American Journal ofPathology, Vol. 136, No. 6, June 1990 Copyright C) American Association ofPathologists

Degradation of Basement Membrane Laminin by Human Neutrophil Elastase and Cathepsin G Louis W. Heck,* Warren D. Blackburn,* Michael H. lrwin,t and Dale R. Abrahamsont From the Department ofMedicine,* Veterans Administration Medical Center, and the Departmenlt of Cell Biology andAnatomyJ University ofAlabama at Birmingham, UAB Station, Birmingham, Alabama

To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manneryielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss oflaminin epitopesfrom basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation. (AmJPathol 1990, 136:1267-1274)

Tissue injury results in the local release of a number of inflammatory mediators that promote an increase in vascular permeability and the recruitment of neutrophils. The directed migration of these cells from the circulation to sites of tissue damage requires passage through the vascular endothelium and subendothelial basement mem-

brane. Although the processes of chemotaxis and diapedesis are not fully understood, the infiltration of large numbers of neutrophils into the subendothelium coupled with release of reactive oxygen metabolites and secretion of neutrophil proteases can lead to tissue destruction.1 Several previous studies have shown that basement membrane degradation mediated by neutrophils occurs mainly as a result of secretion of the serine proteases elastase and cathepsin G.-5 In addition, this enzyme-mediated damage can be augmented by reactive oxygen metabolites from neutrophils, endothelial cells, mesangial cells, and/or injured tissue.6 Although much evidence indicates that collagen type IV is targeted by neutrophil elastase,25 whether other basement membrane components are similarly digested has not been established. The major noncollagenous component of basement membranes is laminin, which is a large multidomain glycoprot6in with a total molecular mass of -850,000.7 The laminin protein originally purified from the mouse Englebreth-HolmSwarm (EHS) tumor is composed of one A chain (Mr -400,000) and two similar B chains (each with Mr '200220,000) assembled as an asymmetric four-armed cross (reviewed in Martin and Timpl8 and in Timpl9). Laminin binds to integral membrane proteins on certain cells and thus appears to serve in basement membranes as a cellsubstrate attachment element. In addition to containing cell-binding domains, laminin also binds specifically to collagen type IV, heparan sulfate proteoglycans, and entactin/nidogen.8-9 Therefore any damage to laminin could conceivably create major structural and functional defects within basement membranes and contribute to tissue disorganization. In the experiments described here, we show that human neutrophil elastase (HNE) and cathepsin G (HNCG) extensively degraded laminin. These experiments were funded by grants from the National Institutes of Health (DK 34972 and DK 39258) and a grant-in-aid from the American Heart Association. M. H. Irwin is the recipient of a fellowship from the American Heart Association, Alabama Affiliate. D.R. Abrahamson is an Established Investigator of the American Heart Association. Accepted for publication January 10, 1990. Address reprint requests to L. W. Heck, MD, Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, LHR 405, UAB Station, Birmingham, AL 35294.



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Materials and Methods Materials and Supplies The following reagents were purchased: phorbol 12-myristate 13-acetate (PMA) from Consolidated Midland Chemical Co., Brewster, NY; diisopropylfluorophosphate (DFP), ethylenediamine tetraacetic acid (EDTA), and bovine serum albumin (BSA) from Sigma Chemical Co., St. Louis, MO; Dextran T 400 and Ficoll-Paque from Pharmacia Fine Chemicals, Piscataway, NJ; p-nitrophenol, N-succinyl-(L-alanine)3-p-nitroanilide, succinyl-(L-alanyl)2L-prolyl-L-phenylalanine-p-nitroanilide, benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide, and p-nitrophenyl p'guanidinobenzoate (p-NPGB) from Calbiochem, San Diego, CA; bovine trypsin from Worthington Co., Freehold, NJ; Dulbecco's phosphate buffered saline (PBS) without magnesium and calcium, Hank's balanced salt solution (HBSS) without phenol red, and 0.4% Trypan Blue solution from Grand Island Biological Co., Grand Island, NY; reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from Bio-Rad, Richmond, CA.

Preparation of Soluble Laminin and Rat Monoclonal Anti-laminin Antibodies Laminin was purified from the EHS tumor by sequential salt extraction, ion-exchange, and gel-filtration chromatography by a modification of the method of Timpl et al.7 and characterized as previously described.'0 Laminin was quantitated by the Lowry method" with BSA as the protein standard and stored at 40C in 0.5 mol/l (molar) NaCI, 0.05 mol/l TRIS hydrochloride, pH 7.6, containing 0.01% sodium azide. Before digestion assays, laminin was dialyzed against azide-free TRIS buffer. Rat monoclonal antilaminin antibodies designated 5A2, 5C1, and 5D3 were prepared and characterized as described elsewhere.12

Neutrophil Isolation and Activation Thirty-five milliliters of whole blood from healthy adult volunteers were drawn into syringes containing heparin (20 units/ml). After sedimentation in 4.5% dextran for 20 minutes at room temperature, the leukocyte-rich layer was washed in PBS and then layered over Ficoll-Paque. The neutrophils and mononuclear cells were separated by isopycnic centrifugation as described by B6yum.'3 Neutrophils were washed and resuspended in HBSS, counted in a hemocytometer, and adjusted to a concentration of 5 x 106 cells/ml. The final cell preparation contained 94% to 96% viable neutrophils as determined by trypan blue

exclusion. Phorbol 1 2-myristate 13-acetate, previously prepared as a 1 mg/ml solution in dimethyl sulfoxide and stored at -70°C, was thawed immediately before use, diluted, and added to the neutrophils to give a final concentration of 100 ng PMA/ml. This neutrophil-PMA preparation was incubated at 370C for 60 minutes without C02 and tubes were centrifuged at 300g for 15 minutes to pellet cells. The supernatants were further centrifuged at 40,000g for 15 minutes to obtain soluble products from PMA-stimulated neutrophils.

Isolation of HNE, HNCG, and Affinity-purified Antibodies Human neutrophil elastase and HNCG were isolated from human neutrophils as previously described and differentiated from each other by amino acid composition, amino acid sequence analysis, isoelectric points, substrate specificities, and immunoreactivities.14"15 One microgram of purified HNE released 83.4 pmol of p-nitrophenol from N-succinyl-(L-alanine)3-p-nitroanilide/minute at 370C. One microgram of purified HNCG released 103 pmol of p-nitrophenol from -succinyl-(L-alanyl)2-L-prolyl-L-phenylalaninep-nitroanilide/minute at 37°C. Affinity-purified antibodies to HNE (1 mg/ml) and HNCG (0.75 mg/ml) were prepared as described'1516 and stored at -70°C. The ability of these antibodies to inhibit proteolysis was tested in a standard azocasein assay."7 Azocasein digestion by 2 ,ug HNE or HNCG was completely inhibited by preincubation of either enzyme with 100 gg of the appropriate monospecific antibody. On the other hand, anti-HNE failed to inhibit HNCG, and anti-HNCG failed to inhibit HNE.

Preparation of Inorganic Inhibitors A stock EDTA solution was adjusted to pH 7.4 with 6N HCI and diluted to a final concentration of 0.1 mol/l. The following system was used to monitor DFP inhibitory activity. First, active site titration of trypsin using p-NPGB was carried out by the method of Chase and Shaw.'8 This was necessary because the active site molarity of trypsin in solution differs from that expected on the basis of weight. Second, the DFP (stored under N2 at -700C) was thawed, diluted, and demonstrated to inactivate trypsin amidolytic activity irreversibly in a stoichiometric manner.'9

Incubation of Soluble Laminin with Neutrophil Supernatants One hundred microliters of supernatants from PMA-stimulated neutrophils (protein content 4 Ag) was added to 1.5

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ml polypropylene tubes. Inhibitors and antibodies were added to final concentrations as follows: DFP, 10-5 mol/ I; EDTA, 5 X 10-2 mol/l; anti-HNE, 100 ,g/ml; anti-HNCG, 100 jg/ml; a mixture of both antibodies; or control supernatants containing no inhibitor, and tubes were incubated for 30 minutes at 37°C. Fifty microliters (30 ,ug) of laminin was added to each tube and samples were incubated for an additional 8 hours at 37°C. Incubations were stopped by adding 50 ,ul of 0.125 mol/l TRIS hydrochloride (pH 6.8), 4% SDS, 20% glycerol, 1% 2-mercaptoethanol (SDS-PAGE sample buffer) followed by boiling the samples for 2 minutes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed by a modification of the method of Laemmli using a 5% to 20% polyacrylamide gradient as previously described.20

Incubation of Soluble Laminin with Purified HNE and HNCG Laminin (30 jig) was incubated with purified HNE or HNCG at different enzyme:substrate ratios ranging from 1:3 to 1:300 for 30 minutes at 37°C or at a fixed enzyme: substrate ratio (1:30) for varying times (5 to 480 minutes) at 37°C. Samples were then prepared for SDS-PAGE as described above.

Immunoblotting of Digested Laminin Laminin was digested for 1 hour at 37°C with either HNE HNCG at an enzyme:substrate ratio of 1:12. Control laminin containing no enzyme was also incubated for 1 hour at 37°C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer was then added and samples were heated to 100°C for 2 minutes. Samples were electrophoresed and transferred to nitrocellulose by the method of Towbin et al.21 Nonspecific binding sites were blocked by overnight incubation at 4°C in 5% nonfat dry milk in PBS22 containing 1 % bovine serum albumin and 2% normal goat serum. Nitrocellulose membranes were sandwiched into a 16-channel Miniblotter (Immunetics, Cambridge, MA) and treated for 1 hour at room temperature with rat monoclonal anti-laminin IgGs diluted to 100 ,g/ml in blocking buffer. Blots were washed extensively and subsequently reacted for 1 hour with horseradish peroxidase-conjugated goat anti-rat IgG (Organon TeknikaCappel, West Chester, PA), diluted 1:800 in blocking buffer. After extensive washing, bound antibody was detected by reaction with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.01% H202 in 0.05 mol/l phosphate buffer, pH 6.0. Prestained molecular weight standards were obtained from BRL, Bethesda, MD. or

Enzyme Treatment of Kidney Basement Membranes and Fluorescence Microscopy Kidneys were removed from anesthetized mice and wedges of unfixed cortex were rapidly frozen in isopentane chilled in a bath of dry ice and acetone. Sections (4 ztm thick) were cut at -200C in a cryostat and air-dried at room temperature for 1 hour. Sections were then flooded with 70 Al of 100 ,ug/ml HNE or HNCG, in 0.2 mol/l TRIS buffer, pH 7.6, and incubated at 37°C for 5 minutes. Control slides were treated with buffer alone. Slides were then washed with PBS and incubated for 5 minutes with 50 ,g/ ml of rat monoclonal anti-mouse laminin IgG designated 5A2. Slides were again washed with PBS and then treated for 5 minutes with fluorescein-conjugated goat anti-rat IgG (Organon-Teknika Corp., Cappel, West Chester, PA). Washed sections were coverslipped and examined by epifluorescence in a Leitz Orthoplan photomicroscope (Rockleigh, NJ).

Results Laminin Digestion by Supernatants of Stimulated Neutrophils As shown in Figure 1, PMA-activated neutrophils released laminin-cleaving activity into supernatants and both the A and B chains were extensively degraded in our assay system (lane 7). The observed degradation was not due to the presence of contaminating proteinases in the laminin preparation because the A and B chains persisted after prolonged incubation at 37°C in the absence of exogenous enzyme (Figure 1, lane 1). Pretreatment of the neutrophil supernatants with the metalloproteinase inhibitor, EDTA, failed to inhibit the laminin-cleaving activity (lane 2). In contrast, apparently complete inhibition was obtained with addition of the serine proteinase inhibitor, DFP (lane 6). Because HNCG and HNE are the major neutrophil serine proteinases active at physiologic pH, we tested whether inhibition of laminin digestion could be obtained by preincubation of neutrophil supernatants with affinitypurified antibodies against these enzymes. An equal mixture of anti-HNCG and anti-HNE effectively blocked laminin degradation (lane 3), but anti-HNCG alone was generally ineffective (lane 4). On the other hand, anti-HNE alone (lane 5) largely inhibited laminin degradation. As a further test to determine the presence of granule constituents in activated neutrophil supernatants, a solid-phase radioimmunoassay for HNE, HNCG, and lactoferrin was carried out. As shown in Table 1, neutrophils treated with PMA released approximately 3.6 times more HNE and 2.2 times more HNCG than untreated cells.

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polypeptides, including one at -103 kd, were detected after HNCG treatment (lanes 8 to 10), further indicating less extensive digestion with this enzyme.

12 3 4 5 6 7 8 9 S



Digestion of Basement Membrane Laminin


I Figure 1. Coomassie blue-stained SDS polyacrylamide gel showing electrophoretic patterns of lamintin incubated with activated neutrophil supernatanzts. Approximate Mr X 1000 of globular protein standards (S) are shown on right. Samples in lanes 1 to 8 were incubatedfor 8 hours at 37' C before boilintg in SDS-PAGE sample buffer and electrophoresis. Lane 1 contains 30sg laminin alone and A antd B chaints are indicated. Lanes 2 to 7 containt laminin inicubated with 4 tsg of activated neutrophil suipernzatant proteint to which was added EDTA (lane 2) anti-HNCG and anti-HNE (lane 3), atnti-HNCG (lane 4), anti-HNE (lane 5), DFP (lane 6), or no agent (lane 7), as described in Materials and Methods. Lane 8 contains 100 ul of neutrophil supernatant protein and lane 9 con taints laminiiin alonte at zero time. Neither EDTA nor anti-HNCG are effective at inhibiting lamininz degradation. In contrast, anti-HNE and DFP inhibit proteolysis. Note presenice of IgG heavy and light (H and L) chains in lanes 3 to 5.

Laminin Digestion by Purified HNE and HNCG Both HNE and HNCG extensively degraded laminin A and B chains in dose-dependent (Figure 2) and time-dependent manners (Figure 3). In general, more extensive degradation of laminin was observed with HNE than with HNCG and the A chain was more susceptible to degradation than the B chains. With limited incubations, however, both enzymes produced similar partial digests of laminin and fragments with M, of 94 kd and 80 kd were commonly seen (Figure 2, lanes 3 to 6 and 9 to 12; Figure 3, lanes 5 to 7 and 12 to 14).

Immunoblotting of Digested Laminin To determine whether the similarly sized fragments produced after laminin digestion with HNE and HNCG were immunologically related, immunoblots were reacted with monoclonal anti-laminin antibodies. After HNE digestion, all three antibodies recognized a single fragment migrating at 103 kd (Figure 4, lanes 5 to 7). In contrast, several -

To test the effects of purified HNE and HNCG on authentic basement membrane laminin, cryostat sections of unfixed mouse kidney were incubated with HNE, HNCG, or buffer for 5 minutes at room temperature. Sections were then labeled with monoclonal anti-laminin IgG and processed for immunofluorescence microscopy. Brilliant linear basement membrane fluorescence was seen in kidney sections treated with buffer alone (Figure 5a). In contrast, a marked reduction in fluorescence was observed in sections incubated with HNE (Figure 5b) or HNCG (Figure 5c). Once again, however, the laminin-cleaving activities seemed more pronounced with HNE than with HNCG.

Discussion A number of earlier studies have indicated that neutrophil enzymes, particularly the serine proteases elastase and cathepsin G, can mediate basement membrane injury. Although HNE has been shown to degrade type IV collagen,2 the specific molecular substrates for these enzymes in intact basement membranes have not been completely characterized. Our findings clearly show that supernatants obtained from activated neutrophils contained DFP-sensitive proteases capable of rapidly degrading laminin. Furthermore, the ability of monospecific antibodies against HNE and HNCG to block laminin digestion indicates that these enzymes were the most active laminin-degrading substances within the neutrophil supernatants. In addition, the observations that purified HNE and HNCG rapidly degraded soluble laminin and removed epitopes from basement membrane laminin in tissue sections strongly suggests that these enzymes play important roles in basement membrane damage in vivo. In addition to HNE and HNCG, neutrophils contain a number of other proteases potentially capable of degrading basement membrane molecules. However, because Table 1. Release of Granule Constituents in Response to Neutrophils Treated with PA'I Lactoferrin Triggering HNCG Agent HNE (ng/ml) (ng/mI) (ng/ml) None 453± 56 43± 11 28+4.7 PMA 2331 ± 431 154 ± 27 61 ± 6 Cells at 5 x 1 Q6 cells/ml incubated with or without 100 ng/ml of PMA for 60 minutes at 37°C. Standard error of mean of four separate experiments.

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45.A.. 6.s..78.7 B.9 1011l 200 117 94 67 Figure 2. Coomassie blue-stained SDSpolyacrylamide gel shouing electrophoretic patterns after incubation of laminin u'ith

vatying concentrations ofpurifed HNE or HINCG. Protein standards (S) are shown on lcft anid lane 1 contains 30 jig laminin

alone. Lane 2 contains 10 ug HNE alone and lanes 3 to 7 contain laminin incuibated uith 10, 5, 1, 0.5, and 0.1 ig HNE3 respectively. Lane 8 contains 10 ,ig HNCG alone and lanes 9 to 13 contain lamitnin with the same decreasing amounts oJ IINCG. Similar digestion produicts are produced by both enzymes but IINf appears more active in laminin degradation.

43 30 21 14

many of these proteases are active only at acidic pH,23 a role for these enzymes in mediating primary tissue damage is improbable. It is more likely that these acidic proteases are active in the phagolysosome in which molecules ingested by the neutrophil are degraded intracellularly. On the other hand, proteinase 3, a neutral serine protease found in neutrophils, has been shown to degrade elastin.24 Because antibodies specific for HNE and HNCG blocked essentially all of the laminin-degrading activity in neutrophil supernatants, however, we do not believe that proteinase 3 accounted for a significant amount of laminin digestion. Neutrophil metalloproteinases, which are activated by oxygen metabolites, also were shown previously to degrade interstitial and basement membrane collagens in vitro.25-29 However, our observation that EDTA did not affect the observed laminin digestion indicates that any Figure 3. Coomassie blue-stained SDSpolyacr)'lamide gel shou'ing time course of digestion of 30 jig laminin bJy 1 Ig purified lINE or HNCG. Protein standards (S) are shown on left. Lanes 1 and2 contain lami- 200 nin alone incubated for 0 and 480 mimutes, respectively. Lane 3 is HNE alone and lanes 4 to 9 containt laminin incubated u'ith HNE for 5, 15, 60, 120, 240, and 480 117 minutes, respectively. Lane 10 is IINCG 94 alonie and lanes 11 to 16 contain laminin 67 incubated uwith HNCG for 5, 15, 60, 120, 240, anid 480 minutes, respectively. Laminin degradation by both enzymes is evident, but HNE is more effective. 43 30 21 14

metalloproteinases in the neutrophil supernatants had little laminin-degrading ability. Similarly, earlier studies have shown that the antioxidants superoxide dismutase and catalase fail to block fibronectin release from amnionic basement membranes treated with activated neutrophil supernatants.'30 In contrast, inhibitors specific for HNE block fibronectin release.30 Other studies have also shown that neutrophil serine proteases, rather than metalloproteinases, account for most of the glomerular basement membrane collagen degradation seen in vitro.4'28 The laminin B chains were generally more resistant to degradation by HNE and HNCG than the A chain, as seen previously in studies with various pancreatic enzymes, 3132 32plasmin,323 and Pseudomonas elastase and alkaline protease.3 Several comparably sized degradation fragments were produced by both HNE and HNCG, al-

j4 5 6 7 8 9O11121314 1516


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205 103 67 42

Figure 4. Immu noblot demonstrating changes in immunoreactivities after incubating laminin with HNE or HNCG at a 1: 12 enzyme:substrate ratio. Standard molecular weight markers (S) after transfer to nitrocellulose are shown on left. Lanes 1 to 4 show undigested laminin recognized by rat anti-laminin monoclonal antibodies 5A2 (lan e 1), 5D3 (lan e 2), 9D2 (lane 3), and control rat IgG (lane 4). Lanes 5 to 7 show lINE-digested laminin recognized by 5A2 (lane 5), 5D3 (lane 6), and 9D2 (lane 7). Lanes 8 to 10 show HNCG-digested laminin recognized by 5A2 (lane 8), 5D3 (lane 9), and 9D2 (lane 10). More epitope loss results after HNE digestion.


18 15

though immunoblotting studies demonstrated that at least some of the products from the two enzymes possessed different epitopes and therefore were not identical. In addition, all our experiments showed that laminin degradation by HNE was more extensive than that obtained with equal amounts of HNCG. Because HNE is released in vitro in


greater quantities than HNCG after stimulation with rheumatoid factors,34 perhaps much of the laminin degradation that may occur in vivo is mediated primarily by HNE. Our findings discussed here add additional support to two recent studies indicating direct roles for HNE and HNCG in basement membrane pathology. First, perfusion


Figure 5. A: Fluorescence photomicrograph of a section of un4fixed mouse kidney labeled sequentially with rat monoclonal antilaminin IgG 5A2 and anti-rat IgGflluorescein. Brilliant linear basement membrantefluorescence is evident throughout the section. B and C: Sections of mouse kidney treated with HNE(B) or IINCG (C) as described in Materials and Methods before labeling with antilaminin monoclonal 5A2. Unlike that showni in A, there is a marked reduction in biniding of specific IgG to sectiots pretreated with either neutrophil enzyme. Thefluorescent labeling is weakest in sections treated with HNE, X470.

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of rat renal arteries with purified HNE or HNCG results in acute proteinuria.35 Whether the damage mediated by these enzymes in vivo occurs as a result of cleavage of laminin or other substrates remains undetermined. Paradoxically, despite previous work showing that neutrophil serine proteases lyse and detach cultured endothelial cells from substrates,3'37 there was no ultrastructural evidence of renal glomerular capillary wall damage after HNE perfusion in vivo.' Second, when experimental antiglomerular basement membrane disease was produced in beige mice with specific deficiencies in neutrophil elastase and cathepsin G, no albuminuria developed.38 Although HNE and HNCG appear capable of basement membrane degradation, the roles for these or other enzymes in neutrophil diapedesis are uncertain. For example, migration of neutrophils through a basement membranelike extracellular matrix assembled in vitro by cultured umbilical vein endothelial cells is apparently not impeded in the presence of a wide range of serine-, cysteine-, aspartate-, or metallo-proteinase inhibitors.39 Clearly more work needs to be done to clarify mechanisms of pathologic basement membrane damage as well as basement membrane transmigration by inflammatory cells. Nevertheless, our evidence demonstrates that HNE and HNCG derived from activated neutrophils digest laminin. Therefore these proteases, probably in concert with other enzymes and reactive oxygen metabolites, can undoubtedly affect the normal barrier properties and other functions of basement membranes. Furthermore, because laminin has also been shown to be a chemoattractant for neutrophils and to promote the oxidative burst,'41 release of laminin fragments from damaged basement membranes may be proinflammatory.

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79:669-674 2. Davies M, Barreft AJ, Travis J, Sanders E, Coles GA: The degradation of human glomerular basement membrane with purified lysosmal proteinases: Evidence for the pathogenic role of the polymorphonuclear leukocyte in glomerulonephritis. Clin Sci Mol Med 1978, 54:233-240 3. Mainardi CL, Dixit SN, Kang AH: Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules. J Biol Chem 1980, 255:5435-5441 4. Vissers MC, Winterbourn CC, Hunt JS: Degradation of glomerular basement membrane by human neutrophils in vitro. Biochim Biophys Acta 1984, 804:154-160 5. Pipoly DJ, Crouch EC: Degradation of native type IV procollagen by human neutrophil elastase. Implications for leuko-

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109:3477-3491 13. Boyum A: Isolation of mononuclear cells and granulocytes from human peripheral blood. Scand J Clin Lab Invest 1968, 21 (Suppl 97):77-82 14. Heck LW, Darby WL, Hunter FA, Bhown A, Miller EJ, Bennett JC: Isolation, characterization and amino terminal amino acid sequence analysis of human neutrophil elastase from normal donors. Ann Biochem 1985,149:153-162 15. Heck LW, Rostand KS, Hunter FA, Bhown A: Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors. Ann Biochem 1986,158:217-227 16. Dunn TL, Blackburn WD, Koopman WJ, Heck LW: Solidphase radioimmunoassay for human neutrophil elastase: A sensitive method for determining secreted and cell-associated enzyme. Ann Biochem 1985,150:18-25 17. Gordon S, Werb Z, Cohn ZA: Methods for detection of macrophage secretory enzymes. In Bloom BR, David JR, eds. In Vitro Methods in Cell-Mediated and Tumor Immunity. New York, Academic Press, 1976, pp 341-352 18. Chase T, Shaw E: Titration of trypsin, plasmin, and thrombin with p-nitrophenyl p'-guanidinobenzoate HCI. In Perlmann GE, Lorand L, eds. Methods in Enzymology, Vol. 19. New York, Academic Press, 1970, pp 20-27 19. Odegard OR, Lie M, Abildgaard U: Heparin cofactor activity measured with an amidolytic method. Thrombosis Res 1975, 6:287-294 20. Heck LW, Remold-O'Donnell E, Remold H: DFP-sensitive polypeptides of the guinea pig macrophage. Biochem Biophys Res Commun 1978, 83:1576-1583 21. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979, 76:4350-4354


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22. Johnson DA, Gautsch JW, Sportsman JR, Elder JH: Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Techn 1984,1:3-8 23. Cochrane CG: Immunological tissue injury mediated by neutrophil leukocytes. In Dixon FJ, Kunkel HG, eds. Advances in Immunology, Vol. 9. New York, Academic Press, 1968. pp 97-156 24. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR: Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988, 82:1963-1973 25. Weiss SJ, Peppin G, Ortiz X, Ragsdale C, Test S: Oxidative autoactivation of latent collagenase by human neutrophils. Science 1985, 227:747-749 26. Peppin GJ, Weiss SJ: Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils. Proc NatI Acad Sci USA 1986, 83:4322-4326 27. Shah SV, Baricos WH, Basci A: Degradation of human glomerular basement membrane by stimulated neutrophils. Activation of a metalloproteinase(s) by reactive oxygen metabolites. J Clin Invest 1987, 79:25-31 28. Vissers MCM, Winterbourn CC: Gelatinase contributes to the degradation of glomerular basement membrane collagen by human neutrophils. Coll Rel Res 1988, 8:113-122 29. Baricos WH, Murphy G, Zhou Y, Nguyen HN, Shah SV: Degradation of glomerular basement membrane by purified mammalian metalloproteinases. Biochem J 1988, 254:609612 30. Sibille Y, Lwebuga-Mukasa JS, Polomski L, Merrill WW, Ingbar DH, Gee JB: An in vitro model for polymorphonuclearleukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic basement membranes. Am Rev Respr Dis 1986,134:134-140 31. Ott U, Odermatt E, Engel J, Furthmayr H, Timpl R: Protease resistance and conformation of laminin. Eur J Biochem 1982,123:63-72 32. Rao CN, Margulies IMK, Goldfarb RH, Madri JA, Woodley DT, Liotta LA: Differential proteolytic susceptibility of laminin alpha and beta subunits. Arch Biochem Biophys 1982, 219: 65-70 33. Heck LW, Morihara K, Abrahamson DR: Degradation of soluble laminin and depletion of tissue-associated basement



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Acknowledgments The authors thank Patricia L. St. John for assistance in preparation of the photographs and Maxine Rudolph for secretarial help. They also thank Dr. Albert Chung for providing laminin used in some preliminary experiments.

Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.

To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phor...
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