Direct Cytotoxicity of Polymorphonuclear Leukocyte Granule Proteins to Human Lung-derived Cells and Endothelial Cells 1- 4
DEREK G. OKRENT,5 ALAN K. LICHTENSTEIN, and TOMAS GANZ8
Introduction
The polymorphonuclear leukocyte (PMN) is believed to play an important role in the pathogenesis of the adult respiratory distress syndrome (1), emphysema (2), and cryptogenic fibrosing alveolitis (idiopathic pulmonary fibrosis) (3). PMNs may participate in lung injury through their production of reactive oxygen intermediates or release of hydrolytic enzymes and cytocidal proteins from their granules (4, 5). Although the ability of reactive oxygen intermediates to injure the respiratory and other host organ systems is well documented (6-9), relatively little information is available concerning the potential of granule components acting independently of oxygen. Of these, only elastase has been wellstudied and shown to damage lung tissue in vivo and in vitro (2). Human defensins, three recently described small (molecular weight approximately 3900) microbicidal peptides, are among the most abundant components of azurophil granules of PMNs (10). In a recent study (11), a defensin-containing fraction of PMN granule extracts wasthe most damaging to several tumor cell targets, but fractions containing elastase, lysozyme, and other hydro lases were relatively ineffective. The cytotoxic potential of defensins for tumor cells has been confirmed using purified peptide (12,13). Since nonoxidative components of PMN granules may be involved in pulmonary injury and since only elastase has been examined using lung tissue as a target, wedesigned the current study to compare the relative activities of all granule components against pulmonary-derived cells and endothelial cells. Because our previous experience with nonpulmonary targets demonstrated that defensins had the greatest activity, the various granule extract fractions were compared to purified defensins in the current study. The results demonstrate the potent lysisof targets by defensins and defensin-containing granule extract fractions; relatively little
SUMMARY Neutrophlls, In the course of defending the host against microbial Invasion, release a potent arsenal of proteins that can potentially damage host tissues. Defenslns are major peptldes of human polymorphonuclear leukocyte (PMN)granules and are both broadly microbicidal and cytotoxic to several tumor cell lines. Todetermine whether these peptldes could play a role In neutrophllmediated lung Injury, we examined the cytotoxicity of defenslns and other PMN granule proteins in a chromium release assay with human lung-derlved cell lines MRC-5 (lung fetal fibroblast), A549 (lung adenocarcinoma with features of alveolar epithelium), and primary cultures of human umblll· cal vein endothelial cells (HUVEC). Crude fractionation of an acid extract of human PMN granules yielded four fractions A-D. Only fraction D (containing mostly defenslns) was significantly cytotOXic to all three target cells. In contrast, fraction A (containing myeloperoxldase and lactoferrln) and fraction C (containing lysozyme) had little effect, and fraction B (containing chiefly cathepsin G and elastase) was only injurious to endothelial cells. The cytotoxicity of whole PMNgranule extracts on pulmonary epithelial and fibroblast targeta could be completely accounted for by their defensin content. Fraction D- and defensln-medlated cytotoxicity was concentration dependent, required at least 10 to 12 h to become manifest, and was Inhibited by serum. The role of these peptldes In lung damage during acute and chronic Inflammation deserves further study. AM REV RESPIR DIS 1990; 141:179-185
Injury was induced by other granule components. Methods Purification of PMN Human PMN were obtained from a commercial supplier (Hemacare, Van Nuys, CA) in single-donor leukopheresis packs containing 2 x 1010 to 4 X 10'0 cells, with> 90% viable PMN as determined by trypan blue exclusion and Giemsa stain differential count. Contaminating platelets and erythrocytes were subsequently removed by differential centrifugation and hypotonic lysis as described previously (10). PMN and granule preparations were processed at 2 0 C throughout.
Preparation of PMN Granules and Granule Protein Extraction After suspension in Hanks' balanced salt solution (HBSS), pH 7.4 with 2.5 mM MgCl" the cell suspension was sealed in a nitrogen bomb (Parr Instrument Co., Moline, IL) and pressurized to 750 psi for 20 min. The suspension was then released dropwise into HBSS with 5 mM NazEDTA while stirring. Suspensions were centrifuged at low speed (200 x g for 10 min) to remove nuclei and cellular debris. The pellet was examined by phase-contrast microscopy to ensure> 900/0 cellular disruption. The supernatant containing the granules was sedimented at 27,000 x g for 20 min and the pellets stored at -70 0 C.
Granule proteins were extracted, and the extract was fractionated essentially as described by Modrzakowski and coworkers (14, 15).Briefly, the granule sediment was extracted three times in a volume of 0.2 M sodium acetate buffer with 0.01 M caci, (pH 4.0) to yield a final concentration of 2 x 108 cell equivalents per milliliter. Each extraction was done over 12 h at 4 0 C with gentle stirring, and the residue separated by centrifugation
(Received in original form January 19, 1989 and in revised form May 10, 1989) t From the Will Rogers Institute Pulmonary ResearchLaboratory, Department of Medicine,UCLA School of Medicine, and the Department of Medicine, VAWadsworth-UCLA Medical Center, Los Angeles, California. 2 Supported in part by the National Institutes of Health Grant CA 37184 and HL 35640, the American Lung Association of California Grant (CRMEF) A860701, and research funds of the Veterans Administration. 3 Correspondence and requests for reprints should be addressed to Tomas Ganz, Ph.D., M.D., UCLA School of Medicine, Center for the Health Sciences, Los Angeles, CA 90024-1736. • Presented in part at the WesternRegionalMeeting of the American Federation for Clinical Research, Carmel, California, February 1988. 5 Parker B. Francis Research Fellowof the Puritan-Bennett Foundation. • RJR Nabisco Research Scholar.
179
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OKRENT, LICHTENSTEIN, AND GANZ
at 27 000 x g for 20 min. The pooled acetate extra~ts were concentrated by ultrafiltration (YC-Q5 filter; Amicon Corp., Lexington , MA) and placed on a Sephadex G-loo column (2.5 x 150 em), Approximately 70 mg crude granule protein extract was eluted with 0.2 M sodium acetate buffer (pH 4.0) and collected in lO-ml fractions. The fractions were pooled into four fractions (A, B, C, and D) defined by their A. 80 pattern, concentrated by ultrafiltration (Amicon YC-Q5 filter), and dialyzed against phosphate-buffered saline (PBS) in Spectrapore 3 tubing (molecular weight cutoff 3500). Roughly 500/0 of the protein in the original granule extract is recovered by this technique.
Defensins The three defensin peptides, human neutrophil proteins 1-3 (HNPI-3), were individually prepared as described previously (10) and stored as stock solutions of 2 mg/ml in 0.01% acetic acid at -20° C. HNPI-3 were mixed in a ratio of 2:2:1 by weight, and this mixture was tested for lytic activity.
Protein Studies Protein concentration was measured by the BCA (16)protein assay system (Pierce Chemical Company, Rockford, IL) with chicken egg white lysozyme as the standard. A 1 to 4 ug amount of crude granule extract, each fraction, and purified defensins was subjected to electrophoresis on 12.5% acid-urea polyacrylamide gels (PAGE).
Cell Lines Human lung fibroblasts (MRC-5), a nontransformed cell line, and human lung carcinoma with features of alveolar epithelial cells (A549) were obtained from the American Type Culture Collection (Rockville, MA) and maintained in RPMI-I640 with 10% fetal calf serum (FCS). Human umbilical vein endothelial cells (HUVEC) were prepared in primary culture by collagenase treatment of fresh term umbilical cord veins using previously described techniques (17). HUVEC were maintained in M-199 plus HEPES (MA Bioproducts) supplemented with 20% FCS « I ng/ml of endotoxin; Hyclone Laboratories, Logan, UT) , 1 mM pyruvate, 2 mM glutamine, and 90 lig/ ml of heparin plus 20 ug/rnl of endothelial cell growth supplement (Collaborative Research, Bedford, MA) (18). The HUVEC used for the experiments were passages 3 to 8, at confluence displayed uniform cobblestonelike morphology, and 100% of cells stained with 1,1'-dioctadecyl-l,3,3,3',3'tetramethylindocarbocyanine perchlorateLDL (DiI-Ac-LDL; Biomedical Technologies, Inc., Cambridge, MA) (18).
Chromium Release Assay Cell targets wereinitially plated in 96-wellmicrotiter plates at a density of approximately 2 x 104 cells/well and grown to confluence over 24 h. For assays with HUVEC, microtiter wells were precoated with gelatin 0-1890
Control release was determined from targets incubated in medium alone (see RESULTS), and maximal release was achieved by adding 3% Triton X-loo (> 80% incorporated counts). Release from targets incubated in medium containing dilute acetic acid or PBS (the vehicles used in defensin and granule fraction stock solutions, respectively) did not differ from controls incubated in medium alone. The standard deviation of the triplicate samples was always < 5% of the mean.
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(Sigma, St. Louis, MO) by adding 100 iii of I% gelatin in PBS to each well and storing plates at 4° C. Immediately before seeding HUVEC, the wells were warmed to 37° C, gelatin was aspirated, and wells were rinsed with PBS . All targets were labeled by incubating with I to 2 IiCi/well "Cr for 2 h at 37° C. The monolayers were then extensively washed with medium and then incubated with test substances in serum-free (A549 and MRC5) or 0.1% serum (HUVEC) at 37° C. Subsequently, the plates were centrifuged (1,200 rpm for 5 min) to pellet nonadherent cells, and the supernatants counted in a gamma counter. Specific chromium release was calculated as: cpmexp - cpmcontrol x 100. Cpmmaximal - CPfficontrol
1
Results
Composition of Granule Fractions
2
Figure 1 is a typical elution profile from Sephadex G-loo gel filtration, demonstrating the resolution of the crude granule extract into four fractions (A-D) with relative protein contents of approximately 12, 28, 15, and 45070, respectively. According to previous work (14, 19), fraction A contains myeloperoxidase (MPO; 150,000D), fraction B contains proteases including elastase and cathepsin G (27,000 D), and fraction C contains lysozyme (14,000 D) (13). Over 95% of fraction D consists of a mixture of the three defensins: HNPl, HNP2, and HNP3 (3,000 to 4,000 D) (19). Acid-urea PAGE (figure 2) and 10to 20% sodium dodecyl sulfate (SDS) gradient PAGE (not shown) of the fractions obtained confirm the presence of dominant bands that comigrate with purified MPO (fraction A), elastase (fraction B), lysozyme (fraction C), and defensins (fraction D), respectively.
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181
DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS TO WNG AND ENDOTHELIAL CELLS
100
Cytotoxicity of Granule Fractions Figures 3 and 4 illustrate the cytotoxicity induced by granule fractions A-D as well as purified defensins (HNPI-3) to the lung epithelial and fibroblast target cell lines. In figure 3, A549 epithelial cells were mixed with different concentrations of granule proteins for 20 h. As shown, fractions A and B produced a small amount (approximately 20070) of specific release. This modest cytotoxic effect occurred at concentrations of 50 ug/ml but did not increase at higher concentrations. In contrast, fraction D demonstrated cytotoxicity that was concentration dependent, reaching a maximum of 60% at 200 ug/ml, Fraction C was without effect at all concentrations tested. Although rv 95% of fraction D is composed of defensins, purified peptide was more potent than fraction D at lower concentrations (50 and 100 ug/rnl), The difference between fraction D and purified defensins was not significant at a concentration of 200 ug/ml, Spontaneous release of chromium at the end of the 20-h incubation for the A549 epithelial cells was < 30070. In some experiments (9 of 41), rapid (3 h) and complete cell killing was seen with fraction A (data not shown). This activity was not due to the enzymatic action of myeloperoxidase (MPO) as the inhibitors azide (l mM), methimazole (l mM), or aminotriazole (25 mM) did not prevent fraction A cytotoxicity. Furthermore, acid-urea PAGE of fraction A from granule preparations displaying cytotoxicity did not differ from acid-urea PAGE of fractions A not displaying cytotoxicity, indicating the absence of a cytotoxic contaminating protein. Fraction A cytotoxicity was eliminated in granule preparations in which scrupulous attention was paid to maintaining the temperature below 4 0 C during the extraction. Figure 4 demonstrates the dose dependence of granule fraction cytotoxicity against MRC-5 fibroblast targets. During this 20-h assay, peak C induced little chromium release and fractions A and B were minimally active (20% specific release). Both peak D and purified defensins weredistinctly more cytotoxic, achieving roughly 60 to 80% specific release. In addition, the lung fibroblasts were more sensitive than A549 epithelial cells, being killed at a concentration as low as 50 ug/rnl (fraction D) or 25 ug/ml (HNP13) (figure 4). As with the epithelial cells, purified defensins appeared to be slightly more potent releasers of chromium
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than fraction D when used at low concentrations (25 ug/ml), The fibroblasts were also more susceptible to spontaneous chromium release,which rose linearly to 48% by the end of the 20-h incubation. In contrast to the two lung cell lines, human umbilical vein endothelial cells were more resistant to the cytotoxic activity of either fraction D or defensins, but more sensitive to fraction B. Follow-
ing a lO-hincubation, 200 ug/ml of fraction D or defensins resulted in 36 and 56070 specific release, respectively, with essentially no killing at lower concentrations (figure 5). In contrast to either the epithelial or fibroblast cells, human umbilical vein endothelium was exquisitely sensitive to fraction B, manifesting 58% specific release at a concentration of 25 ug/ml. There was no increase in kill-
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ing at higher concentrations of fraction B. At the concentrations tested, fraction A produced minimal chromium release and fraction C produced none.
Time Course of Chromium Release As shown in figure 6, the kinetics ofchromium release from lung epithelial cells was distinctly different for the various granule fractions. The effect of fraction
B was maximal at 3 h. Fraction A reached its maximal chromium release of 20070 by 6 h, at which point cytotoxicity tended to plateau. As stated previously, most preparations of fraction A were devoid of any activity. In contrast, specific release by both purified defensins and fraction D took 12h to be manifested, paralleled each other, and continued to increase until 20 h. Lung fibroblasts (figure
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7) demonstrated minimal specific release when exposed to fractions A, B, or C at any time point. Both purified defensins and fraction D demonstrated identical kinetics as seen with lung epithelial targets. Killing of endothelial cells was time dependent and comparable for fraction D and defensins (figure 8). Significant cytolysis occurred more quickly with the endothelial cells but was not as complete as for the lung cells in the 10 h of incubation. Fraction B killing occurred over a similar time course as defensins and fraction D and was of roughly the same magnitude. Fraction C was essentially inactive at all time points, and fraction A was intermediate between fractions B, D, and defensins and fraction C. Spontaneous release for these experiments varied between 30 and 50%. More complete killing (approximately 100%) was seen in some experiments (data not shown) with 20-h incubations but at the expense of very high (61 to 69%) spontaneous release, which limited the time that the incubations could be carried out.
Cytotoxicity of Whole Granule Extract Compared to Pure Defensins HNPI-3 constitute approximately 25% of the total protein content of granule extracts (10). Specific release from pulmonary epithelial (figure 9) and pulmonary fibroblasts (figure 10)targets exposed to whole granule extracts during a 20-h incubation paralleled the specific release from targets exposed to HNPI-3 at onefourth the protein concentration, especially at the higher concentrations. Some chromium was released from epithelial cells by whole granule extracts in excess of defensins at low concentrations (figure 9). At higher concentrations the curves converged and appeared to increase linearly. Release by fibroblasts was similar at all protein concentrations studied, was reached at lower protein concentrations, and tended to plateau (figure 10).
Inhibition by Serum
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Discussion
Instead of selecting a specific granule component and testing it for activity to
183
DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS TO WNG AND ENDOTHELIAL CELLS
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certain target cell types, we chose first to fractionate whole granule extracts. In this way,we were better able to assess the relative cytotoxicity of the different granule proteins. The major cytotoxic activity of these extracts against lung fibroblast and epithelial cells is confined to the fraction known to contain human neutrophil proteins 1-3 (defensins) as its main constituent. Furthermore, the cy-
totoxicity of whole PMN granule extracts to either the lung epithelial cells or fibroblasts can be accounted for by their defensin content. Compared to the lung epithelial cells and fibroblasts, human umbilical vein endothelial cells wereless susceptible to the cytotoxic action of defensins but were highly sensitive to fraction B (containing the serine proteases elastase and cathep-
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sin G). Simon and coworkers (20) noted similar differential sensitivity of bovine aortic endothelial and rat alveolar epithelial cellsto oxygen-derived cytotoxins, the former being 14-fold more sensitive to H 2 0 2 than the latter. Alveolar epithelium is normally exposed to much higher oxygen tensions than pulmonary vascular endothelium. The unusual susceptibility of endothelial cellsto PMN-derived cytotoxins may contribute to their injury during the early phase of ARDS (21). Under the conditions ofthis assay, the non-defensin-containing peaks displayed minimal cytotoxicity to epithelial cells and fibroblasts. We suspect that the cytotoxicity of fraction A in some experiments may have been due to a detergent effect of phospholipids generated in the purification scheme since the lytic effect occurred rapidly, was not inhibited by MPO inhibitors, and was not caused by the presence of any protein detectable by acid-urea PAGE. Phospholipid generation was probably prevented when extraction and purification wereassiduously performed at 4 0 C. For all three cell types, the elastasecontaining, but not other fractions, markedly decreased the adherence of viable cells to the bottom of the wells. The unusual sensitivity of endothelial cells to the loss of anchorage may have contributed to the observed cytotoxicity of fraction B. The cytocidal effects of granule fraction D are attributable to its content of defensin. First, > 95070 of that fraction's protein consists of defensin. Second, the kinetics of lysis achieved by fraction D is identical to that of purified defensin. Third, the dose-response relationship of the two cytotoxic preparations is almost identical. The minor differences at low concentrations (50 and 100 ug/ml for A549 and 25 ug/ml for MRC-5 targets) may be due to variations in the relative abundance ofthe three peptides, HNP1, 2, and 3, that make up fraction D. The ratio of HNPI :2:3 in purified peptide preparations (2:2:1) is identical to that present on the average in normal PMN granules and granule extracts. Since the fractionation procedures for purified defensins and granule fractions differ somewhat (see reference 10), it is possible that inactivation of some of the peptide occurred, accounting for the decreased activity of fraction D when suboptimal low concentrations are tested. Killing of pulmonary targets by fraction D and purified defensins demonstrated kinetics that were distinct from
184
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Fig. 9. Concentration dependence of lung epithelial cell cytotoxicity induced by purified defensins (abscissa: upper scale) or PMN granule extract (abscissa: lower scale). Points are mean ± SEM; n = 3.
intimate contact with their pulmonary target cells. Such intercellular clefts could exclude inhibitory serum components, such as albumin. Defensins are released by human PMN in response to soluble and particulate stimuli (22). This suggests that they could participate in PMN-mediated extracellular target cell lysis or injury. Theoretical considerations indicate that defensin concentrations could exceed several milligrams per milliliter in sequestered environments, such as clefts between PMN and target cells (22). Additionally, PMN disintegrating at inflammatory sites may release large quantities of defensins. Finally, synergistic cytotoxic activity between defensins and reactive oxygen intermediates, although not addressed in this report, has been observed with leukemia targets (11). Since both cytotoxins would be theoretically released simultaneously by stimulated PMNs, synergistic injury of pulmonary cells may occur
in vivo. The marked cytotoxic effect of defensins demonstrated in these studies, as well as the potential for synergistic action of defensins and H 202 , suggests that defensins may playa role in lung damage. The potential role of defensins as agents of lung injury deserves further study.
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Acknowledgment The authors are most grateful to Julia Tallos for her expert technical assistance.
20
References
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the modest toxicity due to fractions A or B. Cytotoxicity was not evident until 12 h of incubation. These slow kinetics are similar to those we have observed during defensin-mediated lysis of leukemia targets. They suggest that cytotoxicity of pulmonary and endothelial cells follows internalization or modification of HNP and is not simply due to rapid plasma membrane pore formation. Other data (13)indicate that internalization of defensin is required for subsequent lysis of
leukemia cells, and it will be interesting to determine if similar events occur during pulmonary cell injury. The inhibition of defensin killing by serum was not unexpected. Albumin inhibits defensin-mediated cytotoxicity of K562 cells and may also account for the serum-induced suppression we detected. These results suggest that if defensins play a pathologic role in vivo during PMN-mediated inflammatory tissue injury, they must be secreted by PMNs in
1. Weiland JE, Davis WB, Holter JF, Mohammed JR, Dorinsky PM, Gadek JE. Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance. Am Rev Respir Dis 1986; 133:218-25. 2. Janoff A. Elastases and emphysema: current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132:417-33. 3. Keogh BA. Interstitial lung disease of unknown cause. Disorders characterized by chronic inflammation of the lower respiratory tract. N Eng! J Moo 1984; 310:154-66, 235-44. 4. Gallin 11, Malech HL. Current concepts - immunology: neutrophils in human diseases. N Engl J Med 1987; 317:687-94. 5. Lehrer RI, Ganz T, Selsted ME, Babior BM, Curnutte JT. UCLA Conference: neutrophils and host defense. Ann Intern Med 1988; 109:127-42. 6. Johnson KJ, Fantone J C, Kaplan J, Ward PA. In vivo damage of rat lungs by oxygen metabolites. J Clin Invest 1981; 67:983-93. 7. Brigham KL.Role of free radicals in lung injury. Chest 1986; 89:859-63. 8. 'Iate RM, Repine JE. Phagocytes, oxygen radicals and lung injury. In: Pryor W, ed, Free radicals in biology. vol VI. New York: Academic Press, 1984; 199-209. 9. Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL, McCord JM, Harman D. Conference: oxygen radicals and human disease. Ann Intern Med 1987; 107:526-45.
DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS 10 WNG AND ENDOTHELIAL CELLS
10. Ganz T, Selsted ME, Harwig SSL, Szklarek D, Daher K, Lehrer RI. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 1985; 76:1427-35. 11. Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI. Synergistic cytolysis mediated by hydrogen peroxide combined with peptide defensins. Cell Immunol 1988; 114:104-16. 12. Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI. In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes. Blood 1986; 68:1407-10. 13. Lichtenstein AK, Ganz T, Nguyen T, Selsted ME, Lehrer RI. Mechanism of target cytolysis by peptide defensins: target cell metabolic activities possibly involving endocytosis are crucial for expression of cytotoxicity. J Immunol 1988; 140: 2686-94. 14. Modrzakowski MC, Cooney MH, Martin LE,
Spitznagel JK. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes. Infect Immun 1979; 23: 587-91. . 15. Modrzakowski MC, Paranavitana CM. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: role of bacterial membrane lipid. Infect Immun 1981; 32:668-74. 16. Smith PK, Krohn RI, Hermanson GT, et at. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150:76-85. 17. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins:identification by morphologic and immunologic criteria. J Clin Invest 1973; 52:2745-56. 18. Navab M, Hough GP, Berliner JA, Joy AF, Fogelman AM, Haberland ME, Edwards PA. Rab-
185 bit beta-migrating very low density lipoprotein increases endothelial macromolecular transport without altering electrical resistance. J Clin Invest 1986; 78:389-97. 19. Greenwald 01, Ganz T. Defensins mediate the microbicidal activity of human neutrophil granule extract against Acinetobacterca/coaceticus. Infect Immun 1987; 55:1365-8. 20. Simon RH, DeHart PD, Todd RF III. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J Clin Invest 1986; 78:1375-86. 21. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygenradicals mediate endothelial cell damage by complement-stimulated granulocytes: an in vitro model of immune vascular damage. J Clin Invest 1978; 61:1161-7. 22. Ganz T. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect Immun 1987; 55:568-71.
DEREK G. OKRENT,5 ALAN K. LICHTENSTEIN, and TOMAS GANZ8
Introduction
The polymorphonuclear leukocyte (PMN) is believed to play an important role in the pathogenesis of the adult respiratory distress syndrome (1), emphysema (2), and cryptogenic fibrosing alveolitis (idiopathic pulmonary fibrosis) (3). PMNs may participate in lung injury through their production of reactive oxygen intermediates or release of hydrolytic enzymes and cytocidal proteins from their granules (4, 5). Although the ability of reactive oxygen intermediates to injure the respiratory and other host organ systems is well documented (6-9), relatively little information is available concerning the potential of granule components acting independently of oxygen. Of these, only elastase has been wellstudied and shown to damage lung tissue in vivo and in vitro (2). Human defensins, three recently described small (molecular weight approximately 3900) microbicidal peptides, are among the most abundant components of azurophil granules of PMNs (10). In a recent study (11), a defensin-containing fraction of PMN granule extracts wasthe most damaging to several tumor cell targets, but fractions containing elastase, lysozyme, and other hydro lases were relatively ineffective. The cytotoxic potential of defensins for tumor cells has been confirmed using purified peptide (12,13). Since nonoxidative components of PMN granules may be involved in pulmonary injury and since only elastase has been examined using lung tissue as a target, wedesigned the current study to compare the relative activities of all granule components against pulmonary-derived cells and endothelial cells. Because our previous experience with nonpulmonary targets demonstrated that defensins had the greatest activity, the various granule extract fractions were compared to purified defensins in the current study. The results demonstrate the potent lysisof targets by defensins and defensin-containing granule extract fractions; relatively little
SUMMARY Neutrophlls, In the course of defending the host against microbial Invasion, release a potent arsenal of proteins that can potentially damage host tissues. Defenslns are major peptldes of human polymorphonuclear leukocyte (PMN)granules and are both broadly microbicidal and cytotoxic to several tumor cell lines. Todetermine whether these peptldes could play a role In neutrophllmediated lung Injury, we examined the cytotoxicity of defenslns and other PMN granule proteins in a chromium release assay with human lung-derlved cell lines MRC-5 (lung fetal fibroblast), A549 (lung adenocarcinoma with features of alveolar epithelium), and primary cultures of human umblll· cal vein endothelial cells (HUVEC). Crude fractionation of an acid extract of human PMN granules yielded four fractions A-D. Only fraction D (containing mostly defenslns) was significantly cytotOXic to all three target cells. In contrast, fraction A (containing myeloperoxldase and lactoferrln) and fraction C (containing lysozyme) had little effect, and fraction B (containing chiefly cathepsin G and elastase) was only injurious to endothelial cells. The cytotoxicity of whole PMNgranule extracts on pulmonary epithelial and fibroblast targeta could be completely accounted for by their defensin content. Fraction D- and defensln-medlated cytotoxicity was concentration dependent, required at least 10 to 12 h to become manifest, and was Inhibited by serum. The role of these peptldes In lung damage during acute and chronic Inflammation deserves further study. AM REV RESPIR DIS 1990; 141:179-185
Injury was induced by other granule components. Methods Purification of PMN Human PMN were obtained from a commercial supplier (Hemacare, Van Nuys, CA) in single-donor leukopheresis packs containing 2 x 1010 to 4 X 10'0 cells, with> 90% viable PMN as determined by trypan blue exclusion and Giemsa stain differential count. Contaminating platelets and erythrocytes were subsequently removed by differential centrifugation and hypotonic lysis as described previously (10). PMN and granule preparations were processed at 2 0 C throughout.
Preparation of PMN Granules and Granule Protein Extraction After suspension in Hanks' balanced salt solution (HBSS), pH 7.4 with 2.5 mM MgCl" the cell suspension was sealed in a nitrogen bomb (Parr Instrument Co., Moline, IL) and pressurized to 750 psi for 20 min. The suspension was then released dropwise into HBSS with 5 mM NazEDTA while stirring. Suspensions were centrifuged at low speed (200 x g for 10 min) to remove nuclei and cellular debris. The pellet was examined by phase-contrast microscopy to ensure> 900/0 cellular disruption. The supernatant containing the granules was sedimented at 27,000 x g for 20 min and the pellets stored at -70 0 C.
Granule proteins were extracted, and the extract was fractionated essentially as described by Modrzakowski and coworkers (14, 15).Briefly, the granule sediment was extracted three times in a volume of 0.2 M sodium acetate buffer with 0.01 M caci, (pH 4.0) to yield a final concentration of 2 x 108 cell equivalents per milliliter. Each extraction was done over 12 h at 4 0 C with gentle stirring, and the residue separated by centrifugation
(Received in original form January 19, 1989 and in revised form May 10, 1989) t From the Will Rogers Institute Pulmonary ResearchLaboratory, Department of Medicine,UCLA School of Medicine, and the Department of Medicine, VAWadsworth-UCLA Medical Center, Los Angeles, California. 2 Supported in part by the National Institutes of Health Grant CA 37184 and HL 35640, the American Lung Association of California Grant (CRMEF) A860701, and research funds of the Veterans Administration. 3 Correspondence and requests for reprints should be addressed to Tomas Ganz, Ph.D., M.D., UCLA School of Medicine, Center for the Health Sciences, Los Angeles, CA 90024-1736. • Presented in part at the WesternRegionalMeeting of the American Federation for Clinical Research, Carmel, California, February 1988. 5 Parker B. Francis Research Fellowof the Puritan-Bennett Foundation. • RJR Nabisco Research Scholar.
179
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OKRENT, LICHTENSTEIN, AND GANZ
at 27 000 x g for 20 min. The pooled acetate extra~ts were concentrated by ultrafiltration (YC-Q5 filter; Amicon Corp., Lexington , MA) and placed on a Sephadex G-loo column (2.5 x 150 em), Approximately 70 mg crude granule protein extract was eluted with 0.2 M sodium acetate buffer (pH 4.0) and collected in lO-ml fractions. The fractions were pooled into four fractions (A, B, C, and D) defined by their A. 80 pattern, concentrated by ultrafiltration (Amicon YC-Q5 filter), and dialyzed against phosphate-buffered saline (PBS) in Spectrapore 3 tubing (molecular weight cutoff 3500). Roughly 500/0 of the protein in the original granule extract is recovered by this technique.
Defensins The three defensin peptides, human neutrophil proteins 1-3 (HNPI-3), were individually prepared as described previously (10) and stored as stock solutions of 2 mg/ml in 0.01% acetic acid at -20° C. HNPI-3 were mixed in a ratio of 2:2:1 by weight, and this mixture was tested for lytic activity.
Protein Studies Protein concentration was measured by the BCA (16)protein assay system (Pierce Chemical Company, Rockford, IL) with chicken egg white lysozyme as the standard. A 1 to 4 ug amount of crude granule extract, each fraction, and purified defensins was subjected to electrophoresis on 12.5% acid-urea polyacrylamide gels (PAGE).
Cell Lines Human lung fibroblasts (MRC-5), a nontransformed cell line, and human lung carcinoma with features of alveolar epithelial cells (A549) were obtained from the American Type Culture Collection (Rockville, MA) and maintained in RPMI-I640 with 10% fetal calf serum (FCS). Human umbilical vein endothelial cells (HUVEC) were prepared in primary culture by collagenase treatment of fresh term umbilical cord veins using previously described techniques (17). HUVEC were maintained in M-199 plus HEPES (MA Bioproducts) supplemented with 20% FCS « I ng/ml of endotoxin; Hyclone Laboratories, Logan, UT) , 1 mM pyruvate, 2 mM glutamine, and 90 lig/ ml of heparin plus 20 ug/rnl of endothelial cell growth supplement (Collaborative Research, Bedford, MA) (18). The HUVEC used for the experiments were passages 3 to 8, at confluence displayed uniform cobblestonelike morphology, and 100% of cells stained with 1,1'-dioctadecyl-l,3,3,3',3'tetramethylindocarbocyanine perchlorateLDL (DiI-Ac-LDL; Biomedical Technologies, Inc., Cambridge, MA) (18).
Chromium Release Assay Cell targets wereinitially plated in 96-wellmicrotiter plates at a density of approximately 2 x 104 cells/well and grown to confluence over 24 h. For assays with HUVEC, microtiter wells were precoated with gelatin 0-1890
Control release was determined from targets incubated in medium alone (see RESULTS), and maximal release was achieved by adding 3% Triton X-loo (> 80% incorporated counts). Release from targets incubated in medium containing dilute acetic acid or PBS (the vehicles used in defensin and granule fraction stock solutions, respectively) did not differ from controls incubated in medium alone. The standard deviation of the triplicate samples was always < 5% of the mean.
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(Sigma, St. Louis, MO) by adding 100 iii of I% gelatin in PBS to each well and storing plates at 4° C. Immediately before seeding HUVEC, the wells were warmed to 37° C, gelatin was aspirated, and wells were rinsed with PBS . All targets were labeled by incubating with I to 2 IiCi/well "Cr for 2 h at 37° C. The monolayers were then extensively washed with medium and then incubated with test substances in serum-free (A549 and MRC5) or 0.1% serum (HUVEC) at 37° C. Subsequently, the plates were centrifuged (1,200 rpm for 5 min) to pellet nonadherent cells, and the supernatants counted in a gamma counter. Specific chromium release was calculated as: cpmexp - cpmcontrol x 100. Cpmmaximal - CPfficontrol
1
Results
Composition of Granule Fractions
2
Figure 1 is a typical elution profile from Sephadex G-loo gel filtration, demonstrating the resolution of the crude granule extract into four fractions (A-D) with relative protein contents of approximately 12, 28, 15, and 45070, respectively. According to previous work (14, 19), fraction A contains myeloperoxidase (MPO; 150,000D), fraction B contains proteases including elastase and cathepsin G (27,000 D), and fraction C contains lysozyme (14,000 D) (13). Over 95% of fraction D consists of a mixture of the three defensins: HNPl, HNP2, and HNP3 (3,000 to 4,000 D) (19). Acid-urea PAGE (figure 2) and 10to 20% sodium dodecyl sulfate (SDS) gradient PAGE (not shown) of the fractions obtained confirm the presence of dominant bands that comigrate with purified MPO (fraction A), elastase (fraction B), lysozyme (fraction C), and defensins (fraction D), respectively.
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181
DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS TO WNG AND ENDOTHELIAL CELLS
100
Cytotoxicity of Granule Fractions Figures 3 and 4 illustrate the cytotoxicity induced by granule fractions A-D as well as purified defensins (HNPI-3) to the lung epithelial and fibroblast target cell lines. In figure 3, A549 epithelial cells were mixed with different concentrations of granule proteins for 20 h. As shown, fractions A and B produced a small amount (approximately 20070) of specific release. This modest cytotoxic effect occurred at concentrations of 50 ug/ml but did not increase at higher concentrations. In contrast, fraction D demonstrated cytotoxicity that was concentration dependent, reaching a maximum of 60% at 200 ug/ml, Fraction C was without effect at all concentrations tested. Although rv 95% of fraction D is composed of defensins, purified peptide was more potent than fraction D at lower concentrations (50 and 100 ug/rnl), The difference between fraction D and purified defensins was not significant at a concentration of 200 ug/ml, Spontaneous release of chromium at the end of the 20-h incubation for the A549 epithelial cells was < 30070. In some experiments (9 of 41), rapid (3 h) and complete cell killing was seen with fraction A (data not shown). This activity was not due to the enzymatic action of myeloperoxidase (MPO) as the inhibitors azide (l mM), methimazole (l mM), or aminotriazole (25 mM) did not prevent fraction A cytotoxicity. Furthermore, acid-urea PAGE of fraction A from granule preparations displaying cytotoxicity did not differ from acid-urea PAGE of fractions A not displaying cytotoxicity, indicating the absence of a cytotoxic contaminating protein. Fraction A cytotoxicity was eliminated in granule preparations in which scrupulous attention was paid to maintaining the temperature below 4 0 C during the extraction. Figure 4 demonstrates the dose dependence of granule fraction cytotoxicity against MRC-5 fibroblast targets. During this 20-h assay, peak C induced little chromium release and fractions A and B were minimally active (20% specific release). Both peak D and purified defensins weredistinctly more cytotoxic, achieving roughly 60 to 80% specific release. In addition, the lung fibroblasts were more sensitive than A549 epithelial cells, being killed at a concentration as low as 50 ug/rnl (fraction D) or 25 ug/ml (HNP13) (figure 4). As with the epithelial cells, purified defensins appeared to be slightly more potent releasers of chromium
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than fraction D when used at low concentrations (25 ug/ml), The fibroblasts were also more susceptible to spontaneous chromium release,which rose linearly to 48% by the end of the 20-h incubation. In contrast to the two lung cell lines, human umbilical vein endothelial cells were more resistant to the cytotoxic activity of either fraction D or defensins, but more sensitive to fraction B. Follow-
ing a lO-hincubation, 200 ug/ml of fraction D or defensins resulted in 36 and 56070 specific release, respectively, with essentially no killing at lower concentrations (figure 5). In contrast to either the epithelial or fibroblast cells, human umbilical vein endothelium was exquisitely sensitive to fraction B, manifesting 58% specific release at a concentration of 25 ug/ml. There was no increase in kill-
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ing at higher concentrations of fraction B. At the concentrations tested, fraction A produced minimal chromium release and fraction C produced none.
Time Course of Chromium Release As shown in figure 6, the kinetics ofchromium release from lung epithelial cells was distinctly different for the various granule fractions. The effect of fraction
B was maximal at 3 h. Fraction A reached its maximal chromium release of 20070 by 6 h, at which point cytotoxicity tended to plateau. As stated previously, most preparations of fraction A were devoid of any activity. In contrast, specific release by both purified defensins and fraction D took 12h to be manifested, paralleled each other, and continued to increase until 20 h. Lung fibroblasts (figure
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7) demonstrated minimal specific release when exposed to fractions A, B, or C at any time point. Both purified defensins and fraction D demonstrated identical kinetics as seen with lung epithelial targets. Killing of endothelial cells was time dependent and comparable for fraction D and defensins (figure 8). Significant cytolysis occurred more quickly with the endothelial cells but was not as complete as for the lung cells in the 10 h of incubation. Fraction B killing occurred over a similar time course as defensins and fraction D and was of roughly the same magnitude. Fraction C was essentially inactive at all time points, and fraction A was intermediate between fractions B, D, and defensins and fraction C. Spontaneous release for these experiments varied between 30 and 50%. More complete killing (approximately 100%) was seen in some experiments (data not shown) with 20-h incubations but at the expense of very high (61 to 69%) spontaneous release, which limited the time that the incubations could be carried out.
Cytotoxicity of Whole Granule Extract Compared to Pure Defensins HNPI-3 constitute approximately 25% of the total protein content of granule extracts (10). Specific release from pulmonary epithelial (figure 9) and pulmonary fibroblasts (figure 10)targets exposed to whole granule extracts during a 20-h incubation paralleled the specific release from targets exposed to HNPI-3 at onefourth the protein concentration, especially at the higher concentrations. Some chromium was released from epithelial cells by whole granule extracts in excess of defensins at low concentrations (figure 9). At higher concentrations the curves converged and appeared to increase linearly. Release by fibroblasts was similar at all protein concentrations studied, was reached at lower protein concentrations, and tended to plateau (figure 10).
Inhibition by Serum
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Discussion
Instead of selecting a specific granule component and testing it for activity to
183
DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS TO WNG AND ENDOTHELIAL CELLS
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certain target cell types, we chose first to fractionate whole granule extracts. In this way,we were better able to assess the relative cytotoxicity of the different granule proteins. The major cytotoxic activity of these extracts against lung fibroblast and epithelial cells is confined to the fraction known to contain human neutrophil proteins 1-3 (defensins) as its main constituent. Furthermore, the cy-
totoxicity of whole PMN granule extracts to either the lung epithelial cells or fibroblasts can be accounted for by their defensin content. Compared to the lung epithelial cells and fibroblasts, human umbilical vein endothelial cells wereless susceptible to the cytotoxic action of defensins but were highly sensitive to fraction B (containing the serine proteases elastase and cathep-
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sin G). Simon and coworkers (20) noted similar differential sensitivity of bovine aortic endothelial and rat alveolar epithelial cellsto oxygen-derived cytotoxins, the former being 14-fold more sensitive to H 2 0 2 than the latter. Alveolar epithelium is normally exposed to much higher oxygen tensions than pulmonary vascular endothelium. The unusual susceptibility of endothelial cellsto PMN-derived cytotoxins may contribute to their injury during the early phase of ARDS (21). Under the conditions ofthis assay, the non-defensin-containing peaks displayed minimal cytotoxicity to epithelial cells and fibroblasts. We suspect that the cytotoxicity of fraction A in some experiments may have been due to a detergent effect of phospholipids generated in the purification scheme since the lytic effect occurred rapidly, was not inhibited by MPO inhibitors, and was not caused by the presence of any protein detectable by acid-urea PAGE. Phospholipid generation was probably prevented when extraction and purification wereassiduously performed at 4 0 C. For all three cell types, the elastasecontaining, but not other fractions, markedly decreased the adherence of viable cells to the bottom of the wells. The unusual sensitivity of endothelial cells to the loss of anchorage may have contributed to the observed cytotoxicity of fraction B. The cytocidal effects of granule fraction D are attributable to its content of defensin. First, > 95070 of that fraction's protein consists of defensin. Second, the kinetics of lysis achieved by fraction D is identical to that of purified defensin. Third, the dose-response relationship of the two cytotoxic preparations is almost identical. The minor differences at low concentrations (50 and 100 ug/ml for A549 and 25 ug/ml for MRC-5 targets) may be due to variations in the relative abundance ofthe three peptides, HNP1, 2, and 3, that make up fraction D. The ratio of HNPI :2:3 in purified peptide preparations (2:2:1) is identical to that present on the average in normal PMN granules and granule extracts. Since the fractionation procedures for purified defensins and granule fractions differ somewhat (see reference 10), it is possible that inactivation of some of the peptide occurred, accounting for the decreased activity of fraction D when suboptimal low concentrations are tested. Killing of pulmonary targets by fraction D and purified defensins demonstrated kinetics that were distinct from
184
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Fig. 9. Concentration dependence of lung epithelial cell cytotoxicity induced by purified defensins (abscissa: upper scale) or PMN granule extract (abscissa: lower scale). Points are mean ± SEM; n = 3.
intimate contact with their pulmonary target cells. Such intercellular clefts could exclude inhibitory serum components, such as albumin. Defensins are released by human PMN in response to soluble and particulate stimuli (22). This suggests that they could participate in PMN-mediated extracellular target cell lysis or injury. Theoretical considerations indicate that defensin concentrations could exceed several milligrams per milliliter in sequestered environments, such as clefts between PMN and target cells (22). Additionally, PMN disintegrating at inflammatory sites may release large quantities of defensins. Finally, synergistic cytotoxic activity between defensins and reactive oxygen intermediates, although not addressed in this report, has been observed with leukemia targets (11). Since both cytotoxins would be theoretically released simultaneously by stimulated PMNs, synergistic injury of pulmonary cells may occur
in vivo. The marked cytotoxic effect of defensins demonstrated in these studies, as well as the potential for synergistic action of defensins and H 202 , suggests that defensins may playa role in lung damage. The potential role of defensins as agents of lung injury deserves further study.
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Acknowledgment The authors are most grateful to Julia Tallos for her expert technical assistance.
20
References
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the modest toxicity due to fractions A or B. Cytotoxicity was not evident until 12 h of incubation. These slow kinetics are similar to those we have observed during defensin-mediated lysis of leukemia targets. They suggest that cytotoxicity of pulmonary and endothelial cells follows internalization or modification of HNP and is not simply due to rapid plasma membrane pore formation. Other data (13)indicate that internalization of defensin is required for subsequent lysis of
leukemia cells, and it will be interesting to determine if similar events occur during pulmonary cell injury. The inhibition of defensin killing by serum was not unexpected. Albumin inhibits defensin-mediated cytotoxicity of K562 cells and may also account for the serum-induced suppression we detected. These results suggest that if defensins play a pathologic role in vivo during PMN-mediated inflammatory tissue injury, they must be secreted by PMNs in
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DIRECT CYTOTOXICITY OF PMN GRANULE PROTEINS 10 WNG AND ENDOTHELIAL CELLS
10. Ganz T, Selsted ME, Harwig SSL, Szklarek D, Daher K, Lehrer RI. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 1985; 76:1427-35. 11. Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI. Synergistic cytolysis mediated by hydrogen peroxide combined with peptide defensins. Cell Immunol 1988; 114:104-16. 12. Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI. In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes. Blood 1986; 68:1407-10. 13. Lichtenstein AK, Ganz T, Nguyen T, Selsted ME, Lehrer RI. Mechanism of target cytolysis by peptide defensins: target cell metabolic activities possibly involving endocytosis are crucial for expression of cytotoxicity. J Immunol 1988; 140: 2686-94. 14. Modrzakowski MC, Cooney MH, Martin LE,
Spitznagel JK. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes. Infect Immun 1979; 23: 587-91. . 15. Modrzakowski MC, Paranavitana CM. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: role of bacterial membrane lipid. Infect Immun 1981; 32:668-74. 16. Smith PK, Krohn RI, Hermanson GT, et at. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150:76-85. 17. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins:identification by morphologic and immunologic criteria. J Clin Invest 1973; 52:2745-56. 18. Navab M, Hough GP, Berliner JA, Joy AF, Fogelman AM, Haberland ME, Edwards PA. Rab-
185 bit beta-migrating very low density lipoprotein increases endothelial macromolecular transport without altering electrical resistance. J Clin Invest 1986; 78:389-97. 19. Greenwald 01, Ganz T. Defensins mediate the microbicidal activity of human neutrophil granule extract against Acinetobacterca/coaceticus. Infect Immun 1987; 55:1365-8. 20. Simon RH, DeHart PD, Todd RF III. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J Clin Invest 1986; 78:1375-86. 21. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygenradicals mediate endothelial cell damage by complement-stimulated granulocytes: an in vitro model of immune vascular damage. J Clin Invest 1978; 61:1161-7. 22. Ganz T. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect Immun 1987; 55:568-71.
