Activation of tumor cell matrix metalloproteinase-2 by neutrophil proteinases requires expression of membrane-type 1 matrix metalloproteinase

Activation of tumor cell matrix metalloproteinase-2 by neutrophil proteinases requires expression of membrane-type 1 matrix metalloproteinase Jess D. Schwartz, MD, Peter Shamamian, MD, Sara Monea, MS, David Whiting, BA, Stuart G. Marcus, MD, Aubrey C. Galloway, MD, and Paolo Mignatti, MD, New York, NY

Background. Matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor invasion, is secreted as an inactive proenzyme and requires interaction with membrane-type 1 MMP (MT1-MMP) for activation. We have previously demonstrated that polymorphonuclear neutrophils (PMNs) release a soluble factor(s) that activates pro-MMP-2. Therefore, we tested the hypothesis that PMN-derived proteinases act in concert with MT1-MMP to activate pro-MMP-2. Methods. Human HT-1080 cells transfected with MT1-MMP cDNA (HT-SE) or the corresponding antisense cDNA (HT-AS) or an empty vector (HT-V), which expressed differing levels of MT1-MMP, were incubated with serum-free, human PMN-conditioned medium with or without proteinase inhibitors. The culture supernatants were analyzed by gelatin zymography. Results. HT-1080 cells expressing basal (HT-V) or low levels (HT-AS) of MT1-MMP secreted MMP-2 in proenzyme form (72 kd). HT-1080 cells with high levels of MT1-MMP (HT-SE) secreted proMMP-2 and a 68 kd intermediate activation product. Addition of PMN-conditioned medium to either HT-SE or HT-V clones resulted in dose-dependent generation of active, 62 kd MMP-2. In contrast, when PMNconditioned medium was added to HT-AS clones, no MMP-2 activation occurred. Conclusions. PMN-derived serine proteinases act in concert with MT1-MMP to activate proMMP-2. This finding indicates a potential role for inflammatory cells in promoting extracellular matrix breakdown during tumor invasion. (Surgery 1998;124:232-8.) From the New York University School of Medicine, Department of Surgery, S. Arthur Localio Surgical Research Laboratory, New York, NY

THE INVASIVE PROCESSES that occur during tumor progression—local invasion, intravasation, extravasation, and metastasis formation—involve extracellular matrix (ECM) degradation by serine and matrix metalloproteinases (MMPs). MMPs are a family of enzymes that are involved in a variety of physiologic and pathologic processes that require ECM degradation, such as organogenesis, wound repair, angiogenesis, tumor invasion, and metastasis.1-3 MMPs are zinc-dependent endopeptidases that are secreted as inactive proenzymes and actiSupported by funds from the S.A. Localio Laboratory for General Surgery Research. Presented at the Fifty-ninth Annual Meeting of the Society of University Surgeons, Milwaukee, Wis., Feb. 12-14, 1998. Reprint requests: Aubrey C. Galloway, MD, 530 First Ave, Suite 9V, New York, NY 10016. Copyright © 1998 by Mosby, Inc. 0039-6060/98/$5.00 + 0

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vated by limited proteolytic cleavage.1,2 MMP-2 and MMP-9 (type IV collagenases/gelatinases) play important roles in these invasive processes.1,2 The physiologic mechanisms that control proMMP-2 activation remain unclear. Several studies implicate the cell membrane as a requirement for pro-MMP-2 activation.2-5 Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a 63 kd transmembrane protein that converts 72 kd pro-MMP-2 into an active form of 64/62 kd via an intermediate of 68/66 kd.6,7 High levels of MT-1-MMP and the presence of active MMP-2 have been described in a variety of malignant tumors, suggesting that MT-1-MMP expression can facilitate tumor invasiveness and neovascularization via the activation of MMP-2.8-10 However, experiments demonstrating MT1-MMP-mediated processing of proMMP-2 have required overexpression of MT1-MMP by transfection with MT1-MMP cDNA or by the nonphysiologic treatment of cells with concanavalin

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A or phorbol esters, suggesting that other factor(s) may be involved in MMP-2 activation in vivo.7,11,12 Components of the urokinase (uPA)-plasmin system are involved in the control of type IV collagenase activity on the cell surface.5 Activation of MMP2 by plasmin requires MMP-2 binding to MT1-MMP; expression of basal levels of MT1-MMP requires plasmin activity for MMP-2 activation.13 Tumor growth and angiogenesis are frequently associated with an intense inflammatory response.14 Tumors secrete growth factors and cytokines that are chemoattractants for macrophages and polymorphonuclear leukocytes (PMNs).15 However, these inflammatory cells are not sufficient to eradicate the tumor. In fact, their presence has been correlated with a greater tumor aggressiveness by promoting the tumors’ ability to degrade the ECM.16,17 We have previously demonstrated that PMNs secrete a soluble factor(s) capable of activating pro-MMP-2.18 Because PMNs have been associated with tumor invasiveness and contain a variety of proteinases with substrate specificity similar to plasmin, we hypothesized that PMN-derived proteinases can act in concert with MT1-MMP to activate pro-MMP-2. MATERIAL AND METHODS Material. Gelatin-Sepharose was purchased from Pharmacia Biotech AB (Uppsala, Sweden), gelatin from Merck (Darmstadt, Germany), aprotinin, 1,10phenanthroline, E-64, eglin c peptide, pepstatin A and histopaque (H-1077) from Sigma (St. Louis, Mo), and α1 antitrypsin from Athens Research Technologies (Athens, Ga). Batimastat (BB-94) was provided by British Bio-technology Ltd. (Oxford, UK). Rabbit antibody to a 26-residue synthetic peptide corresponding to the C-terminal, intracellular domain of MT1-MMP (amino acid residues 557-582) of human MT1-MMP was a gift from Dr J. Keski-Oja (University of Helsinki, Finland).12 HT-1080 fibrosarcoma cells and culture medium. Human HT-1080 fibrosarcoma cells were used for our studies because they produce high levels of pro MMP-2.5 The cells were grown in RPMI (Gibco, Life Technologies, Gaithersburg, Md) supplemented with 10% fetal bovine serum (FBS), L-glutamine 2 mmol/L, 100 units/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (Gibco, BRL) Clones of HT-1080 cells transfected with either the sense or antisense cDNA for human MT1-MMP or with the empty vector have been described.13 These cells were grown in medium containing 250 µg/mL of geneticin (G418) (Sigma). Expression of MT1-MMP was characterized by Western blotting. PMN isolation. A modification of the technique

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described by Boyum was used for PMN isolation.19 Blood samples (40 mL) from healthy volunteers were mixed (9:1) with 3.8% sodium citrate, and 5mL aliquots were layered over a 2-mL discontinuous gradient of histopaque (H-1077) and 5 mL of PMN isolation solution (Robbins Scientific Corp, Sunnyvale, Calif). After centrifugation at 500g for 30 minutes at room temperature, the lower band, corresponding to the PMN layer, was isolated and contaminating erythrocytes removed by hypotonic lysis. The PMN pellet was resuspended in RPMI with 0.25% bovine serum albumin (BSA) at a final concentration of 5 × 106 PMNs/mL. Purity (Diff Quick differential kit) and viability (trypan blue exclusion test) of PMN preparations were greater than 95%. Isolated cells were kept on ice and used immediately. Preparation of cell extracts and conditioned medium. HT1080 cell extract and conditioned medium were prepared as described.5 HT-1080 cells were seeded into 6-well flat bottom culture dishes at a density of 5 × 104 cells/well in 2 mL of growth medium. When the cultures were confluent, the cells were washed twice with serumfree medium (RPMI with 0.25% BSA) to remove residual FBS. Serum-free medium conditioned by PMNs (5 × 106 PMNs/mL) for 2 hours was added to HT-1080 clones for 16 hours with or without the indicated proteinase inhibitors. The culture supernatants were centrifuged at 500g for 10 minutes at 22° C. The cells were washed twice with PBS, lysed for 10 minutes on ice with 50 µL/well of Triton X-100 0.5% (v/v) in Tris-HCl 0.1 mol/L, pH 8.1 under constant shaking, and scraped with a rubber policeman. The cell lysates were centrifuged at 800g for 10 minutes at 4° C. Conditioned media and cell extracts were stored at –20° C until they were analyzed by gelatinzymography and/or Western blotting. Western blotting. Cell extracts (80 µg) were electrophoresed in a reducing sodium dodecyl sulfate–10% polyacrylamide gel and electroblotted to a nitrocellulose membrane (Hybond-C Extra; Amersham). The membrane was prehybridized at 4° C overnight in Tris base 20 mmol/L, NaCl 150 mmol/L, 0.1% Tween 20, pH 7.4 (TBS-T) containing 5% milk (Carnation), hybridized at 22° C for 1 hour in TBS-T containing 5% milk and the indicated rabbit antibody diluted 1:500, and incubated at 22° C for 45 minutes in TBS-T containing horseradish peroxidase-labeled anti-rabbit immunoglobulin G. Each step was followed by extensive washing in TBST (4 mL/cm2) at 22° C. After removing the TBS-T buffer, the membrane was incubated for 1 minute at 22° C with 0.125 mL/cm2 of enhanced chemolumi-

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Fig. 1. MT1-MMP expression by HT1080 cell transfectants. HT1080 cells transfected with either the empty vector (HT-V) or antisense (HT-AS) or sense cDNA for MT1-MMP (HT-SE) were incubated in serum-free medium or in medium conditioned by PMNs (PMN-CM) for 16 hours. Cell extracts were analyzed by Western blotting with antibody to MT1-MMP. The molecular masses of the different forms of MT1-MMP are shown in kd on the left.

Fig. 2. Characterization of the gelatinases expressed by PMNs. PMN-conditioned medium was analyzed by gelatin zymography. Arrows point to the different Mr forms of MMP-9. The position of Mr markers is shown in kd on the right.

nescence detection solution (Boehringer Mannheim) and exposed to films (Hyperfilm MP; Amersham) for 10 seconds to 5 minutes. RESULTS MT1-MMP expression and pro-MMP-2 activation in HT-1080 cells. Western blotting analysis of the MT1-MMP transfectants showed bands with MrS 63 kd, 60 kd, 58 kd, and 43 kd (Fig. 1). The 63 kd and the 60 kd bands represent pro and active MT1MMP, respectively, the 58 kd and the 43 kd bands degradation products.12,13 Cells transfected with the vector alone (HT-V cells) constitutively expressed 60 kd and 58 kd MT1-MMP. In cells trans-

fected with the antisense cDNA (HT-AS cells), the levels of these bands were dramatically reduced. Some HT-AS clones had no detectable 58 kd MT1MMP. In addition to these bands, cells transfected with the sense cDNA (HT-SE cells) showed the 63 kd proenzyme band and a broad, intense band of 43 kd, consistent with the hypothesis that the generation of this peptide is associated with overproduction of MT1-MMP.12,13 The transfected cells and PMNs were also characterized for gelatinase expression by gelatin zymography. Human PMNs spontaneously released large amounts of 92 kd progelatinase B (MMP-9) (Fig. 2), in addition to 220 kd and 130 kd gelati-

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Fig. 3. Effect of PMN-conditioned medium on pro-MMP-2 activation. HT1080 cells transfected with either the empty vector (HT-V) or with antisense (HT-AS) or sense cDNA for MT1-MMP (HT-SE) were grown in serum-free medium with or without the indicated dilutions of PMN-conditioned medium (PMN-CM). The culture supernatant was analyzed by gelatin zymography. Arrows point to MMP-9 and the different Mr forms of MMP-2.

nase bands likely representing homodimers of proMMP-9 and complexes with lipocalin, respectively.20, 21 No MMP-2 was detected in PMN-conditioned medium. Pro-MMP-9 was also present in the conditioned medium of all the HT1080 cell transfectants. In contrast, the pattern of MMP-2 forms showed significant differences among different clones. HT-V cells, which constitutively expressed active MT1-MMP, and HT-AS cells, which expressed very low amounts of MT1-MMP, secreted only 72 kd pro-MMP-2. In contrast, HT-SE, overexpressing MT1-MMP, secreted intermediate activation forms of MMP-2 with MrS 68/66 kd in addition to 72 kd pro-MMP-2 (Fig. 3). PMN-mediated activation of pro-MMP-2. To test the effect of PMNs on pro-MMP-2 activation HT1080 cells were incubated with PMN-conditioned medium. Gelatin zymography showed that treatment of HT-V cells with serial dilutions of PMN-conditioned medium resulted in the conversion of pro-MMP-2 (72 kd) to its active 62 kd form in a dose-dependent manner. Similarly, treatment of HT-SE cells resulted in the conversion of the 72 kd and 68/66 kd bands to active 62 kd MMP-2 in a dose-dependent manner (Fig. 3). In contrast, PMNconditioned medium had no effect on the proMMP-2 secreted by HT-AS cells, which expressed very low levels of MT1-MMP. To test whether PMN-mediated activation of proMMP-2 requires the activity of PMN proteinases, we characterized the effect of various proteinase inhibitors. Inhibitors of plasmin (aprotinin, 100 µg/mL), acid proteinases (pepstatin A, 10 µg/mL), or cysteine proteinases (E-64, 40 µg/mL) did not block pro-MMP-2 activation (Fig. 4). In contrast, the pro-MMP-2 activating activity of PMN-conditioned

medium was inhibited by the serine proteinase inhibitors antitrypsin (20 µg/mL) (Fig. 4), pefabloc (4 mmol/L) (not shown), or SBTI (5 mg/mL) (not shown), implicating PMN serine proteinases (elastase, cathepsin G and proteinase-3) as potential activators of MMP-2. Addition of the metalloproteinase inhibitors 1,10-phenanthroline (25 µg/mL; not shown) or Batimastat (1 µmol/L) to HT-SE cells resulted in the disappearance of the 68/66 kd band (Fig. 5), consistent with the finding that the processing of pro-MMP-2 to 68/66 kd is MT1-MMPdependent.7 In the presence of Batimastat, PMNconditioned medium retained the ability to convert 72 kd and 68/66 kd MMP-2 to the 62 kd active form, indicating that catalytically active MT1-MMP is not required for PMN-mediated activation of pro-MMP2 (Fig. 6). In contrast, α1 antitrypsin (20 µg/mL) blocked pro-MMP-2 activation by PMN-conditioned medium (Fig. 6). Addition of α1-antitrypsin alone had no effect on the MT1-MMP-mediated generation of the 68/66 kd intermediate form of MMP-2 (not shown). Thus, PMN-conditioned medium strongly accelerates the generation of 64/62 kd MMP-2 from the 68/66 kd form generated by MT1MMP and from the 72 kd proenzyme. DISCUSSION The data reported show that PMN serine proteinases and MT1-MMP act in concert to activate pro-MMP-2, and that pro-MMP-2 activation by PMN serine proteinase requires the presence but not the catalytic activity of MT1-MMP. These conclusions are based on the following observations. Clones of HT-1080 cells that expressed high levels of MT1-MMP cDNA (HT-SE cells) constitutively activated MMP-2. However, cells transfected

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Fig. 4. Effect of proteinase inhibitors on proMMP-2 activation. Clones of HT1080 cells transfected with the empty vector (HT-V) were incubated for 16 hours in serum-free medium or with PMN-conditioned medium (PMN-CM) in the absence or in the presence of the indicated proteinase inhibitors. At the end of the incubation, the culture supernatants were analyzed by gelatin zymography. Inhibitor concentrations: aprotinin, 100 µg/mL; pepstatin A, 10 µg/mL; E-64, 40 µg/mL; antitrypsin, 20 µg/mL. ProMMP-2 activation was blocked only by the addition of α1-anti-trypsin. Arrows point to MMP-9 and the different Mr forms of MMP-2.

with an empty vector (HT-V cells) expressed significant levels of MT1-MMP but no active MMP-2. Addition of PMN-conditioned medium to these clones resulted in pro-MMP-2 activation. In contrast, with clones of cells transfected with antisense cDNA (HT-AS cells) that expressed very low levels of MT1-MMP, activation of pro-MMP-2 by PMN-conditioned medium did not occur. Thus, to activate pro-MMP-2 PMN serine proteinases require the expression of “basal” levels of MT1MMP; conversely, basal levels of MT1-MMP require PMN proteinases for pro-MMP-2 activation. The concurrent presence of MT1-MMP and PMN serine proteinases is required for pro-MMP-2 activation. Our observation that PMN-mediated activation of pro-MMP-2 occurred in the presence of the metalloproteinase inhibitor Batimastat is consistent with previous reports that plasmin activation of pro-MMP-2 does not require the action of metalloproteinases.5 The cleavage of pro-MMP-2 by MT1-MMP into a 68/66 kd intermediate activation product has been proposed to trigger autocatalytic activation of the gelatinase to the fully active 64/62 kd form. 7 However, PMN-conditioned medium generated 62 kd MMP-2 in the

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Fig. 5. Effect of proteinase inhibitors on MT1-MMP-mediated activation of pro-MMP-2. HT1080 cells transfected with MT1-MMP cDNA (HT-SE cells) were incubated in serum-free medium in the absence or presence of Batimastat (1 µmol/L) for 16 hours. At the end of the incubation, the culture supernatants were analyzed by gelatin zymography. Arrows point to MMP-9 and the different Mr forms of MMP-2. HT-SE cells, which express high levels of MT1-MMP, partially convert 72 kd proMMP2 to the 68 kd intermediate activation form. This effect is completely abolished by addition of Batimastat.

presence of Batimastat. Thus, whereas the presence of MT1-MMP on the cell surface is necessary, its catalytic activity and autoactivation do not appear to be required for PMN-mediated activation of pro-MMP-2. Although MMP-1, MMP-3, and MMP-7 can activate pro-MMP-2, our finding that PMN-conditioned medium activates pro-MMP-2 in the presence of Batimastat shows that pro-MMP-2 activation by PMN-conditioned medium does not require the action of other MMPs.22,23 As the catalytic activity of MT1-MMP is not required for PMN-mediated activation of pro-MMP-2, our results suggest that a primary role for MT1-MMP may be to serve as a membrane binding site for MMP-2/TIMP-2 complex. A consistent body of clinicopathologic evidence has shown a correlation between inflammation and tumor progression.14-17 Our results may provide a possible explanation for this observation. Tumor cells can grow in the absence of vascularization only up to nodules in the range of 1 to 2 mm in diameter; necrosis, a stimulus for inflammatory cell recruitment, ensues if this size is exceeded.24 In addition, tumors secrete a variety of molecules that can attract inflammatory cells.15

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Surgery Volume 124, Number 2 REFERENCES

Fig. 6. PMN-mediated activation of proMMP-2 is independent of catalytically active MT1-MMP. HT1080 cells transfected with MT1-MMP cDNA (HT-SE cells) were incubated in control, serum-free medium or in PMN-conditioned medium (PMN-CM) in the presence or in the absence of the indicated proteinase inhibitors for 16 hours. At the end of the incubation the culture supernatants were analyzed by gelatin zymography. Arrows point to MMP-9 and the different Mr forms of MMP-2. HT-SE cells incubated in control medium show partial conversion of pro-MMP-2 to the 68 kd intermediate activation form. Addition of PMN-CM converts both pro-MMP-2 (72 kd) and the 68 kd form to active, 62 kd MMP-2. In the presence of Batimastat, PMN-CM retains the ability to activate pro-MMP-2. In contrast, in the presence of α1-antitrypsin (20 µg/mL) the generation of active, 62 kd MMP-2 does not occur; as in control medium, only the 68 kd, intermediate activation form is generated.

PMNs are mediators of acute inflammation that are brought to sites of necrosis by a variety of chemoattractants. On reaching the source of the stimulus, PMNs become activated and release the contents of their granules, including the serine proteinases elastase, cathepsin G, and proteinase 3.25 The release of these proteinases affords high local concentrations of proteolytic enzymes. Although basal levels of MT1-MMP expressed by tumors in vivo may be insufficient to activate proMMP-2 directly, they may afford gelatinase activation by providing a cell membrane binding site that permits the activation of pro-MMP-2 by PMN serine proteinases. This interaction will promote the ECM degradation required for tumor invasion and angiogenesis.

1. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73:161-95. 2. Kleiner DE, Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteinases. Curr Opin Cell Biol 1993;5:891-7. 3. Strongin AY, Manner BL, Grant GA, Goldberg GI. Plasma membrane dependent activation of the 72-kDa type IV collagenase is prevented by complex formation with TIMP2. J Biol Chem 1993;268:14033-9. 4. Ward RV, Atkinson SJ, Reynolds JJ, Murphy G. Cell surfacemediated activation of progelatinase A: demonstration of the involvement of the C-terminal domain of progelatinase A in cell surface binding and activation of progelatinase A by primary fibroblasts. Biochem J 1994;304:263-9. 5. Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, et al. Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J 1997;16:2319-32. 6. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994;370:61-5. 7. Strongin AY, Collier I, Bannikov G, Mariner BL, Grant GA, Goldberg GI. Mechanism of cell surface activation of 72kDa type IV collagenase. J Biol Chem 1995;270:5331-8. 8. Yamamoto M, Mohanam S, Sawaya R, Fuller GN, Seiki M, Sato H, et al. Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro. Cancer Res 1996;56:384-92. 9. Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, et al. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl Acad Sci U S A 1995;92:2730-4. 10. Nomura H, Sato H, Seiki M, Mai M, Okada Y. Expression of membrane-type matrix metalloproteinase in human gastric carcinomas. Cancer Res 1995;55:3263-6. 11. Atkinson SJ, Crabbe T, Cowell S, Ward RV, Butler MJ, Sato H, et al. Intermolecular autolyric cleavage can contribute to the activation of progelatinase A by cell membranes. J Biol Chem 1995;270:30479-85. 12. Lohi J, Lehti K, Westermarck J, Kähäri VM, Keski-Oja J. Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12-myristate 13acetate. Eur J Biochem 1996;239:239-47. 13. Monea S, Lehti K, Schwartz J, Shamamian P, Marcus S, Galloway AC, et al. Requirement for plasmin and membrane type I matrix metalloproteinase in the cell surface activation of gelatinase A (MMP-2) [abstract]. Mol Biol Cell 1997;8:434. 14. Lee AH, Happerfield LC, Borrow LG, Millis RR. Angiogenesis and inflammation in ductal carcinoma in situ of the breast. J Pathol 1997;181:200-6. 15. Jackson JR, Seed MP, Kircher CH, Willoughby DA, Winkler JD. The codependence of angiogenesis and chronic inflammation. FASEB J 1997;11:457-65. 16. Yamashita J, Ogawa M, Sakai K. Prognostic significance of three novel biologic factors in a clinical trial of adjuvant therapy for node-negative breast cancer. Surgery 1995;117:601-8. 17. Scholl SM, Pallud C, Beuvon F, Hacene K, Stanley ER, Rohrschneider L, et al. Anticolony-stimulating factor-1 antibody staining in primary breast adenocarcinoma correlates

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with marked inflammatory cell infiltrates and prognosis. J Natl Cancer Inst 1994;86:120-6. 18. Schwartz JD, Monea S, Marcus SG, Patel S, Eng K, Mignatti P, et al. Soluble factor(s) released from neutrophils activates endothelial cell matrix metalloproteinase 2. J Surg Res In press. 19. Boyum A, Lovvhaug D, Tresland L, Nordlie EM. Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand J Immunol 1991;34:697-712. 20. Goldberg GI, Collier IE, Eisen AZ, Grant GA, Marmer BL, Wilhelm SM. Mosaic structure of the secreted ECM metalloproteases and interaction of the type IV collagenases with inhibitors. Matrix Suppl 1992;1:25-30. 21. Kjeldsen L, Bainton DF, Sengelov H, Borregaard N. Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils. Blood 1994;83:799-807. 22. Miyazaki K, Umenishi F, Funahashi K, Koshikawa N, Yasumitsu H, Umeda M. Activation of TIMP-2/progelatinase A complex by stromelysin. Biochem Biophys Res Commun 1992;185:852-9. 23. Crabbe T, O’Connell JP, Smith BJ, Docherty AJ. Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A. Biochemistry 1994;33:14419-25. 24. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4-6.

Surgery August 1998 25. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1988;300:365-76.

DISCUSSION Dr Gary B. Nackman (New Brunswick, NJ). One of your conclusions is that neutrophils produce a serine proteinase that is partially responsible for the activation of MMP-2. On one of the gels you showed aprotinin in one of the lanes, which is actually a nonspecific serine inhibitor, and yet the MMP-2 was still being activated. Dr Schwartz. We have as yet unreported information indicating that neutrophils have a number of different serine proteinases. In fact, 3 different serine proteinases that neutrophils secrete are capable of activating MMP-2. Why aprotinin did not block it is curious. We do know that the aprotinin was active because we did control experiments using pure plasmin, and previous work from our laboratory has demonstrated that plasmin is able to work, and that the aprotinin in this case was able to block plasmin alone. Perhaps the specificity of aprotinin for blocking the mixture of neutrophil serine proteinases is not very strong. One may need to use the stronger or more physiologic serine proteinase inhibitor, α1-antitrypsin, to see this effect