MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ACTIVE OXYGEN METABOLITES AND THROMBOXANE IN PHORBOL MYRISTATE ACETATE TOXICITY TO THE ISOLATED, PERFUSED RAT LUNG BY Laurie J. Carpenter A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1988 ABSTRACT Active Oxygen Metabolites and Thromboxane in Phorbol Myristate Acetate Toxicity to the Isolated, Perfused Rat Lung by Laurie J. Carpenter When administered intravenously or intratracheally to rats, rabbits and sheep, phorbol myristate acetate (PMA) produces changes in lung morphology and function which are similar to those seen in humans with the adult respiratory distress syndrome (ARDS). Therefore, it is thought that information about the mechanism of ARDS development can be gained from experiments using PMA- treated animals. Currently, the mechanisms by which PMA causes pneumotoxicity are unknown. Results from other studies in rabbits and in isolated, perfused rabbit lungs suggest that PMA-induced lung injury is mediated by active oxygen species from neutrophils (PMN), whereas studies in sheep and rats suggest that PMN are not required for the toxic response. The role of PMN, active oxygen metabolites and thromboxane (TxAZ) in PMA-induced injury to isolated, perfused rat lungs (PLs) was examined in this thesis. To determine whether PMN were required for PMA to produce toxicity to the IPL, lungs were perfused for 30 min with buffer containing various concentra- tions of PMA (in the presence or absence of PMN). When concentrations _>_57 ng/ml were added to medium devoid of added PMN, perfusion pressure and lung weight increased. When a concentration of PMA (14-28 ng/ml) that did not by itself cause lungs to accumulate fluid was added to the perfusion medium Laurie J. Carpenter containing PMN (1x108), perfusion pressure increased, and lungs accumulated fluid. These results indicate that high concentrations of PMA produce lung injury which is independent of PMN, whereas injury induced by lower concentrations is PMN-dependent. To examine whether active oxygen species were involved in mediating lung injury induced by PMA and PMN, lungs were ceperfused with the oxygen radical scavengers SOD and/or catalase. Coperfusion with either or both of these enzymes totally protected lungs against injury caused by PMN and PMA. These results suggest that active oxygen species (the hydroxyl radical in particular), mediate lung injury in this model. To determine whether TxA2 was involved in toxicity induced by PMN and PMA, lungs were coperfused with the cyclooxygenase inhibitor, indomethacin or the thromboxane synthase inhibitor, Dazmegrel. Experiments were also per- formed using lungs and/or PMN that had been pretreated with aspirin. These drug treatments had little effect, if any, on the pressure increase; however, they protected lungs against edema development. These results suggest that TxAz may participate in the pathogenesis of edema by some other mechanism than by increasing vascular pressure. In conclusion, results from studies perform ed in this thesis suggest that both active oxygen species and thromboxane are involved in toxicity to the isolated rat lung induced by PMA and PMN. How both of these interact to produce lung injury is a question which remains to be answered. To Richard and Gloria, who taught me the meaning of hard work and perseverance ACKNOWLEDGEMENTS I am extremely grateful to my advisor, Dr. Robert A. Roth, for his ideas, encouragement, patience and friendship. I am equally as grateful to my husband, Jim Deyo, for providing emotional support. I also thank Lonnie Dahm, Jim Hewett, Susan White, Jim Wagner, Cindy Hoorn, Leon Bruner, Patti Ganey and Terry Ball for making B346 Life Sciences a fun place to work. Without the people mentioned in this paragraph, obtaining my degree would have been a lot tougher than it was. During the past few years I have received wonderful technical support from Eric Johnson, Traci Banjanin and Eric Shobe. Their willingness to perform radioimmunoassays of massive size is to be commended. I also thank Dr. Kent Johnson and Ms. Robin Kunkel for performing services which could not be performed in 8346 Life Sciences. Special thanks are due to Diane Hummel, who has masterfully typed everything I have written in the past five years (including this thesis). I also thank Drs. Theodore Brody, James Bennett and N. Edward Robinson for serving on my thesis committee. Each one of these individuals has contributed to this research by aiding in the interpretation of the results. Finally, I would like to thank those who have provided financial support for myself and my research. My stipend and travel allowance came from NRSA Training Grants GM07392 and HL07404, NIEHS grant E804139 and NHLBI grant I-IL32244. Funds for my research came from NHLBI grant HL32244. iii LIST OF TABLES LIST OF FIGURES INTRODUCTION I. PMA A. B. C. TABLE OF CONTENTS General Mechanism of action of PMA Effects of PMA on inflammatory cells 1. 4. Neutrophils a. NeutrOphil function b. Effect of PMA on PMN 1) Introduction 2) Release of active oxygen species 3) Release of lysosomal enzymes 4) Release of arachidonic acid metabo- lites 5) Other mediators released by PMA- stimulated PMN Macrophages a. Macrophage ftmction b. Effect of PMA on macrophages Platelets a. Platelet ftmction b. Effect of PMA on platelets Mast cells a. Mast cell function h. Effect of PMA on mast cells Toxic effects of mediators released from PMA-stimu- lated inflammatory cells 1. 2. Active oxygen metabolites Lysosomal enzymes iv Page xii xiv D-J \O\O~O 0010101 11 12 13 13 13 14 14 15 16 16 16 16 16 17 TABLE OF CONTENTS (continued) 3. Arachidonic acid metabolites a. Cyclooxygenase metabolites b. Lipoxygenase metabolites 4. Histamine 5. 5-Hydroxytryptamine (Serotonin) 6. Platelet activating factor (PAF) E. E vivo toxicity of PMA 1. Skin toxicity a. Skin irritation and inflammation b. Tumor promotion 2. Glomerular injury 3. Pleuritis 4. Pneumotoxicity a. Effects on pulmonary hemodynamics b. Alterations in lung fluid balance c. Development of edema (1. Increase in epithelial and endothelial cell permeability e. Morphologic changes f. Changes in endothelial cell fimction g. Changes in respiratory mechanics F. Summary II. Adult Respiratory Distress Syndrome A. General B. Pathologic changes C. Therapy D. Mechanism of ARDS development 1. Involvement of PMN a. Sequestration of PMN in lungs of ARDS patients b. Involvement of complement in PMN seque- stration in lungs of ARDS patients c. Occurrence of neutrophil-derived mediators 'm BAL fluid of ARDS patients 2. Evidence against involvement of PMN in ARDS development 3. Other potential mechanisms of ARDS development a. Abnormalities in surfactant b. Involvement of platelets 4. Animal models of ARDS 34 34 36 37 38 38 39 40 TABLE OF CONTENTS (continued) 111. The Phorbol Myristate Acetate Model of ARDS A. Similarities between PMA pneumotoxicity and ARDS B. Potential mechanisms of PMA-induced pulmonary injury 1. Involvement of PMN a. Studies i_n_ vivo b. Studies in isolated, perfused lungs 1) Use of the IPL preparation 2) Role of the PMN in PMA toxicity to the IPL 2. Involvement of active oxygen metabolites in PMA pneumotoxicity 3. Role of cyclooxygenase metabolites in PMA pneumotoxicity 4. The role of lysosomal enzymes in PMA pneumo- toxicity 5. Other potential mediators of toxicity derived from PMN 6. Role of other cells in PMA pneumotoxicity C. Summary D. Specific Aims METHODS I. Animals II. Preparation of PMN III. Preparation of PMA IV. The Isolated Perfused Limg A. B. C. Surgery Apparatus and conditions of perfusion General protocol for experiments V. Evaluation of Cytotoxicity to the IPL A. B. C. LW/BW ratio (relative lung weight) Change in perfusion pressure Measurement of albumin in BAL fluid, edema fluid and perfusion medium 1. Lavage procedure 2. Preparation of samples for analysis 3. Measurement of albumin in fluids from lungs Determination of LDH activity Morphology Disposition of serotonin (SHT) perfused through the vasculature vi 45 47 50 51 52 53 53 54 56 56 56 59 59 59 61 61 63 63 63 64 64 64 64 64 65 66 TABLE OF CONTENTS (continued) Production of Prostanoids by Isolated Lungs A. B. Samples Analysis of TxB and 6-Keto-PGF a by RIA 2 1 Effect of PMA on the IPL A. B. C. D. PMA dose/response Effect of oxygen radical scavengers on toxicity medi- ated by PMA Involvement of thromboxane in toxicity mediated by PMA 1. Prostanoid production 2. Effect of indomethacin on PMA pneumotoxicity 3. Effect of a specific thromboxane synthetase inhi- bitor Involvement of vasoconstriction in edema induced by PMA Effect of PMN on PMA Toxicity to the IPL A. B. C. Preliminary experiments Perfusions with PMN and a concentration of PMA which is not toxic in the absence of PMN Involvement of active oxygen species in edema induced by perfusion with PMN and PMA 1. Release of O - _into the perfusion medium/ confirmation otZO scavenging by SOD 2. Effect of SOD andzcatalase on pneumotoxicity 3. Effect of SOD on pneumotoxicity 4. Effect of catalase on pneumotoxicity Involvement of vasoconstriction in edema induced by perfusion with PMN and PMA 1. Perfusion with papaverine 2. Effect of papaverine on PMN function Involvement of thromboxane in edema induced by per- fusion with PMN and PMA 1. Production of prostanoids in lungs perfused with PMN and PMA 2. Effect of indomethacin 3. Effect of a thromboxane synthetase inhibitor (Dazmegrel) vii Page 67 67 67 69 69 69 70 70 7O 71 71 72 72 72 73 73 74 74 74 75 75 75 75 75 75 76 TABLE OF CONTENTS (continued) RESULTS 4. Experiments to determine if indomethacin or Dazmegrel scavenge active oxygen species Source of TxB and 6-keto-PGF in lungs perfused with PMN and IZMA l. Involvement of oxygen radical production in pro- stanoid synthesis 2. Release of prostanoids from PMA-stimulated PMN 1o 3. Effect of aspirin pretreatment of lungs or PMN on pneumotoxicity a. Rationale for experiment b. Preparation of drugs and chemicals c. Preliminary experiments in isolated lungs 1) Confirmation of ASA washout in lungs 2) Confirmation of cyclooxygenase inhi- bition in lungs (1. Preliminary experiments with PMN 1) Confirmation of ASA washout in PMN 2) Confirmation of cyclooxygenase inhi- bition in PMN 3) Effect of ASA which remained in lung vasculature after washout on prosta- noid production by PMN 4) Effect of ASA pretreatment on PMN function e. Experimental protocol Analysis of Data Miscellaneous A. B. Sources of chemicals Preparation of buffers Effect of PMA on the IPL A. B. C. Dose/Response of PMA in the IPL Histopathology of ltmgs perfused with a high concentra- tion of PMA Lack of effect of SOD and catalase on PMA-mediated toxicity to the IPL viii 76 77 77 78 78 78 78 79 79 8O 8O 80 80 81 81 81 84 85 85 88 89 89 89 94 TABLE OF CONTENTS (continued) E. Involvement of thromboxane in toxicity mediated by a high concentration of PMA 1. Prostanoid production 2. Relationship between prostanoid production and the relative lung weight or the increase in perfu- sion pressure 3. Effect of indomethacin on pneumotoxicity a. Indices of lung injury b. Confirmation of inhibition of cyclooxygen- ase 4. Effect of Dazmegrel on pneumotoxicity a. Indices of lung injury b. Confirmation of inhibition of thromboxane synthase Involvement of vasoconstriction in edema induced by PMA 11. Effect of PMN on PMA Toxicity to the IPL A. B. .0 Preliminary experiments Toxicity produced by perfusion with PMN and PMA or the respective controls 1. Effect on relative lung weight 2. Effect on perfusion pressure 3. Effect of perfusion with PMN and PMA on SHT removal and metabolism Histopathology of lungs perfused with PMN and PMA Involvement of active oxygen species in edema induced by perfusion with PMN and PMA 1. Generation of O - by PMA-stimulated PMN 2. Effect of SOD and catalase on pneumotoxicity 3. Effect of SOD on pneumotoxicity 4. Effect of catalase on pneumotoxicity Involvement of vasoconstriction in edema induced by perfusion with PMA and PMN 1. Effect of papaverine on pneumotoxicity 2. Effect of papaverine on PMN function ix Page 98 98 98 103 103 105 105 105 106 108 108 108 111 111 111 117 117 124 124 130 130 130 133 133 133 TABLE OF CONTENTS (continued) Involvement of thromboxane in edema induced by per- fusion with PMN and PMA 1. 2. 3. 4. 5. Prostanoid production Relationship between prostanoid production and relative lung weight or increase in perfusion pres- sure Effect of indomethacin on pneumotoxicity a. Markers of lung injury b. Confirmation of inhibition of cyclooxygen- ase Effect of Dazmegrel on pneumotoxicity a. Markers of lung injury b. Confirmation of inhibition of thromboxane synthase Effect of indomethacin and Dazmegrel on PMN function Source of TxB and 6-keto-PGF1G in lungs perfused with PMN and IZMA 1. Effect of SOD on synthesis of TxB and 6-keto- PGF1 2 Effecgt of catalase on synthesis of Tsz and 6- keto-PGF Prostanoidcproduction from PMA-stimulated PMN Experiments with aspirin pretreatment of lungs or PMN a. Preliminary experiments in isolated lungs 1) Confirmation of ASA washout from lungs 2) Confirmation of cyclooxygenase inhi- bition in lungs b. Preliminary experiments with PMN 1) Confirmation of ASA washout from PMN 2) Confirmation of cyclooxygenase inhi- bition in PMN 3) Effect of ASA which remained in the lung vasculature after washout on pro- stanoid production by PMN 4) Effect of ASA pretreatment on PMN function 137 137 137 140 140 143 143 143 148 148 155 155 155 159 159 159 159 164 164 164 166 166 166 TABLE OF CONTENTS (continued) DISCUSSION c. Effect of aspirin pretreatment of lungs or PMN on PMA pneumotoxicity d. Effect of aspirin pretreatment of lungs and/or PMN on prostanoid production 1) TxB synthesis 2) 6-Keto-PGF1a synthesis I. Ability of PMA to Produce PMN-Independent or -Dependent Lung Injury II. Mechanism of PMN-Independent Injury A. B. C. Lack of effect of SOD and catalase Involvement of a cyclooxygenase metabolite(s) Effect of papaverine on PMA pneumotoxicity III. Mechanism of Neutrophil-Dependent Lung Injury A. m 00 E. Involvement of active oxygen species Involvement of vasoconstriction Involvement of cyclooxygenase metabolites Experiments to determine the source of TxAz and PGI2 1. Role of active oxygen species in prostanoid pro- duction 2. Studies with PMN perfused through the apparatus 3. Studies with aspirin Mechanism of action of thromboxane SUMMARY AND CONCLUSIONS BIBLIOGRAPHY xi 170 175 175 180 184 184 188 188 189 192 193 193 196 197 201 201 204 205 209 212 214 Table 10 11 12 13 14 15 LIST OF TABLES Effect of PMA on various cell types Mechanisms of formation of active oxygen species Advantages and disadvantages of isolated lung preparations Changes seen in isolated lungs perfused with PMA and PMN Crossreactivities of antibodies used in radioimmunoassays Effect of SOD and catalase on toxicity induced by PMA in the absence of PMN Effect of indomethacin on injury to the IPL mediated by PMA in the absence of PMN Effect of Dazmegrel on injury to the IPL mediated by PMA in the absence of PMN Effect of papaverine on toxicity induced by PMA in the absence of PMN Effect of PMN on PMA pneumotoxicity Effect of SOD and catalase on toxicity produced by perfu- sion with PMN and PMA Effect of SOD on pneumotoxicity induced by PMN and PMA Effect of catalase on lung injury induced by PMN and PMA Effect of papaverine on toxicity induced by perfusion with PMN and PMA Effect of indomethacin on toxicity induced by perfusion with PMA and PMN xii 44 46 87 97 104 107 109 110 131 132 134 135 144 LIST OF TABLES (Continued) Table 16 17 18 19 Effect of Dazmegrel on injury to the IPL mediated by PMA and PMN Effect of Dazmegrel on H202 Confirmation of irreversible inhibition of prostanoid synthe- sis by pretreatment of PMN with ASA Effect of ASA ptreatment of lungs or PMN on the response of the IPL to PMA or PMA vehicle xiii 147 156 168 174 10 11 12 13 14 15 16 LIST OF FIGURES Structure of PMA Inositol phospholipid turnover and signal transduction Pathways of arachidonic acid metabolism Products released by PMN stimulated with PMA Procedure for isolation of PMN from the rat peritoneum Surgical procedure for lung isolation Diagram of isolated, perfused lung apparatus Protocol for aspirin experiment Effect of PMA concentration on relative weight of the IPL Effect of PMA concentration on inflow pressure of the IPL Ultrastructural findings in lungs perfused with a toxic concentration of PMA in the absence of PMN Effect of a toxic concentration of PMA in the absence of PMN on prostanoid production in the IPL Correlations between prostanoid production and indices of injury in lungs perfused with a toxic concentration of PMA in the absence of PMN - Effect of perfusion with PMN and PMA on relative weight of the IPL Effect of perfusion with PMN and PMA on inflow pressure of the IPL Representative inflow pressure recordings of ltmgs perfused with PMN and PMA or the respective vehicles xiv 10 42 57 60 62 82 95 99 101 112 114 116 LIST OF FIGURES (Continued) Figge 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Effect of PMA and PMN on SHT removal and metabolism by the IPL Electron micrograph from a lung perfused with vehicles for PMN and PMA Electron micrograph from a lung perfused with PMN and PMA vehicle Electron micrograph from a lung perfused with PMA and PMN vehicle Electron micrograph from a lung perfused with PMA and PMN Production of Oz- by PMA-stimulated PMN perfused through the IPL apparatus Effect of papaverine on 02 production by PMA-stimulated PMN Effect of perfusion with PMN and PMA on prostanoid production Correlations between prostanoid production and indices of injury in lungs perfused with PMA and PMN Effect of indomethacin on prostanoid production by lungs perfused with PMA and PMN Effect of Dazmegrel on prostanoid production by lungs perfused with PMA and PMN Effect of indomethacin and Dazmegrel on 02- production by isolated PMN Effect of Dazmegrel on production of 02- by lungs perfused with PMN and PMA Effect of SOD on prostanoid production by lungs perfused with PMN and PMA 120 122 125 127 129 136 138 141 145 149 151 153 157 LIST OF FIGURES (Continued) FigEe 31 32 33 34 35 36 37 38 39 40 Effect of catalase on prostanoid production by lungs per- fused with PMN and PMA Effect of PMA on prostanoid production by isolated PMN Confirmation of inhibition of thromboxane synthesis in lungs of ASA-treated rats Confirmation of inhibition of thromboxane synthesis by ASA-pretreated PMN Effect of ASA remaining in the lungs on thromboxane production by PMN Effect of ASA pretreatment of PMN on 02.- production Effect of ASA pretreatment of lungs or PMN on toxicity induced by perfusion with PMN and PMA Effect of ASA pretreatment of lungs and/or PMN on prosta- noid production by lungs co-perfused with PMA Effect of ASA pretreatment of lungs or PMN on TxB production by lungs co-perfused with PMA or PMA vehicle Effect of ASA pretreatment of lungs or PMN on 6-keto- PGF production by lungs co-perfused with PMA or PMA vehiéi’é 160 162 165 167 169 171 172 176 178 181 AA ACE ALM AMP ARDS ASA ATI BAL BSA DMSO 5HT FMLP HBSS H202 HETE HPETE IPL i.t. i.v. KRBSA LIST OF ABBREVIATIONS arachidonic acid angiotensin-converting enzyme alveolar macrophages adenine monophosphate adult respiratory distress syndrome aspirin angiotensin I bronchoalveolar lavage bovine serum albumin dimethylsulfoxide S-hydroxytryptamine formyl-methionyl-leucyl-phenylalanine Hank's balanced salt solution hydrogen peroxide hydroxyeicosatetraenoic acid hydroperoxyeicosatetraenoic acid isolated, perfused lung intratracheal intravenous Krebs-Ringer Bicarbonate Buffer with 4% bovine serum albumin lactate dehydrogenase luteinizing hormone leukotriene norepinephrine hydroxyl radical singlet oxygen superoxide anion pulmonary artery platelet activating factor PAP PBS PG PGIz PKC PMA PMN PVR RBC RIA 6-keto-PGF SN SOD TRH TxAz, BZ V 1a pulmonary arterial pressure phosphate-buffered saline prostaglandin prostacyclin protein kinase C phorbol myristate acetate neutrophils pulmonary vascular resistance red blood cells radioimmunoassay 6-keto-prostaglandin F1 on neutrophil supernatant superoxide dismutase thyrotropin releasing hormone thromboxane A2, BZ aspirin vehicle xviii INTRODUCTION I. EMA A. General Phorbol myristate acetate (PMA), or 12-O-tetradecanoyl-phorbol—13- acetate, is one of the toxic phorbol esters found in the oil from the seeds of the croton tiglium L plant which is indigenous to India and Ceylon. Its structure is shown in Figure 1. In pure form or even as a component of the seed oil in which it occurs naturally, PMA can induce a number of biochemical changes in a variety of different cells and tissues. As shown in Table l, the effects of PMA on cells can be divided into three major categories. These are (1) regulation of cell growth and differentia- tion, 2) modulation of hormone release, and 3) activation of inflammatory cells. Because information about the effects of PMA on hormone secretion or cell growth and differentiation is not relevant to this thesis, these properties will not be discussed. For more information about these topics, the reader should refer to the references listed in the table. B. Mechanism of Action of PMA Because PMA is lipOphilic, it was first suggested that its biological activity was due to its ability to interact nonspecifically with cell membranes (Van Duuren, 1969). However, more recent studies suggest that PMA exerts its effects by binding to a receptor and activating a specific Ca2+- and phospholipid- dependent enzyme known as protein kinase C (Ashendel, 1985; Castagna et 11., Figure 1. Structure of phorbol myristate acetate (PMA) TABLE 1 Effect of PMA on Various Cells and Biological Systems Reference Eggplation of Cell Growth] Differentiation lymphocyte mitogenesis HL-60 cell differentiation mouse skin tumor promotion mitogenesis of 3T3 cells differentiation of mouse keratino- cytes reduced binding of EGF Effects on the Endocrine System release of LH from pituitary cells inhibition of prolactin release release of human placental lactogen release of aldosterone secretion of insulin secretion of TRH Activation of Inflammatory Cells PMN aggregation and adherence superoxide release from PMN, macrophages (ALM) and eosinophils release of lysosomal enzymes from PMN and ALM eicosanoid production from PMN, ALM and mast cells release of histamine from PMN and mast cells Touraine _e_t_ a_l., 1977; Abb §_t_a_l., 1979 Rovera 9; fly 1979 Van Duuren, 1969 Dicker and Rozengurt, 1978 Yaspa, 1984 Lee and Weinstein, 1978 Smith and Vale, 1981; Smith and Conn, 1984 Harman 3t_31., 1986a Harman SEQ-v 1986b Kojima 33., 1983 Zawalich e_t_§., 1983 Martin and Kowalchyk, 1984 O'Flaherty 3t__a_l., 1980; Harlan 3t 31., 1985a; Diener §§., 1985 Repine g 33., 1974; Hoidal 93 31., 1978; Petreccia g g” 1987 Estensen 3331., 1974; Bonney 3113., 1980 Brune e_t_§., 1978; Ward 3t_ 31., 1985; Heiman and Crews, 1985b Schleimer 93 3., 1980; Heiman and Crews, 1985a,b TABLE 1 (continued) Reference platelet aggregation and release of serotonin release of platelet activating factor from PMN Other Effects of PMA inhibition of cytotoxic T cell function stimulation of fatty acid synthe- sis by hepatocytes inhibition of action of alpha adrenergic agonists contraction of smooth muscle Zucker 3t fl” 1974; Kaibuchi £31., 1983 Betz and Henson, 1980 Orosz £31., 1983 Vaartjes and deHaas, 1985 Corvera _e__t_ 31., 1986 Rasmussen e_t_ 33., 1984 1982). When activated, this enzyme catalyzes the phosphorylation of specific proteins, which in turn, initiate cell functions such as secretion and proliferation. Protein kinase C is widely distributed in tissues and organs of many species of vertebrates, including lower species of insects (Ashendel, 1985). Structurally, the receptor consists of a single polypeptide chain (MW 77,000) that comprises both a hydrOphilic and a hydrophobic domain. It is thought that the hydrophilic domain is the catalytically active portion of the enzyme and that the hydrophobic end binds to membranes (Nishizuka, 1984). In 1973, Rohrschneider and Boutwell hypothesized that phorbol esters acted by simulating the biological activity of an endogenous substance. This substance was identified as diacylglycerol by Takai and coworkers in 1979. Under normal conditions, very little diacylglycerol is present in membranes. However, when stimulated by certain agonists, i.e. hormones, cells produce diacylglycerol from inositol phospholipids according to the scheme in Figure 2. Like PMA, diacylglycerols activate protein kinase C by increasing the affinity of the enzyme for Ca2+ (Kishimoto 3t 31., 1980). However, unlike PMA, diacylglycerols are rapidly metabolized (Bell, 1986). Diacylglycerols are also much less potent than PMA in activating protein kinase C (Bell, 1986). C. Effects of PMA on Inflammatory Cells l. Neutrthils a. Neutr0phil ftmction Polymorphonuclear leukocytes, or neutrophils (PMN), are phagocytic cells which defend the host against infection. PMN are formed in the bone marrow and circulate in the bloodstream. They are attracted to a site of inflammation or bacterial invasion by chemotactic factors formed by the interac- tion of antigens or pathogens with serum proteins (Gallin and Quie, 1978; Becker and Ward, 1980). When exposed to a chemotactic gradient, PMN undergo .0 mmmcfix :fimuoum .Uxm noumnmmonmocoa onwcwuao .mzo «Houoohama>omfin oumcmmonmfip wcflcfluxo .oa.moo .mumzmmocmufluu mcficfiuao .meo «Baum Oacwumnmmonm rocusu Uflmflaocmmonm aouamocH .N musmwm 3:033. 1530—31—5 Alllli :o:o~:50<<+~ou NL?.:»:H If (v x23; : . _2_ 9; m” a a as. v :2; S. (easel!) .b mpu _m «0 asuk m, -. .54. .:< ma< .=< 8: . l. 8 «5.3.5.2.. 2.3%... Ill-ll... h <53 Ecou< alterations in shape, become morphologically oriented toward the gradient, adhere to vascular endothelial cells, and migrate through the vascular endothelium into tissues which contain the activating insult (Boxer 3]; 31., 1985). When PMN encounter foreign particles, they engulf them and internalize them within a phagosome. Lysosomes then fuse with the phagosome, and potent enzymes from specific or azurophilic granules are dis- charged. Enzymes which are released from specific granules include lysozyme, lactoferrin and collagenase, and from azurophilic granules include myeloperoxi- dase, lysozyme, elastase, cationic proteins and cathepsins. Some of those enzymes, such as lysozyme, lactoferrin, myeloperoxidase and cationic proteins, are bactericidal, whereas others function to modulate the inflammatory response (Arnold g a_l., 1982; Spitznagel, 1984; Spitznagel and Shafer, 1985; Falloon and Gallin, 1986). Upon recognition of a phagocytic stimulus, PMN also experience a "respiratory burst" which is characterized by an increase in oxygen consumption and the generation of active oxygen species such as the superoxide anion (02-), singlet oxygen ('02), hydrogen peroxide (H202) and the hydroxyl radical (OI-1‘). These species are formed according to the reactions in Table 2. The superoxide anion is formed by the reduction of molecular oxygen by a membrane-bound NADPH oxidase (Patriarca g 31., 1971; McPhail 3t_ 31., 1976; Nakamura _e_£ Q” 1981). H202 is formed by the spontaneous dismutation of 02- (Root and Metcalf, 1977), and it has been hypothesized that OH“ is formed by the interaction of H202 and Oz- with iron via a modified Haber-Weiss, or Fenton, reaction (Fenton, 1894; Haber and Weiss, 1934; McCord and Day, 1978). Singlet oxygen is formed when one of the electrons in Oz undergoes spin inversion and orbital transition (Wasserman and Murray, 1979). Generation of these active Oxygen species appears to be obligatory for bacterial killing, for PMN which TABLE 2 Proposed mechanisms of formation of oxygen-derived radicals and their metabolites 1. One electron reduction of oxygen 02 + e -—-——-9' O2 2. Spontaneous dismutation of 02' + 1 202 + 2H ———> H202 + 02 (or oz) 3. Haber-Weiss reaction 1 .- 4. Modified Haber-Weiss reaction (Fenton Reaction) 3+ _______ 2+ 02 + Fe > Fe + 02 Fe2+ + H202——-> Fe3+ + OH' + OH. 02 + H202 ——> 02 + OH + OH from Fantone and Ward, 1985 cannot undergo a respiratory burst cannot destroy most types of bacteria (Boxer £31., 1985; Quie £31., 1967). When phagocytizing material such as opsonized zymosan, PMN also release arachidonic acid (Waite £ a_l., 1979). As shown in Figure 3, after AA is liberated from membrane phospholipids via the action of phospho- lipases A2 or C, it can be metabolized by cyclooxygenase or lipoxygenases to prostanoids or leukotrienes, respectively. Many of these eicosanoids are involved in regulating phagocytosis and/or the accompanying inflammatory response. b. Effect of PMA on PMN 1) Introduction. When exposed to PMA i_n__ vitro, PMN aggregate, adhere to endothelial cells and undergo morphologic and biochemical changes which are associated with phagocytosis (O'Flaherty £ 31., 1980; Harlan £ 31., 1985a; Diener £33., 1985). Although not directly chemotactic for PMN, PMA enhances chemotaxis induced by formyl-methionyl-leucyl—phenylalanine (FMLP) (Estensen £31., 1973). 2) Release of active oxygn species. As shown in Table l, PMA stimulates PMN to release active oxygen metabolites (Repine £ 31., 1974; Hoidal £ 31., 1978; Petreccia £ 31., 1987). Associated with the activation of NADPH oxidase and the subsequent release of 02,- is a depolarization of the plasma membrane and increases in Oz consumption and glucose metabolism via the hexose monophosphate (HMP) shunt. Although the exact mechanism whereby PMA elicits these responses is unknown, considerable evidence exists which suggests that activation of protein kinase C (PKC) is involved (Cox £ 31., 1985; Fujita £31., 1986; Gerard £31., 1986; Berkow £31., 1987; Tauber, 1987). 3) Release of lysosomal enzymes. After exposure to PMA, the cytoplasm of PMN contains a large number of vacuoles (Repine £ 31., 1974). Associated with the increase in size and number of cytoplasmic granules is 10 Emwaonmumz cfloc oficocfinonuc mo m>mznumm E E E omnuonuczn ~xomw~ NH owczwmaxoawm m . / _ _ x - — :~o< o~zoo~=o H202 +02 (1) znzoZ catalase >oZ + ZHZO (2) The concentrations of enzymes used in this experiment were greater than those shown to protect isolated endothelial cells and isolated rat or rabbit lungs from injury produced by enzymically-generated 02- and H202 (Tate 3t 33., 1982; Steinberg 913., 1982; Autor 33 g” 1984). C. Involvement of Thromboxane in Toxicity Mediated by PMA 1. Prostanoid production To assess whether TxA2 and PGI2 were produced by lungs perfused with a high concentration of PMA in the absence of perfused PMN, lungs were perfused with medium containing PMA (57 ng/ml) or an equivalent volume of DMSO vehicle. Samples of effluent medum were collected 10, 20 and 30 minutes (if possible) after the addition of PMA or DMSO to the medium and were analyzed for TxB and 6-keto-PGF a as previously described (Section VI). 2 l 2. Effect of indomethacin on PMA pneumotoxicity To determine if a cyclooxygenase metabolite was involved in the pathogenesis of edema produced by a high concentration of PMA in the absence of perfused PMN, indomethacin (10 11M) was added to the perfusion medium immediately before PMA (57 ng/ml). The concentration of indomethacin used was 3 orders of magnitude greater than that shown to inhibit synthesis of Tsz and 6- keto-PGFIG in isolated rabbit lungs perfused with H202 (Burghuber 33 33., 1984). A perfusion with indomethacin and one with the vehicle for indomethacin (100 pl ethanol) was carried out in the same day. Experiments were performed in this manner so that PMA from the same vial would be used both in a control and in a 71 lung perfused with indomethacin. The order in which lungs were perfused with indomethacin or with vehicle was alternated so that perfusions with indomethacin were not always the first perfusions performed on a given day. Experiments with drugs described below were also performed in a similar, paired manner. To confirm that the concentration of indomethacin which was used inhibited cyclooxygenase, samples of effluent medium were collected 10, 20 and 30 min (if possible) after the addition of PMA for analyses of TxB and 6- 2 keto-PGF1 a as described above (Section VI). 3. Effect of a specific thromboxane synthetase inhibitor To determine if TxA2 was involved in the pathogenesis of edema produced by a high concentration of PMA in the absence of PMN, Dazmegrel (50 11M) or the vehicle for Dazmegrel (0.1 N NaOI-I, 100 11L) was added to the recirculating buffer 5 min before the addition of PMA (57 ng/ml). Dazmegrel is a selective inhibitor of thromboxane synthetase in man (Fischer 313 a_l., 1983). To confirm that the concentration of Dazmegrel which was used selectively inhibited thromboxane synthetase, samples of effluent medium were collected 10, 20 and 30 min (if possible) after the addition of PMA and were subsequently analyzed for Tsz and 6-keto-PGF1 D. Involvement of Vasoconstriction in Edema Induced by PMA (1 by RIA (see Section VI). To determine if edema in lungs perfused with a high concentration of PMA in the absence of PMN resulted from the increased perfusion pressure, lungs were perfused with PMA (57 ng/ml) in the presence and absence of papaverine hydrochloride (0.5 mM). Papaverine is a vasodilator which reportedly acts by inhibiting phosphodiesterase (Needleman and Johnson, 1980). Papaverine was added immediately before PMA, and perfusions were carried out for 30 min (or less if fluid accumulated in the tracheal cannula). 72 VIII. Effect of PMN on PMA Toxicity to the IPL A. Preliminary Experiments To assess whether PMN altered the response to PMA in the IPL, lungs which had been perfused with KRBSA buffer in a single-pass manner for 10 min were perfused in a recirculating manner for 30 min with buffer (70 ml) containing 7, 1x108 or 2x108 PMN. 1x107 PMN were chosen for 7 or 14 ng/ml PMA and 1x10 use in these experiments because this approximates the number of PMN which circulate in a 300 g rat (Cocchetto and Bjornsson, 1983; Benirschke e_t_ a_l., 1978). 1x108 PMN were also used since, when expressed on a concentration basis, 1x108 PMN/70 ml buffer better approximates the concentration of PMN which is present in the blood of a 300 g rat. After 30 min of perfusion with PMN and PMA, the relative lung weight and increase in pressure was assessed. Based on the results of this study, 1x108 PMN were used in subsequent experiments. Supernatant fluid (SN) obtained by spinning 1x108 PMN at 700 g for 10 min in a centrifuge was used as a vehicle control for some experiments. B. Perfusions with PMN and a Concentration of PMA which is not Toxic in the Absence of PMN To assess whether PMN altered the response of the IPL to a concen- tration of PMA which did not produce edema by itself, PMN (1x108) or the supernatant (SN) from a PMN preparation from the same animals was added to the reservoir. In all experiments PMA (14 ng/ml) or an equivalent volume of DMSO vehicle was added immediately after the PMN or SN. Perfusions were then carried out for 30 minutes, if possible (see Section IV, C). 73 C. Involvement of Active Oxygen Species in Edema Induced by Perfusion with PMN and PMA 1. Release of 0.,- into the perfusion medium/confirmation of 02 scavenging by‘SOD Release of 02- by PMA-stimulated PMN in the perfusion medium was determined according to a variation of methods previously described (Babior 3_t_ 33., 1973; Ward 33 Q” 1983). First, ferricytochrome C (70 mg) was added to a 70-ml reservoir of KRBSA containing PMN (1x108) or SN and PMA (14 ng/ml) or DMSO vehicle. Sham perfusions were then conducted in a manner similar to that of the experiments described above, except that lungs were not included in the circuit. To confirm that 02- was the species which reduced ferricytochrome C, sham perfusions with KRBSA containing ferricytochrome C, SOD (500 U/ml), PMN and PMA were also performed. In addition, to determine if KRBSA reduced ferricytochrome C independently of O - formation, sham perfu- 2 sions with KRBSA containing only ferricytochrome C and SOD (500 U/ml) were performed. For all systems, aliquots of perfusion medium (0.9 ml) were removed from the reservoir at various times and were added to 0.1 ml of solution containing SOD (86 U) in test tubes to quench ferricytochrome C reduction. Total volume in these test tubes was brought up to 1.8 ml with KRBSA buffer. After centrifugation at 700 g for 5 min, the absorbance of the supernatant fluid at 550 nm was determined using a Beckman Model 42 spectrophotometer. Since perfusions with ferricytochrome C, SOD and KRBSA revealed that a small amount of ferricytochrome C reduction was not OZ--dependent, absorbance values of these samples were subtracted from absorbance values of samples from the other 1 1wasused to media described above. An extinction coefficient of 18.5 cm- mM- convert absorbance of ferricytochrome C to nanomoles 02- (Margoliash and Frohwirt, 1959). 74 2. Effect of SOD and catalase on pneumotoxicity To determine if active oxygen species mediate injury in this model, as they do in isolated rabbit lungs perfused with PMA and human PMN, SOD (500 U/ml) and catalase (400 U/ml) were added to the perfusion medium of some lungs before the addition of PMN (1x108) and PMA (21 ng/ml). A perfusion with and without the scavenging enzymes was carried out in the same day to ensure that PMN from the same animals and PMA from the same vial were used in a control lung and in a lung perfused with the enzymes. The order in which lungs were perfused with PMN and PMA or with PMN, PMA, SOD and catalase was alternated so that perfusions with SOD and catalase were not always the first perfusions performed on a given day. Experiments with all other drug treatments described below were performed in a similar paired and alternating manner. 3. Effect of SOD on pneumotoxicity To determine if 02- was the species involved in pneumotoxicity, SOD (500 U/ml) was added to the perfusion medium of some lungs before the addition of PMN (1x108) and PMA (21 ng/ml). Lungs were also perfused with PMA (21 ng/ml) to confirm that at this concentration, PMA did not produce edema in the absence of perfused PMN. 4. Effect of catalase on pneumotoxicity To determine if H202 was the species involved in pneumotoxi- city, catalase (400 U/ml) was added to the perfusion medium of some lungs before the addition of PMN (1x108) and PMA (21 or 28 ng/ml). On any given day, a perfusion with PMA (28 ng/ml) was performed first to determine if this concen- tration of PMA produced edema. Any lungs which had a relative weight greater than 2 standard deviations (1.103) above the mean of lungs perfused with no PMA (4.25) were considered to be edematous. If this concentration proved to cause edema, experiments with PMN in the absence or presence of catalase were then 75 performed with 21 ng/ml PMA. If 28 ng/ml did not induce edema, this concentration was used for experiments described above. D. Involvement of Vasoconstriction in Edema Induced by Perfusion with PMN and PMA 1. Perfusion with papaverine To determine if edema in lungs perfused with PMN and PMA resulted from the increased perfusion pressure, lungs were perfused with PMN (1x108) and PMA (21 ng/ml) in the presence or absence of papaverine (0.5 mM). Papaverine was added immediately before PMN and PMA were added to the perfusion medium, and perfusions were carried out for 30 min. As described in Section VIILC, a perfusion with and without papaverine was performed on a given day. The order in which perfusions with or without papaverine were done also varied from day to day. 2. Effect of papaverine on PMN function PMN treated with or without papaverine (0.5 mM) were incu- bated for 30 min at 37°C with PMA (2, 10, 20, 200 ng/ml) and cytochrome C (1 mg/ml) in the presence or absence of SOD (86 U), and release of 02- was determined as previously described (see Section VIII.C). E. Involvement of Thromboxane in Edema Induced by Perfusion with PMN M 1. Production of prostanoids in lugsperfused with PMN and PMA To assess whether prostanoid release was greater in lungs perfused with PMN and PMA than in vehicle-perfused lungs, samples of effluent medium from lungs perfused in Section VIII.B. were collected at various times and were later analyzed for '1‘sz and 6-keto-PGF1G as described in Section VLB. 2. Effect of indomethacin To assess whether a cyclooxygenase metabolite(s) of arachidonic acid mediated lung injury in this model, indomethacin (10 uM) or its vehicle (100 76 111 ethanol) was added to the recirculating perfusion medium immediately before the PMN (1x108) and PMA (21 ng/ml). To confirm that the concentration of indomethacin which was used inhibited cyclooxygenase, samples of effluent medium were obtained at various times during the perfusions and were analyzed for Tsz and 6-keto-PGF1a according to Section VLB. 3. Effect of a thromboxane gynthetase inhibitor (Dazmeig-el) To determine whether thromboxane was involved in the patho- genesis of lung injury induced by PMA and PMN, lungs were perfused with PMN (1x108), PMA (21 ng/ml) and Dazmegrel (10 1.1M) or its vehicle (0.1 N NaOH). Dazmegrel (or vehicle) was allowed to circulate through the lungs for 2 min before the addition of PMN and PMA. Samples of effluent medium were collected and analyzed for Tsz and 6-keto-PGF1a as previously described to confirm inhibition of Tsz synthesis. 4. Experiments to determine if indomethacin or Dazmegrel scavenge active oxygen Species Superoxide generation by PMN incubated with indomethacin or Dazmegrel was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C as described in Section VIII.C. PMN (2x106/ml) used in this assay were preincubated with indomethacin (final concentration, 10 mM) or the vehicle for indomethacin (ethanol) or with Dazmegrel (final concentration, 10 11M) or the vehicle for Dazmegrel (0.1 N NaOl-I) for 5 min at room temperature before being incubated for 30 min at 37°C with ferricytochrome C (1 mg/ml) and PMA (2, 10, 20, or 200 ng/ml) in the presence or absence of SOD (86 U). After 30 min, SOD was added to the tubes which did not previously contain SOD to stop ferricytochrome C reduction. All samples were then spun in a centrifuge at 700 g for 10 min, and the amount of 02 in the supernatant fluid was determined as previously described. 77 To determine if 02- production was inhibited in lungs perfused with Dazmegrel, ferricytochrome C (1 mg/ml) was added to the perfusion medium 5 min before the addition of PMN (1:108), PMA (21 ng/ml) and Dazmegrel (10 11M) or vehicle (0.1 N NaOH). At various times after the addition of PMA, samples of perfusion medium (0.9 ml) were collected into tubes containing SOD (86 U). These samples were spun in a centrifuge at 700 g for 10 min, and the supernatant was then assayed for O - as described in Section VlII.C. 2 To determine if Dazmegrel could scavenge H202, 3 ml solutions containing H O2 (19 mM in KH P04, pH 7.0) and Dazmegrel vehicle (100 111 2 2 KHZPO4), H202 and Dazmegrel (10 11M), or H202 and catalase (l U/ml) were placed in a spectrophotometer and the decrease in absorbance at 240 nm (at 25°C) was followed over a course of 10 min. The concentration of H202 in the samples before and 10 min after the addition of vehicle, Dazmegrel or catalase was determined using an extinction coefficient of 43.6 M.1 cm-1. The amount of H202 which was scavenged was calculated as the difference between the initial amount of H202 present and the amount which remained in each sample after 10 min of incubation. F. Source of TxB and 6-keto-PGF 2 in Lungs Perfused with PMN and PMA 1a l. Involvement of oxyjen radical production in prostanoid synthesis To determine if generation of 02 or H202 was required for synthesis of TxB or 6-keto-PGF from lungs perfused with PMN and PMA, 2 samples of effluent medium from lungs which were perfused with PMA or with 1d PMN (1x108) and PMA (21 or 28 ng/ml) in the presence or absence of SOD or catalase (see Section VIII.C) were collected at various times during perfusions and were subsequently analyzed for TxBZ and 6-keto-PGF by RIA (see Section VI). 101 78 2. Release of prostanoids from PMA-stimulated PMN To determine if TxB2 or 6-keto-PGF1 a were released from PMA-stimulated PMN, KRBSA containing PMN (1x108) and PMA (21 ng/ml) or DMSO vehicle was circulated through the perfusion apparatus (without a lung) at 37°C for 30 min, and samples of effluent medium were collected at various times for subsequent analysis of Tsz and 6-keto-PGF1G by RIA as described in Section VLB. 3. Effect of aspirin pretreatment of lungs or PMN on pneumotoxi- city a. Rationale for experiment To examine whether the lungs or PMN were the source of TxA2 in lungs perfused with PMN and PMA, experiments were performed utilizing PMN and/or limgs from rats that had been pretreated with the irreversible cyclooxygenase inhibitor, aspirin (ASA). I hypothesized that if the lung was the source of TxA2 in this model, pretreatment of lungs with ASA should inhibit TxBZ synthesis and lung injury. Conversely, if TxBZ was produCed by PMN, pretreat- ment of PMN with ASA should produce these effects. Furthermore, if synthesis from both the lungs and PMN was required for the development of lung injury, pretreatment of both the lungs and PMN with ASA would be required to attenuate edema. b. Prgparation of drugs and chemicals Acetyl[carboxyl-14C] salicylic acid (MC-ASA, sp. activity 0.31 mCi/mg) was diluted with H O to an activity of 100 uCi/ml and was stored 2 'm 30 ul aliquots at -4°C. Arachidonic acid (AA) was diluted to a concentration of 50 ng/ml with ethanol and stored at -4°C imder N2. Immediately before use, it was converted to the sodium salt by addition of NaOH. Dilutions to the prOper concentration were then made with saline (0.9%). 79 An aspirin (ASA) preparation suitable for p.o. administra- tion was made by dissolving 100 mg ASA in ethanol (0.5 ml). This solution was then diluted to a concentration of 100 mg/ml with a solution containing 1.5% ethanol in saline (dilute ethanol). Propylene glycol (1 ml) was added to aid in solubilization. A solution containing identical volumes of ethanol, dilute ethanol, and propylene glycol (0.5:0.5:l) was used as a vehicle control. ASA used to treat PMN was dissolved in ethanol (50 ml) and diluted with PBS (0.1 M) to a concentration of 100 nM. A similar, dilute ethanol solution was used as a vehicle control. c. Preliminary elmeriments in isolated lung_s_ 1) Confirmation of ASA washout in lungp. Lungs were isolated from fasted (15-24 hours) male, Sprague-Dawley rats as described in Section IV. One hour prior to surgery, they were treated with ASA (100 mg/kg, 14'C-ASA (10 uCi/mg). An aliquot of this solution was p.o.) which was spiked with saved for later analysis of 14C content. After being suspended in the perfusion apparatus, the lungs were perfused with KRBSA in a single-pass manner for 10, 15 or 20 min to clear the vasculature of blood and ASA. They were then perfused with KRBSA in a recirculating manner for 30 min. Samples of effluent medium were collected at various times during the single-pass perfusion, and a sample of perfusion medium was taken from the reservoir after the perfusion was termi- nated. One milliliter of each of the samples was added to vials containing 15 m1 ACSR. Radioactivity in these vials was then determined for 15 min in a 14C. scintillation counter (Beckman Model LS-3150P) with the window set for The counts were then used to calculate the concentration of ASA in the perfusion medium. Based on the results of this study, a washout time of 20 min was chosen for use in subsequent experiments. 80 2) Confirmation of cyclooxygenase inhibition in lungp. One hour prior to surgery, fasted (15-24 hours), male, Sprague-Dawley rats were treated with ASA (100 mg/kg, p.o.) or vehicle. ng3 which were isolated were perfused in a single-pass manner with KRBSA for 20 min to rid the vasculature of ASA. They were then perfused with KRBSA in a recirculating manner for 30 min. After 30 min had elapsed, they were perfused in a single-pass manner with buffer (KRB) which did not contain BSA. The lungs were then statically inflated with 2-3 cc room air and 80 11M AA was infused (0.1 ml/min) into the pulmonary artery for 5 min using a Harvard Apparatus Compact Infusion pump (Millis, MA). Samples of effluent medium were taken during the infusion and were subsequently analyzed for TxB by RIA (see Section VI). 2 d. Preliminary experiments with PMN 1) Confirmation of ASA washout in PMN. PMN used in the study were isolated from the peritoneal cavities of male, Sprague-Dawley, retired breeder rats as described in Section II and were treated with PBS (0.1 M) 14C-ASA. After incubation for 10 min at 37°C, containing 100 11M ASA and 1 uCi cells were spun in a centrifuge at 300 g for 7 min. One milliliter of the supernatant was then added to a vial containing 15 ml ACSR, and radioactivity in the vial was measured in a Beckman Model LS-3150P scintillation counter. After being resuspended in 50 ml PBS (0.1 M), the cells were again spun in a centrifuge for 300 g for 7 min, and radioactivity in the superantant was measured as described above. This procedure was repeated until background activity was detected in the supernatant fluid. Based on results of this study, all PMN were washed twice with 50 ml PBS before being used in an experiment. 2) Confirmation of cyclooxygpnase inhibition in PMN. PMN were isolated as described above and were incubated in PBS (0.1M) containing 100 11M ASA or vehicle for 10 min at 37°C. The cells were then 81 washed twice with 50 ml PBS to remove residual ASA. In preliminary experi- ments, these PMN were incubated with AA (100 11M) for 30 min at 37°C and synthesis of TxBZ was assessed by RIA. This experiment was also repeated at the end of each day using ASA and vehicle-pretreated PMN which were not used in experiments described in Section e. below. For these cells, synthesis of both Tsz and 6-keto-PGF1 a was determined. 3) Effect of ASA which remained in lung vasculature after washout on prostanoid production by PMN. To determine if the amount of ASA which remained in the lung vasculature after a 20-min washout period (1 uM) could inhibit prostanoid production by PMN which were perfused through the lungs, PMN treated with 1 1.1M ASA in HBSS or with vehicle (0.0005% EtOH in HBSS) were incubated with 100 11M AA for 30 min at 37°C. Synthesis of TxB2 from cells treated with ASA was then compared with that of cells treated with vehicle. 4) Effect of ASA pretreatment on PMN function. Superoxide release from ASA (100 1.1M) or vehicle-pretreated PMN (2x106) stimulated with PMA (21 ng/ml) for 30 min at 37°C was determined by measuring the superoxide dismutase-inhibitable reduction of ferricytochrome C (see Section VIII.E., part 4.). e. Experimental protocol The protocol used for this experiment is illustrated in Figure 8. Lungs were isolated from fasted (15-24 hours), male, Sprague-Dawley rats which had been treated with ASA (100 mg/kg, p.o.) or vehicle one hour prior to surgery (as described above). After being suspended in the perfusion apparatus, they were perfused with KRBSA in a single-pass manner for 20 min. Lungs from vehicle-pretreated rats were then perfused in a recirculating manner for 30 min with KRBSA containing either 1) PMA (21 ng/ml), 2) PMA and vehicle-pretreated 82 .coflmsuumm mo .cfla on young pommmmmo who: >u=ncfi mafia no mooflpcfl pas .coflmaduod on... usocmsounu “Knowing: 0.33 ousmmoum 303:“ ppm mfimonucmam mm ocoxonfiouce ...moomm.mz: ou onwpuooon 22m pounouuoum iV daoand> pan 42m sum: .33 5.33 pomsmumm who: mun.“ pousouuoumlzms can—ammo Ho A>v oaowno> Eouu mmcsq .ucmafluomxm cfluwdmo Mom Hooououm .m ouzmwm 83 m ousmwm I v daoazm> V daoazm> .m A>C oaoazo> A>V oaoazo> .m 22m o: A>V oaoazo> .a xx mum zzm x: mum mcsq cassnas panama 42d 22d 55 on wax? consummum ##o owl mcauoasoufloom mmom mamcwm 84 PMN (1x108) or with 3) PMA and ASA-pretreated PMN. Lungs from ASA- pretreated rats were also perfused with either 4) PMA and vehicle-pretreated PMN or with 5) PMA and ASAdpretreated PMN. One perfusion from each of the treatment groups (with the exception of group 5) was carried out in the same day. The order in which lungs from each of the groups were perfused was also alternated on a daily basis. Samples of effluent medium were taken at various times during each perfusion for analysis of TxB and 6-keto-PGF1a by RIA (see 2 Section VI). Immediately after perfusions were terminated, lungs were weighed and lavaged. Experiments were also performed in lungs from ASA vehicle-pre- treated rats perfused with 1) PMA vehicle (DMSO), with 2) DMSO and ASA/vehicle-pretreated PMN, or with 3) DMSO and ASA-pretreated PMN, and in 4) lungs from ASA-pretreated rats perfused with DMSO and ASA vehicle- pretreated PMN to determine if ASA pretreatment alone of either the lung or the PMN produced toxicity to isolated lungs. Samples of effluent medium from these lungs were also collected for subsequent prostanoid analysis. IX. Analysis of Data PMA dose/response data (see VILA) were analyzed using a one-way analysis of variance (ANOVA), and Dunnett's test was used to compare means (Steel and Torrie, 1980). In experiments with a high concentration of PMA (57 ng/ml) in the absence or presence of SOD and catalase (see Section VII. B), lung injury data were compared using the Student's t-test. In all other experiments involving comparisons between two groups, data (with the exception of prostanoid data) were analyzed using a paired t-test. In experiments involving comparisons among three groups, lung injury data were analyzed using a randomized block ANOVA, and means were compared using the least significant difference (lsd) test. Lung 85 injury data from experiments with PMN and PMA or the respective controls or from experiments with ASA were analyzed using a completely random ANOVA. Group means from each of these experiments were also compared using the lsd test. Prostanoid data from lungs perfused with PMN and PMA or the respective controls or from experiments with ASA were analyzed using a mixed-design ANOVA. Prostanoid data from experiments with 57 ng/ml PMA were analyzed using a one-way ANOVA, and those from PMN incubated with PMA or DMSO were analyzed using a blocked factorial ANOVA. A blocked factorial ANOVA was also used to analyze prostanoid data from lungs perfused with PMN, PMA and indomethacin, Dazmegrel, SOD or catalase. Superoxide data from PMN incubated with indomethacin, Dazmegrel or papaverine were also analyzed using a blocked factorial ANOVA. In each of the experiments described in this paragraph, group means were compared using the lsd test. When variances were non-homogenous, log transformations were performed to render them so. Pearson's product moment correlation coefficient (r) was used to determine whether relative lung weight or perfusion pressure correlated with prostanoid synthesis. Data are presented as the mean 1'. SE. The criterion for significance was p < 0.05. X . Miscellaneous A. Sources of Chemicals The phorbol myristate acetate (PMA, l2—O-tetradecanoyl-phorbol-l3- acetate, MW 616) which was used in the initial studies was purchased from Consolidated Midland Corporation (Brewster, NY). Halfway through this project, the company sold the rights to this compound to L.C. Services Corporation 86 (Woburn, MA). Thereafter, PMA was obtained from this company. Having to use PMA from a different source appeared to have no effect on the studies. Bovine serum albumin (BSA; Fraction V) was purchased from Miles Biochemicals (Elkhart, IN). Before being used in an experiment, each lot of BSA was tested for monoamine oxidase (MAO) activity using 14C-SHT as the substrate. Glycogen (Type II from oyster), trypan blue, arachidonic acid (free acid, from porcine liver), ferricytochrome C (Type III from horse heart), catalase (2890 U/mg protein, from bovine liver), propylene glycol, NADH, soduim pyruvate solution, indomethacin, aspirin and papaverine hydrochloride were obtained from Sigma Chemical Corp. (St. Louis, MO). Superoxide dismutase (SOD; 3800 U/mg protein, from bovine liver) and dimethylsulfoxide (DMSO) were purchased from Diagnostic Data (Mountainview, CA) and EM Sciences (Gibbstown, NJ), respec- tively. Dazmegrel (UK384585) was a gift from the Pfizer Corp. (Sandwich, Kent, England). Antibodies and standards for 6-keto-PGF a and TxB were purchased 1 2 from Advanced Magnetics (Boston, MA), and 3H--Tsz ([5,8,9,11,12,l4,15-3H]- 3 thromboxane B2; specific activity, 471 mCi/mg) and H-6-keto-PGF (6- 1a [5,6,8,9,11,12,l4,15-3H]Reta-prostaglandin F ; specific activity, 445 mCi/mg) is were obtained from Amersham (Arlington Heights, IL). The crossreactivities of the antibodies to various lipoxygenase and cyclooxygenase metabolites of AA are listed in Table 5. Gelatin which was used to make PBS was obtained from Difco Laboratories, Detroit, ML The decolorizing carbon (NoritR) and dextran (T70) which were used to make dextran-coated charcoal were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Research Products Intl. (Mt. Prospect, IL), was the source of the scintillation cocktail used for detection of tritium (Safety SolveR). The 5- hydroxy [side chain-2-14C1tryptamine creatinine sulfate (MC-5HT; specific 87 TABLE 5 Cross Reactivities of Antibodies* Anti Thromboxane 82 Anti 6-Keto-PGF1a Thromboxane 82 100% <0.01% 6-Keto-PGFla _57 ng/ml, Figure 9) also 89 90 .<=u acorns: pmmzuumm mmcsa Eouw ucmuowmfio .Sucsowuwcmwm an .hn.o H mm.m mmz dzm usocuwz pomsuumm manna mo unmfi03 0>fiusamu one .mucwom >2 pmusomno mus mush Hound .mommo meow :H .mmCDH duos no owns» 593 muasmou mo mm H cams on» uncommudou ucfiom comm ...m.uq:mmm: Mops: pousofipCH omwzuonuo mmoacs cofimsuuom mo ad: on nouns pmcflgouop mums magmas: 9:5 ...moomemz= Hoops confluomop ms $5 an?» moss—ma mamasasoufioou n ca pomsuuod mum3 mmcsq .dzm mo mc0wusuucoocoo msofiun> nu“: pomsuumm mmcsa up.“ pouoaomfi mo Aoooa x samba: aromas m5; 0>fiunaom .m ousmwm 91 m musmwm Asexmco om mHucm0flmwcmwm .<2m uponufiz pmmsuumm mmcsa Ca mm 55 m.o H m.o >3 nanomhoom musmmoua .cflE cm can OH nuon um .mm H some on» mucomoumou ucwom comm .mucmHOQ >2 pmusomno our mush uouum .momoo mEom :H .Amaoafigm comov pas an no Amaone>n pomoaov 0H Mow :oflmsmuom nouns urn» can ufio>uomou one on poops ms: ass c023 ousmmoum :onsuuom onu :003uon 00:0H0MMflp on“ ma ousmmmum cfl omsouocfi one .a shaman ou pcomoa on» :H ponfiuomop ms «Sm :ufl3 ornamumd 0u03 mmcsq .42m mo mCOausuucoocoo unoHus> cud: pwmsuuom mmcsa you pmusaomw mo ousmmoum Howumuus >uscoaasm .OH ousmfih 93 oH Guzman m<2n3 ma AHE\mco om eunssaud ut espauoul 94 produced an increase in perfusion pressure. Furthermore, a concentration of PMA which did not cause an increase in the relative weight (21 ng/ml) produced a significant increase in perfusion pressure by 30 min of perfusion. Since perfusions with 71 and 143 ng/ml were terminated before 30 minutes, the increase in pressure at this time could not be obtained for lungs at these concentrations. B. Histopathology of Lungs Perfused with a High Concentration of PMA Electron micrographs of lungs which were perfused with a high concentration of PMA are shown in Figure 11. As shown in Figure 11A, perfusion with PMA produced endothelial cell injury. Although there are no gaps in the endothelial cell lining of the vessel, there are extensive areas of uplifting of the endothelium from basement membranes (arrows). As shown in Figure 11B, perfusion with PMA also produced marked interstitial (IS) edema. These changes are not seen in lungs perfused with DMSO vehicle (see Figure 18). C. Lack of Effect of SOD and Catalase on PMA-Mediated Toxicity to the IPL To determine if toxicity mediated by a high concentration of PMA was dependent on the presence of oxygen radicals, lungs were perfused with PMA (57 ng/ml) or with PMA, SOD (500 U/ml) and catalase (400 U/ml). As shown in Figure 9, this concentration of PMA produced lung injury. As illustrated in Table 6, SOD and catalase altered neither the increase in relative lung weight nor the elevation in perfusion pressure induced by this toxic concentration of PMA. Average body weights of rats used as lung donors and initial perfusion pressures also did not differ between the two experimental groups. 95 Figure 11. Representative ultrastructural findings in isolated lungs perfused with PMA (57 ng/ml) in the absence of added PMN. (A) Vascular changes such as endothelial cell blebbing (arrows) were observed, as well as (B) marked interstitial (IS) edema (4250x). Figure 11 97 TABLE 6 EFFECT OF SOD AND CATALASE ON INJURY TO THE IPL MEDIATED BY A HIGH CONCENTRATION OF PMA Treatment PMA PMA + SOD/CAT Body Weight (g) 298.2 1 7.6 305.0 1 10.2 Initial Pressure 4.5 i 0.2 5.1 i 0.3 (mm Hg) LW/BW x 103 19.6 i 7.4 30.8 i 5.6 Change in Pressure 17.4 i 0.9 19.3 i 1.7 (mm Hg) Lungs were perfused with PMA (57 ng/ml) or with PMA, SOD (500 U/ml) and catalase (400 U/ml) as described in "Methods". Some perfusions with or without SOD and catalase were terminated before 30 minutes due to fluid accumulation in the tracheal cannula. Time of perfusion averaged 26.6 i 1.2 min and did not differ betwgen the two groups. Relative lung weight (LW/BW x 10 ) was determined when perfusions were terminated. Change in perfusion pressure was determined at 20 min for both groups. 98 D. Involvement of Thromboxane in Toxicity Mediated by a High Concen- tration of PMA l. Prostanoid production To determine if a cyclooxygenase metabolite(s) was associated with edema formation induced by a toxic concentration of PMA, I first investigated whether concentrations of cyclooxygenase metabolites were elevated in effluent media from lungs perfused with a toxic concentration of PMA (57 ng/ml). The amount of TxBZ in effluent media collected at various times after the addition of PMA (57 ng/ ml) or dilute DMSO vehicle to the perfusion medium is illustrated in Figure 12A. A significantly greater amount of Tsz was produced by lungs perfused with 57 ng/ml PMA than by controls by 20 min of perfusion. 6- Keto-PGFla production also increased throughout the duration of perfusion with PMA (Figure 12B); however, with respect to control, the amount of 6-keto-PGF1a produced in lungs perfused with PMA was not significantly elevated until 30 min of perfusion. 2. Relationship between prostanoid production and the relative lung weight or the increase in perfusion pressure To determine if the increase in perfusion pressure or relative weight of lungs perfused with 57 ng/ml were related to the release of arachidonic acid metabolites, I examined whether these indices of lung injury correlated with prostanoid formation. A significant correlation existed between the amount of TxB detected in media after 30 min of perfusion and the relative lung weight 2 (Figure 13A). The final increase in perfusion pressure correlated with the amount of TxB formed after both 20 (r=0.75) and 30 minutes (Figure 13B) of perfusion. A 2 correlative relationship did not exist between the amount of TxB formed at 10 2 minutes and the increase in pressure (r=0.49). At 10 and 20 minutes of perfusion, the amount of Tsz formed also did not correlate with the relative lung weight (r=0.39 and 0.59, respectively). 99 Figure 12. Effect of a toxic concentration of PMA in the absence of neutrophils on prostanoid production in isolated, perfused rat lungs. Lungs were perfused with PMA (57 ng/ml) or with DMSO vehicle as described under "METHODS”. Samples of effluent medium were collected at various times after the addition of PMA or DMSO vehicle to the reservoir and were analyzed for (A) TxBZ and (B) 6-Keto-PGF a by radioimmunoassay. Open circles, lungs per used with DMSO tyehicle; closed circles, lungs perfused with PMA. Significantly different from lungs perfused with DMSO vehicle. 100 - u 2 Amsxmco mmxe 1--3_ so 20 TIME (min) Assxmcooauuuoooxm -- ‘ 30 20 TIME (min) Figure 12 101 .czm cums comsuuoa manna .moaoufio comoHo “0H0w50> Omzo cum: momsuumm mmcsa .moaouflo coco .coflmafiuom mo ad: on us :ofiucuucoocoo amouioudxio ”Engaged uo composers. n no 3039, 085 so 53 cufi3 cofiusuuom mo nae on moans musmmoum cofimsuuod :H musoHOCH Any can ucmfioz 93H 0>Husamm 6v .consuuoa no cg cm us coHumuucoocoo mmxe ucoszuo mo cofluocad n no 0H0fi20> Omzc no 5.3 cu“: cofimsuuod no ad: on nouns ousmmoum :oflmsuuoa ca mmc0u0:H Amy can unmfio3 ocsn 0>fiucamm :3 .rmooremzr on 656.8006 3029, 09.5 no :56: E 53 5a: is on How comswuoa mums wmcsq .<:m no :oflucuucoocoo no“: a cuw3 cousuumc manna ca ousmmoum cowm3uuom :H 0mmmu0c« no AoOOH x 3m\3av ucmfi03 m:=H paragraph can :ofiuospoum pflocsunoum possumn afinmcofiusaom .3 056mm 102 25?; o _ uuuooozm ma ousmfim O.N m4 O4 md 06 u n a w o NI o m. m. :N a“ w :Q MD M I. so swung :0 (H B :O— a m :N— a i?— o a_sxmco mmxe 90.0... sanssaud u; asoeusux a_sxmco o.uuuouoxm o.~ m._ 6.. m.o o.o vi a a a o 20— ..m_ ..°N ..m~ . l . om a_sxmco meh m.o m.o e.o ~.o o.o u u n u D Cod-.- 0001 x A8/M1 0001 x A8/M1 103 In contrast, the relative lung weight and the final increase in perfusion pressure did not correlate significantly with 6-keto-PGF1a concentra- tions in effluent samples obtained after 10 (r = -0.15 and -0.12, respectively), 20 (r = 0.16 and 0.24, respectively) or 30 min (Figures 13C and 13D, respectively) of perfusion. A correlative relationship also did not exist between the ratio of the concentration of Tsz to 6'-lHucsofiuacmfim u 0 .Qsoum zm 0:3 42m Eouu uc0u0umwc >Hucsofiuficmflm u n .Qsoum 22m mafia Omzo Eoum uc0u0uuwc haucsocficmfim n c .mmcDH 0>C ummma us Eoum muHsm0u mo mm H :00... uc0m0um0u musm ...moomm.mz.. u0cc: c0uo: no .53 HO 1 cut; cofimsuu0n mo .cfl: ascofiufipcs ca u0umn 602250006 0H0: munmw0r 0:54 Shoo—Lama: ou mcficuooos Azmv 0H0ac0> acouscu0msm no 223 cuwz can 0H0Hc0> Omzo no 423 cu“: CHE em you c0msuu0a 0u03 mmcsq .mmcsa you c0msuu0a .p0unaona mo mucmfi03 0>wuna0u co 03.39590: can 55 mo uo0uum .va 05:10am 113 <23 223 Zm ea 0usmwm it omzm zzu Zm 0001 X MB/MW 114 .Qsoum zm mafia 0H0fi:0> Omzo Eon» »c0u0uuwc >H»c00HuHcmfim u 0 .msoum zm mafia czm Eouu »c0u0umwc >H»c00fiuwcmflm u : .msoum 22m mafia 0H0H:0> Omzo soum »:0u0mm«c >a»:00wuacmfim u 0 .mm H mcm0s »c0m0um0u mucm nuwo>u0m0u 0:». o» .c0ccs 0M0: m0H0fi:0>. 0>H»00Qm0u uw0:». no zzm 6:0 4:3 c0:3 0usmm0um 0:» can cofl09uu0a 8mm ”De L 0:» Mo unacch0: 0:» »c 05600.5 0:» c003»0: 00c0u0wuflc 0:» ma 0urmm0um am 00005:; 0:9 .ea 0usmflm :w pc0m0~ 0:» o» mcwcuoooc m0H0w:0> 0>w»00dm0u 0:» no 223 can cad :»fi3 c0m=uu0m 0u03 mmcsq .mmc:H »0u c0msuu0m .c0»saoma mo 0u=mm0um 303.: so 03:90.350: can 53 mo »00uum .mH 055?: C'J t—t 115 L0 (Swa) aunssaud ut esoauoul PMN SN PMN SN DMSO PMA Figure 15 116 10 mmHg DMSOISN 1 W o A 10 mm Hg DMSOliPMN 0 B 10 meHg 1 “‘_‘--"—' F——' m0 C ~10 mm Hg PMAIPMN " l ...o D g L l l I I l o lo 20 30 TIME (min) Figure 16. Inflow pressure recordings of lungs perfused with PMA and/or PMN. Perfusion pressures of lungs. perfused with. PMA and. PMN or 'the respective vehicles were recorded on a Grass MOdel 7B polygraph. Representative recordings from lungs perfused with (A) DMSO vehicle plus SN, (B) DMSO vehicle plus PMN, (C) PMA plus SN, and (D) PMA plus PMN. ' 117 significantly different from that of lungs perfused with DMSO plus SN (Figure 16A). However, in these lungs there was an early increase in inflow pressure between 5 and 10 min which was similar to that seen in lungs perfused with PMN plus PMA. In contrast to lungs perfused with PMN and PMA, the increase in pressure in lungs perfused with PMN and DMSO was reversible. This early, reversible elevation did not occur when PMN were absent from the medium (Figure 16A). The change in perfusion pressure of lungs perfused with PMA and SN (Figure 16C) was significantly greater than that of lungs perfused with DMSO and SN (Figure 16A). This pressure increase in lungs perfused with PMA and SN was gradual and occurred over the full duration of the perfusion. 3. Effect of perfusion with PMN and PMA on 5HT removal and metabolism To determine if the metabolic function of lungs perfused with PMN and PMA was altered, the disposition of perfused [14C]5HT was deter- mined. Neither the removal nor the metabolism of 5HT was altered by perfusion with PMA or PMN or with the two together (Figure 17). Approximately 81% of 5HT was removed in a single-pass and 53% was metabolized by all lungs after 10 min of perfusion with this amine. The percentage of perfused 5HT removed or metabolized after 5 min of perfusion with it also did not differ among groups (data not shown). C. Histopathology of ngp Perfused with PMN and PMA Electron micrographs of lungs perfused with PMA (14 ng/ml) or DMSO vehicle and with PMN (1x108) or SN vehicle are shown in Figures 18 through 21. As shown in Figure 18, lungs which were perfused with DMSO vehicle plus PMN supernatant appeared normal. The vascular endothelium (arrow) is intact, and there is no evidence of blebbing. The insterstitium (IS) around the vessel also does not appear to be edematous. Figure 19 is an electron micrograph taken from a lung perfused with PMN and DMSO vehicle. A PMN which has not undergone 118 Figure 17. Effect of PMA and neutrophils on 5HT removal and metabolism from isolated, perfused lungs. The fraction of 5HT removed (A) and (B) metabolized by isolated lungs perfused as described in the legend of Figure 14 was determined according to "METHODS". Bars represent means 1; SE of results from four or more lungs. 119 oo>oeom ezm N SN PMN PMN DMSO SN PMA 60" _ . mu 4 nonasonoooz arm N SN PMN PMN DMSO SN PMA Figure 17 120 Figure 18. Representative electron micrograph taken from a lung perfused with PMA vehicle (DMSO) and neutrophil vehicle (SN) as described in "METHODS". The vascular endothelium (arrows) and interstitium (IS) of these lungs appeared fairly normal (7000x). n 03% («is . v. m .99 .127}... tats gure 18 F1 122 Figure 19. Representative electron micrograph taken from a lung perfused with PMA vehicle (DMSO) and PMN as described under "METHODS". PMN were present in capillaries (open arrow). Some areas of the endothelium were lifted off from the basement membrane (solid arrow). The interstitium (IS) appeared normal (7000x). 123 19 igure F 124 degranulation is present in the vessel. A red blood cell is also present in the vessel; however, this was a rather uncommon finding. Although the majority of the endothelium appears to be intact, there is uplifting of endothelial cells from basement membranes in some areas of the vessel (arrow). There is, however, little evidence of interstitial (IS) edema. The representative findings of lungs perfused with PMA and SN vehicle are depicted in Figure 20. The appearance of this vessel is similar to that of the vessel perfused with both vehicles (Figure 18). There is no evidence of interstitial edema or injury to the endothelium. As shown in Figure 21, marked damage to endothelial cells, with blebbing (arrows) and exposed basement membranes occurred in lungs perfused with PMN and PMA. Interstitial (IS) edema is also evident. One of the neutrophils within this vessel has degranulated (open arrow). This PMN appears to be in contact with areas of endothelium which are damaged. D. Involvement of Active Oxygen Species in Edema Induced by Perfusion with PMN and PMA 1. Generation of 01.3by PMA-stimulated PMN To determine whether 02- was released into the perfusion medium by stimulated PMN, sham perfusions with PMN (1x108) and PMA (14 ng/ml) or their respective vehicles were conducted as described in "METHODS". By 10 min of perfusion, more 02- was detected in medium containing PMN (1x108) and PMA (14 ng/ml) than in medium containing either PMN or PMA (Figure 22). By 30 min, the amount of o ' formed in the PMA plus PMN system 2 was 13 nmol/108 cells, compared to less than 3 nmol/lO8 cells produced in the other groups. Reduction of cytochrome C by PMN plus PMA was due to Oz- formation, since addition of SOD (500 U/ml) totally abolished cytochrome C reduction. 125 Figure 20. Representative electron micrograph taken from a lung perfused with PMA and PMN vehicle (SN) as described under "METHODS". There was little evidence of injury to vascular endothelium (arrows) or interstitial (IS) edema (9000x). 126 rFigure 20 127 Figure 21. Representative electron micrograph taken from a lung perfused with PMA and PMN as described under "METHODS". Degranulated PMN (open arrow) were present in capillaries. Marked damage to endothelial cells (arrows) and interstitial (IS) edema were also present (5500x). Figure 21 129 02— (nmol/IUB cells) (ZQ 15-- 02.68 + +-- 10-- 5-- + _ , 1.. 7‘ I:2 7’ 0'" +--r + 0T I 10 V 20 I 30 TIME (min) Figure 22. Production of O ' by PMN stimulated with PMA. Sham perfusions with medium containing cytochrome C plus PMA and/or PMN were performed according to "METHODS". Sham perfusions with medium containing PMA, PMN, cytochrome C, and SOD also were performed to confirm that O " was the species which reduced the cytochrome C. he amount of 02' formed in each system was calculated according to "METHODS". .Values represent means from two separate experiments. 130 2. Effect of SOD and catalase on pneumotoxicity After determining that active oxygen metabolites were being formed in the IPL system, I examined whether these metabolites were responsible for lung damage. Lungs were perfused with PMN and PMA in the absence or presence of SOD and catalase according to "METHODS". Perfusion with SOD and catalase prevented the increase in relative lung weight, but not the increase in perfusion pressure caused by perfusion with PMN and PMA (Table 11). As stated in this table, average body weights of rats used as lung donors and initial perfusion pressures did not differ between experimental groups. 3. Effect of SOD onpneumotoxicipy To determine if 02- was the injurious oxygen species, lungs were perfused with PMA (21 ng/ml), with PMA and PMN (1x108) or with PMA, PMN and SOD (500 U/ml) according to "METHODS". As stated in Table 12, the relative weight, change in perfusion pressure and amount of albumin and LDH in BAL of lungs perfused with PMA, PMN and SOD were significantly less than those of lungs perfused with PMA and PMN and were not different from those of lungs perfused only with PMA. Average initial pressures and body weights of animals from which lungs were isolated were not different between experimental groups. 4. Effect of catalase on pneumotoxicity To determine if H202 was involved in the toxic response, lungs were perfused with PMA (21 or 28 ng/ml), with PMA and PMN (1x108) or with PMN, PMA and catalase (400 U/ml) according to "METHODS". As with other experiments, initial inflow pressures and weights of rats used as lung donors were not different among the 3 groups. The results of this study were similar to those of the experiment with SOD. In llmgs which were co-perfused with catalase, the relative weight and the amount of albumin in BAL were significantly less than those of llmgs perfused with PMN and PMA and were not significantly different 131 TABLE 11 EFFECT OF SOD AND CATALASE ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment PMA/PMN PMA/PMN + SOD/CAT Body Weight (g) 305.4 1 10.3 324.3 : 11.0 Initial Pressure 4.6 i 0.2 4.1 i 0.1 (mm Hg) LW/BW x 103 11.8 i 3 2 5.2 i o 4* Change in Pressure 12.2 i 1.1 12.1 i 1.0 (mm Hg) Lgngs were perfused with PMA (21 ng/ml) and PMN (1 x 10 )or with PMA, PMN, SOD (500 U/ml) and catalase (400 U/ml) as desc§ibed in "Methods". Relative lung weight (LW/BW x 10 ) and change in perfusion pressure ere determined after 30 min. of perfusion. Significantly different from. PMA plus PMN-perfused group. 132 TABLE 12 EFFECT OF SOD ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment Body Weight (g) 285.8 1 7.5 293.8 1 6.6 299.5 : 11.0 Initial Pressure 4.8 i 0.2 4.9 i 0.2 5.1 i 0.2 (mm Hg) Time of Perfusion 26.9 i 1.5 30.0 i 0.0 30.0 i 0.0 (min) LW/Bw x 103 25.1 : 4.8ab 5 9 i o 8 5 1 i 0 5 hpressure 14.1 : 1.9ab 9.3 i o 9 7 2 1 0 (mm Hg) Albumin (mg) 70.8 : 28.2ab 5.1 + 2 1 4 0 i 1 3 LDH (Units) 47.9 : 7.3ab 23.3 i 3.8 24.7 i 5.3 Lungs we e perfused with PMA (21 ng/ml), with PMA and PMN (1 x 10 )or with PMA, PMN, and SOD (500 U/ml) as de cribed in "Methods". Relative lung weight (LW/BW x 10 ), change in perfusion pressure (A‘Pressure) and the total amount of albumin and lactate dehydrogenase (LDH) in BAL and edema fluid in the lungs were assessed after perfusions were terminated. n= 7 for each group. a bSignificantly different from PMA group Significantly different from PMA/PMN + SOD group 133 from those of lungs perfused with only PMA (Table 13). As in lungs perfused with SOD, the perfusion pressure of lungs perfused with catalase was significantly less than that of lungs perfused with PMN and PMA. However, in contrast to lungs perfused with SOD, the inflow pressure of lungs co-perfused with catalase was significantly greater than that of lungs perfused only with PMA. Also, in contrast to the experiment with SOD, measurement of LDH in lavage fluid did not appear to be a good indicator of toxicity induced by PMN and PMA. E. Involvement of Vasoconstriction in Edema Induced by Perfusion with PMA and PMN 1. Effect of papaverine on pneumotoxicity To determine if the increase in pressure was responsible for edema produced by PMN and PMA, lungs were perfused with PMN (1x108) and PMA (21 ng/ml) or with PMN, PMA and papaverine (0.5 mM) as described in "METHODS". Average weights of rats used as lung donors and initial pressures of isolated lungs did not differ between experimental groups (Table 14). As shown in this table, perfusion with papaverine attenuated the increases in both pressure and relative lung weight induced by perfusion with PMN and PMA. As indicated in Section 1. part E, perfusion with papaverine alone did not change perfusion pressure or produce edema in the isolated lung. 2. Effect of papaverine on PMN function To determine if papaverine affected the release of active oxygen metabolites from PMN, PMN which were incubated with or without papaverine (0.5 mM) were stimulated with various concentrations of PMA and release of 02 was assessed according to "METHODS". Figure 23 shows that treatment of PMN with papaverine had no effect on their ability to release 02 134 TABLE 13 EFFECT OF CATALASE ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment PMA/PMN PMA/PMN PMA + Catalase Body Weight (g) 302.1 1 7.4 306.1 i 8.4 312.8 + 8 5 Initial Pressure 4.8 i 0.2 5.0 i 0 2 4.9 i o 3 (mm Hg) LW/BW x 103 13.3 : 4.1ab 5.6 i 0 3 5.0 i 0 2 APressure 15.1 i 2 0ab 9 6 : 0.5a 6 8 i o 9 (mm Hg) Albumin (mg) 42.6 : 22.2ab 5.0 i 0 8 4.9 i 0 5 LDH (Units) 32.5 i 3.8 29.8 i 1.5 29.9 i 2.2 Lungs were per used with PMA (21 or 28 ng/ml), with PMA and PMN (1 x 10 )or with PMA, PMN, and Catalase (400 U/ml) as depcribed in "Methods". Relative lung weight (LW/BW x 10 , change in perfusion pressure (dPressure) and the total amount of albumin and lactate dehydrogenase (LDH) in BAL and edema fluid in the lungs were assessed after 30 min. of perfusion. n= 9 for each group. 3Significantly different from PMA group bSignificantly different from PMA/PMN + Catalase group 135 TABLE 14 EFFECT OF PAPAVERINE ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment PMA/PMN PMA/PMN + Papaverine Body Weight (g) 300.8 i 9.9 309.8 i 11.5 Initial Pressure 6.0 i 0.7 4.9 i 0 2 (mm Hg) LW/BW x 103 6.2 i 0 4 5.0 i 0 2* Change in Pressure 11.2 i 0.6 8.4 i 0.4* (mm Hg) Lqus were perfused with PMA (21 ng/ml) and PMN (1 x 10 )or with PMA, PMN and papaverine (0.5 mM) as degcribed in "Methods". Relative lung weight (LW/BW x 10 ) and change in perfusion pressure were determined after 30 min. of perfusion. Significantly different from PMA plus PMN-perfused group. 136 02— (nmol/ml) 25y 'i 20-- $ 30 __ 4—0 is- /.e M J 10-- l ' 5.- T O O 1' i i 'r 2 10 20 2000 PMA (ng/mi) Figure 23. Effect of papaverine on release of active oxygen metabolites from PMN. PMN treated with or without papaverine were stimulated with various concentrations of PMA and O ' production was determined as described under "METHO S". Closed circles, 02' produced by untreated PMN. Open circles, 02' produced by PMN treated with papaverine. n = 3 for each group. 137 F. Involvement of Thromboxane in Edema Induced by Perfusion with PMN and PMA 1. Prostanoid production To determine if a cyclooxygenase metabolite(s) was involved in lung toxicity produced by PMN (1x108) and PMA (14 ng/ml), I first examined whether concentrations of some of these metabolites were greater in effluent media from isolated lungs perfused with PMN and PMA than in that of controls. These samples were collected from the same lungs used for experiments described in Figure 14. As shown in this figure, edema occurred in lungs perfused with PMA and PMN, but did not occur in lungs perfused with PMA or PMN alone. The amount of Tsz in effluent media from these lungs is illustrated in Figure 24A. Little TxBZ was released from lungs perfused only with PMA or with PMN. By contrast, substantially more TxB was released from lungs 2 perfused with both PMA and PMN. The amount of Tsz formed by these lungs was significantly greater than that of controls by 10 min of perfusion. The concentration of 6-keto-PGF1G produced by lungs described in the preceding paragraph is illustrated in Figure 248. In lungs perfused with PMN and/or PMA, 6-keto-PGF release was significantly greater than that of 1a lungs which were not perfused with PMN or PMA by 10 min of perfusion. In contrast to lungs perfused with PMN or PMA, 6-keto-PGF1G release increased with time in lungs perfused with PMN and PMA. However, the concentration of 6-keto-PGF1a in effluent samples from these lungs was not significantly greater than that of lungs perfused with PMA or PMN until 30 min of perfusion. 2. RelationshiLbetween prostanoid production and relative lung weight or increase in perfusion pressure To determine if relative lung weights or the increase in perfusion pressure were related to the release of arachidonic acid metabolites, Iexamined whether correlations existed between these indices of lung injury and prostanoid 138 Figure 24. Effect of PMA and neutrophils on prostanoid production by isolated, perfused lungs. Lungs were perfused with PMA or DMSO vehicle and with PMN or supernatant vehicle as described in the legend to Figure 14. Samples of effluent were collected at various times after the addition of PMA or DMSO to the perfusion medium reservoir and were analyzed for (A) Tx82 and (B) 6-Keto-PGF1a by radioimmunoassay. In some cases, error bars are obscured by points. a = Significantly different from all other treatment groups. b a Significantly different from lungs perfused with both vehicles. 139 A O n n — b - b b - q _ q q a - T“ 2 1 8 6 4 L Assxmcv mmxe L . . . . . J 2 D .+. . —Azn_ + + . +. . (IQ + . + 20 TIME (min) 1b Alsxmcoosuoaoumxm TIME (min) Figure 24 140 formation from lungs perfused with PMN and PMA or the respective vehicles. These relationships are depicted in Figure 25. A significant positive correlation existed between the amount of Tsz detected in medium from lungs perfused for 30 min with PMN and PMA or the respective vehicles and the relative lung weight (Figure 25A). The increase in perfusion pressure also correlated with the amount of Tsz produced after 30 min of perfusion (Figure ZSB). Relative lung weights and final increases in pressure also correlated with the amount of TxBZ produced after 10 min (r = 0.91 and 0.76, respectively) or 20 min (r = 0.94 and 0.88, respectively) of perfusion with PMA and PMN or the respective vehicles. Significant correlations existed between the amount of 6-keto- PGF1 a produced at 30 min of perfusion with PMN and PMA or the respective vehicles and both the relative weight and the final increase in perfusion pressure (Figures 25C and D, respectively). The amount of ('i-keto-PGF1ch produced after 20 min of perfusion also correlated with the relative lung weight (r=0.76) but did not correlate with the increase in pressure (r=0.67). Correlations did not exist in between either of these variables and the concentration of 6-keto-PGF1G medium collected after 10 min of perfusion (r = 0.46 and 0.39, respectively). There were no significant correlations between the ratio of TxBZ to 6-keto- PGF1 a calculated for data obtained at 10, 20 or 30 min and either the relative lung weight (r = 0.0, 0.17 and 0.24, respectively) or perfusion pressure (r = -0.09, 0.32 and 0.15, respectively). 3. Effect of indomethacin on pneumotoxicity a. Markers of Inniiniury To determine if a cyclooxygenase metabolite participated in edema formation in lungs perfused with PMN (1x108) and PMA (21 ng/ml), lungs were perfused with PMA, PMN and indomethacin (10 11M) or with PMN, PMA and indomethacin vehicle (100 ml EtOH). As indicated in Section I. part D, the volume 141 .zxm mafia Oman and: oomsunom mmcsa .mmamconnu cwmoHo “2m moan mannzm> Oman :uw3 pom: non mmcsa .mmamconnu ammo .c0nmsunom mo .92: on an conuonucmocoo ngnoumxlo unmanumw no :Onuocsn o no mononno> m>nuomnmmn on» no 23m can czm zunz COnmsunon no .c«& on nouns onsmmmnm conmounmm an onmonocn any can unmno3 0:5n m>nuonmm on .COHmsunmm no ans on no conumnucoocoo mea ucmsnumm no acnuocsm o no nonunnm> o>nuomammn can no 22m can 42m mafia codmsunom «0 .:n3 om nouns onsmmmnm scamsmnmm an mmomnocn Amy can unmno3 mafia manna—3mm 23 .3” can ma .3 monsmnm an nonwnomop mucmEHnonm Eonu nonnmuno one; onsOHM mncu an own: sumo .zzm no\oco «an mafia ommsunma mocsa an onsmmmnm :Onmsunwm an ommmno:w no Aoooa x 3m\34V usmno3 mafia o>wuonmn can counuoscona unocoumonm .303qu gnaw—noduoaom .mm one—mam «- 142 n_a\mco o.c - d a_uumouoxm o.~ ‘- n_e\mco mmxn D.— m.o mm mnsmflm (DH mu) annssead u: esoeaoul ..N— :v- .YD— ..m_ BJHSOBJd U! BODIJOUI a_sxmco a_uomosoxm o.v o.~ o.o “ l o q- Q ocfix o o 1..m.. o .vou ..m— .. ON A.s\mco mmxn m.o 0.0 u o D 00.0.; 1 3m— .DN ..mm 000! x A8/M1 000! x M8/A1 143 of ethanol used was not sufficient to induce an increase in perfusion pressure or relative weight of isolated lungs. Initial perfusion pressures and body weights of rats used as lung donors were not different between the two groups. Addition of indomethacin to the medium prevented the increase in relative weight and delayed the pressure increase induced by perfusion with PMN and PMA (Table 15). Two-thirds of the way through the perfusion, the inflow pressure of lungs co-perfused with indomethacin was significantly less than that of lungs perfused with PMN and PMA; however, final pressures were not different. b. Confirmation of inhibition of gclooxygenase Production of TxB and 6-keto-PGF by lungs perfused 2 10. with PMN and PMA and indomethacin or with PMN, PMA and indomethacin vehicle is depicted in Figure 26. In contrast to lungs which were perfused with indomethacin, prostanoid production increased with time in lungs perfused with PMN, PMA and the vehicle for indomethacin. The amount of TxB2 and 6-keto- PGF1 a produced by lungs which were not co-perfused with indomethacin was significantly greater than that of lungs which were perfused with indomethacin by 10 and 30 min of perfusion, respectively. As seen previously, indomethacin appeared to have a greater inhibitory effect on Tsz synthase than on prosta- cyclin synthase. 4. Effect of Dazmegrel on pneumotoxicity a. Markers of lung_injury To determine whether thromboxane mediated lung edema induced by PMN and PMA, lungs were perfused with PMA, PMN and either Dazmegrel (10 1.1M) or its vehicle as described in "METHODS". Like indometha- cin, Dazmegrel prevented the increase in relative lung weight and delayed the increase in perfusion pressure caused by PMN and PMA (Table 16). Two-thirds of 144 TABLE 15 EFFECT OF INDOMETHACIN ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment PMA/PMN PMA/PMN + Indomethacin Body Weight (9) 309.0 i 9.2 309.4 i 7.4 Initial Pressure 5.5 i 0.3 5.9 i 0.6 (mm Hg) Time of Perfusion 29.6 i 0.4 30.0 i 0 0 (min)a LW/BW x 103 b 23.6 i 6.8 6.0 + 0.5* * AP 20 min (mm Hg)c 13.6 i 1.4 8.8 i 1.1 AP final (mm Hg)d 7.0 i 1.4 12.2 i 1.4 Lungs were perfused with PMA (21 ng/ml), PMN (1 x 108) and indomethacin (10 uM) or with PMA, PMN and the vehicle for indomethacin (etha 01) in a paired manner as described in "Methods". Significantly different from PMA/PMN-perfused group. n=8 for each group. aPerfusions were terminated at 30 minutes or sooner if fluid became visible in the tracheal cannula. bRelative lung weight (LW/BW x 103) was determined when the perfusion was terminated. C APZO min is the difference between the pressure at 20 minutes and the pressure immediately before PMN and PMA were added to the reservoir. d APf nal is the difference between the pressure at the ‘termination. of each. perfusion and the pressure immediately before PMN and PMA were added to the reservoir. 145 Figure 26. Effect of indomethacin on prostanoid production by isolated rat lungs perfused with PMA and PMN. Samples of effluent medimm from lungs described in Table 15 were collected at various times after the addition of PMA and PMN to the perfusion medium and were analyzed for (A) Tx82 and (B) 6-Keto-PGF1 content by radioimmunoassay. In some cases, error inars are obscured by points. Closed circles, lungs perfused with PMA, PMN and indomethacin vehicle. Open circles, ungs perfused with PMA, PMN and indomethacin. Significantly different from lungs perfused with PMA, PMN and indomethacin. 146 Ansxmco mmxn TIME (min) Anexmcoonuomxm 30 2b TIME (min) 10 Om-r Figure 26 147 TABLE 16 EFFECT OF DAZMEGREL ON INJURY TO THE IPL MEDIATED BY PMA PLUS PMN Treatment PMA/PMN PMA/PMN + Dazmegrel Body Weight (9) 307.3 i 15.2 310.5 i 16.5 Initial Pressure 5.2 i 0.2 5.0 i 0.2 (mm Hg) Time of Perfusion 26.8 i 2.0 30.0 i 0 0 (min)a LW/BW x 103 b 20.2 i 6.8 6.1 i o 8* 1% AP 20 min (mm Hg)° 14.1 i 2.4 8.2 i o 4 AP final (mm Hg)d 14.3 i 2.6 9.6 i o 6 ungs were perfused with PMA (21 ng/ml), PMN (1 x 10 ) and Dazmegrel (10 uM) or with PMA, PMN and the vehicle for Dazmegrel (0.1 N EPOH) in a paired manner as described in "Methods". Significantly different from PMA/PMN-perfused group. n=6 for each group. aPerfusions were terminated at 30 minutes or sooner if fluid became visible in the tracheal cannula. bRelative lung weight (LW/BW x 103) was determined when the perfusion was terminated. c AP min is the difference between the pressure at 20 minutes and the pressure immediately before PMN and PMA were added to the reservoir. AP 'nal is the difference between the pressure at the 'termination. of each. perfusion and the pressure immediately before PMN and PMA were added to the reservoir. 148 the way through the perfusion, the inflow pressure of lungs perfused with Dazmegrel was significantly less than that of lungs perfused only with PMN and PMA; however, final pressures were not different. As indicated in this table, weights of rats used as lung donors and initial inflow pressures of lungs did not differ between the two groups. b. Confirmation of inhibition of thromboxane synthase As indicated in Figure 27A, effluent samples collected at 30 min from lungs perfused with Dazmegrel contained 90% less Tsz than samples from lungs which were perfused with the vehicle for this drug. Dazmegrel did not have an inhibitory effect on PGIZ synthase, for no statistical differences were noted between 6-keto-PGF a production at any time point in these lungs and in 1 lungs which were perfused with the vehicle for Dazmegrel (Figure 27B). 5. Effect of indomethacin and Dazmegrel on PMN function To determine if indomethacin or Dazmegrel affected the release of active oxygen metabolites from PMN, PMN which were incubated with indomethacin (10 1.1M), Dazmegrel (10 11M) or the respective vehicles were stimulated with various concentrations of PMA and release of 02- was assessed according to "METHODS". As shown in Figure 28, PMN which were incubated with indomethacin (Figure 28A) or Dazmegrel (Figure 288) produced as much 02- as PMN which were not incubated with these drugs. To assess whether superoxide production in the isolated lung was inhibited by Dazmegrel, lungs were perfused with cytochrome C, PMN, PMA and either Dazmegrel or Dazmegrel vehicle and synthesis of 02- was assessed according to "METHODS". As shown in Figure 29, production of 02‘ by PMA- stimulated PMN within the lung vasculature was not altered by co-perfusion with Dazmegrel. 149 Figure 27. Effect of Dazmegrel on prostanoid production by isolated rat lungs perfused with PMA and PMN. Samples of effluent medium from lungs described in Table 16 were collected at various times after the addition of PMA and PMN to the perfusion medium and were analyzed for (A) '1'sz and (B) 6-Keto—PGF1a by radioimmunoassay. In some cases, error bars are obscured by points. Closed circles, lungs perfused with. PMA, PMN and Dazmegrel vehicle: open circles, lungs perfused with PMA, PMN and Dazmegrel. Significantly different from lungs perfused with PMA, PMN and Dazmegrel. 150 Ansxmco mmxn ‘3 so 20 TIME (min) 10 4 1. ad .1 Ansxmcoo_uunxm n. my 3b 26 TIME (min) fig 1 Ifimme 27 151 Figure 28. Effect of indomethacin and Dazmegrel on PMN function in vitrg. PMN treated with (A) indomethacin or (B) Dazmegrel were stimulated with. various concentrations of PMA and 02' production was assessed according to "METHODS". Closed circles, 02' produced by PMN treated with the corresponding vehicles. Open circles, 02’ produced by PMN treated with (A) indomethacin or (B) Dazmegrel. n - 3 for each group. 152 > 25~ T 20-- I/ \2 15-- 4// 10" / r 5.- 2 10 20 2000 PMA (mg/ml) 02— (nmol/ml) 30‘- 25-- 4 l 15«- l 10.- 02— (nmol/ml) 2 10 20 2000 PMA (rig/ml) Figure 28 153 .moono 2060 now «u c .Honmoanoo 5:3 communmm manna .mmaonno :omo «gnaw—non, nuns communom nmcaa .mmnonno oomono .:maomew:: nous: omnanomop no INO non pauhnoso ono3 mug—non can» msonno> um oouomnaoo 33.60... ucooauuo no mmHg.—om .oHOncoc, nonmosuoo no nonmmauno no mocmmmnm can an 53 can .58 .O oeonnooucao 5:3 ~395an onw3 mmczq .mcsa oouonomn on» anions» ommsunom 22m Eonu .. o no omooHon so Honmosuoo no voouum .mm onsmnm 154 mm mnsmnm AEEV 0E; om ON or _ _ _ o .60 Q :m _ m/ a w i: A \ w... 0 < o :2 155 To determine if Dazmegrel was a H202 scavenger, a solution containing H202 (19 mM) was incubated with vehicle, Dazmegrel (10 11M) or catalase (1 U/ml) for 10 min, and the disappearance of H202 from the solution was determined spectrophotometrically. As shown in Table 17, in contrast to samples containing catalase (l U/ml), H202 did not disappear in samples containing Dazmegrel or Dazmegrel vehicle. G. Source of TxB and 6-keto-PGF 2 in Lungs Perfused with PMN and PMA lo 1. Effect of SOD on synthesis of TxB and 6-keto-PGF 2 As stated in "METHODS", samples of medium from lungs which 16. were perfused with PMA, with PMN and PMA, or with PMN, PMA and SOD (as described in Table 12) were collected and subsequently analyzed for prostanoid content. The amount of Tsz and 6-keto-PGF1a produced by these lungs is illustrated in Figure 30. By 10 min of perfusion, a significantly greater amount of Tsz was produced by lungs perfused with PMA, PMN and SOD than by ltmgs perfused with PMA alone. Synthesis of Tsz by lungs perfused with PMA and PMN was also greater than that of lungs perfused with PMA by 10 min; however, it was not significantly greater than that produced by lungs co-perfused with SOD until 30 min (Figure 30A). In contrast, the amount of 6-keto-PGF produced by lungs 10. perfused with PMA, PMN and SOD did not differ from that of lungs perfused with only PMA at any time point (Figure 30B). By 20 min of perfusion, synthesis of 6- keto-PGF a by lungs perfused with PMA and PMN was significantly greater than 1 that of lungs co-perfused with SOD or with PMA in the absence of PMN. 2. Effect of catalase on mthesis of TxB and 6-keto-PGF 2 The amount of Tsz and 6-keto-PGF1a perfused with PMA, with PMA and PMN, or with PMA, PMN and catalase is 10. produced by lungs 156 TABLE 17 EFFECT or DAZMEGREL ON H202 Treatment H202 scavenged (mM) at Catalase 3.90 i 0.25 Vehicle 0.00 i 0.04 Dazmegrel - 0.02 i 0.05 Samples containing H202 (19 mM) and Catalase (1 U/ml), Dazmegrel (10 uM) or Dazmegrel vehicle were placed in a spectrophotometer and the absorbance of H O at 240 nanometers was assessed. The concentration of 31202 immediately and 10 min after. the addition of these substances to the samples was determined according to "METHODS". The amount of H 02 scavenged is the difference between the concentragaons obtained at the two different time points. Significantly different from vehicle-treated samples. n = 3-4 for each group. 157 Figure 30. Effect of 02' on prostanoid production by lungs perfused with PMN and PMA. Synthesis of (A) TxB and (B) 6-Keto-PGF a from lungs described in Table 13 was determined as escribed under "METHODS". Circles, lungs perfused with PMA and PMN. Triangles, lungs perfused with PMA, PMN and SOD. Inverted triangles, lungs perfused with PMA alone. In some cases, error bars are obscured by symbols. a = Significantly different from lungs perfused with PMA. b = Significantly different from lungs perfused with PMA, PMN and SOD. 158 O \l/ .m o O m 1 0 "All i%( 6 TH O 16 Vim T l l l 1. o. 5. o. 5 o A_E\©5 mmvfi 12.0" ob 0 TI .r n u l o. o. o. o. o. O 8 6 4. 2 9885 Econ 88. c If. I 20 Time (min) AT “ 0. 0 30 :0 Figure 30 159 illustrated in Figure 31. The effect of catalase on TxBZ and 6-keto-PGFh1 production was similar to that of SOD. By 10 min of perfusion, a significantly greater amount of Tsz was produced by lungs perfused with PMA and PMN in the absence or presence of catalase than by lungs perfused only with PMA (Figure 31A). The amount of Tsz produced by lungs perfused with PMN and PMA was not significantly greater than that of lungs co-perfused with catalase until 30 min of perfusion. The amount of 6-keto-PGF10L produced by these lungs also was not significantly greater than that produced by lungs co-perfused with catalase or perfused with only PMA until 30 min of perfusion (Figure 31B). As in lungs perfused with PMA, PMN and SOD, lungs which were perfused with PMA, PMN and catalase did not release greater concentrations of 6-keto-PGF1a than lungs perfused only with PM A. 3. Prostanoid production from PMA-stimulated PMN When circulated through the perfusion apparatus at 37°C, PMN which were stimulated with PMA (21 ng/ml) or DMSO produced prostanoids (Figure 32). Although Tsz production by PMA stimulated PMN increased with respect to time, by 30 min of circulation the amount of Tsz produced by these cells was not significantly different from unstimulated cells (Figure 32A). In contrast, by 30 min of circulation a significantly greater amount of 6-keto-PGF1a was produced by PMN stimulated with PMA than by unstimulated PMN (Figure 323). 4. Experiments with aspirin pretreatment of lungs or PMN a. Preliminary experiments in isolated lung_s_ 1) Confirmation of ASA washout from lungg. As stated 6 in "METHODS", lungs from rats which had been treated with 1‘I'C-aspirin (2.8x10 dpm; 100 mg/kg, p.o.) were isolated and perfused in a single-pass manner for 10, 15 and 20 min to determine the length of time needed to wash ASA out of the 160 Figure 31. Effect of H202 on prostanoid production by lungs perfused with PMA and PMN. Synthesis of (A) TxB and (B) 6-Keto-PGF a from lungs described in Table 1% was determined as escribed under "METHODS". Circles, lungs perfused with PMA and PMN. Triangles, lungs perfused with PMA, PMN and catalase. Inverted triangles, lungs perfused with PMA alone. In some cases, error bars are obscured by symbols. a = Significantly different from lungs perfused with PMA. b - Significantly different from lungs perfused with PMA, PMN and catalase. 161 10 .0 o o T) O l 7%.. v o TOT .v O A? V . .r) m l .6 _ n J. 0 5 0 5 0 5 O 6 2 o. 7. 5 2 o. 1 1. 0 0 O 0 2.6.6 cv NmXH 30 20 Time (min) B 12.0-5 ob l l l . l o. o. o. o. o. O 8 5 4. 2 9885 2.8.“. Box 6 Q “ O. 0 30 20 Time (min) 0 Figure 31 162 Figure 32. Effect of PMA on prostanoid production by isolated PMN. Medium containing PMN and PMA or PMN and DMSO vehicle was circulated through the perfusion apparatus and samples of effluent medium were analyzed for (A) TxB and (B) 6-Keto-PGF a as described in "METHODS". ungs were not inclu ed in the circuit. Closed circles, medium containing PMN and PMA. Open circles, medium containing PMN and DMSO vehicle. In some cases, error bars are obscured by points. n - 9 for each group. *Significantly different from. medium containing PMN and DMSO. 163 111.1315 1 w \./ .n Till. no m .IGII I. 2 ( e m“ .Tllm..||.f. i O s~ Au :0 no .3 no 2 2 l 1.. 0. 0. A_E\ocV meh 4 nm “ .0 no ‘2 no . 1|" I I CID nu my coins Econ. 23. c n~ no 20 30 Time (min) 10 Figure 32 164 lungs. The amount of radioactivity which remained in the medium after 10, 15 and 20 min of perfusion through the lungs was approximately 50, 41 and 41 dpm/ml, respectively. Background radioactivity was 28 dpm/ml. If lungs had been perfused in a single-pass manner for 25 or 30 min, perhaps more ASA would have been washed from the vasculature. However, to cut down on perfusion time, a washout period of 20 min was chosen for use in subsequent experiments. Based on the amount of radioactivity detected in the perfusion medium and in the gavage solution, the amount of ASA which remained in the lung vasculature was calculated. After a 20-min washout period, approximately 1 11M ASA was detected in the perfusion medium. 2) Confirmation of cyclooxygenase inhibition in lung_s_. To determine if pretreatment of rats with 100 11M ASA (p.o.) 1 hr prior to lung isolation inhibited cyclooxygenase, lungs from aspirin or vehicle-pretreated rats were infused with AA (80 pM) as described in "METHODS", and samples of effluent medium were collected for analysis of Tsz. As indicated in Figure 33, after 5 min of infusion with AA, approximately 90% less Tsz was produced by lungs from ASA-pretreated rats than by lungs from vehicle-treated rats. Based on this result, it was concluded that the chosen treatment regimen inhibited cyclooxygenase. b. Preliminary experiments with PMN 1) Confirmation of ASA washout from PMN. As stated in "METHODS", washout of ASA from PMN was determined by counting the radioactivity which remained in the SN after cells which had been spiked with 14C-ASA (100 1.1M, 6.6x104 dpm/ml) were washed. After one wash with 50 ml PBS, approximately 77 dpm/ml remained in the SN. However, after two washes, the amount of radioactivity detected in the supernatant fluid (23 dpm/ml) was approximately equal to background (22 dpm/ml). Based on this result, aspirin or 165 20» e 1 a * E 15“ o//1 c J V N 10-- CD X 1— 5__ T ‘ o O (EB/Q 9 1 o 1 2 3 51 ‘ Time (min) Figure 33. Effect of aspirin pretreatment on synthesis of thromboxane from isolated lungs. Lungs from rats pretreated with ASA or with ASA vehicle were infused with AA and synthesis of Tsz was assessed as described under "METHODS". Closed circles, lungs from rats pretreated with ASA vehicle; open circles, lungs from rats pretreated with ASA. In some cases, error bars are obscured by points. 11 - 3 for each group. Significantly different from lungs of rats pretreated with ASA. 166 vehicle-treated PMN were washed twice with 50 ml PBS before being used in studies. 2) Confirmation of cyclooxygenase inhibition in PMN. To determine if pretreatment of PMN with 100 11M ASA inhibited cyclooxygenase, ASA or vehicle-pretreated PMN were incubated for 30 min at 37°C with AA (100 1.1M) as described in "METHODS". As seen in Figure 34, after 30 min of incubation with 100 1.1M AA, approximately 95% less TxBZ was released by ASA-pretreated PMN than by vehicle-pretreated PMN. These results indicate that the aspirin pretreatment protocol almost totally blocked synthesis of thromboxane by PMN. To determine if cyclooxygenase was still inhibited in ASA-pretreated PMN which were used at the end of an experimental day, ASA- and vehicle-pretreated PMN which were not used in experiments were saved and AA-induced synthesis of '1‘sz and 6-keto-PGF1a by these cells was assessed. As shown in Table 18, synthesis of Tsz and 6-keto-PGF1a was inhibited by greater than 94% and 87%, respectively, in cells which had been pretreated with ASA several hours prior to being used for the last perfusion on a given day. 3) Effect of ASA which remained in the lung vascula- ture after washout on prostanoid production by PMN. As indicated above, approximately 1 11M ASA remained in the lung vasculature after a 20-min washout period. As shown in Figure 35, TxBZ synthesis by AA-stimulated PMN was not inhibited by co-incubation with l 1.1M ASA. These results suggest that although a small amount of ASA remained free in the vasculature after a 20-min washout period, the amount which remained was not sufficient to inhibit Tsz synthesis by PMN. 4) Effect of ASA pretreatment on PMN function. To determine if ASA pretreatment affected the release of active oxygen metabolites from PMN, vehicle or ASA-pretreated PMN were stimulated with PMA (21 ng/ml) 167 TXBZ (“Q/ml) 20 -— t . q- 1 5 " \ I * 1 ‘ i 10" 5-- O Q Q o O 10 20 30 T1 m e (m i n ) Figure 34. Effect of aspirin pretreatment on thromboxane synthesis by isolated neutrophils. PMN pretreated with ASA or ASA vehicle were stimulated with AA and synthesis of TxB was determined as described under "METHODS". Closed circles, Tx82 synthesized from vehicle-pretreated PMN; open circles, Tsz synthesized from ASA-pretreated PMN. In some cases, error bars are gbscured by points. 11 - 4 for each group. Significantly different from PMN pretreated with ASA. 168 TABLE 18 EFFECT OF ASPIRIN PRETREATMENT ON PRODUCTION OF PROSTANOIDS BY NEUTROPHILS USED IN EXPERIMENTS Treatment Prostanoid ASA vehicle ASA szz (ng/ml) 18.36 i 4.29 1.05 i 0.21* 6-Keto-PGF1a (ng/ml) 1.87 i 0.44 0.24 i 0.04* At the end of an experimental day, vehicle or ASA- pretreated. PMN were incubated. with .AA (100 uM) as described in "METHODS" and.*synthesis of TxB and 6-Keto-PGF a was assessed. Significantly dif erent from PMN which were pretreated with ASA vehicle. 169 TxB2 (rig/ml) 50-— 40-- 30-- ——- I __ \+ 1 1 9 20-- 10~- O 1 z 1 0 10 20 30 Time (min) Figure 35. Effect of the concentration of aspirin which remains in aspirin-pretreated lungs on thromboxane synthesis by isolated neutrophils. PMN incubated with ASA (1 uM) or ASA vehicle were stimulated with AA (80 uM) and synthesis of TxB was assessed as described under "METHODS". Closed circles, Tsz synthesized by vehicle-treated PMN. Open circles, Tx32 synthesized by ASA-treated PMN. 170 and release of 02- was assessed according to "METHODS". As shown in Figure 36, the ability of PMN to produce O 2 was not inhibited by pretreatment of PMN with ASA. c. Effect of aspirin pretreatment of lungs or PMN on PMA pneumotoxicity Lungs from rats pretreated with ASA or ASA vehicle were perfused with PMA (21 ng/ml), with PMA and vehicle-pretreated PMN (1x108) or with PMA and ASA-pretreated PMN according to "METHODS". The effect of these pretreatments on pneumotoxicity induced by PMN and PMA is illustrated in Figure 37. As shown in this figure, increases in relative lung weight and albumin content of BAL produced by perfusion with PMN and PMA were attenuated by pretreatment of either the PMN or the lung donors with ASA. Relative lung weights and albumin content of BAL of these lungs were not significantly different from lungs of vehicle-pretreated rats which were perfused only with PMA. They were also not different from those of aspirin-pretreated lungs perfused with PMN and PMA vehicle (DMSO) or of lungs perfused with ASA- pretreated PMN and DMSO (Table 19). In contrast, the perfusion pressures of these lungs were significantly greater than that of lungs from vehicle-pretreated rats which were perfused only with PMA (Figure 37C) or of the corresponding lungs perfused with PMN and DMSO (Table 19). The use of both ASA-pretreated PMN and lungs from ASA-pretreated rats in the experiment did not lead to a further reduction in relative lung weight, albumin content of lavage or perfusion pressure (data not shown). Respectively, these values were 5.73:0.74, 6.013.? mg and 9510.8 mmHg for these lungs. 171 15.0-- [:1 V W ASA c: i E 1 > 10.0-- (D E CI V I 51)" CV 0 Oil Figure 36. Effect of aspirin pretreatment on production of active oxygen metabolites from PMN. PMN pretreated with either ASA or ASA vehicle were stimulated with PMA for 30 min. and synthesis of 02" was determined as described under "METHODS". Open bar, 02' produced by PMN pretreated with ASA vehicle. Hatched bar, 02' produced by PMN pretreated with ASA. 172 Figure 37. Effect of aspirin pretreatment of lungs or neutrophils on (A) relative lung weight (LW/BW x 103), (B) albumin content of lavage fluid and (C) increase in perfusion pressure of isolated rat lungs perfused with PMA. Lungs from rats pretreated with ASA or vehicle were perfused with PMA in the presence or absence of PMN pretreated with ASA or ASA vehicle according to "METHODS”. a = Significantly different from vehicle- pretreated lungs perfused with vehicle-pretreated PMN and PMA. b = Significantly different from vehicle- pretreated lungs perfused with only PMA. 173 O 0 2 0 .00 was n 9 x 2m}: 0.0 V LUNG: V PMN: NONE B ‘1 60.0 50.0 . 0 . . 0 m w. m m 3.5 5632 so: 866.. V LUNG: V ASA PMN: NONE V r» 15.0 “- Aa:_tso 053mmwun— C_ ”mowing:— V PMN: NONE V LUNG: Figure 37 174 TABLE 19 EFFECT OF ASPIRIN ON LUNG INJURY INDUCED BY PMN/PMA 3 Treatment LW/BW x 10 AP (mm Hg) Albumin (mg) a a a PMA/PMN 14.91 i 5.48 13.1 i 2.2 38.0 i 24.0 DMSO/PMN 4.47 i 0.21 0.4 i 0.2 2 l i 0 5 a PMA/ASA PMN 5.42 i 0.39 8.9 i 0.7 2 6 + o 8 DMSO/ASA PMN 4.44 i 0.07 0.8 i 0.4 o 9 i 0 3 PMA/PMN a ASA LUNGS 5.63 i 0.35 10.8 i 0.6 3 4 i 0 8 DMSO/PMN 5.19 i 0.27 0.9 i 0 2 4 0 i o 6 ASA LUNGS a PMA 4.67 i 0.12 6.2 i 0.7 2 5 i 0 6 DMSO 4.32 i 0.15 0.0 i 0.1 1 8 i 0.2 a = significantly different from corresponding DMSO- perfused group 175 (1. Effect of aspirin pretreatment of lungs and/or PMN on prostanoid production 1) TxB2 sy_nthesis. TxBZ synthesis from lungs described in Figure 37 is illustrated in Figure 38A. When cyclooxygenase from either the lungs or PMN was inhibited (inverted triangles and triangles, respectively), the amount of Tsz produced by lungs perfused with PMN and PMA was approximate- ly equal to that produced by lungs perfused with only PMA (diamonds). By five minutes of perfusion, the amount of Tsz produced by all of the lungs mentioned above was significantly greater than that produced by preparations in which both lung and PMN cyclooxygenase were inhibited (squares) and significantly less than that produced by preparations in which neither lung nor PMN cyclooxygenase was inhibited (circles). At any particular time point (other than 30 min), the amount of TxB2 produced by these lungs (circles) was approximately equal to the sum of the amount formed by preparations in which either lung (inverted triangles) or PMN cyclooxygenase (triangles) was inhibited. The amount of '1'sz produced by corresponding DMSO controls is depicted in Figure 39. TxB synthesis from PMA-treated lungs 2 is also illustrated in this figure for comparative purposes. At all time points, synthesis of Tsz from lungs perfused with PMN and DMSO was attenuated by pretreating the PMN, but not the lung donors with ASA. By contrast, synthesis of Tsz from lungs perfused with PMN and PMA was attenuated by either pretreat- ment of PMN or lung donors with ASA. By 20 min of perfusion (Figure 39B), synthesis of TxB from lungs which were perfused with PMA and ASA-pretreated PMN (bar 2 with downsloping line) was greater than that of the corresponding lungs which were perfused with DMSO. Synthesis from lungs of rats perfused with PMA and vehicle-pretreated PMN (black bars) was not significantly different from the 176 Figure 38. Effect of aspirin pretreatment of the lungs and/or PMN on prostanoid production from isolated lungs. Synthesis of (A) TxB2 and (B) 6-Keto-PGF1 from lungs described in Figure 37 was assesse by radioimmunoassay as described under "METHODS". Circles, lungs from.*vehicle-pretreated. rats ‘perfused with vehicle-pretreated PMN and PMA. Inverted triangles, lungs from ASA-pretreated rats perfused with vehicle-pretreated PMN and PMA. Triangles, lungs from vehicle-pretreated rats perfused ‘with ASA-pretreated PMN and PMA. Diamonds, lungs from vehicle-pretreated rats perfused with. PMA. Squares, lungs from ASA- pretreated rats perfused with ASA-pretreated PMN and PMA. In some cases, error bars are obscured by symbols. *Significantly different from all other treatment groups at this and subsequent time points. 1.5-- 1.0-- Tx82 (ng/ml) 0.0 -- 10.0-- 8.0~- 6 Keto PCFIO (ng/ml) 177 [ Lung PMN v v r 1. .L * L ;, ASA v ‘ f- 5—4—5 t:i V ASA _ 5 i V NONE ————. F a ASA ASA 10 20 30 Time (min) 10 20 30 Time (min) Figure 38 178 Figure 39. Effect of cyclooxygenase inhibition of lungs or PMN on TxB production from lungs perfused with PMA or with OH O vehicle. Lungs from ASA- or vehicle-pretreated rats were perfused with ASA- or vehicle-pretreated PMN and DMSO vehicle as described in "METHODS" and samples of medium collected at (A) 10, (B) 20 and (C) 30 min were analyzed for Tsz. Data from corresponding PMA-perfused lungs, which were described in Figure 38, are included for comparative purposes. Note that the scales in graphs A, B and C differ from each other. Open bars, lungs from vehicle pretreated rats. Solid bars, lungs from vehicle- pretreated rats perfused with vehicle-pretreated PMN. Downsloping hatched bars, lungs from vehicle-pretreated rats perfused with ASA-pretreated PMN. Upsloping hatched bars, lungs from ASA-pretreated rats perfused with vehicle-pretreated PMN. a 8 Significantly different from vehicle-pretreated lungs perfused with vehicle-pretreated PMN and PMA. b ,=- Significantly different from corresponding DMSO control. c = Significantly different from vehicle-pretreated lungs perfused with vehicle-pretreated PMN and DMSO. 179 A é_ 0 1E_ C” n. VAV n VVVA w A Eu.” 01% 0.5 -r 0.4 .- 9685 men DMSO PMA B 0.75 -- 0 O A_E\@cv mm: 0.00 DMSO PMA nlié DMSO 1% 1.75 -- s. 0 s. 0 .2 0 ;J 5 no 0 1.50 ‘1' 0 25 0.00 eras we; PMA Figure 3 9 180 corresponding DMSO controls until 30 min of perfusion (Figure 39C). No significant differences were noted between Tsz synthesis from lungs perfused with only PMA or DMSO (white bars) or between synthesis from ASA-pretreated rats perfused with PMN and PMA or with PMN and DMSO (bars with upsloping hatched lines) at any time point. 2) 6-Keto-PGF1a synthesis. 6-Keto-PGF1G synthesis from lungs described in Figure 37 is illustrated in Figure 38B. Since the amount of 6-keto-PGF1a produced by preparations in which both lung and PMN cyclooxy- genase was inhibited was equal to that produced by preparations in which either PMN or lung cyclooxygenase was inhibited, data from these lungs are not included in the graph. By 30 min of perfusion, synthesis of 6-keto-PGF1a from preparations in which neither lung nor PMN cyclooxygenase was blocked (circles) was significantly greater than that of all other lungs (Figure 38B). 6- keto-PGFla synthesis was attenuated when cyclooxygenase from PMN was blocked (triangles) and almost totally inhibited when cyclooxygenase from the lungs was blocked (inverted triangles). The amount of 6-keto-PGF1a produced by corre- sponding DMSO controls is depicted in Figure 40. For comparative purposes, 6- lteto-PGIF1 a synthesis from PMA-treated lungs is also illustrated in this figure. As in lungs perfused with PMN and PMA, 6-keto-PGF1 a synthesis from lungs perfused with PMN and DMSO was attenuated when lung cyclooxygenase was inhibited (bars with upsloping lines). However, in contrast to lungs perfused with PMN and PMA, by 30 min, synthesis from lungs perfused with PMN and DMSO was not attenuated when PMN cyclooxygenase was inhibited (bars with downsloping lines). 181 Figure 40. Effect of cyclooxygenase inhibition of lungs or PMN on 6-Keto—PGF production from lungs perfused with PMA or with D 0 vehicle. Lungs from ASA- or vehicle-pretreated rats were perfused with ASA- or vehicle-pretreated PMN and DMSO vehicle as described in "METHODS" and samples of effluent medium collected at (A) 10, (B) 20 and (C) 30 min were analyzed for 6- Keto-PGF a' Data from corresponding PMA—perfused lungs, which were described in Figure 38, are included for comparative purposes. Note that the scales in graphs A, B and C differ from each other. Open bars, lungs from vehicle pretreated rats. Solid bars, lungs from vehicle-pretreated rats perfused with vehicle- pretreated PMN. Downsloping hatched bars, lungs from vehicle-pretreated rats perfused with ASA-pretreated PMN. Upsloping hatched bars, lungs from ASA-pretreated rats perfused with vehicle-pretreated PMN. a = Significantly different from vehicle-pretreated lungs perfused with vehicle-pretreated PMN and PMA. b = Significantly different from corresponding DMSO control. c =2 Significantly different from vehicle- pretreated lungs perfused with vehicle-pretreated PMN and DMSO. d = Significantly less than that of all other lungs perfused with PMA. e a Significantly less than that of all other lungs perfused with DMSO. 182 11 0 0 1 S e . 111g M S . 1 m N e A M mVSV D. n A A n 3 MT. 1.. V‘ I an n w b M . _.h...\.\kh\kh\.\.\h1htwhhw.hb W a r P .3 . -1111 C“ .D .1111 PM :.3 1“ no 7“. .H. Q My :.c 3.. .u 7.. O n.U n.u n.u nuu AW 0 0 0 0 0 0 0 0 .1. C. 0. 0. 0 0 0. Am 6. 4. Z 0. o. A 2.355 nod 28. c 8 9885 Sued 28. c c acids Sued 28. 8 DMSO Figure 40 P MA 183 At all time points, lungs perfused with PMA in the absence or presence of vehicle-pretreated PMN produced more 6-keto-PGF1a than the corresponding DMSO controls (white and black bars, respectively). In contrast, when either PMN or lung cyclooxygenase was inhibited, the amount of 6- keto--PGF1 (1 produced by lungs perfused with PMN and PMA was not significantly different from that of the corresponding DMSO controls (bars with downsloping and upsloping lines, respectively). DISCUSSION I. Ability of PMA to Produce PMN-Independent or -Dependent LungInjurl Results of studies reported in this thesis indicate that PMA produced injury to the isolated, perfused rat lung whether or not PMN were added to the perfusion medium. The ability of PMA to produce toxicity in the absence of added PMN was concentration-dependent. When perfused with medium containing only a high concentration of PMA (3 57 ng/ml), lungs exhibited a large increase in pressure, edema and vascular injury. At low concentrations (< 28 ng/ml), PMA induced a smaller increase in pressure which was not accompanied by an increase in lung weight. However, when lungs were co-perfused with rat PMN and a concentration of PMA which did not by itself produce an increase in relative lung weight, perfusion pressure increased markedly and fluid accumulation and morphologic changes in endothelial cells occurred. These results suggest that at low concentrations PMA produces PMN-dependent lung injury, whereas at high concentrations, PMA produces PMN-independent lung injury. The results obtained in the isolated rat lung system with a low dose of PMA and rat PMN were similar to those obtained in isolated rat or rabbit lungs perfused with PMA and human PMN (Shasby g 1.1., 1982; Jackson e_t_ a_l., 1986; Ismail at al., 1987; McDonald gt Q” 1987). In these studies, edema developed in lungs perfused with both PMA and PMN but did not develop in lungs perfused only with PMA. However, the concentration of PMA which was used in many of these studies was greater than that which produced PMN-independent lung injury in my system. Rabbit lungs have been perfused for 30 min with KRBSA containing as 184 185 much as 120 ng/ml without developing edema in the absence of PMN. In my isolated rat lung system a concentration of 120 ng/ml PMA induced rapid fluid accumulation. One possible reason for the differing results is that the threshold for toxicity may differ among species. However, Ismail and coworkers (1987) perfused lungs from Long-Evans rats at 6-9 ml/min for 40 min with buffer containing 50 ng/ml PMA and did not find any evidence of lung injury. In my studies, when perfused at 10 ml/min for 30 min with buffer containing 57 ng/ml PMA, lungs from Sprague-Dawley rats became extremely edematous. The reason for the differing results is not clear at this time. However, in the two studies, different rates of flow, as well as different osmotic stabilizers, were used. Rats of different strain were also used. Furthermore, in the study by Ismail §_t_ 31. (1987), lungs were co-perfused with a protein which is not normally present within the vasculature (cytochrome C). Thus, it is possible that the threshold concentra- tion for toxicity'in the absence of perfused PMN may depend on the rate of perfusion, binding of the toxicant to perfusion medium constituents, or on other factors. As shown in Section II below, edema induced by PMA is attenuated by a drug which decreases perfusion pressure. Since pressure is proportional to flow, it may very well be that development of edema in this model is dependent on the rate of perfusion. Experiments using various flows may help determine whether the ability of PMA to produce PMN-independent lung injury is dependent on flow as well as concentration. The results obtained with a high concentration of PMA are similar to those obtained in normal and in neutrophil-depleted sheep (Dyer and Snapper, 1986). In this study, neutrophil depletion did not protect sheep from lung injury induced by PMA. Since PMA treatment led to an increase in lung lymph flow and a decrease in the lymph/plasma ratio in normal and in neutrophil-depleted sheep, PMA appa- rently produced leaky vessels by increasing hydrostatic pressure, not by increasing 186 vascular permeability. Since perfusion pressure increased substantially in isolated rat lungs perfused with a high concentration of PMA in the absence of PMN, fluid accumulation in this preparation may also have been produced by an increase in pressure. However, since endothelial cells appeared to be injured in these lungs, it is also possible that an increase in vascular permeability could have contributed to the development of edema. Because the index of injury which was used in my studies only measured bulk fluid flux, I could not determine the extent to which an increase in vascular permeability and/or an increase in pressure contributed to the development of edema in this preparation. Although the role of the increase in pressure in edema development is addressed in Section II below, experiments to examine the possibility that an increase in vascular permeability was responsible for fluid accumulation were not performed. Additional studies should be performed to address this question. As stated above, both in rat lungs perfused with a high concentration of PMA in the absence of PMN and in lungs perfused with rat PMN and a low concentration of PMA, there was morphologic evidence of altered endothelial cell integrity. This also occurs in rats or rabbits after i.v. or i.t. administration of PMA (Johnson and Ward, 1982; Taylor gt a_l., 1985) and in isolated rat lungs perfused with PMA and human PMN (Ismail gt a_l., 1987). PMA-stimulated PMN also produce injury to cultured endothelial cells (Martin, 1984). Because of this, one might think that metabolic functions of endothelial cells would be compro- mised. Indeed, clearance of 5HT is impaired in rabbits 2 hours after the intratracheal instillation of a pneumotoxic dose of PMA (Havill g: a_l., 1986). One hour after intravenous injection of PMA, rabbits also exhibit decreases in the transpulmonary metabolism of substrates for ACE and 5'-nucleotidase (McCor- mick 3311., 1987). 187 In contrast, results of studies reported in this thesis indicate that disposition of 5HT by rat lungs perfused with PMA and rat PMN is not impaired. Presently, the basis for the differences in results cannot be discerned with certainty. However, when endothelial cells are extremely injured, uptake of 5HT by the cells may occur independently of a carrier-mediated mechanism (J.P. Catravas, perso- nal communication). Perhaps this is what occurred in my system. Thus, to determine if metabolic functions of endothelial cells were indeed compromised in lungs perfused with PMN and PMA, experiments with other substances which are metabolized or removed by endothelial cells (i.e., ATI, AMP, or NE) should be performed. Prior to beginning any experimental manipulation with the isolated lungs, each preparation was perfused for several minutes to clear the vasculature of blood. However, since lungs E 35in contain a large pool of marginated PMN (Cohen and Rossi, 1983), it is possible that some PMN remained sequestered in the vasculature after the washout period. If so, the possibility exists that injury produced by toxic concentrations of PMA may have been mediated by these sequestered PMN. However, as seen below, the mechanisms of toxicity of PMA in the absence or presence of perfused PMN appear to be quite different. Thus, it seems unlikely that PMN which remained in the lungs were responsible for toxicity induced by a high concentration of PMA. In conclusion, experiments in the isolated, perfused rat lung indicate that a high concentration of PMA produces PMN-independent lung injury, whereas a low concentration of PMA requires PMN to produce toxicity. Prior to these experiments, the ability of PMA to produce both neutrophil-dependent and -independent injury to the same preparation after the same route of administra- tion had not been reported. Previous studies have demonstrated that PMN are required for intravenously administered PMA to produce toxicity to the rabbit 188 (Shasby gt 11., 1982) but not to the sheep (Dyer and Snapper, 1986). PMN are also not required for PMA to produce lung injury in rats or rabbits after intratracheal administration (Johnson and Ward, 1982; Schaufstatter _e_t 92°: 1984; Pitt _e__t_ al., 1987). Possible explanations for the contrasting results are that the mechanism of PMA toxicity may be dependent on the route of administration or on the species used. However, results obtained in my isolated rat lung preparation provide an alternate explanation; they suggest that, depending on the concentration used, PMA can produce either PMN-dependent or PMN-independent lung injury. Thus, the nature of the injury obtained in previous studies may have been influenced by the concentration of PMA which was used in addition to or in lieu of other factors. 11. Mechanism of PMN-Independent Injury A. Lack of Effect of SOD and Catalase Results of the study with SOD and catalase suggest that toxicity mediated by a high concentration of PMA in the absence of PMN is not mediated by intravascularly generated active oxygen species. However, because I did not demonstrate that the concentrations of SOD and catalase which were used effectively scavenged oxygen radicals which may have been generated in this system, it might be argued that active oxygen species were still present in the vasculature of lungs co-perfused with SOD and catalase. However, as noted in Section III below, the concentrations of enzymes which were used in these experiments protected lungs against pneumotoxicity caused by perfusion with PMN and PMA. The concentration of SOD which was used also scavenged 02- which was produced by PMA-stimulated PMN which were circulated through the perfusion apparatus. Furthermore, the amount of SOD used in the lungs was an order of magnitude greater than that which effectively scavenged 02" produced 189 i_n_ 11:32 by PMN stimulated with 57 ng/ml PMA (personal observation). Thus, it is likely that the concentrations of enzymes which were used in this study scavenged active oxygen species which were generated within the vasculature of lungs perfused with a pneumotoxic concentration of PMA. Although the results of this study suggest that intravascularly gene- rated active oxygen metabolites are not involved in toxicity produced by a high concentration of PMA in the absence of PMN, they do not totally rule out the possibility that active oxygen species participate in the toxic response. As stated previously, PMA can stimulate macrophages and endothelial cells i_n_ 23332 to release 02" (Hoidal gt a_l., 1978; Mastubara and Ziff, 1986). Therefore, in lungs perfused with PMA, it is possible that active oxygen species were generated from these cells. By being perfused through the vasculature, SOD and catalase may not have had access to oxidants being produced by macrophages or on the basement membrane side of endothelial cells. Thus, the possibility exists that oxidants produced by these cells mediated injury in this model. To determine if macrophages participated in edema development, experiments utilizing lungs from macrophage-depleted animals should be performed. B. Involvement of a Cyclooxygenase Metabolite(s) When perfused with a high concentration of PMA in the absence of PMN, lungs produced greater amounts of '1‘sz and 6-keto-PGFm than controls. Furthermore, the fact that relative weight and perfusion pressure correlated with the amount of Tsz produced by 30 min of perfusion suggested that 'I‘xA2 might participate in the development of edema or may be produced as a result of lung injury. TO determine if T832 was involved in the pathogenesis of lung injury induced by a high concentration of PMA, lungs were perfused with PMA and either indomethacin or Dazmegrel. Results of theses studies suggest that thromboxane 190 contributed to injury induced by PMA in the absence of PMN. However, although Dazmegrel and indomethacin prolonged perfusions and attenuated increases in perfusion pressure and relative weight, they did not protect lungs entirely from injury. These data suggest that thromboxane contributes to or hastens the formation of edema in lungs perfused with a high concentration of PMA (in absence of added PMN) but that other factors also contribute to edema formation. If TxAZ was the only cyclooxygenase metabolite involved in toxicity induced by a high concentration of PMA, one might expect that Dazmegrel and indomethacin would afford a similar degree of protection against toxicity. However, relative weights of lungs co-perfused with indomethacin appeared to be less than those of lungs co-perfused with Dazmegrel. These data suggest that a cyclooxygenase metabolite(s) other than TxA may be involved in the pathogene- 2 sis of PMA toxicity. Since the perfusion pressure increase was also attenuated to a greater degree in lungs perfused with indomethacin than in those perfused with Dazmegrel, it is possible that a vasoconstrictive prostanoid, such as PGF , may 20 participate in edema development. Additional studies utilizing a specific receptor antagonist for PGFZQ may help determine the role of this prostanoid in lung injury in this model. Since the index of edema which was used in this study only measured bulk flow of fluid, it is unknown whether thromboxane participated in edema development by increasing vascular pressure or by increasing vascular permeabi- lity. Q yi_v;g, thromboxane can promote edema formation by either of these mechanisms (Noonan g a_l., 1983; Malik gt_ g” 1985c; Garcia-Szabo gt_ g” 1983a,b). However, since the ability of TxA2 to increase vascular permeability is thought to be PMN—dependent (Garcia-Szabo g gl_., 1983a,b; Malik gt_ a_l., 1985c), it seems unlikely that the TxA formed in lungs perfused with PMA in the absence 2 191 of PMN produced edema via this mechanism. The role of the increase in pressure in the development of edema induced by PMA is discussed in Section C below. From my studies it cannot be determined which cell types were responsible for prostanoid production in lungs perfused with a high concentration of PMA. However, possible sources of prostacyclin may be inferred from the results. In other models of lung injury, PGI2 is released from endothelial cells in response to an increase in pulmonary arterial pressure (Gerber gt_ _a_l_., 1980) or to injury (Demling gt_ gl., 1981; Brox and Nordoey, 1982). Since an increase in 6- keto-PGFIG. synthesis occurred after the perfusion pressure of the lungs had increased substantially, it is possible that in this preparation, PGIZ was released from endothelial cells in response to the pressure increase. However, since there was morphologic evidence of injury to endothelial cells in these lungs, it is also possible that PGIZ was released in response to this injury. Injured endothelial cells from the pulmonary microcirculation can also synthesize and release substantial quantities of TxA (McDonald g gl_., 1983; 2 Ingerman g; gl., 1980). However, since TxB2 synthesis increased before lungs became edematous, it is possible that cells other than injured endothelium produced TxA _Ip vitro, PMA stimulates the release of prostanoids from mast 2' cells and macrophages (Ohuchi e_t_ fl“ 1985; Heiman and Crews, 1985b; Ecker and Ferber, 1984). Since these cells may be present in the isolated lung preparation, it is possible that they contributed to the release of TxAz and/or PGIZ. Whether PMA can induce the release of prostanoids from endothelial cells has not been reported. However, since PMA can stimulate prostanoid release from cells other than phagocytes (Beaudry gt §_l., 1985; Levine and Xiao, 1985), it is possible that it can also stimulate the release of AA metabolites from endothelial cells. This possibility warrants further investigation. 192 C. Effect of a Papaverine on PMA Pneumotoxicity Whereas perfusion with indomethacin and Dazmegrel did not totally protect lungs from edema induced by a high concentration of PMA in the absence of PMN, perfusion with papaverine did. At present, the mechanism by which papaverine prevented edema development induced by PMA is unknown. Since an increase in venous pressure can promote edema development (Malik g3 g1., 1985c), it is possible that papaverine prevented edema by decreasing venous pressure. Since the drug decreases inflow pressure, this may indeed be the mechanism whereby papaverine protects lungs from fluid accumulation. Results obtained in isolated rabbit lungs perfused with an oxygen radical-generating system are consistent with the hypothesis that papaverine attenuates edema by decreasing venous pressure (Tate gt a_l., 1982). In this study, the protective effect of papaverine was eliminated by increasing left atrial pressure. To determine if papaverine protected against PMA-induced lung injury in my preparation by decreasing venous pressure, experiments with elevated left atrial pressure should be performed. Although thromboxane contributes to the increase in pressure induced by PMA, other factors may be involved in the increase. For example, the increase may have been mediated by lipoxygenase metabolites of arachidonic acid, since some of these mediators, such as LTC4 and D 4, are potent vasoconstrictors (Voelkel g; gl__., 1984; Noonan g; _a_l_., 1985). However, at concentrations similar to those used in this study, PMA produces a direct, calcium-dependent contraction of vascular smooth muscle '2 gill-g (Danthuluri and Deth, 1984; Rasmussen gt_ gl_., 1984; Forder gt_ _a_l., 1985; Chatterjee and Tejada, 1986). Thus, PMA may also have produced an increase in pressure in the isolated rat lung via this mechanism. Additional experiments with a lipoxygenase and a PKC inhibitor may help 193 determine whether the increase in pressure is mediated by leukotrienes or by a direct effect of PMA on vascular smooth muscle. III. Mechanism of Neutrophil-Dependent Lung Injury A. Involvement of Active Oxygen Species As stated previously, results of several investigations suggest that active oxygen metabolites are involved in PMA-induced pneumotoxicity E yiyg and in isolated lungs co-perfused with PMN (Shasby e_t_ _a_l_., 1982; Canham e_t _a_._l_., 1983; Jackson Q Q” 1986; Ismail gt a_l., 1987; Kuroda gt_ flu 1987; McDonald gt_ gl., 1987). However, there is some disagreement as to which active oxygen species produce lung injury in this model. To determine whether 0 -, H202 or OH' mediated injury in isolated rat lungs perfused with PMA and rat PMN, lungs were co-perfused with SOD and/or catalase. SOD catalyzes the formation of H202 and 02 from 02-, and catalase catalyzes the conversion of H202 to Oz and H20 (McCord and Fridovich, 1969; Fantone and Ward, 1982). Since formation of OH' requires participation of O - and H O (Fenton, 1894), perfusion with both SOD and catalase would be 2 2 2 expected to inhibit OH' formation. As shown in Tables 11, 12 and 13, SOD and catalase, either alone or in combination, protected lungs against toxicity caused by perfusion with PMN and PMA. These data suggest that superoxide, hydrogen peroxide and the hydroxyl radical are somehow involved in the pathogenesis of PMA pneumotoxicity. Q yijgg, the conversion of 02- to H202 can proceed spontaneously or it can be catalyzed by SOD, which increases the rate of dismutation by a factor of 108 to 109 (Fridovich, 1983; Hammond g a_l., 1984; Hess and Manson, 1984). Therefore, in isolated lungs perfused with PMN, PMA and SOD, it is likely that H202 was formed at an extremely fast rate. Since results with catalase suggest 194 that H202 mediates toxicity in the isolated lung system, at first glance it is puzzling why SOD (which theoretically increases H202 production) provides protection against lung injury. Furthermore, since catalase does not scavenge Oz’ (Babior gt 31., 1973), 02. was probably produced in these lungs. Since experiments with SOD suggest that 02 is toxic, why then is 02 unable to produce lung injury in the presence of catalase? One possible explanation for these results is that production of both 02 and H202 is required for the manifestation of edema. Since production of both 02- and H202 is required for the synthesis of the highly reactive species, OH' (Fenton, 1898; McCord and Day, 1978; Hess and Manson, 1984), production of this species is inhibited with SOD or 2 or H202 were mediated indirectly through the production of OH'. Additional studies using hydroxyl radical scavengers or iron chelators may help to answer this catalase (Cohen, 1985). Therefore, it is possible that the cytotoxic effects of 0 question. In addition to or in lieu of inhibiting OH' synthesis, SOD may have also protected 11mgs against injury by inhibiting the influx of neutr0phils into the lungs. Experiments performed by Fligiel e_t_ g. (1984b) suggest that this is the mechanism whereby SOD protects rats from dermal vasculitis induced by intradermal injection of immune complexes. Since experiments to examine whether SOD inhibited sequestration of PMN in my isolated rat lung system were not per- formed, I cannot dismiss this possibility. Results obtained in rabbits given PMA intravenously and in isolated rat or rabbit lungs perfused with PMA and human PMN also suggest that the OH' radical mediates the pneumotoxic response (Jackson a gl_., 1987; McDonald gt gl_., 1987; Kuroda gt _a_l., 1987). However, in contrast to the results obtained in my isolated lung preparation, administration of either SOD or catalase i_xl 3132 does not protect rabbits from lung toxicity induced by PMA (Kuroda gt g” 1987). At 195 present, the reason why these enzymes protect against development of lung injury in my system but do not :12 yiyg is unknown. One possible explanation for the apparent inability of SOD to protect against PMA pneumotoxicity in the rabbit is that the concentration of enzyme which was used may not have scavenged the 02- which was formed. Since the half-life of SOD _ig 21533. is approximately 8 min (Freeman 3 Q” 1985), in experiments of long duration, SOD must be given frequently. In the experiment of Kuroda gt g. (1987), SOD (6000 U/kg) was given at 15 min intervals throughout the 4-hr experiment. Therefore, it is possible that the treatment regimen which was used scavenged all of the 02- which was formed in the animals. However, these investigators did not demonstrate that the 2 which was produced i_n_ vivo. Because of the possibility that the treatment regimen was ineffective, conclusions treatment regimen effectively scavenged 0 about the involvement of 02- in PMA toxicity i_n_ vivo should not be based solely on the results of this study. Additional studies using concentrations of SOD that - should be performed before concluding that O - is not demonstrably scavenge O 2 2 involved in mediating PMA pneumotoxicity E vivo. Another possibility for the apparent lack of involvement of 02- in PMA toxicity in the rabbit is that several endogenous reducing agents other than 3" (Cohen, 1985; Youngman, 1984). Therefore, 02- (i.e., ascorbate) can reduce Fe i_n_ yiyg, production of 02- may not be necessary for the generation of OH'. If this is the case, treatment of animals with SOD would have no effect on the generation of OH' or on the toxic response. The fact that agents other than 02- can reduce Fe3+ also suggests that the requirement of 02- for the development of PMA pneumotoxicity in the isolated lung may be due to the absence of endogenous reducing agents in this system. To determine if this is why SOD protects against toxicity in the isolated lung and not in vivo, experiments using isolated lungs 196 perfused with SOD plus ascorbate and/or other reducing agents should be performed. As stated previously, removal of H202 with catalase inhibits OH' production ip fltgg (Cohen, 1985). Therefore, treatment of animals with catalase should protect them from OH'-induced lung injury. However, catalase attenuates, but does not totally inhibit edema development in PMA-treated rabbits (Kuroda gt _a_l., 1987). In this study, experiments were performed to determine if H202 was scavenged in animals treated with catalase. Results of these experiments suggested that the amount of H202 present in the lungs of rabbits treated with PMA and catalase was less than that of animals treated with PMA alone, but was greater than that of controls. These results suggest that treatment of the animals with catalase scavenged some, but not all of the H202 which was produced. Therefore, it is likely that OH' was formed in these animals. This could account for the inability of catalase to inhibit pneumotoxicity in this study. To determine if the ability of catalase to attenuate, but not inhibit lung injury, was due to the use of a treatment regimen which did not adequately scavenge H202, experiments should be repeated using a regimen which is proven to be effective. B. Involvement of Vasoconstriction In lungs perfused with PMN and PMA, perfusion pressure increased to a much larger extent than in lungs perfused with PMN or PMA. Since a large increase in pressure can promote fluid accumulation in lungs (Malik gt a_l., 1985c), I hypothesized that this was the mechanism whereby perfusion with PMN and PMA produced edema in the isolated lung. To investigate this possibility, lungs were perfused with PMN and PMA in the presence or absence of the vasodilator, papaverine. Perfusion with papaverine attenuated both the increase in pressure and lung weight caused by perfusion with PMN and PMA. These results suggest that the increase in pressure may mediate edema formation in these lungs. 197 Since papaverine did not scavenge 02- produced by PMA-stimulated PMN _i_n y_i_t1_-g, it is likely that papaverine did not attenuate lung injury by acting as a scavenger of 02-. However, since I did not perform experiments to determine whether papaverine scavenged H202 or OH', it is possible that this drug protected lungs against injury by this mechanism. Although the exact molecular mechanism whereby papaverine causes vasodilation is unknown, it has been suggested that its action on vascular smooth muscle is due to its ability to inhibit phosphodiesterase, and therefore increase cAMP (Needleman and Johnson, 1980). Since other drugs which increase cyclic AMP (i.e., P612 and PGE2) also inhibit PMN chemotaxis, adherence and aggrega- tion (Riukin gt _a_._l_., 1975; Weksler gt gl_., 1977; Boxer 3% gt, 1980) it is possible that papaverine also has these properties. Since PMN adherence to endothelium is obligatory for PMN to injure endothelial cells via an oxygen radical-dependent mechanism (Shasby gt 2.1., 1983), and since injury to the isolated lung perfused with PMN and PMA is oxygen radical-dependent, it is possible that papaverine attenuated lung injury by inhibiting PMN adherence. Therefore, the conclusion that papaverine protected against lung injury by acting as a vasodilator may not be correct. To prove or disprove the hypothesis that vasoconstriction is involved in toxicity to the IPL caused by perfusion with PMN and PMA, additional experiments with vasodilators which do not act by increasing CAMP (i.e., nitroglycerin) should be performed. C. Involvement of Cycloogjgenase Metabolites When perfused with PMA and PMN, lungs produced greater amounts of '1‘sz and 6-keto-PGF perfusion pressure and relative lung weight correlated with TxBZ concentrations la than controls. The observation that increases in in effluent medium from lungs after 10 min of perfusion with PMN and/or PMA suggested the possibility that TxBZ mediated or exacerbated lung injury. That 198 Dazmegrel provided virtually complete protection from injury produced by PMA- stimulated PMN supports this hypothesis. The results also suggest that cyclo- oxygenase metabolites other than thromboxane are not mediators of lung injury in this model since indomethacin and Dazmegrel afforded identical degrees of protection against lung injury. Dazmegrel and indomethacin also attenuated the increase in perfusion pressure to a similar extent, suggesting that TxAZ is the major cyclooxygenase metabolite that is responsible for the pressure increase induced by perfusion with PMA and PMN. However, since these inhibitors had a small, if any, inhibitory effect on the pressure increase, other substances may have been formed which contributed to the increase. I}; yttrg, PMA stimulates the release of vasoconstric- tive substances such as histamine, PAF and leukotrienes from activated PMN (Schleimer gtgt., 1980, 1982; Betz and Henson, 1980; Volpi gt gl_., 1985; McColl gt a_l., 1986; Liles gt gl_., 1987). Thus, it is possible that any or all of these compounds could have contributed to the pressure increase. I}; gig-g, PMA also stimulates the release of 5HT from platelets (White and Estensen, 1974b; Zucker gt a_l., 1974). Thus, it is also possible that 5HT from platelets which may have remained in the IPL could have participated in the pressure increase. Further- more, at concentrations similar to those used in this study, PMA produces a direct, calcium-dependent contraction of vascular smooth muscle i_n_ _v_i_t_1_'_9_ (Dan- thuluri and Deth, 1984; Rasmussen fl _a_l., 1984). Therefore, PMA may also have produced an increase in pressure via this mechanism. Clearly, further studies are necessary to determine the exact mechanism whereby PMA causes a pressure increase in this model. Because thromboxane concentrations correlated with the increase in perfusion pressure and relative lung weight, I hypothesized that TxAZ might promote or accelerate the formation of pulmonary edema by increasing 199 intravascular hydrostatic pressure. Results from experiments with Dazmegrel support this hypothesis. Dazmegrel delayed the increase in perfusion pressure and attenuated the increase in relative weight of lungs perfused with PMN and PMA. However, since Dazmegrel almost completely protected lungs from fluid accumu- lation induced by perfusion with PMN and PMA without attenuating final inflow pressure, TxA2 may mediate lung injury by some other mechanism. The mechanism by which TxA may have produced lung injury in this model is 2 discussed in detail in part E below. In contrast to the results obtained in this study, a cyclooxygenase metabolite(s) of arachidonic acid does not appear to mediate PMA-induced edema in intact sheep (Newman gt gl_., 1984). One possible explanation for the differing results is that some extrapulmonary factor(s) which is present 13 yiyg but absent in the isolated lung may override the protective effect of cyclooxygenase inhibition. As stated previously, it is also possible that in animals treated with meclofenamate, a protective effect of cyclooxygenase inhibition was overridden by the ability of the drug to shunt metabolism of AA to toxic lipoxygenase metabolites such as LTB4, C4 or D4 (Engineer gt Q" 1978; Hitchcock, 1978). Another possibility is that the use of meclofenamate may have blocked the formation of a cyclooxygenase metabolite which may have provided protection against pneumotoxicity. Although the results in isolated rat lungs perfused with PMN, PMA and indomethacin suggest that production of the antiinflammatory prostanoid PGIZ is not necessary for isolated lungs to be protected against PMA toxicity, it is possible that PGI2 has protective effects against PMA toxicity _'_u_1 yi_v_q. Because of the possibility that treatment of animals with meclofenamate altered the production of eicosanoids which promote or inhibit edema formation, conclusions about the role of thromboxane in PMA toxicity should not be based solely on the results of this study. To determine whether TxAz is an important 200 mediator of PMA toxicity '3 2:19, experiments with a specific thromboxane synthetase inhibitor should be performed. As described in part A above, oxygen radical scavengers prevented edema formation in isolated lungs perfused with PMA and PMN. Thus, the possibility existed that Dazmegrel may have attenuated edema formation by inhibiting release of 02. from PMN or by acting as an oxygen radical scavenger. To test this possibility, 02- production from PMA-stimulated PMN in the absence or presence of Dazmegrel was assessed. To determine if Dazmegrel inhibited 02- production in isolated lungs perfused with PMA and PMN, samples of medium from lungs perfused with PMA and PMN in the presence and absence of Dazmegrel were also assayed for 02 content. Results of these experiments suggested that Dazmegrel is not a 02- scavenger. As shown in Table 17, experiments ip gttgg were also performed to determine if Dazmegrel was a H202 scavenger. Results of this study suggest that Dazmegrel does not scavenge this active oxygen species either. From the latter two studies, it is evident that Dazmegrel did not protect lungs from toxicity by scavenging 02. or H202. Since experiments to determine if Dazmegrel was a scavenger of OH' were not performed, the possibility that the drug protected lungs from injury by acting as a scavenger of OH' cannot be dismissed. However, as shown in this thesis, in addition to Dazmegrel, two other drugs which inhibited TxB synthesis (indomethacin and 2 aspirin) also protected lungs against injury. In the ASA experiments, the drug was removed from the lungs and the PMN before perfusions with PMA were per- formed. At concentrations which remained in the lungs, ASA does not inhibit release of 02', H202 or OH' from PMN (Sagone e_t Q” 1980; Sagone and Husney, 1987). As shown in section D below, pretreatment of PMN with ASA also does not inhibit production of 02. . These results suggest that ASA protected lungs by 201 acting as a cyclooxygenase inhibitor, not by acting as an oxygen radical scavenger. The results of studies with indomethacin support this conclusion. Furthermore, results of studies with Dazmegrel suggest that this metabolite is TxAz. As stated above, the amount of 02- produced in lungs perfused with PMA, PMN and Dazmegrel was equal to that produced in lungs perfused with PMN and PMA. However, as stated previously, lungs were totally protected against lung injury by perfusion with Dazmegrel. These results suggest that in the absence of thromboxane release, the superoxide radical (and possibly other active oxygen species) are not capable of inducing lung injury in this model. Thus, both thromboxane and active oxygen species interact somehow to produce injury in lungs perfused with PMA and PMN. D. Experiments to Determine the Source of TxA, and PGI, 1. Role of active oxyggn species in prostanoid production In isolated rabbit lungs, reactive oxygen metabolites which are enzymically generated by xanthine oxidase stimulate thromboxane production (Tate gt gl_., 1984). Perfusion with a hydrogen peroxide-generating system also stimulates TxB2 and PGI'Z production from isolated, perfused lungs (Burghuber gt _a_l_., 1984). In contrast, H202 inhibits the release of P612 from cultured endothelial cells (Whorton gt gl_., 1985). Since PMA-stimulated PMN release active oxygen metabolites, it was possible that active oxygen metabolites modu- lated prostanoid generation in lungs perfused with PMA and PMN. If this occurred, the protective effect of SOD and/or catalase against lung injury may have been due to an inhibition of TxAz synthesis and/or stimulation of P612 synthesis. To examine these possibilities, perfusion medium samples from lungs which were perfused with PMA, or with PMN and PMA in the absence or 202 presence of SOD or catalase were analyzed for Tsz and 6-keto-PGF1a content. At all time periods, TxB synthesis from lungs perfused with PMN and PMA in the 2 presence or absence of SOD or catalase was significantly greater than that of lungs perfused with PMA alone. Furthermore, synthesis in lungs perfused with PMN and PMA was not significantly different from that of lungs co-perfused with SOD or catalase until 30 min of perfusion. These results suggest that at early time points, Tsz synthesis in lungs perfused with PMN and PMA is not dependent on the production of 02’ H202 or OH'. However, at later times, Tsz synthesis is partially dependent on the production of these species. Results of recent studies by McDonald and coworkers (1987) support the hypothesis that OH' is involved in TxBZ synthesis by lungs perfused with PMN and PMA. From my studies, it cannot be determined if the additional amount of Tsz which was produced in lungs which were not co-perfused with the oxygen radical scavengers was released from cells as the result of injury by active oxygen species or if these species stimulated the release of TxBZ from lung cells without producing overt cell injury. The observation that TxB2 synthesis by lungs perfused with PMN and PMA was not significantly greater than that of lungs co- perfused with SOD or catalase until 11mgs perfused with PMN and PMA were edematous suggests that some TxA was probably produced as a result of injury 2 induced by active oxygen species. Furthermore, these findings suggest that SOD or catalase did not protect lungs against edema development by inhibiting TxA2 production. In lungs perfused with PMN, PMA and with SOD or catalase, 6- keto-PGF synthesis was not significantly greater than that of lungs perfused 101 with PMA at any time point. These results suggest that, in contrast to TxAZ, synthesis of 6-keto-PGF is totally dependent on the production of 02 and 101 H 0 Since 6-keto-PGF 2 release in lungs perfused with PMN and PMA was not 2' 10 203 significantly greater than that of lungs co-perfused with either SOD or catalase until 20 or 30 min, respectively, it is likely that 6-keto-PGF1a is produced as the 2 and/or H202. stated previously, results of my experiments suggest that lung injury in this model result of lung damage induced directly or indirectly by 0 As may be mediated by OH', which is formed via the interaction of H202 and 02- with iron. Therefore, it is likely that in lungs perfused with PMN and PMA, 6- Reta-4’61“l a is produced as the result of cellular injury induced by OH' and not 02- or H202. However, since various peroxides can accelerate cyclooxygenase activity (Hemler gt gl_., 1978, 1979) it is possible that H202 induced production of 6-keto-PGF1G from lung cells and/or PMN via this mechanism. The finding that active oxygen species stimulate PGIz synthesis in lungs perfused with PMN and PMA is in direct contrast to the findings of Whorton and coworkers (1985), which suggest that H202 inhibits endothelial cell production of PGIZ. They also differ from the results of Miller gt a_l. (1985) which suggest that active oxygen species from activated PMN do not induce or inhibit PGIZ release from cultured endothelial cells. One possible reason for the differing results is that endothelial cell injury occurred in my isolated lung system, whereas it did not occur in the other studies. As stated previously, injured endothelial cells release PGIZ (Demling gt gt, 1981; Brox and Nordoey, 1982). One other explanation for the different results is that the ability of H202 to stimulate PGIZ synthesis from endothelial cells may be dependent on the increase in vascular pressure which occurs in isolated lungs perfused with PMA and PMN. The fact that PGIz is released from endothelial cells in response to an increase in pressure (Gerber gt g, 1980) supports this hypothesis. Finally, it could be that cells other than endothelial cells, which are present in the isolated lung system but not in the cultured cell systems, are sources of PGIZ. Additional experiments should be performed to explore these possibilities. 204 2. Studies with PMN perfused through the apparatus To determine if 6-keto-PGF and TxB2 synthesis by lungs 101 perfused with PMA and PMN was mediated by a direct effect of PMA on the PMN, medium containing PMN and either PMA or DMSO was circulated through the IPL system (in the absence of a lung), and samples were collected for prostanoid analysis. Results of this study suggest that PMA stimulates the release of PGIZ but does not stimulate the release of TxA from PMN. 2 The results of this experiment are similar to those of Ward and coworkers (1985), which suggest that PMA is a relatively poor stimulus for the release of Tsz from rat PMN. These investigators also found that at concentra- tions below 0.2 ug/ml, PMA does not stimulate the release of Tsz from rat PMN. Since synthesis of PGI2 from PMN was not assessed in the study of Ward gt gt. (1985), it is not known if PMA stimulated the release of PGI2 from cells used in this experiment. However, results of my studies suggest that PMA can stimulate the release of small, but significant amounts of PGI2 from rat PMN. Since much larger quantities of 6-keto-PGF1a are produced by lungs perfused with PMN and PMA than by cells stimulated with PMA, it is evident that cells other than PMN are reSponsible for the majority of the 6-keto- PGF1 (1 released from this preparation. Since the amount of TxBZ synthesized by unstimulated or by PMA—stimulated PMN is similar to that produced by lungs perfused with PMA and PMN, PMN may be the source of most, if not all of the TxB that is produced in the IPL system. However, since isolated lungs remove 2 circulating ’1‘sz (Ganey, 1986), it is possible that more Tsz was produced in lungs perfused with PMA and PMN than by PMN stimulated with PMA. Experi- ments which address whether the PMN or the lung is the source of the Tsz and/or 6-keto-PGF1a that is produced in isolated lungs perfused with PMN and PMA are discussed below. 205 3. Studies with aspirin As stated previously, results of experiments with indomethacin and Dazmegrel suggest that TxAZ is an important mediator of toxicity to the isolated rat lung caused by PMA and PMN. Since large quantities of Tsz were produced by PMN when circulated through the perfusion apparatus, it was possible that all of the TxAz which was required to induce lung injury in the IPL was produced by PMN when perfused through the lungs. Alternatively, it was possible that some or all of the TxA2 which was required for edema development was produced by the lungs. To determine whether the lung and/or PMN were the source of TxAz, experiments were performed utilizing PMN and/or lungs from rats that had been pretreated with the irreversible cyclooxygenase inhibitor, aspirin. When cyclooxygenase from either PMN or the lungs was inhi- bited, TxB synthesis by lungs perfused with PMN and PMA was attenuated, but 2 not totally eliminated. Furthermore, pretreatment of either lungs or PMN with aspirin protected lungs against injury. These data suggest that in lungs perfused with PMN and PMA, 1‘sz is synthesized from both the lung and PMN. In addition, they suggest that TxAZ synthesis from both of these sources is required for the manifestation of lung injury in this model. In previous studies, I demonstrated that perfusion of lungs with medium containing PMA (14 ng/ml) in the absence of PMN does not elicit release of Tsz from lungs. Furthermore, in the current study no significant difference in '1‘sz production was noted between lungs perfused only with PMA (21 ng/ml) or PMA vehicle (DMSO). These results suggest that PMA does not stimulate the release of '1‘sz from the lungs. However, when compared to the amount of Tsz produced by lungs perfused with DMSO vehicle and aspirin-pretreated PMN, the amount of Tsz produced by lungs perfused with PMA and aspirin-pretreated PMN 206 is significantly greater. These results suggest that in the presence of PMN, PMA can stimulate the release of Tsz from the lungs. Whether the effect of PMA is direct or mediated by a product(s) released from PMA-stimulated PMN is not known. However, results from studies with SOD and catalase suggest that active oxygen species from PMA-stimulated PMN elicit release of TxB from the lungs. Since ASA pretreatment of PMN did 2 not inhibit the release of O - from PMA-stimulated PMN i_n_ vitro, it is likely that 2 this radical was present in isolated lungs perfused with PMA and ASA-pretreated PMN. Furthermore, since the concentration of ASA used to treat PMN (100 uM) does not inhibit release of H202 or OH' from PMN (Sagone gt a_l., 1980; Sagone and Husney, 1987), it is likely that these species were also present in lungs perfused with PMA and ASA-pretreated PMN. Therefore, it is possible that active oxygen species from PMN mediate the release of '1‘sz from lungs perfused with PMA and ASA-pretreated PMN. Whether other products from PMA— stimulated PMN (i.e., lysosomal enzymes) also induce release of TxBZ from these lungs cannot be determined from these experiments. Additional experiments should be performed to examine this possibility. The results obtained in this study suggest that in addition to being produced by the lungs, TxBZ is produced by PMN in lungs perfused with PMN and PMA. However, since the amount of TxB2 produced by lungs of ASA- pretreated rats perfused with PMA and PMN was not significantly different from that of ASA-pretreated rats perfused with DMSO and PMN, it does not appear that PMA stimulates the release of TxBZ from PMN being perfused through the lungs. Furthermore, these data suggest that PMA does not stimulate the release of a factor(s) from the lungs which stimulates the release of TxBZ from PMN- From the results of this experiment, is is evident that '1‘sz synthesis from both PMN and the lung is required for edema development in lungs 207 perfused with PMA and PMN. Why synthesis from both of these sources is necessary for lung injury to occur is unknown. One possibility is that a certain amount of TxA2 must be produced to promote edema formation. By eliminating TxAz synthesis from either the lungs or PMN, perhaps the threshold concentration could not be reached. Perhaps if the lungs were perfused with a high enough concentration of PMA, they would synthesize enough Tsz so production from PMN would not be required for the particular threshold to be reached. Converse- ly, if lungs were perfused with larger numbers of PMN, perhaps enough TxAZ would be produced so that synthesis from the lungs would not be necessary for edema development. However, by increasing the number of PMN or the concentration of PMA, it is likely that the model of lung injury will be no longer dependent on the presence of both PMN and PMA. For example, when perfused with PMA at concentrations of _>_57 ng/ml, lungs develop edema which is not dependent on the presence of PMN or on the generation of TxA or active oxygen 2 metabolites. Since there is evidence of some endothelial cell blebbing in lungs perfused with PMN (1x108) in the absence of PMA, it is also possible that perfusion with increased numbers of PMN could induce edema in the absence of PMA. Therefore, by perfusing lungs with PMN (1x108) and a greater concentra- tion of PMA, or with a nontoxic concentration of PMA (21 ng/ml) and larger numbers of PMN, it is likely that lung injury will occur which is not dependent on the presence of both PMN and PMA. Therefore, results of these experiments would not be applicable to the particular model of lung injury which I am studying. As seen in section 2 above, PMA can induce the release of small, but significant amounts of 6-keto-PGF1a from rat PMN. To determine if 6-keto- PGFla was produced by, the lung or the PMN in lungs perfused with PMA and PMN, samples of medium from all lungs used in this experiment were analyzed for 6-keto-PGF1C1 content. Results of this experiment suggest that most of the 208 6-keto-PGF10 which is produced in lungs perfused with PMN and PMA comes from the lung. Since synthesis from lungs perfused with only PMA is significantly greater than that of lungs perfused with PMA vehicle (DMSO), some of the 6- keto-PGFla which is present in lungs perfused with PMA is produced by the interaction of PMA with the lung. At early time points, it appears as if the majority of the 6-keto-PGF which is released from lungs perfused with PMN la and PMA is induced via the action of PMA on the lung. However, at later time points a greater amount of 6-keto-PGF1a is produced by lungs perfused with PMN and PMA than with PMA alone. These results suggest that a factor produced by the interaction of PMA with PMN stimulates the release of 6-keto-PGF a from 1 the lungs. As mentioned previously, results from experiments with SOD and catalase suggest that active oxygen species stimulate the release of 6-keto- PGF1 a from the lungs. Therefore, it is likely that active oxygen species from PMA-stimulated PMN were responsible for some, if not all, of the 6-keto-PGFla released from lungs perfused with PMN and PMA in the current study. The results obtained in this study support the hypothesis that the production of both active oxygen species and thromboxane is required for the manifestation of edema in this preparation. As stated above, it is likely that active oxygen species are present in lungs from ASA-pretreated rats perfused with PMN and PMA and in lungs perfused with ASA-pretreated PMN and PMA. However, when thromboxane synthesis is attenuated, these species are not capable of producing lung injury. Previous results obtained in experiments with Dazmegrel and indomethacin also support this hypothesis. Why the presence of both thromboxane and active oxygen species may be required for lung injury is discussed below. 209 E. Mechanism of Action of Thromboxane In experimental animals, a syndrome resembling ARDS can also be induced by intravenous infusion of zymosan-activated plasma (ZAP), endotoxin or thrombin or by intratracheal administration of 0.1 N HCl (Casey g gl_., 1982; McDonald gt gl_., 1983; Garcia-Szabo gtgl_., 1983b; Huval fl gl__., 1983a). In each of these models, experiments have been performed to determine the role of thromboxane in development of the lung injury. Treatment of sheep, primates and cats with a specific thromboxane synthase inhibitor prevents pulmonary hyperten- sion induced by endotoxin but does not attenuate the increase in vascular permeability which develops (Casey gt a_l., 1982; Ball gt a_l., 1983; Kubo and Kobayashi, 1985). Pretreatment of sheep with aspirin also attenuates the increase in PAP that occurs after infusion of zymosan-activated serum (McDonald gt gt, 1983). However, inhibition of TxAz synthesis does not protect against the increase in vascular permeability which is caused by this agent (Gee gt gl_., 1986). These results suggest that thromboxane may induce edema formation by increas- ing microvascular pressure and not by increasing vascular permeability. By contrast, thromboxane synthase inhibition prevents the thrombin- induced increase in pulmonary transvascular protein clearance in a sheep lung fistula preparation (Garcia-Szabo gt g, 1983b). Furthermore, in dogs given 0.1 N HCl i.t., treatment with ketoconazole inhibits edema formation without attenuat- ing the increase in PAP or pulmonary arterial wedge pressure (Huval gt gl_., 1983a). These results suggest that thromboxane may promote edema formation by increasing vascular permeability. Since the index of edema which was used in the experiments with Dazmegrel measured bulk flow of fluid, I cannot differentiate whether the effects of thromboxane in isolated lungs perfused with PMN and PMA were predominantly due to elevated vascular pressure or to increased vascular permeability. How 210 ever, the fact that final inflow pressure in lungs co-perfused with indomethacin or Dazmegrel was not significantly different from that of lungs perfused with PMN and PMA suggests that TxAZ may have promoted edema formation by some other mechanism than by increasing vascular pressure. Furthermore, the observation that ASA pretreatment of the lungs prevented leakage of albumin from the vasculature into the airways and did not attenuate the increase in pressure caused by perfusion with PMN and PMA suggests that T‘xA2 may induce edema in this model by increasing vascular permeability. Results from experiments in isolated dog lobes perfused with blood containing PMA also support this hypothesis (Allison gt 31., 1986). At present, the mechanism by which TxA2 promotes an increase in vascular permeability in some models of ARDS is not clear. However, results of several studies suggest that its effects may be mediated via a PMN-dependent mechanism. In animals treated with 0.1 N HCl or thrombin, inhibition of cyclooxygenase or thromboxane synthase prevents sequestration of PMN in the lungs (Utsunomiya gt a_l., 1982; Malik gt gt, 1985b). Since lung injury in these models is PMN-dependent, it has been suggested that TxAz may participate in edema development by causing PMN entrapment in the lungs. I_n_ gig-g, thrombox- ane also promotes the adherence of PMN to endothelial cells (Spagnuolo gt g, 1980). Since PMN adherence is obligatory for oxygen radicals from PMN to produce an increase in permeability of endothelial cell monolayers (Shasby gt gl_., 1983), it is possible that thromboxane may indirectly induce an increase in vascular permeability by promoting PMN adherence. Since results of my studies suggest that production of both active oxygen species and thromboxane are necessary for edema deve10pment in isolated lungs perfused with PMN and PMA, this may very well be the mechanism whereby thromboxane participates in edema development in my model. The fact that PMN adherence is required for 211 PMA-stimulated PMN to increase permeability of endothelial cell monolayers and to promote edema in isolated perfused rat and rabbit lungs (Shasby gt gt, 1983; Ismail gt gt, 1987) lends support to this hypothesis. Although not reported in this thesis, experiments to determine whether thromboxane induced edema formation by increasing vascular permeabi- lity or by increasing vascular pressure were attempted. Unfortunately, technical problems arose which could not be resolved. In these experiments, the response of lungs perfused with PMN, PMA and Dazmegrel or with PMN, PMA and papaverine to a venous pressure challenge was assessed. I hypothesized that if thromboxane caused lung injury by acting as a vasoconstrictor, lungs co-perfused with Daz- megrel would become as edematous as lungs perfused with papaverine in response to an equal increase in venous pressure. However, if thromboxane caused lung injury by increasing vascular permeability, lungs co-perfused with Dazmegrel would not become edematous in response to a similar pressure challenge. Similar experiments have been performed to determine if active oxygen species induce edema formation in isolated rabbit lungs by increasing permeability or pressure (Tate gtgg 1982; Jackson gtgt, 1986). In addition to the experiments mentioned above, studies were also performed to determine if thromboxane induced lung injury by promoting PMN adherence. In this study, technical difficulties also arose which could not be resolved within a reasonable length of time. Since results from these experiments were not obtained, I cannot conclude that thromboxane caused lung injury in my model by increasing permeability by a PMN-dependent mechanism. Therefore, this hypothesis remains to be tested. SUMMARY AND CONCLUSIONS The studies described in this thesis were undertaken to examine the role of active oxygen Species and TxAZ in PMA-induced injury to the isolated, perfused rat lung. Since studies using various concentrations of PMA in the absence or presence of PMN revealed that PMA could produce both PMN-dependent and -independent lung injury, the role of these mediators in both of these models of lung injury was assessed. At high concentrations (in the absence of PMN), PMA produces lung injury which is characterized by a large increase in perfusion pressure and fluid accumulation. Intravascularly generated active oxygen species do not mediate the increase in pressure or lung injury in this model. The increase in perfusion pressure is necessary for the development of edema, since lung injury is attenuated by perfusion with a vasodilator. Although thromboxane appears to participate somewhat in the increase in pressure and the development of edema, other unknown factors also contribute to these responses. Since PMA directly stimulates smooth muscle contraction, it could be that this is the mechanism whereby PMA induces an increase in pressure and edema. Additional studies which examine this possibility may prove to be fruitful. At low concentrations, PMA produces lung injury which is dependent on the presence of PMN. Generation of both active oxygen species (in particular, OH“) and thromboxane are necessary for edema development. Furthermore, thrombox- ane synthesis from both PMN and the lungs are required for lung injury to occur. 212 213 Although the exact mechanism whereby thromboxane induces lung injury is unknown, the results suggest that it may act by increasing vascular permeability. In conclusion, the results of these studies suggest that TxAZ mediates injury to isolated rat lungs caused by perfusion with PMN and PMA. Therefore, it is possible that TxAz is involved in the pathogenesis of PMA-induced respiratory distress _'_u_1_ _v__i_go_. Furthermore, since it is possible that ARDS may develop by a mechanism similar to that of PMA-induced lung injury, TxAZ may be involved in the pathogenesis of the human disease. Therefore, drugs that block the synthesis or biological actions of TxA2 may be effective therapies for the disease. Clearly, further investigation into the role which TxAz plays in PMA-induced toxicity jg yjyg and in other models of respiratory distress is necessary to determine whether clinical trials with these drugs should be performed. BIBLIOGRAPHY BIBLIOGRAPHY Abb, J., Bayliss, G.J. and Deinhardt, F.: Lymphocyte activation of the tumor- promoting agent l2-O-tetradecanoyl-phorbol-13-acetate (TPA). J. Immunol. 1_2__2_: 1639-1642, 1979. Adams, D.O. and Hamilton, T.A.: The cell biology of macrophage activation. Ann. Rev. Immunol. 2_: 283-318, 1984. Albert, R.K., Greenberg, G., Guest, R.J., Luchtel, D. and Henderson, W.R.: Leukotrienes C and D do not increase filtration coefficent of excised perfused guinea pig ltmgs. J. Appl. Physiol. 2.2.: 1-19, 1987. 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