American Journal ofPathology, Vol. 136, No. 6, June 1990 Copyright © American Association ofPathologists
Phospholipase A2-induced Pathophysiologic Changes in the Guinea Pig Lung Stephen K. Durham* and William M. Seligt From the Departments of Toxicology and Patholog)* and Pharmacology and ChemotherapytJ Hoffmann-La Roche,
Nutley, NewJersey
The pathophysiology of lung injury induced by phospholipase A2 (PLA2), a lipolytic enzyme implicated in a variety of pulmonary diseases, was examined in the guinea pig. One hundred Iul ofsaline or 10 units ofPLA2 suspended in saline was given as a bolus injection into either the trachea orjugular vein. Intratracheal pressure and mean arterial blood pressure were continuously monitored. The lungs were examined by light and transmission electron microscopy at 1, 10, and 30 minutes after administration. Pulmonary morphologic and physiologic changes were only observed in animals that received PLA2 via the trachea. Significant increases in peak intratrachealpressure occurred as early as 1 minute after intratracheal PLA2 administration. Morphologic evidence of airway constriction, accompanied by blebbing of the apical cytoplasm of airway epithelium, was also observed at this time. A transient increase in mean arterial blood pressure occurred 5 minutes after challenge. At 10 minutes after intratracheal PLA2, there was marked swelling of airway epithelial cells, pronounced blebbing of the apical cytoplasm, and a resultant decrease in size of the airway lumen. Morphologic changes in alveolar cell populations were initially observed 10 minutes after intratracheal PLA2. Interalveolar septa were hypercellular and multifocally thickened. There was prominent perivascular edema and alveolar spaces contained abundant proteinaceous material and occasional hemorrhage. Ultrastructurally, there was marked cell swelling and fragmentation of type I alveolar epithelium resulting in a denuded basal lamina. Sequestration of neutrophils and eosinophils, many of which lacked secretory granules, within alveolar capillaries was accompanied by aggregates of platelets and was observed in close proximity to injured endothelium. Morphologic changes indicative of cell injury were also observed in type II alve-
olar epithelium. Similar, but morefrequent and severe, morphologic injury occurred 30 minutes after intratracheal PLA2. It is concluded that PLA2 induces pronounced morphologic and physiologic changes in the guinea pig and that the route of administration is important in the development of PLA2-induced lung injury. (Am JPathol 1990, 136: 1283-1291)
Phospholipase A2 (PLA2) is a lipolytic enzyme that is ubiquitous in biologic systems and has been implicated in the pathogenesis of a variety of pulmonary diseases, including asthma and adult respiratory distress syndrome (ARDS).'2 PLA2 is responsible for hydrolysis of the 2-acyl position of glycerophospholipids, resulting in the generation of equimolar amounts of two classes of potent biologically active lipids, lysophospholipids, such as lysophosphatidylcholine (lysoPC), and free fatty acids, such as arachidonic acid.34 Two distinct forms of PLA2 exist in biologic systems. Membrane-bound PLA2 is normally associated with the plasmalemma and organelle membranes, whereas soluble PLA2 is normally present in lysosomes, and perhaps in cytosol.2 These products resulting from PLA2-induced hydrolysis may contribute to inflammatory conditions by serving as amphiphiles and/or by regulating transformation of potent proinflammatory lipid mediators. Amphiphiles are molecules having both hydrophilic and hydrophobic properties and are capable of inducing deleterious effects on cells resulting in cytolysis either by inserting into or perturbing components of the cell membrane, or by extracting membrane lipids.5 LysoPC is the principal lysophospholipid present in the lung and has also been identified as an intermediate in one pathway of surfactant synthesis.6'7 Because of their potent cytolytic properties, the tissue concentrations of lysophospholipids must be highly regulated.7 PLA2-induced lipolysis also appears to function in a regulatory role in eicosanoid and platelet-activating factor formation in a wide variety of cell populations Accepted for publication January 11, 1990. Address reprint requests to Dr. Stephen K. Durham, Department of Toxicology and Pathology, Hoffmann-La Roche, 340 Kingsland Ave., Bldg. 100/3, Nutley, NJ 07110-1199.
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having proinflammatory activities in the lung, including leukocytes, alveolar macrophages, and platelets.3'4 The precise mechanism(s) responsible for PLA2-induced pulmonary pathophysiologic changes associated with respiratory diseases remain to be elucidated. Few studies dedicated to PLA2-induced morphologic change have been performed in experimental animals. Limited morphologic evidence in both rabbit and canine experimental models suggest that PLA2-induced pulmonary pathophysiologic changes involve polymorphonuclear leukocyte infiltration with the development of tissue necrosis and edema formation.8'9 The objectives of this study were (1) to describe the temporal sequence of PLA2-induced pulmonary morphologic changes in anesthetized, mechanically ventilated guinea pigs, and (2) to determine the effects of PLA2 on intratracheal pressure and mean arterial blood pressure.
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One Minute After Inoculation Morphologic evidence of injury were observed in airway epithelium of anesthetized, mechanically ventilated guinea pigs as early as 1 minute after receiving intratracheal PLA2. The airway epithelium were markedly elongated perpendicular to the basement membrane and toward the center of the lumen (Figure 2b). There were many prominent blebs on the apical cytoplasmic surface
Figure 4. Transmission electron micrograph of bronchial epithelium 10 minuzites after initratracheal PLA2. Ciliated cells are markedly suollen and hate increased cytoplasmic lucency accompanied bh high volumetric swelling of mitochondria, dilated to vesiculated endoplasmic reticulum, and marked apical cytoplasmic blebbing (lead citrate and uranYl acetate, X 4300).
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quently accompanied denuded basal laminas in animals receiving intratracheal PLA2 (Figure 6). Several neutrophils lacked secretory granules and had prominent, undulating cytoplasmic processes (Figure 7). Alterations in the endothelium 10 minutes after intratracheal PLA2 included focal cell swelling, undulating cytoplasmic processes, and large numbers of pinocytotic vesicles. Thirty Minutes After Inoculation: Airways and Parenchyma
Figure 5. LPM of guineia pig alveolar parenchyma 10 minutes after intratracheal PLA2. There is pronounced perivascular edema characterized by separation of perivascular coyInnective tissue by large amounts ofproteiinaceous material accompanied by dilated lymphatics. 7here are manly neutrophils presentt in1 the vascularspace. The interstitium is h percellular and mnultifocally, thicketned. Alveolar spaces contain variable amounts ofproteinaceous material (Toluidine blue, X300).
Morphologic evidence of marked airway constriction and prominent perivascular and alveolar edema observed at previous time periods were still present at 30 minutes in animals receiving intratracheal PLA2. The alveolar spaces contained many sloughed and swollen alveolar epithelial cells, variable numbers of erythrocytes, and abundant fibrogranular material (Figure 8). By electron microscopy, widespread alveolar edema accompanied by fragmentation and vesiculation of type alveolar epithelium resulting in denuded basal lamina was observed (Figure 9). Morphologic changes indicative of cell injury, including dilatation of endoplasmic reticulum and detachment from the basal lamina, also occurred in type 11 alveolar epithelium 30 minutes after intratracheal PLA2 challenge. Many platelets, several of which have lost their discoid contour and
sometimes lacked secretory granules and contained large electron lucent cytoplasmic vacuoles.
Ten Minutes After Inoculation: Parenchyma Initial changes in alveolar parenchymal morphology were observed by light microscopy at 10 minutes after intratracheal PLA2 challenge. There was pronounced perivascular edema characterized by separation of perivascular connective tissue by large amounts of proteinaceous material accompanied by dilated lymphatics (Figure 5). Many neutrophils were present in the vascular space. The interstitium was hypercellular and multifocally thickened, and alveolar spaces contained variable amounts of proteinaceous material (Figure 5). By electron microscopy, the type alveolar epithelial cell injury was the parenchymal cell population having the most pronounced morphologic change after intratracheal PLA2. The spectrum of alterations in type alveolar epithelial cells was diverse, ranging from dilatation of the endoplasmic reticulum and perinuclear envelope to marked cell swelling and vesiculation. Microsequestration of neutrophils in capillaries and small-caliber vessels was widespread and fre-
Figure 6. Transmission electron micrograph of interalveolar septum 10 minutes after intratracheal PLA2. Many neutrophils (N) are present in the vascular space. The type I alveolar epithelial cell is swollen andfragmented (arrow) (lead citrate and uranyl acetate, X5950).
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branes or indirectly by lipid proinflammatory mediators produced by PLA2, such as lysophospholipids, arachidonic acid, and platelet-activating factor (PAF). Morphologic changes suggestive of a direct surfaceactive phenomenon resulting in altered membrane permeability and membranolysis, as anticipated with amphiphilic agents such as the lysophospholipids, were observed in airway epithelia immediately after the administration of intratracheal PLA2. Morphologic changes observed in airway epithelia indicative of altered membrane permeability included marked blebbing of the apical cytoplasm, cell swelling, increased electron-lucency of the cytoplasm, and dilated, electron-lucent cisternae of the endoplasmic reticulum. The later changes most likely represent an ingress of water. However, these morphologic alterations are not pathognomonic for amphiphilic activity. Acetylation of lysophospholipid at the 2-hydroxy group results in another potent lipid mediator, PAF.16 Many of the morphologic changes observed in the present study can be ascribed to PAF-induced effects. In a previous study, the intratracheal instillation into rabbits of 1 -0-octa-
decyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) Figure 7. Transmission electron micrograph of interalteolar septumn 10 minutes after intratracheal PLA2. A neutrophil (N), lacking secretorjy granules and having prominent, unldlullati7g cytoplasmic processes is present within the capillary space. There is loss of the alveolar epitheliumn resulting in a denluded basal lamina (arrowheads). Fibrin aggregates are present in the alveolarspace (lead citrate ancd uran?l acetate, X 11, 750).
or native PAF was shown to induce a dose-dependent acute pulmonary inflammation characterized by the accumulation of macrophages in the alveolar space, degener-
secretory granules, were observed in close proximity to capillary endothelial cells having increased cytoplasmic lucency, dilated endoplasmic reticulum, and swollen mitochondria (Figure 10). Additional changes observed in endothelium at this time included cell swelling, fragmentation, and separation and loss of endothelial cell cytoplasmic processes from the underlying basal lamina.
Discussion The results of this study indicate that a single intratracheal instillation of PLA2 into the anesthetized, mechanically ventilated guinea pig causes airway constriction and cellular injury initially to the airway epithelium and subsequently to the alveolar epithelium. Endothelial and epithelial injury were associated with neutrophil and platelet aggregation in alveolar capillaries and perivascular and alveolar edema. A wide variety of pathophysiologic reactions occur after PLA2 enzyme activation, either via a direct action or through subsequent transformation of its products into several potent biologically active substances. Thus the changes described in this study may be mediated by the direct effects of PLA2 on cell mem-
Figure 8. Light photomicrograph ofguinea pig alteolar parenchvma 230 minutes after intratracheal PLA2. The alveolar spaces conitain many sloughed swollen alveolar epithelial cells (arrow), occasional erythrocytes, and abundant amounts of fibrogranular material (*) (Toluidine blue, X600).
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injury was negligible in the isolated, perfused guinea pig lung, whereas severe endothelial cell injury and marked pulmonary edema occurred in vivo. Widespread neutrophil microsequestration and degranulation, accompanied by platelet aggregation, was frequently observed in close proximity to injured endothelium in vivo. These findings help formulate the hypothesis that cell populations normally present within the vasculature and recruited into the lung after intratracheal PLA2 challenge play an important role in the development of endothelial cell injury and pronounced pulmonary edema. Those findings in this study, which are similar to those described in the isolated, perfused guinea pig lung after intratracheal PLA222 include the initial changes in airway epithelium. In addition, in both the in vivo and in the isolated, perfused guinea pig lung airway epithelium and type alveolar epithelium were the cell populations most severely affected after intratracheal PLA2. Two predominant mechanisms may be responsible for the absence of PLA2-induced pulmonary pathophysiologic effects following intravascular injection as opposed to airway instillation. The first possibility is the difference in the response of cell populations exposed by the various
Figure 9. Tranismission electront micrograph of interalveolar septum 30 minutes after intratracheal PLA2 There is marked swellinig andfragmentation oj'a tjpe I alveolar epithelial cell (I) resulting int a deniuded basal laminia (arrowheads). Alveolar spaces conitain abunidanit proteinaceous material andfbrin aggregates (lead citrate and uranyl acetate, X 10, 020).
ative and necrotic changes of the alveolar epithelium, and accumulation of polymorphonuclear leukocytes and platelets in the alveolar capillary lumens with degenerative changes of endothelium.17 In guinea pigs, PAF has been recognized as an inducer or contributor of various promnflammatory reactions, including chemotaxis, platelet aggregation, and increased vascular permeability. 1819 Platelet-activating factor has also been implicated in airway hyperreactivity and PAF produc4s a platelet-dependent bronchoconstriction in guinea pigSY202' All the previously described phenomena occurred in the present study. Preliminary investigations that evaluated the effects of various enzyme inhibitors and receptor antagonists on PLA2induced physiologic changes in the guinea pig documented that the PAF antagonist, WEB 2086, partially reduced PLA2-induced increases in mean arterial blood pressure and intratracheal pressure (unpublished obser-
vations). In addition to the obvious differences in the degree of participation of cell populations normally present within the vasculature and recruited into the lung after injury, the predominant difference observed between the isolated, perfused lung and in vivo after intratracheal PLA2 was the severity and extent of endothelial cell injury and subsequent development of pulmonary edema. Endothelial cell
Figure 10. Transmission electron micrograph of interalveolar septum of a guinea pig 30 minutes after intratracheal PLA2. Many platelets (P) are present in the vascular lumen. Several platelets have lost their discoid contour and secretory granules. An inijured endothelial cell has disrupted and swollen cytoplasmic processes with increased cYtoplasmic lucency, dilated endoplasmic reticulum, and swollen mitochondria (arrow). Compare damaged mitochondria (arrow) with unremarkable mitochondria (arrowhead) of adjacent endothelial cell (lead citrate and urantl acetate, X8300).
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routes of administration. Intravascular injection of PLA2 would most likely have its initial effect primarily on the endothelium, whereas intratracheal PLA2 would have initial access to the surfactant-containing monolayer and cell populations lining and in close proximity to airways, including airway epithelium, smooth muscle, and resident proinflammatory cell populations. Previous studies performed in perfused rabbit lungs demonstrated that the intravascular infusion of PLA2 (100 units/minute for 15 minutes) had no effect on pulmonary hemodynamics and did not result in morphologic or physiologic evidence of lung injury.23 These investigators also documented elevations of 6-keto-PGF, the stable metabolite of prostacyclin, and proposed that prostacyclin played a major role in the failure of the development of lung injury after the infusion of PLA2 via the vascular system.23 Prostacyclin is widely recognized as a potent antiinflammatory and vasodilator agent derived primarily from the endothelial cell.24 The second possibility for route-dependent effects may be differences in bioavailability or bioactivity of PLA2 or its metabolic products following administration. The most plausible explanation is the presence of substances within the plasma that would abrogate PLA2-induced effects. Other investigators have documented that albumin present in plasma abrogates or diminishes phospholipase A2-induced activity.25 Other substances within plasma, as yet unidentified, may also have a similar effect. The possibility of any diluting effect by the plasma in abrogating or diminishing phospholipase A2-induced activity after intravascular administration is unlikely because a bolus injection of 100 times (1000 units) the amount of PLA2 used in this study failed to result in changes in arterial pressure (unpublished observations). Another plausible explanation may be the length of contact between PLA2 and endothelium. PLA2 may be rapidly dispersed by the circulation, whereas longer contact would be anticipated after intratracheal administration. The present study documented that intratracheal PLA2 induced transient increases in mean arterial blood pressure and persistent increases in intratracheal pressure in the guinea pig, whereas intravascular PLA2 had no effect on the monitored variables. Morphologic evidence of vasoconstriction was not observed in the current study, and most likely resides in the fact that the sampling times for histopathologic evaluation did not coincide with the time frame in which there were transient increases in mean arterial blood pressure.
References 1. Vadas P, Pruzanski W: Role of extracellular phospholipase A2 in inflammation. Advances in Inflammation Research
1984, 7:51-59
2. Vadas P, Pruzanski W: Role of secretory phospholipase A2 in the pathobiology of disease. Lab Invest 1986, 55:391399 3. Chang J, Musser JH, McGregor H: Phospholipase A2: Function and pharmacologic regulation. Biochem Pharmacol 1987,36:2429-2436 4. O'Flaherty JT: Phospholipid metabolism and stimulus-response coupling. Biochem Pharmacol 1987,36:407-412 5. Helenius A, Simon K: Solubilization of membranes by detergents. Biochem Biophys Acta 1975,415:29-79 6. Morgan TE, Finley TN, Fialkow H: Comparison of the composition and surface activity of "alveolar" and whole lung lipids in the dog. Biochem Biophys Acta 1965,106:403-413 7. van Golde LMG: Metabolism of phospholipids in the lung. Am Rev Respir Dis 1976,114:977-1000 8. Shaw JO, Roberts MF, Ulevitch RJ, Henson P, Dennis EA: Phospholipase A2 contamination of cobra venom factor preparations. Biologic role in complement-dependent in vivo reactions and inactivation with p-bromophenacyl bromide. Am J Pathol 1978, 91:517-530 9. Morgan AP, Jenny ME, Haessler H: Phospholipids, acute pancreatitis, and the lungs: Effect of lecithinase infusion on pulmonary surface activity in dogs. Ann Surg 1968, 167: 329-335 10. Zar JH: Biostatistical Analysis. Englewood Cliffs, NJ, Prentice Hall, 1984, pp 162-205 11. Alkondon M, Ray A, Sen P: Nonstereoselective aspects of propranolol pharmacodynamics. Can J Physiol Pharmacol 1986, 64:1455-1462 12. Barnes PJ: Endogenous catecholamines and asthma. J Allergy Clin Immunol 1986, 77:791 -795 13. Karnovosky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 1965, 27:137A-138A 14. Kay JM: Pulmonary vasculature and nerves. Comparative morphologic features of the pulmonary vasculature in mammals. Am Rev Respir Dis 1983,128:S53-S57 15. McLaughlin FR: Bronchial artery distribution in various mammals and in humans. Am Rev Respir Dis 1983,128:S57-S58 16. Barnes DM, Chung KF, Page CP: Platelet-activating factor as a mediator of allergic disease. J Allergy Clin Immunol
1988, 81:919-934 17. Camussi G, Pawlowski I, Tetta C, Roffinello C, Alberton M, Brentjens J, Andres G: Acute lung inflammation induced in the rabbit by local instillation of 1 -0-octa-decyl-2-acetyl-snglyceryl-3-phosphorylcholine or of native platelet-activating factor. Am J Pathol 1983,112:78-88 18. Dewar A, Archer CB, Paul W, Page CP, MacDonald DM, Morley J: Cutaneous and pulmonary histopathologic responses to platelet-activating factor (Paf-acether) in the guinea pig. Journal of Pathology 1984, 44:25-34 19. Evans TW, Chung K, Rodgers DF, Barnes PJ: Effect of platelet-activating factor on airway vascular permeability: Possible mechanisms. J Appl Physiol 1987, 63:479-484 20. Vargaftig BB, Lefort J, Chignard M, Benveniste J: Plateletactivating factor induces a platelet-dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives. Eur J Pharmacol 1980, 65:185-192
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21. Barnes PJ, Grandordy BM, Page CP, Rhoden KJ, Robertson DN: The effect of platelet-activating factor on pulmonary beta-adrenceptors. Br J Pharmacol 1987, 90:709-715 22. Durham SK, Selig WM: Phospholipase A2-induced pathophysiologic changes in the isolated, perfused guinea pig lung. Exp Lung Res (In press) 23. Littner MR, Kazmi GM, Lott FD: Effects of A23187, exogenous phospholipase A2 and exogenous arachidonic acid on pulmonary vascular resistance in isolated rabbit lung. J Pharmacol Exp Therap 1987, 242:974-980 24. Gryglewski RJ, Botting RM, Vane JR: Mediators produced by the endothelial cell. Hypertension 1988, 12:530-548
25. Luzzio AJ: Inhibitory properties of serum proteins on the enzymatic sequence leading to lysis of red blood cells by snake venom. Toxicon 1967, 5:97-103
Acknowledgments The authors thank Joel E. Tocker and Thomas Valentine for their excellent technical assistance provided throughout this study, and Dr. Timothy Anderson and Janet Becker for manuscript review and suggestions.
Phospholipase A2-induced Pathophysiologic Changes in the Guinea Pig Lung Stephen K. Durham* and William M. Seligt From the Departments of Toxicology and Patholog)* and Pharmacology and ChemotherapytJ Hoffmann-La Roche,
Nutley, NewJersey
The pathophysiology of lung injury induced by phospholipase A2 (PLA2), a lipolytic enzyme implicated in a variety of pulmonary diseases, was examined in the guinea pig. One hundred Iul ofsaline or 10 units ofPLA2 suspended in saline was given as a bolus injection into either the trachea orjugular vein. Intratracheal pressure and mean arterial blood pressure were continuously monitored. The lungs were examined by light and transmission electron microscopy at 1, 10, and 30 minutes after administration. Pulmonary morphologic and physiologic changes were only observed in animals that received PLA2 via the trachea. Significant increases in peak intratrachealpressure occurred as early as 1 minute after intratracheal PLA2 administration. Morphologic evidence of airway constriction, accompanied by blebbing of the apical cytoplasm of airway epithelium, was also observed at this time. A transient increase in mean arterial blood pressure occurred 5 minutes after challenge. At 10 minutes after intratracheal PLA2, there was marked swelling of airway epithelial cells, pronounced blebbing of the apical cytoplasm, and a resultant decrease in size of the airway lumen. Morphologic changes in alveolar cell populations were initially observed 10 minutes after intratracheal PLA2. Interalveolar septa were hypercellular and multifocally thickened. There was prominent perivascular edema and alveolar spaces contained abundant proteinaceous material and occasional hemorrhage. Ultrastructurally, there was marked cell swelling and fragmentation of type I alveolar epithelium resulting in a denuded basal lamina. Sequestration of neutrophils and eosinophils, many of which lacked secretory granules, within alveolar capillaries was accompanied by aggregates of platelets and was observed in close proximity to injured endothelium. Morphologic changes indicative of cell injury were also observed in type II alve-
olar epithelium. Similar, but morefrequent and severe, morphologic injury occurred 30 minutes after intratracheal PLA2. It is concluded that PLA2 induces pronounced morphologic and physiologic changes in the guinea pig and that the route of administration is important in the development of PLA2-induced lung injury. (Am JPathol 1990, 136: 1283-1291)
Phospholipase A2 (PLA2) is a lipolytic enzyme that is ubiquitous in biologic systems and has been implicated in the pathogenesis of a variety of pulmonary diseases, including asthma and adult respiratory distress syndrome (ARDS).'2 PLA2 is responsible for hydrolysis of the 2-acyl position of glycerophospholipids, resulting in the generation of equimolar amounts of two classes of potent biologically active lipids, lysophospholipids, such as lysophosphatidylcholine (lysoPC), and free fatty acids, such as arachidonic acid.34 Two distinct forms of PLA2 exist in biologic systems. Membrane-bound PLA2 is normally associated with the plasmalemma and organelle membranes, whereas soluble PLA2 is normally present in lysosomes, and perhaps in cytosol.2 These products resulting from PLA2-induced hydrolysis may contribute to inflammatory conditions by serving as amphiphiles and/or by regulating transformation of potent proinflammatory lipid mediators. Amphiphiles are molecules having both hydrophilic and hydrophobic properties and are capable of inducing deleterious effects on cells resulting in cytolysis either by inserting into or perturbing components of the cell membrane, or by extracting membrane lipids.5 LysoPC is the principal lysophospholipid present in the lung and has also been identified as an intermediate in one pathway of surfactant synthesis.6'7 Because of their potent cytolytic properties, the tissue concentrations of lysophospholipids must be highly regulated.7 PLA2-induced lipolysis also appears to function in a regulatory role in eicosanoid and platelet-activating factor formation in a wide variety of cell populations Accepted for publication January 11, 1990. Address reprint requests to Dr. Stephen K. Durham, Department of Toxicology and Pathology, Hoffmann-La Roche, 340 Kingsland Ave., Bldg. 100/3, Nutley, NJ 07110-1199.
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having proinflammatory activities in the lung, including leukocytes, alveolar macrophages, and platelets.3'4 The precise mechanism(s) responsible for PLA2-induced pulmonary pathophysiologic changes associated with respiratory diseases remain to be elucidated. Few studies dedicated to PLA2-induced morphologic change have been performed in experimental animals. Limited morphologic evidence in both rabbit and canine experimental models suggest that PLA2-induced pulmonary pathophysiologic changes involve polymorphonuclear leukocyte infiltration with the development of tissue necrosis and edema formation.8'9 The objectives of this study were (1) to describe the temporal sequence of PLA2-induced pulmonary morphologic changes in anesthetized, mechanically ventilated guinea pigs, and (2) to determine the effects of PLA2 on intratracheal pressure and mean arterial blood pressure.
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One Minute After Inoculation Morphologic evidence of injury were observed in airway epithelium of anesthetized, mechanically ventilated guinea pigs as early as 1 minute after receiving intratracheal PLA2. The airway epithelium were markedly elongated perpendicular to the basement membrane and toward the center of the lumen (Figure 2b). There were many prominent blebs on the apical cytoplasmic surface
Figure 4. Transmission electron micrograph of bronchial epithelium 10 minuzites after initratracheal PLA2. Ciliated cells are markedly suollen and hate increased cytoplasmic lucency accompanied bh high volumetric swelling of mitochondria, dilated to vesiculated endoplasmic reticulum, and marked apical cytoplasmic blebbing (lead citrate and uranYl acetate, X 4300).
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quently accompanied denuded basal laminas in animals receiving intratracheal PLA2 (Figure 6). Several neutrophils lacked secretory granules and had prominent, undulating cytoplasmic processes (Figure 7). Alterations in the endothelium 10 minutes after intratracheal PLA2 included focal cell swelling, undulating cytoplasmic processes, and large numbers of pinocytotic vesicles. Thirty Minutes After Inoculation: Airways and Parenchyma
Figure 5. LPM of guineia pig alveolar parenchyma 10 minutes after intratracheal PLA2. There is pronounced perivascular edema characterized by separation of perivascular coyInnective tissue by large amounts ofproteiinaceous material accompanied by dilated lymphatics. 7here are manly neutrophils presentt in1 the vascularspace. The interstitium is h percellular and mnultifocally, thicketned. Alveolar spaces contain variable amounts ofproteinaceous material (Toluidine blue, X300).
Morphologic evidence of marked airway constriction and prominent perivascular and alveolar edema observed at previous time periods were still present at 30 minutes in animals receiving intratracheal PLA2. The alveolar spaces contained many sloughed and swollen alveolar epithelial cells, variable numbers of erythrocytes, and abundant fibrogranular material (Figure 8). By electron microscopy, widespread alveolar edema accompanied by fragmentation and vesiculation of type alveolar epithelium resulting in denuded basal lamina was observed (Figure 9). Morphologic changes indicative of cell injury, including dilatation of endoplasmic reticulum and detachment from the basal lamina, also occurred in type 11 alveolar epithelium 30 minutes after intratracheal PLA2 challenge. Many platelets, several of which have lost their discoid contour and
sometimes lacked secretory granules and contained large electron lucent cytoplasmic vacuoles.
Ten Minutes After Inoculation: Parenchyma Initial changes in alveolar parenchymal morphology were observed by light microscopy at 10 minutes after intratracheal PLA2 challenge. There was pronounced perivascular edema characterized by separation of perivascular connective tissue by large amounts of proteinaceous material accompanied by dilated lymphatics (Figure 5). Many neutrophils were present in the vascular space. The interstitium was hypercellular and multifocally thickened, and alveolar spaces contained variable amounts of proteinaceous material (Figure 5). By electron microscopy, the type alveolar epithelial cell injury was the parenchymal cell population having the most pronounced morphologic change after intratracheal PLA2. The spectrum of alterations in type alveolar epithelial cells was diverse, ranging from dilatation of the endoplasmic reticulum and perinuclear envelope to marked cell swelling and vesiculation. Microsequestration of neutrophils in capillaries and small-caliber vessels was widespread and fre-
Figure 6. Transmission electron micrograph of interalveolar septum 10 minutes after intratracheal PLA2. Many neutrophils (N) are present in the vascular space. The type I alveolar epithelial cell is swollen andfragmented (arrow) (lead citrate and uranyl acetate, X5950).
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branes or indirectly by lipid proinflammatory mediators produced by PLA2, such as lysophospholipids, arachidonic acid, and platelet-activating factor (PAF). Morphologic changes suggestive of a direct surfaceactive phenomenon resulting in altered membrane permeability and membranolysis, as anticipated with amphiphilic agents such as the lysophospholipids, were observed in airway epithelia immediately after the administration of intratracheal PLA2. Morphologic changes observed in airway epithelia indicative of altered membrane permeability included marked blebbing of the apical cytoplasm, cell swelling, increased electron-lucency of the cytoplasm, and dilated, electron-lucent cisternae of the endoplasmic reticulum. The later changes most likely represent an ingress of water. However, these morphologic alterations are not pathognomonic for amphiphilic activity. Acetylation of lysophospholipid at the 2-hydroxy group results in another potent lipid mediator, PAF.16 Many of the morphologic changes observed in the present study can be ascribed to PAF-induced effects. In a previous study, the intratracheal instillation into rabbits of 1 -0-octa-
decyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) Figure 7. Transmission electron micrograph of interalteolar septumn 10 minutes after intratracheal PLA2. A neutrophil (N), lacking secretorjy granules and having prominent, unldlullati7g cytoplasmic processes is present within the capillary space. There is loss of the alveolar epitheliumn resulting in a denluded basal lamina (arrowheads). Fibrin aggregates are present in the alveolarspace (lead citrate ancd uran?l acetate, X 11, 750).
or native PAF was shown to induce a dose-dependent acute pulmonary inflammation characterized by the accumulation of macrophages in the alveolar space, degener-
secretory granules, were observed in close proximity to capillary endothelial cells having increased cytoplasmic lucency, dilated endoplasmic reticulum, and swollen mitochondria (Figure 10). Additional changes observed in endothelium at this time included cell swelling, fragmentation, and separation and loss of endothelial cell cytoplasmic processes from the underlying basal lamina.
Discussion The results of this study indicate that a single intratracheal instillation of PLA2 into the anesthetized, mechanically ventilated guinea pig causes airway constriction and cellular injury initially to the airway epithelium and subsequently to the alveolar epithelium. Endothelial and epithelial injury were associated with neutrophil and platelet aggregation in alveolar capillaries and perivascular and alveolar edema. A wide variety of pathophysiologic reactions occur after PLA2 enzyme activation, either via a direct action or through subsequent transformation of its products into several potent biologically active substances. Thus the changes described in this study may be mediated by the direct effects of PLA2 on cell mem-
Figure 8. Light photomicrograph ofguinea pig alteolar parenchvma 230 minutes after intratracheal PLA2. The alveolar spaces conitain many sloughed swollen alveolar epithelial cells (arrow), occasional erythrocytes, and abundant amounts of fibrogranular material (*) (Toluidine blue, X600).
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injury was negligible in the isolated, perfused guinea pig lung, whereas severe endothelial cell injury and marked pulmonary edema occurred in vivo. Widespread neutrophil microsequestration and degranulation, accompanied by platelet aggregation, was frequently observed in close proximity to injured endothelium in vivo. These findings help formulate the hypothesis that cell populations normally present within the vasculature and recruited into the lung after intratracheal PLA2 challenge play an important role in the development of endothelial cell injury and pronounced pulmonary edema. Those findings in this study, which are similar to those described in the isolated, perfused guinea pig lung after intratracheal PLA222 include the initial changes in airway epithelium. In addition, in both the in vivo and in the isolated, perfused guinea pig lung airway epithelium and type alveolar epithelium were the cell populations most severely affected after intratracheal PLA2. Two predominant mechanisms may be responsible for the absence of PLA2-induced pulmonary pathophysiologic effects following intravascular injection as opposed to airway instillation. The first possibility is the difference in the response of cell populations exposed by the various
Figure 9. Tranismission electront micrograph of interalveolar septum 30 minutes after intratracheal PLA2 There is marked swellinig andfragmentation oj'a tjpe I alveolar epithelial cell (I) resulting int a deniuded basal laminia (arrowheads). Alveolar spaces conitain abunidanit proteinaceous material andfbrin aggregates (lead citrate and uranyl acetate, X 10, 020).
ative and necrotic changes of the alveolar epithelium, and accumulation of polymorphonuclear leukocytes and platelets in the alveolar capillary lumens with degenerative changes of endothelium.17 In guinea pigs, PAF has been recognized as an inducer or contributor of various promnflammatory reactions, including chemotaxis, platelet aggregation, and increased vascular permeability. 1819 Platelet-activating factor has also been implicated in airway hyperreactivity and PAF produc4s a platelet-dependent bronchoconstriction in guinea pigSY202' All the previously described phenomena occurred in the present study. Preliminary investigations that evaluated the effects of various enzyme inhibitors and receptor antagonists on PLA2induced physiologic changes in the guinea pig documented that the PAF antagonist, WEB 2086, partially reduced PLA2-induced increases in mean arterial blood pressure and intratracheal pressure (unpublished obser-
vations). In addition to the obvious differences in the degree of participation of cell populations normally present within the vasculature and recruited into the lung after injury, the predominant difference observed between the isolated, perfused lung and in vivo after intratracheal PLA2 was the severity and extent of endothelial cell injury and subsequent development of pulmonary edema. Endothelial cell
Figure 10. Transmission electron micrograph of interalveolar septum of a guinea pig 30 minutes after intratracheal PLA2. Many platelets (P) are present in the vascular lumen. Several platelets have lost their discoid contour and secretory granules. An inijured endothelial cell has disrupted and swollen cytoplasmic processes with increased cYtoplasmic lucency, dilated endoplasmic reticulum, and swollen mitochondria (arrow). Compare damaged mitochondria (arrow) with unremarkable mitochondria (arrowhead) of adjacent endothelial cell (lead citrate and urantl acetate, X8300).
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routes of administration. Intravascular injection of PLA2 would most likely have its initial effect primarily on the endothelium, whereas intratracheal PLA2 would have initial access to the surfactant-containing monolayer and cell populations lining and in close proximity to airways, including airway epithelium, smooth muscle, and resident proinflammatory cell populations. Previous studies performed in perfused rabbit lungs demonstrated that the intravascular infusion of PLA2 (100 units/minute for 15 minutes) had no effect on pulmonary hemodynamics and did not result in morphologic or physiologic evidence of lung injury.23 These investigators also documented elevations of 6-keto-PGF, the stable metabolite of prostacyclin, and proposed that prostacyclin played a major role in the failure of the development of lung injury after the infusion of PLA2 via the vascular system.23 Prostacyclin is widely recognized as a potent antiinflammatory and vasodilator agent derived primarily from the endothelial cell.24 The second possibility for route-dependent effects may be differences in bioavailability or bioactivity of PLA2 or its metabolic products following administration. The most plausible explanation is the presence of substances within the plasma that would abrogate PLA2-induced effects. Other investigators have documented that albumin present in plasma abrogates or diminishes phospholipase A2-induced activity.25 Other substances within plasma, as yet unidentified, may also have a similar effect. The possibility of any diluting effect by the plasma in abrogating or diminishing phospholipase A2-induced activity after intravascular administration is unlikely because a bolus injection of 100 times (1000 units) the amount of PLA2 used in this study failed to result in changes in arterial pressure (unpublished observations). Another plausible explanation may be the length of contact between PLA2 and endothelium. PLA2 may be rapidly dispersed by the circulation, whereas longer contact would be anticipated after intratracheal administration. The present study documented that intratracheal PLA2 induced transient increases in mean arterial blood pressure and persistent increases in intratracheal pressure in the guinea pig, whereas intravascular PLA2 had no effect on the monitored variables. Morphologic evidence of vasoconstriction was not observed in the current study, and most likely resides in the fact that the sampling times for histopathologic evaluation did not coincide with the time frame in which there were transient increases in mean arterial blood pressure.
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Acknowledgments The authors thank Joel E. Tocker and Thomas Valentine for their excellent technical assistance provided throughout this study, and Dr. Timothy Anderson and Janet Becker for manuscript review and suggestions.
