Effects of Activated Polymorphonuclear Leukocytes upon Pulmonary Surfactant In Vitro Stephen F. Ryan, Y. Ghassibi, and Deng F. Liau Pulmonary Research Group, Department of Pathology and Pulmonary Division of the Department of Medicine, St. Luke's-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York
Current evidence suggests that products of activated inflammatory cells cause or contribute to the acute lung injury of the adult respiratory distress syndrome (ARDS). To assess the possibility that these products may impair surfactant function during ARDS, we exposed surfactant in vitro to polymorphonuclear leukocytes (PMN) activated by phorbol myristate acetate and to the oxidant-producing pair ferric chloride/ascorbate (FeCl,/ASC). After incubation of surfactant with 8 to 32 X 106 activated PMN for 1 to 4 h or with FeCl,/ASC for 16 h, its isopycnic density (d), minimum surface tension ('Ymio), time course of adsorption, compressibility (SC), and stability index (SI) were determined. We found progressive decreases of d, adsorption, and SI and progressive increases of 'Ymio and SC after exposure to activated PMN in increasing numbers or for longer time periods. Superoxide dismutase completely inhibited all of these effects except the decreased adsorption, which it did not significantly inhibit. Similar changes in all of these parameters occurred after exposure of surfactant to FeCI,/ASC. Polyacrylamide gel electrophoresis of surfactant after exposure to activated PMN showed a decrease of the major apoprotein that progressed with exposure time and was associated with the appearance of several bands with both lower and higher molecular weights than that of the apoprotein. The data show that activated PMN are capable of impairing surfactant function in vitro and of degrading the major apoprotein. They suggest that the effects upon d, 'Ymin, SC, and SI are mediated largely if not exclusively by oxidant radicals. While oxidants may contribute to delayed adsorption, proteolysis appears to play the principal role in this effect.
The pathogenesis ofthe acute lung injury in the adult respiratory distress syndrome (ARDS) remains unclear, but accumulating evidence, both experimental and clinical, suggests that toxic products of activated inflammatory cells, including oxygen radicals and proteases, playa major role. Polymorphonuclear leukocytes (PMN) activated by any one of several proinflammatory signals become increasingly adherent to endothelial cells and to other PMN and produce a large variety of products, including toxic oxygen radicals, proteases, and hypochlorous acid. These toxic products are used to kill phagocytized microorganisms, but they are also (Received in original form January 31, 1990 and in revised form June 6, 1990) Address correspondence to: Stephen F. Ryan, M.D., Pulmonary Research Group, Department of Pathology & Pulmonary Division, Department of Medicine, St. Luke's-Roosevelt Hospital Center, Amsterdam Avenue at 114th Street, New York, NY 10025. Abbreviations: adult respiratory distress syndrome, ARDS; ascorbate, ASC; dimethyl sulfoxide, DMSO; ferric chloride, FeCh; phosphatebuffered saline, PBS; malondialdehyde, MDA; phospholipid, PL; phorbol myristate acetate, PMA; polymorphonuclear leukocyte, PMN; sodium dodecyl sulfate-polyacrylamide minislab gel electrophoresis, SDS-PAGE; superoxide dismutase, SOD; thiobarbituric acid, TBA. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 33-41, 1991
released into the extracellular milieu, where they may cause tissue injury by initiating peroxidation and proteolysis (1-3). Experimentally, intravascular activation of PMN is followed by their sequestration in the lung and by injury to its capillary endothelial cells, which can be mitigated by antioxidants or by PMN depletion (4-8). Increased numbers of marginated or aggregated PMN have been found in the pulmonary capillaries of patients with ARDS by several investigators (9, 10), and others have reported marked increases in numbers of PMN recovered by bronchoalveolar lavage from these patients (11, 12). A recent study has presented evidence that oxidants are generated in the lungs of patients with ARDS by demonstrating that a fraction of the - 40
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Figure 4. Time course of surface adsorption of surfactant exposed
Figure 5. Representative surface tension versus area diagrams of
to FeCh/ascorbate (ASC). Samples, containing 2 mg PL, were exposed to FeCh/ASC at 37° C for 16 h. Control 1, unexposed; control 2, mixed with FeCh/ASC immediately before measurement; control 3, incubated alone at 37° C for 16 h. FeCI3/ASC are significantly lower than controls (n = 4 for all groups; P < 0.001).
surfactant exposed to FeCl3/ascorbate (ASC). Each sample, after measurement of surfaceadsorption (Figure 4), wascompressedand expanded for 15 cycles. Diagrams shown were recorded at fifth cycle (broken line) and fifteenthcycle (unbroken line) of compression and expansion of the surface.
Ryan, Ghassibi, and Liau: Effects of Activated PMN on Surfactant
because the numbers of PMN that could be practically isolated required quantities of surfactant that were too small to assay by this method. We attempted to use ratios of activated PMN to quantities of surfactant that would be plausible in the alveoli of patients during ARDS. For this calculation, we used the lavage data of Fowler and coworkers (12) to estimate the total number of alveolar PMN during ARDS and our own quantification of total alveolar surfactant in the normal dog lung (38). Our figure, based on these data, of 1.77 X 106 PMN/mg surfactant PL probably underestimates the number of PMN to which the surfactant is exposed during ARDS because of incomplete recovery of cells by alveolar lavageand because lavage recovers alveolar PMN at one time point, whereas, during ARDS, they are probably continually recruited to the alveoli, exhausted, and lysed over a period of many hours. We therefore used 4, 8, 12, and 16 X 106 PMN/mg surfactant PL with relatively short exposure times of I to 4 h. We also examined the effects of strong oxidants upon the surfactant by exposing it to the oxidant pair FeCliASC. In this system, the surfactant was exposed only to oxidants rather than to the large number of products released by activated PMN. Comparisons of the densities and surface properties of surfactant after exposure to oxidants alone with those after exposure to activated PMN allow some preliminary proposals concerning possible causes of surfactant abnormalities induced by activated PMN. The data show that exposure of surfactant to activated PMN under these conditions caused a decrease in its isopycnic density and abnormality in each of the surface properties studied. Furthermore, there was a progressive decrease in density and a progressive increase in degree of abnormality in each of the parameters of surface function as the product of numbers of PMN (xl()6) and hours of exposure (nPMN X h) increased. The decrease in isopycnic density was maximized at 1.045 from a normal of 1.051 after npMN X h of 32 (Table 1). The decrease was completely inhibited by addition of SOD to the reaction mixture, and exposure to FeCb/ASC caused a similar decrease in density. Thus, oxidative alteration of surfactant apoproteins or lipids appears to be the major cause of decreased density of surfactant after exposure to activated PMN. A similar but greater decrease in density was found by Petty and coworkers in the major surface active lipoprotein fraction in alveolar lavage fluid from a patient with posttraumatic ARDS (39). The lipid/protein ratio of the abnormallow-density fraction was greater than that of the normal fraction, and it was speculated that the former was a degradation product of the latter. Our data support this hypothesis. The progressive decrease in density of the surfactant in vitro precludes the alternative possibilities that the low-density fraction is a newly synthesized product of injured or regenerating type II cells or that it results from contamination of surfactant by plasma lipids. The progressive increase of ')Imin developed by the surfactant after exposure to increasing npMN X h was also completely prevented by SOD, and exposure to FeCb/ASC caused increases in ')Imin similar to those caused by maximum npMN X h. These observations again suggest that oxidative processes are at least mostly responsible for increased ')Imin after exposure to activated PMN. Delayed
39
adsorption cannot explain this increase because when equal quantities of normal and of PMN-exposed surfactant were sprayed on the surface in the balance, ')Im;n was increased only in the PMN-exposed surfactant and the degree of increase was similar to that recorded after placing the surfactant suspension in the trough and allowing adsorption to occur prior to determination of ')Im;n as described above. After exposure to lower n pMN X h, there was a significant decrease in ')Im;n between the fifth and the fifteenth compression cycles, which was not seen after exposure to higher npMN X h. This failure to reduce ')Im;n with repeated compression after greater npMN X h may have resulted from greater oxidation of polyunsaturated fatty acids of unsaturated phosphatidylcholine species or of other lipids containing polyunsaturated fatty acids resulting in an alteration in the liquid-gel transition temperature (40). Surface film adsorption kinetics and reentry and respreadability of the surfactant lipids may thus have been affected, resulting in impaired refinement of the stable phospholipids on the surface believed to occur during cyclic compression and expansion (41). SC and Sf are widely accepted measures of the rigidity and stability of the surfactant film in the Wilhelmy balance. Their abnormalities after exposure to activated PMN, which trend with ')Im'n and adsorption, provide further evidence of profound functional impairment of the surfactant. The apoproteins of the surfactant are believed to be largely responsible for its rapid surface adsorption (42, 43), and the marked reduction of adsorption of surfactant after exposure to activated PMN suggests that the apoprotein has been altered. Because SOD failed to significantly reduce the impairment of adsorption, degradation of the apoprotein by PMN proteases appears to be the principal cause of its impaired adsorption. Support for this suggestion is provided by the findings on SDS-PAGE. After exposure of surfactant to 16 X 1()6 PMN for 2 h, the quantity of SP-A was clearly decreased and this decrease was not inhibited by SOD (Figure 3; lanes C and D). After exposure to 32 X 1()6 PMN for 1 h, several bands with mol wt lower than those of SP-A appeared (Figure 3, lane E), and after exposure for 2 and 3 h, the quantities of SP-Aand of the low-molecular-weight bands progressively decreased (Figure 3, lanes F and G). Exposure of the canine apoprotein complex to bacterial collagenase has recently been shown to generate several bands with mol wt similar to these (44). In addition to the bands with mol wt lower than those of SP-A, several bands with higher mol wt also appeared and progressively decreased with exposure time. The progressive decrease in quantity of SP-A with exposure time suggests that at least some of the low-molecular-weight bands were degradation products of SP-A, which in their own turn were further degraded, but it is possible that some or all of them were fragments of degraded proteins from PMN. The bands with mol wt higher than SP-A may be aggregates of degradation products of SP-Aor of contaminating fragments derived from PMN. Further study will be necessary to clarify this. If fragments of PMN-derived proteins did contaminate the filtered surfactant, it seems very unlikely that they impaired surface function of the surfactant because SDS-PAGE of surfactant proteins after separation by continuous sucrose
40
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
density gradient centrifugation demonstrated that all of the minor bands have been removed and yet surface function remained abnormal. The alterations of in vitro surface function after exposure to FeCb/ASC were more severe than those reported by Seeger and associates after exposure of surfactant to a variety of strong oxidants including FeCb/ASC (45). One possible explanation for this discrepancy is that we used partially purified surfactant, whereas Seeger and associates used crude alveolar lavage fluid for their experiments. Such material does not reduce I'm'n to below about 30 dynes/em and therefore is not useful for studying altered I'min under abnormal conditions. The changes reported by Seeger and associates in other parameters of surfactant function after exposure to oxidants, for example, reduction of hysteresis area and delayed reduction of surface tension during compression, are similar to those described here after exposure to FeCI3/ASC. Although our data suggest that delayed surface adsorption induced by activated PMN is more likely due to proteolysis than to oxidative processes, the results with FeCI 3/ASC demonstrate that oxidants are capable of profoundly impairing adsorption. It thus remains possible that PMN may impair adsorption by oxidative mechanisms if present in sufficient numbers in the lung. While our data show that products of activated PMN are capable of altering surfactant function in vitro, the relevance of these findings to such effects in vivo is speculative. SOD activity has been reported in extracellular fluid (46), and antiproteases are abundant in the alveoli of some patients with ARDS (47, 48). These inhibitors may reduce or prevent adverse effects of PMN upon surfactant in vivo. Alterations of the physical and chemical properties of the surfactant during ARDS have not been analyzed sufficiently to permit detailed comparisons with the changes induced in vitro by activated PMN, but the changes in surface properties so far described are very similar to those found in our study (16, 17). On the other hand, this mechanism cannot explain the reported decrease in surfactant disaturated phosphatidylcholine during ARDS (16), and it seems probable that if alteration by PMN does occur, it is only one of multiple processes that may impair surfactant synthesis or function during ARDS. Nevertheless, it seems possible that the surfactant may be damaged by products of activated inflammatory cells during ARDS, and this possibility deserves further investigation. If inflammatory cells cause abnormality of surfactant function as suggested by our observations, then it will be important to learn not only the potential magnitude of the injury but also the molecular mechanisms involved. If surfactant replacement in patients with ARDS is ever to be employed successfully, these mechanisms must be understood. If oxidative or proteolytic damage is indeed important, then instilled surfactant may become rapidly altered or degraded and be of no benefit. It seems possible that, in that case, therapeutic strategies such as addition of antioxidants or antiproteases to the instilled surfactant could be developed. Acknowledgments: The writers gratefully acknowledge the excellent technical assistance of Giselle Cemansky, Jin Huang, and Martin Torni and thank Dr. A. L. Loomis Bell for his encouragement and for his critical review of this manuscript. This work was supported in part by a grant from the Stoney Wold Foundation of New York. Parts of this study were presented at the 55th Annual Scientific Assembly, XVI World Congress on Disease of the Chest (Boston, MA, October 30, 1989).
References 1. Weiss, S. 1. 1989. Tissue destruction by neutrophils. N. Eng/. J. Med. 320:365-376. 2. Henson, P. M., and R. B. Johnston, Jr. 1987. Tissue injury in inflammation: oxidants, proteinases and cationic proteins. J. Clin. Invest. 79: 669-674. 3. Fantone, J. C., and P. A. Ward. 1985. Polymorphonuclear leukocytemediated cell and tissue injury: oxygen metabolites and their relation to human disease. Hum. Pathol. 16:973-978. 4. Hosea, S., E. Brown, C. Hammer, and M. Frank. 1980. Role ofcomplement activation in a model of adult respiratory distress syndrome. J. Clin. Invest. 66:375-382. 5. Till, G. 0., K. J. Johnson, R. Kunkel, and P. A. Ward. 1982. Intravascular activation of complement and acute lung injury: dependency on neutrophils and toxic oxygen metabolites. J. Clin. Invest. 69:1126-1135. 6. Brigham, K. L., R. Bowers, and 1. Haynes. 1979. Increased sheep lung vascular permeability caused by E. coli endotoxin. Ore. Res. 45:292297. 7. Shasby, D. M., K. M. Vanbenthuysen, R. M. Tate, S. S. Shasby, 1. F. McNurty, and J. E. Repine. 1982. Granulocytes mediate acute edematous lung injury in rabbits and isolated rabbit lung perfused with phorbol myristate acetate: role of oxygen radicals. Am. Rev. Respir. Dis. 125: 443-447. 8. Tvedten, H. W., G. O. Till, and P. A. Ward. 1985. Mediators of lung injury in mice following systemic activation of complement. Am. J. Pathol. 119:92-100. 9. Pratt, P. C. 1978. Pathology of the adult respiratory distress syndrome. 1978. In The Lung Structure, Function and Disease. Williams and Wilkins, Baltimore MD. 43-57. 10. Bachofen, M., and E. R. Weibel. 1977. Alterations of the gas exchange apparatus in adult respiratory insufficiency associated with septicemia. Am. Rev. Respir. Dis. 116:589-615. 11. Weiland, J. E., W. B. Davis, J. F. Holter, J. R. Mohammed, P. M. Dorinsky, and J. E. Gadek. 1986. Lung neutrophils in the adult respiratory distress syndrome: clinical and pathologic significance. Am. Rev. Respir. Dis. 133:218-252. 12. Fowler, A. A., T. M. Hyers, B. J. Fisher, D. E. Bechard, R. M. Centor, and R. 0. Webster. 1987. The adult respiratory distress syndrome: cell population and soluble mediators in the air spaces of patients at high risk. Am. Rev. Respir. Dis. 136:1225-1231. 13. Cochrane, C. G., R. Spragg, and S. D. Revak. 1983. Pathogenesis of the adult respiratory distress syndrome. J. Clin. Invest. 71:754-761. 14. Lee, C. T., A. M. Fein, M. Lippman, H. Holtzman, P. Kimbel, and G. Weinbaum. 1981. Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory distress syndrome. N. Engl. J. Med. 304: 192-196. 15. Christner, P., A. Fein, S. Goldberg, M. Lippmann, W. Abrams, and G. Weinbaum. 1985. Collagenase in the lower respiratory tract of patients with adult respiratory distress syndrome. Am. Rev. Respir Dis. 131: 690-695. 16. Hallman, M., R. Spragg, J. H. Harrell, K. M. Mose, and L. Gluck. 1982. Evidence of lung surfactant abnormality in respiratory failure. J. Clin. Invest. 70:673-683. 17. Petty, T. L., G. W. Silvers, and G. W. Paul. 1979. Abnormalities in lung elastic properties and surfactant function in adult respiratory distress syndrome. Chest 75:571-574. 18. Barrett, C. R., A. L. L. Bell, and S. F. Ryan. 1979. Alveolar epithelial injury causing respiratory distress in dogs. Chest 75:705-711. 19. Liau, D. F., C. R. Barrett, A. L. L. Bell, andS. F. Ryan. 1987. Functional abnormalities of lung surfactant in experimental acute alveolar injury in the dog. Am. Rev. Respir. Dis. 135:395-401. 20. Repine, J. E., J. W. Eaton, M. W. Anders, J. R. Hos, andR. B. Fox. 1978. Generation of hydroxyl radical by enzymes, chemicals, and human phagocyte in vitro: detection with the anti-inflammation agent, dimethyl sulfoxide. J. Clin. Invest. 64: 1642-1651. 21. Weiss, S. J., J. Young, A. F. LoBuglio, A. Slivka, andN. F. Nimeh. 1981. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68:714-721. 22. Curnutte, J. T., D. M. Whitten, and B. M. Babior. 1974. Defective superoxide production by granulocyte from patients with chronic granulomatous disease. N. Engl. J. Med. 290:593-597. 23. Dumelin, E. E., and A. A. Tappe!. 1977. Hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides. Lipids 12:894-900. 24. Buege, J. A., and S. D. Aust. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310. 25. Baenziger, N. L., and P. W. Majerus. 1974. Isolation of human platelets and platelet surface membranes. Methods Enzymol. 31:149-155. 26. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Bioi. Chem. 226:497-509. 27. Beveridge, J. M. R., andS. E. Johnson. 1949. The determination of phospholipid phosphorus. Can. J. Res. 27:159-163.
Ryan, Ghassibi, and Liau: Effects of Activated PMN on Surfactant
28. Laemmli, U. K., and M. Favre. 1973. Maturation of the head ofbacteriophage T4.1. DNA packaging events. J. Mol. BioI. 80:575-599. 29. Liau, D. F., C. R. Barrett, A. L. L. Bell, and S. F. Ryan. 1985. Normal surface properties of phosphatidylglycerol deficient surfactant from dogs after acute lung injury. J. Lipid Res. 26:1338-1344. 30. Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods. Iowa State University Press, Ames, IA. 31. Possmayer, F. 1988. A proposed nomenclature for pulmonary surfactant-associated proteins. Am. Rev. Respir. Dis. 138:990-998. 32. Repine, J. E., J. G. White, C. C. Clawson, and B. M. Holmes. 1974. The influence of phorbol myristate acetate on oxygen consumption by polymorphonuclear leukocytes. J. Lab. Clin. Med. 83:911-920. 33. DeChatelet, L. R., P. S. Shirley, and R. B. Johnston. 1976. Effect of phorbol myristate acetate on the oxidative metabolism of human polyrnorphonuclear leukocytes. Blood 47:545-554. 34. O'Flaherty, J. T., S. Cousart, A. S. Lineberger et al. 1980. Phorbol myristate acetate-in vivo effects upon neutrophils, platelets, and lung. Am. J. Pathol. 101:79-92. 35. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka, 1982. Direct activation of calcium activated, phospholipiddependent protein kinase by tumor promoting phorbol esters. J. Biol. Chem. 257:7847-7851. 36. Aust, S. D., and B. C. White. 1985. Iron chelation prevents tissue injury following ischemia. Adv. Free Rad. Biol. I: 1-17. 37. Bird, R. F., S. S. O. Hung, M. Hadley, and H. H. Draper. 1983. Determination of malonaldehyde in biological materials by high-pressure liquid chromatography. Anal. Biochem. 128:240-244. 38. Ryan, S. F., S. A. Hashim, G. Cernansky, C. R. Barrett, A. L. L. Bell, and D. F. Liau. 1980. Quantification of surfactant phospholipids in the dog lung. J. Lipid Res. 21:1004-1014. 39. Petty, T. L., O. K. Reiss, G. W. Paul, G. W. Silvers, and N. D. Elkins.
40. 41. 42. 43. 44.
45.
46. 47. 48.
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1977. Characteristics of pulmonary surfactant in adult respiratory distress syndrome associated with trauma and shock. Am. Rev. Respir. Dis. 115:531-536. Verma, S. P., and D. F. H. Wallach. 1982. Action of ionizing radiation on lipid-protein interaction. Biochem. Biophys. Res. Commun. 105: 105-109. Morley, C., and A. Bangham. 1981. Physical properties of surfactant under compression. Prog. Respir. Res. 15:188-193. King, R. J., and M. C. Macbeth. 1979. Physicochemical properties of dipalmitoylphosphatidylcholine after interaction with an apolipoprotein of pulmonary surfactant. Biochim. Biophys. Acta 557:86-101. Hawgood, S., B. J. Benson, and R. L. Hamilton. 1985. Effects of surfactant-associated protein and calcium ions on the structure and surface activity of lung surfactant lipids. Biochemistry 24:184-190. Hawgood, S., H. Efrati, J. Schilling, and B. Benson. 1985. Chemical characterization of lung surfactant apoproteins: amino acid composition, N-terminal sequence and enzymic digestion. Biochem. Soc. Trans. 13: 1092-1096. Seeger, W., H. Lepper, H. R. D. Wolf, and H. Neuhof. 1985. Alteration of alveolar surfactant function after exposure to oxidative stress and to oxygenated and native arachidonic acid in vitro. Biochim. Biophys. Acta 835:58-67. Marklund, W. W., E. Holme, and L. Heiner. 1982. Superoxide disrnutase in extracellular fluid. Clin. Chim. Acta 126:41-51. McGuire, W. W., R. G. Spragg, A. B. Cohen, and C. G. Cochrane. 1982. Studies on the pathogenesis of the adult respiratory distress syndrome. J. Clin. Invest. 69:543-553. Wewers, M. D., D. J. Herzyk, andJ. E. Gadek. 1988. Alveolar fluid neutrophil elastase activity in the adult respiratory distress syndrome is complexed to alpha-2-macroglobulin. J. Clin. Invest. 82: 1260-1267.
Current evidence suggests that products of activated inflammatory cells cause or contribute to the acute lung injury of the adult respiratory distress syndrome (ARDS). To assess the possibility that these products may impair surfactant function during ARDS, we exposed surfactant in vitro to polymorphonuclear leukocytes (PMN) activated by phorbol myristate acetate and to the oxidant-producing pair ferric chloride/ascorbate (FeCl,/ASC). After incubation of surfactant with 8 to 32 X 106 activated PMN for 1 to 4 h or with FeCl,/ASC for 16 h, its isopycnic density (d), minimum surface tension ('Ymio), time course of adsorption, compressibility (SC), and stability index (SI) were determined. We found progressive decreases of d, adsorption, and SI and progressive increases of 'Ymio and SC after exposure to activated PMN in increasing numbers or for longer time periods. Superoxide dismutase completely inhibited all of these effects except the decreased adsorption, which it did not significantly inhibit. Similar changes in all of these parameters occurred after exposure of surfactant to FeCI,/ASC. Polyacrylamide gel electrophoresis of surfactant after exposure to activated PMN showed a decrease of the major apoprotein that progressed with exposure time and was associated with the appearance of several bands with both lower and higher molecular weights than that of the apoprotein. The data show that activated PMN are capable of impairing surfactant function in vitro and of degrading the major apoprotein. They suggest that the effects upon d, 'Ymin, SC, and SI are mediated largely if not exclusively by oxidant radicals. While oxidants may contribute to delayed adsorption, proteolysis appears to play the principal role in this effect.
The pathogenesis ofthe acute lung injury in the adult respiratory distress syndrome (ARDS) remains unclear, but accumulating evidence, both experimental and clinical, suggests that toxic products of activated inflammatory cells, including oxygen radicals and proteases, playa major role. Polymorphonuclear leukocytes (PMN) activated by any one of several proinflammatory signals become increasingly adherent to endothelial cells and to other PMN and produce a large variety of products, including toxic oxygen radicals, proteases, and hypochlorous acid. These toxic products are used to kill phagocytized microorganisms, but they are also (Received in original form January 31, 1990 and in revised form June 6, 1990) Address correspondence to: Stephen F. Ryan, M.D., Pulmonary Research Group, Department of Pathology & Pulmonary Division, Department of Medicine, St. Luke's-Roosevelt Hospital Center, Amsterdam Avenue at 114th Street, New York, NY 10025. Abbreviations: adult respiratory distress syndrome, ARDS; ascorbate, ASC; dimethyl sulfoxide, DMSO; ferric chloride, FeCh; phosphatebuffered saline, PBS; malondialdehyde, MDA; phospholipid, PL; phorbol myristate acetate, PMA; polymorphonuclear leukocyte, PMN; sodium dodecyl sulfate-polyacrylamide minislab gel electrophoresis, SDS-PAGE; superoxide dismutase, SOD; thiobarbituric acid, TBA. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 33-41, 1991
released into the extracellular milieu, where they may cause tissue injury by initiating peroxidation and proteolysis (1-3). Experimentally, intravascular activation of PMN is followed by their sequestration in the lung and by injury to its capillary endothelial cells, which can be mitigated by antioxidants or by PMN depletion (4-8). Increased numbers of marginated or aggregated PMN have been found in the pulmonary capillaries of patients with ARDS by several investigators (9, 10), and others have reported marked increases in numbers of PMN recovered by bronchoalveolar lavage from these patients (11, 12). A recent study has presented evidence that oxidants are generated in the lungs of patients with ARDS by demonstrating that a fraction of the - 40
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Figure 4. Time course of surface adsorption of surfactant exposed
Figure 5. Representative surface tension versus area diagrams of
to FeCh/ascorbate (ASC). Samples, containing 2 mg PL, were exposed to FeCh/ASC at 37° C for 16 h. Control 1, unexposed; control 2, mixed with FeCh/ASC immediately before measurement; control 3, incubated alone at 37° C for 16 h. FeCI3/ASC are significantly lower than controls (n = 4 for all groups; P < 0.001).
surfactant exposed to FeCl3/ascorbate (ASC). Each sample, after measurement of surfaceadsorption (Figure 4), wascompressedand expanded for 15 cycles. Diagrams shown were recorded at fifth cycle (broken line) and fifteenthcycle (unbroken line) of compression and expansion of the surface.
Ryan, Ghassibi, and Liau: Effects of Activated PMN on Surfactant
because the numbers of PMN that could be practically isolated required quantities of surfactant that were too small to assay by this method. We attempted to use ratios of activated PMN to quantities of surfactant that would be plausible in the alveoli of patients during ARDS. For this calculation, we used the lavage data of Fowler and coworkers (12) to estimate the total number of alveolar PMN during ARDS and our own quantification of total alveolar surfactant in the normal dog lung (38). Our figure, based on these data, of 1.77 X 106 PMN/mg surfactant PL probably underestimates the number of PMN to which the surfactant is exposed during ARDS because of incomplete recovery of cells by alveolar lavageand because lavage recovers alveolar PMN at one time point, whereas, during ARDS, they are probably continually recruited to the alveoli, exhausted, and lysed over a period of many hours. We therefore used 4, 8, 12, and 16 X 106 PMN/mg surfactant PL with relatively short exposure times of I to 4 h. We also examined the effects of strong oxidants upon the surfactant by exposing it to the oxidant pair FeCliASC. In this system, the surfactant was exposed only to oxidants rather than to the large number of products released by activated PMN. Comparisons of the densities and surface properties of surfactant after exposure to oxidants alone with those after exposure to activated PMN allow some preliminary proposals concerning possible causes of surfactant abnormalities induced by activated PMN. The data show that exposure of surfactant to activated PMN under these conditions caused a decrease in its isopycnic density and abnormality in each of the surface properties studied. Furthermore, there was a progressive decrease in density and a progressive increase in degree of abnormality in each of the parameters of surface function as the product of numbers of PMN (xl()6) and hours of exposure (nPMN X h) increased. The decrease in isopycnic density was maximized at 1.045 from a normal of 1.051 after npMN X h of 32 (Table 1). The decrease was completely inhibited by addition of SOD to the reaction mixture, and exposure to FeCb/ASC caused a similar decrease in density. Thus, oxidative alteration of surfactant apoproteins or lipids appears to be the major cause of decreased density of surfactant after exposure to activated PMN. A similar but greater decrease in density was found by Petty and coworkers in the major surface active lipoprotein fraction in alveolar lavage fluid from a patient with posttraumatic ARDS (39). The lipid/protein ratio of the abnormallow-density fraction was greater than that of the normal fraction, and it was speculated that the former was a degradation product of the latter. Our data support this hypothesis. The progressive decrease in density of the surfactant in vitro precludes the alternative possibilities that the low-density fraction is a newly synthesized product of injured or regenerating type II cells or that it results from contamination of surfactant by plasma lipids. The progressive increase of ')Imin developed by the surfactant after exposure to increasing npMN X h was also completely prevented by SOD, and exposure to FeCb/ASC caused increases in ')Imin similar to those caused by maximum npMN X h. These observations again suggest that oxidative processes are at least mostly responsible for increased ')Imin after exposure to activated PMN. Delayed
39
adsorption cannot explain this increase because when equal quantities of normal and of PMN-exposed surfactant were sprayed on the surface in the balance, ')Im;n was increased only in the PMN-exposed surfactant and the degree of increase was similar to that recorded after placing the surfactant suspension in the trough and allowing adsorption to occur prior to determination of ')Im;n as described above. After exposure to lower n pMN X h, there was a significant decrease in ')Im;n between the fifth and the fifteenth compression cycles, which was not seen after exposure to higher npMN X h. This failure to reduce ')Im;n with repeated compression after greater npMN X h may have resulted from greater oxidation of polyunsaturated fatty acids of unsaturated phosphatidylcholine species or of other lipids containing polyunsaturated fatty acids resulting in an alteration in the liquid-gel transition temperature (40). Surface film adsorption kinetics and reentry and respreadability of the surfactant lipids may thus have been affected, resulting in impaired refinement of the stable phospholipids on the surface believed to occur during cyclic compression and expansion (41). SC and Sf are widely accepted measures of the rigidity and stability of the surfactant film in the Wilhelmy balance. Their abnormalities after exposure to activated PMN, which trend with ')Im'n and adsorption, provide further evidence of profound functional impairment of the surfactant. The apoproteins of the surfactant are believed to be largely responsible for its rapid surface adsorption (42, 43), and the marked reduction of adsorption of surfactant after exposure to activated PMN suggests that the apoprotein has been altered. Because SOD failed to significantly reduce the impairment of adsorption, degradation of the apoprotein by PMN proteases appears to be the principal cause of its impaired adsorption. Support for this suggestion is provided by the findings on SDS-PAGE. After exposure of surfactant to 16 X 1()6 PMN for 2 h, the quantity of SP-A was clearly decreased and this decrease was not inhibited by SOD (Figure 3; lanes C and D). After exposure to 32 X 1()6 PMN for 1 h, several bands with mol wt lower than those of SP-A appeared (Figure 3, lane E), and after exposure for 2 and 3 h, the quantities of SP-Aand of the low-molecular-weight bands progressively decreased (Figure 3, lanes F and G). Exposure of the canine apoprotein complex to bacterial collagenase has recently been shown to generate several bands with mol wt similar to these (44). In addition to the bands with mol wt lower than those of SP-A, several bands with higher mol wt also appeared and progressively decreased with exposure time. The progressive decrease in quantity of SP-A with exposure time suggests that at least some of the low-molecular-weight bands were degradation products of SP-A, which in their own turn were further degraded, but it is possible that some or all of them were fragments of degraded proteins from PMN. The bands with mol wt higher than SP-A may be aggregates of degradation products of SP-Aor of contaminating fragments derived from PMN. Further study will be necessary to clarify this. If fragments of PMN-derived proteins did contaminate the filtered surfactant, it seems very unlikely that they impaired surface function of the surfactant because SDS-PAGE of surfactant proteins after separation by continuous sucrose
40
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
density gradient centrifugation demonstrated that all of the minor bands have been removed and yet surface function remained abnormal. The alterations of in vitro surface function after exposure to FeCb/ASC were more severe than those reported by Seeger and associates after exposure of surfactant to a variety of strong oxidants including FeCb/ASC (45). One possible explanation for this discrepancy is that we used partially purified surfactant, whereas Seeger and associates used crude alveolar lavage fluid for their experiments. Such material does not reduce I'm'n to below about 30 dynes/em and therefore is not useful for studying altered I'min under abnormal conditions. The changes reported by Seeger and associates in other parameters of surfactant function after exposure to oxidants, for example, reduction of hysteresis area and delayed reduction of surface tension during compression, are similar to those described here after exposure to FeCI3/ASC. Although our data suggest that delayed surface adsorption induced by activated PMN is more likely due to proteolysis than to oxidative processes, the results with FeCI 3/ASC demonstrate that oxidants are capable of profoundly impairing adsorption. It thus remains possible that PMN may impair adsorption by oxidative mechanisms if present in sufficient numbers in the lung. While our data show that products of activated PMN are capable of altering surfactant function in vitro, the relevance of these findings to such effects in vivo is speculative. SOD activity has been reported in extracellular fluid (46), and antiproteases are abundant in the alveoli of some patients with ARDS (47, 48). These inhibitors may reduce or prevent adverse effects of PMN upon surfactant in vivo. Alterations of the physical and chemical properties of the surfactant during ARDS have not been analyzed sufficiently to permit detailed comparisons with the changes induced in vitro by activated PMN, but the changes in surface properties so far described are very similar to those found in our study (16, 17). On the other hand, this mechanism cannot explain the reported decrease in surfactant disaturated phosphatidylcholine during ARDS (16), and it seems probable that if alteration by PMN does occur, it is only one of multiple processes that may impair surfactant synthesis or function during ARDS. Nevertheless, it seems possible that the surfactant may be damaged by products of activated inflammatory cells during ARDS, and this possibility deserves further investigation. If inflammatory cells cause abnormality of surfactant function as suggested by our observations, then it will be important to learn not only the potential magnitude of the injury but also the molecular mechanisms involved. If surfactant replacement in patients with ARDS is ever to be employed successfully, these mechanisms must be understood. If oxidative or proteolytic damage is indeed important, then instilled surfactant may become rapidly altered or degraded and be of no benefit. It seems possible that, in that case, therapeutic strategies such as addition of antioxidants or antiproteases to the instilled surfactant could be developed. Acknowledgments: The writers gratefully acknowledge the excellent technical assistance of Giselle Cemansky, Jin Huang, and Martin Torni and thank Dr. A. L. Loomis Bell for his encouragement and for his critical review of this manuscript. This work was supported in part by a grant from the Stoney Wold Foundation of New York. Parts of this study were presented at the 55th Annual Scientific Assembly, XVI World Congress on Disease of the Chest (Boston, MA, October 30, 1989).
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