteolytic enzymes (11). The exact role that proteolytic enzymes play in these models of acute inflammation is not well understood. In a recent study it was demonstrated that SLPI and TIMP-2 were both capable of partially inhibiting neutrophil-mediated lung injury (e.g., injury induced by IgG-containing immune complexes) (12). The two agents together produced greater inhibition than did either separately. Because these two peptides exhibit narrow inhibitory specificities and high affinity constants for their respective proteinases (allowing for pseudo-irreversible inhibition), the data suggest that both serine proteinases and MMPs contribute to neutrophil-induced lung injury. The present study extends this line of research. Here it is shown that TIMP-2 also produces significant inhibition of inflammatory lung injury in models that are primarily macrophagedependent. A role for macrophage-derived oxidants in these models has previously been shown (29), but this is the first evidence that proteinases are also involved. These findings raise several additional questions to be addressed. First is the source of the enzymes responsible for injury. Although human macrophages are known to elaborate MMPs (13–17), and other human parenchymal cells in the lung also express some of the same enzymes, the critical characterizations have not been done for cells of the rat lung. To begin addressing this question, we compared the gelatinolytic and caseinolytic profiles of BALF from normal and injured rats with activities present in culture fluid from lung macrophages, type II epithelial cells, fibroblasts, and endothelial cells. The gelatinolytic enzyme profile of rat plasma was also examined because extravasation of plasma proteins into BALF occurs as a consequence of injury. Only the macrophage profile (i.e., high level of gelatinolytic activity in the 92-kD region, a low level of 72-kD activity, and caseinolytic activity in the 35 to 22–kD region) matched that seen in BALF from injured rats. Thus, we suggest that lung macrophages themselves are responsible for much of the MMP activity observed in the BALF of injured rats. This does not, of course, rule out the involvement of MMPs produced by other cell types. Another important issue is the identities of the enzymes involved. Macrophages from a number of sources have been partially characterized with regard to MMP elaboration at the protein level. However, with the exception of one study demonstrating 92-kD gelatinolytic activity in alveolar macrophages from bleomycin-injured rats (45), rat lung macrophages have not been investigated. The accompanying article (38) provides a detailed characterization of the proteolytic enzyme activities recovered from culture fluids of rat lung macrophages. Characterization studies indicate which enzymes are represented (and

therefore potential candidates), but because all of the MMPs are sensitive to TIMP-2 inhibition it is impossible to rule specific MMPs in or out as mediators of injury. Specific inhibitors, blocking antibodies, or genetically altered animals will be needed to demonstrate definitively a role for individual MMPs in the injury process. It should be noted that most of the observable MMPs in BALF, as well as the majority of enzymes in the cell-culture fluids, was in the latent form. If these enzymes are mechanistically involved in lung injury, then it might be expected that active as well as latent forms of the enzymes would be detected. There are a number of possible explanations for the lack of detectable MMP activity. First, active enzyme forms are generally present as a small fraction of the total enzyme pool. It may be that the concentration of cells used to condition the culture medium and the amount of saline instilled in the lung as part of the lavage protocol are such that the minor enzyme forms were diluted to a point where they could not be detected. In support of this, we can, in fact, detect active forms of MMP-9 in BALFs after 50-fold concentration. Second, active forms of the MMPs have high affinity for both substrate (matrix) and inhibitors (46–48). This leads to a situation where the activated enzyme would be preferentially adsorbed and may, therefore, not show up in BALF until the potential “sink” was overwhelmed. This might be of particular concern in the animal models used here because the amount of immune complexes or the amount of LPS instilled into the lung was adjusted to produce detectable, but not lethal, injury. Virtually all of the animals injured to such degree and then left alone recover fully. When injury is more severe, a much greater amount of enzyme can be detected in the BALF, and under such conditions, active enzyme forms are also seen (unpublished observation). Active forms of the 72- and 92-kD gelatinases have also been reported in the BALF of animals exposed to 100% oxygen (26). Such animals succumb to injury within 3 d. Finally, the mechanism(s) by which MMPs contribute to acute lung injury in the macrophage-dependent models needs to be addressed. One mechanism supported by our data involves a direct role for MMPs in neutrophil recruitment. In the LPS injury model, in which both neutrophils and macrophages play roles, the intratracheal instillation of TIMP-2 at the time of injury strikingly reduced the number of neutrophils that entered into the alveolar space. Likewise, in our previous studies with the IgG immune complex (neutrophil-dependent) model of injury, there was an inhibition of neutrophil influx into the lungs of animals treated with intratracheal instillation of TIMP-2 (12). The presence of exogenous MMP inhibitor in the alveolar space may limit neutrophil influx in a variety of possible ways. The presence of the inhibitor may prevent damage to the basement membrane, which is necessary for neutrophil transmigration across the vessel wall. Such an effect might also account for the reduction in PI. Another possibility is that the proteolytic enzymes might be directly responsible for injury to the resident epithelial cells and endothelial cells. Injury to resident cells in the alveolar unit is a pathogenic feature of acute inflammatory lung injury. Cellular injury has been directly attributed to proteolytic enzymes in some studies, whereas other studies have dem-


onstrated cell injury resulting from combinations of oxidants and proteolytic enzymes (reviewed in 49). Certainly, other mechanisms are also possible. For example, it was recently shown in IgG immune complex–induced injury (neutrophil-dependent) that neutrophil influx was mediated primarily by the complement chemotactic peptide C5a. The levels of this chemotactic peptide in the BALF were inversely correlated with TIMP-2 levels (50). Additional studies will be required to address the relevance of these findings to macrophage-dependent injury.

21.

22. 23.

24.

Acknowledgments: This study was supported in part by grants HL42607 and CA60958 from the U.S. Public Health Service. The authors thank Beverly Schumann for her help in the preparation of this manuscript.

25.

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