Effect of acute ozone exposure on the proteinase—Antiproteinase balance in the rat lung

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Effect of Acute Ozone Exposure on the Proteinase-Antiproteinase Balance in the Rat Lung’ J. A.PICKRELL,~ R.E. GREGORY, D.J. COLE, F. E HAHN, AND R. F. HENDERSON Lovelace inhalation

Toxicology Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87185 Received July 21, 1986, and in revisedform October 15, 1986

Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.O, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a i4C-globin substrate. The 48-hr exposures to O3 at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm 0, resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65- 120% by exposure to 1 or 1.5 ppm 0,, which correlated with inflammatory cells noted histologically. At 1.5 ppm O,, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if 0, exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema.

INTRODUCTION Elastase degrades lung elastin, leading to destruction of lung connective tissue and the development of emphysema (Janoff, 1972; Starkey, 1977). In the healthy lung, polymorphonuclear leukocyte elastase activity is inhibited by binding to a,-proteinase inhibitor (a,PI)3 (Laskowski and Kato, 1980; Travis and Salvesen, 1983). In contrast, macrophage elastase is inhibited primarily by a,-macroglobulin (a,M) or tissue inhibitor of metalloproteinase (Laskowski and Sealock, 1971; Laskowski and Kato, 1980; Stone et al., 1982; Starkey and Barrett, 1977; Werb, 1986). Proteinolytic destruction of lung can result if inhibitors are reduced or proteinase activity is increased. Ozone (0,) exposure may lead to an apparent excess of elastase from an influx of inflammatory cells in response to oxidative lung injury. Both neutrophils and macrophages are rich in proteinolytic and elastolytic enzymes (Janoff, 1972; Las* The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. Research was supported by the U.S. Environmental Protection Agency via Interagency Agreement 79-D-X5003 under U.S. Department of Energy Contract DE-ACO4-76EV01013 and conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. 2 To whom all correspondence should be addressed. 3 Abbreviations used: a,PI, a,-proteinase inhibitor; a,M, cu,-macroglobulin; PPE, porcine pancreatic elastase; SAPNA, succinyl-trialanyl-p-nitroanalide; BAPNA, a-N-benzoyl-oL-arginine-p-nitroanalide; EIC, elastase inhibitory capacity; TIC, trypsin inhibitory capacity. 168 0014-4800/87 $3.00

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kowski and Sealock, 1971; Laskowski and Kato, 1980; Starkey, 1977; Starkey and Barrett, 1977; Travis and Salvesen, 1983). This excess may be potentiated by oxidative destruction of methionyl residues at the active site of the major elastase inhibitor, (riPI, as indicated by a reduction of elastase inhibitory capacity/trypsin inhibitory capacity measured using ester substrates (Travis and Salvesen, 1983; Johnson and Travis, 1978, 1979; Matheson et al., 1979; Carp and Janoff, 1977, 1979; Janoff et al., 1979; Gadek et al., 1979; Carp et al., 1982; Johnson, 1980; Beatty et al., 1982; Leh et al., 1978). In moderate to severe pulmonary disease, vascular injury and edema will develop and azM will transmigrate into the alveolar and interstitial space and form complexes with tissue proteinases. It is not clear whether such complexes continue to attack lung elastin (Galdston et al., 1979; Kueppers et al., 1981; Roberts, 1982). In the present study we investigated the effect of acute 0, exposure on the proteinase-antiproteinase balance in the rat lung by measuring proteinolytic, antielastolytic, and antitrypsin activities after exposure to 0,. METHODS Materials. Seventy barrier maintained female Fischer-344 rats from the Institute’s breeding colony, 12-14 weeks of age, were used for these studies. Except during exposure to ozone, the animals were housed in polycarbonate cages supplied with aspen bedding and filter tops. They were fed a pelleted ration (Wayne Lab Blox, Allied Mills, Chicago, IL) and tap water was available ad libitum. Elastin-rhodamine B and porcine pancreatic elastase (PPE) were purchased from Elastin Products, Pacific, Missouri. Succinyl-trialanyl-p-nitroanalide (SAPNA), ol-N-benzoyl-DL-arginine-p-nitroanalide (BAPNA), and trypsin were purchased from Sigma Chemical Company, St. Louis, Missouri. All other chemicals used were reagent grade or better. Ozone exposures. Animals were randomized by weight into seven exposure groups of 10 rats/group (5 O,-exposed groups and 2 air-exposed control groups). Groups of rats were exposed to the following 0, 0.5, 1.0, or 1.5 ppm 0, concentrations. In the 1 ppm exposure group animals were observed after 24 and 48 hr of exposure and after 48 hr of exposure and 48 hr recovery. All other exposure groups were observed at the end of a 48-hr exposure. Rats were exposed in a 2.2-m3 stainless-steel multitiered exposure chamber (H-2000, Hazelton Systems, Aberdeen, MD). The animals were allowed feed and water as described above throughout the exposures. Ozone was generated by passing oxygen through a silent discharge ozonizer (Erwin-Sander, Eltze, West Germany) and metered into filtered chamber supply air to the desired concentrations. Chamber 0, concentrations were continuously monitored with an ultraviolet O3 monitor (Model 1003AH, Dasibi Environmental Instruments, Glendale, CA). Chamber temperature and relative humidity were maintained at 25 2 2°C and 40 + lo%, respectively. Animal sacrifice and tissue preparation. The animals were anesthetized with carbon dioxide and bled via the axillary vessels. Whole blood was collected and serum prepared. After bleeding, the animals were sacrificed by cervical dislocation. The heart-lung block was excised and the lungs were intratracheally lavaged with three, 5-ml aliquots of 0.15 M NaCl at 20°C. Lavage fluid (referred throughout this paper as airway fluid) was centrifuged at 300g for 10 min to remove cells and cell debris and was maintained at 4°C until assayed. The lungs

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were intravascularly perfused with 0.15 M NaCl and the individual lung lobes were separated and weighed. Left lung lobes were homogenized in a motordriven tissue homogenizer (Tekmar, Cincinnati, OH) for 30 set in a total of 8.0 ml of 50 mM Tiis-HCl, pH 7.4, containing 0.25 M sucrose. Lung homogenates were centrifuged at 300g for 10 min and the lung homogenate supernatants (referred to throughout this paper as lung) filtered through gauze and maintained at 4°C until assayed. The lungs from two animals from each exposure group were used for histopathological examination. After excision of heart-lung block, the left bronchus and blood vessels were clamped; the right lung was lavaged with three 2-ml aliquots of 0.15 M NaCl at 20°C and perfused as described above. The right bronchus and blood vessels were then ligated and, after the clamp was removed, the left lung was intratracheally perfused with 10% neutral buffered formalin. After fixation and paraffin embedding the left lung lobe was sectioned at 5 pm, stained with hematoxylin and eosin, and examined microscopically. Assays. Elastase inhibitory capacity (EIC) was determined in serum, lung homogenate, and airway fluid using two types of substrate for the elastase activity: elastin itself and (in some cases) an ester, SAPNA. All assays were modified and run on an automated microcentrifugal analyzer (Instrumentation Labs, Lexington, MA). Elastolytic EIC was determined using a modification of the elastinrhodamine B method (Huebner, 1976). Briefly, 1 pg of PPE in 0.2 M Tris-HCl, pH 8.8, was added to 0, 2.5, 10, and 20 ~1 of serum, or 0, 100, 300, and 500 p.1 of airway fluid, or a I:10 dilution of lung homogenate and brought to a volume of 0.85 ml with the same buffer. The mixture was incubated for 5 min at 20°C after which 3 mg of elastin-rhodamine B in 0.15 ml of 0.2 M Tris-HCl, ph 8.8, was added. The samples were incubated in microcentrifuge tubes for 20 min at 37°C with continuous mixing. Unhydrolyzed substrate was removed by centrifugation and the A,,, of the supernatant determined. To study the mechanism of 0s damage to a,PI esterolytic EIC was determined on selected samples using the method of Bieth et al. (1974) and using SAPNA as a substrate. This was then compared to trypsin inhibitory capacity (method described below) and the ratio of elastase inhibitory capacity to trypsin inhibitory capacity on similar-sized substrates (esters) was calculated. A reduction in this ratio indicates that antielastase has been attacked at its active site (Travis and Salvesen, 1983). Esterolytic trypsin inhibitory capacity (TIC) was determined using a modification of the method of Dietz et al. (1974). Trypsin in (1.2 p,g) 1 mN HCl was added to the same volumes of serum, airway, and lung as described for the esterolytic EIC assay, brought to a final volume of 175 ~1 with 50 mM sodium barbital, pH 8.2, containing 1 mM calcium lactate and 0.5 mM CaCl,, and preincubated at 20°C for 5 min. The BAPNA dissolved in dimethyl sulfoxide (0.021 mg/ml) was diluted 1:50 in the sodium barbital buffer described above. BAPNA (50 ~1, 21 kg) was added and incubated at 37°C for 20 min and the A,,, determined. Protein concentrations of the serum, lung homogenate, and airway fluid samples were determined using the method of Lowry et al. (1951). Proteinolytic activity of the lung was measured by the quantification of the radioactive peptides released upon incubation with 14C-labeled globin substrate. Radioactive globin (sp act = 33,000 cpm/mg), prepared at this Institute, was suspended in 0.1 M citrate/citric acid, pH 4.2 (acid), and a 0.1 M Tris pH 7.6 (neutral) buffer both containing 5 mM CaCl,. A 0.2-ml aliquot of lung was incubated with

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1.5 mg of 14C-labeled globin, at the acid and neutral pH and in the presence of 0.02 M cysteine, for 60 min at 37°C with gentle agitation. After incubation, 9.6 mg of bovine serum albumin (BSA) was added, and unhydrolyzed globin and BSA were precipitated by the addition of trichloracetic acid to a final concentration of 3%. Samples were centrifuged at 1OOOg for 10 min and the 14C activity in the supernatant was determined by liquid scintillation counting. Data evaluation. Elastolytic and esterolytic EIC and esterlytic TIC were calculated as the amount (mg) of purified proteinase (PPE or trypsin) that could be inhibited per milligram of serum protein, lung, or airway protein and as the total amount of proteinase inhibited by the whole lung homogenate or total airway fluid normalized to animal body weight. Statistical differences between the O,-exposed and air-exposed groups were determined using a paired Student t test (Remington and Schortz, 1970). A P value ~0.05 was considered statistically significant. RESULTS Histological changes. Experimental 0s concentrations were maintained within * 5% of the intended level for the duration of each exposure. Ozone exposure (0.5 ppm for 48 hr) produced modest changes in pulmonary histopathology including areas of focal alveolar cell proliferation, pulmonary edema, and accumulations of alveolar macrophages and polymorphonuclear leukocytes (Fig. 1A). The severity of these lesions was markedly increased in those animals exposed to 1.0 ppm 0, for 48 hr (Fig. 1B) or 1.5 ppm 0s for 48 hr (Fig. 1C) as there was a marked increase in alveolar cell proliferation, alveolar thickening, influx of inflammatory cells (macrophages and neutrophils), pulmonary edema, and hemorrhage in the highest concentration. Rats exposed to air (controls) (Fig. 1D) had no distinguishable pulmonary lesions. Effect of 0, exposure concentration. The effect of 48 hr of exposure to 0, concentrations of 0.5, 1.O, and 1.5 ppm was compared to that of controls in Fig. 2. Serum protein concentrations were not consistently changed by 0s exposure (Fig. 2A). Lung soluble protein at the upper two dose levels was increased. Protein from airway washings (Fig. 2A) increased with increasing 0, concentrations, suggesting that significant amounts of serum protein had leaked into pulmonary airways after injury from 0,. The TIC increased in parallel with protein increases in both lung and airway (Fig. 2B). However, serum TIC decreased in 0, exposure. Airway fluid EIC increased markedly as a function of increasing exposure concentration of 0, (Fig. 2C). Lung and serum EIC were reduced at 0.5 and 1 ppm O,, but returned to normal or were increased at 1.5 ppm. Airway esterolytic EICiTIC ratios for exposures to 1.O and 1.5 ppm O3 for 48 hr were decreased to 75 and 50% of the control response (P s 0.05). Lung tissue proteinase activities measured at acid pH increased at 1 and 1.5 ppm 0, exposures conducted for 48 hr (Fig. 3). Proteinases measured at neutral pH were unchanged by 0, exposure (data not shown). Effect of time of exposure to I ppm 0,. The effects of length of both exposure and recovery were examined. The effects of 24 or 48 hr exposure to 1 ppm 0, and 48 hr exposure to 0, followed by a 48-hr recovery period in room air were compared to those of sham-exposed rats. The lung and airway protein EIC and TIC activities increased with the length of exposure to 1 ppm 0, (Fig. 4). Increased

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FIG. 2. (A) Protein, (B) trypsin inhibitory capacity (TIC), and (C) elastase inhibitory capacity (EIC) are shown as a function of increasing concentrations of 0, (0 to 1.5 ppm) for a 48hr exposure (?? k SD). Each parameter is measured on serum, lung, and airway fluid. Protein concentrations are given as mg/ml. Serum concentrations of TIC and EIC are given as milligrams of purified proteinase released per milligram of protein. Concentrations in lung and airway washings are given as milligrams of purified proteinase released per gram of control lung. *P < 0.05, Student’s t. Lung mass of control rats, 1.2 k 0.1 g. Only 1.5 ppm 0, for 48 hr increased lung weight (80% increase).

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amounts of protein were released into lavage fluid for up to 48 hr of exposure to O3 (Fig. 4A). Serum TIC decreased as TIC in lung and airway fluid increased up to 48 hr of exposure to 1 ppm O3 (Fig. 4B). EIC in serum, lung, and lavage fluid was qualitatively similar to TIC (Fig. 4C). Following 48 hr recovery, concentrations of all of these measured parameters had returned to levels near those of controls. DISCUSSION Ozone is a potent environmental oxidant that produces lung injury (Bils and Christie, 1980) at the level of the terminal bronchiole and proximal alveoli (Cavender et al., 1977). This injury includes epithelial cell hyperplasia and hypertrophy, acute inflammation and alveolitis, alveolar septal thickening, macrophage accumulation, pulmonary edema, and, at high exposure levels, pulmonary hemorrhage. Similar histological changes observed in the present study may have resulted from both the proteinase excess or the antiproteinase loss, or both. The dose-dependent and time-dependent pulmonary edema and hemorrhage following 0, exposure probably resulted in increased protein and TIC in the airway washings (Henderson et al., 1979; Mudd et al., 1969). A similar finding has been noted following exposure to NO* (Kleinerman et al., 1985). Most of these changes were reversible as indicated by a return to normal antiproteinase and protein levels following 48 hr recovery. Reduction in lung EIC was probably related to oxidation of a,PI because oxidized OL,PI retains the ability to inhibit trypsin but not elastin (Beatty et al., 1980), leading to a decreased EIC/TIC ratio (Leh et al., 1978) such as that observed in this study. The mechanisms of in viva a,PI oxidation are poorly defined; however, in vitro oxidation of methionine at the active site of a,PI leads to reduction in alPI activity (Travis and Salvesen, 1983; Laskowski and Kato, 1980; Johnson and Travis, 1978, 1979; Matheson et al., 1979; Carp and Janoff, 1977; Janoff et al., 1979; Gadek et al., 1979; Carp et al., 1982; Mudd et al., 1969). The return to normal elastolytic EIC after 48 hr exposure to 1.5 ppm 0, may have been related to transmigration of a,-macroglobulin from blood to lung

* I

1.5 FIG. 3. Soluble lung proteinase activity is shown for lung tissue. *P < 0.05, Student’s t. x * SD.

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FIG. 4. (A) Protein, (B) trypsin inhibitory capacity (TIC), and (C) elastase inhibitory capacity (EIC) are shown as a function of increasing time of exposure to 1 ppm 0, (z k SD). Each parameter is measured in the serum, lung, and airway fluid. Protein concentrations are in mg/ml. Serum concentrations of TIC and EIC are given as milligrams of purified proteinase released per milligram of protein. Concentrations in lung and airway fluid are given as milligrams of purified proteinase released per gram of control lungs. *P < 0.05, Student’s t. Lung mass of control rats, 1.2 f 0.1 g. Only 1.5 ppm 0, for 48 hours increased lung mass (80% increase).

tissue. The presence of hemorrhage and edema in the airway suggested that 02macroglobulin may have crossed the endothelial/epithelial barrier into the pulmonary airways (Mudd et al., 1969; Stone et al., 1982). The role of a ol,M-elastase complexes in chronic lung disease is the subject of much controversy. These complexes have esterolytic but not elastrolytic activity (Travis and Salvesen, 1983; Starkey and Barret, 1977; Roberts, 1982; Kueppers et al., 1981; Stone et al., 1982). ol,M-elastase complexes are cleared very slowly from the lung (Mudd et al., 1969; Stone et al., 1982; Roberts, 1982). It is possible

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that ol,M-elastase complexes may be active in vivo against parts of large native substrates which come into contact with the cY,M-elastase complex (reviewed in Roberts, 1982). To be an important determinant in the development of lung disease, the a,M-elastase complexes should have activity against native elastin or collagen or against tropoelastin, an elastin precursor. Such activity has not been demonstrated. The results presented in this manuscript suggest that ozone exposure produced a time- and dose-dependent response characterized by leakage of protein EIC and TIC from blood into airways and an influx of inflammatory cells. The inflammatory cells released acid proteinase, leading to a proteinase excess relative to the antiproteinases protecting proteins in general (TIC) and elastin (EIC). In addition, the active site of EIC may have been attacked, increasing the inflammatory cellinduced proteinase excess by reducing antiproteinase. These changes may be important in the development of ozone-induced lung diseases, especially emphysema. ACKNOWLEDGMENTS The technical assistance of Ms. E C. Straus and Dr. S. Silbaugh and the reviews of Dr. J. L. Mauderly and Dr. C. H. Hobbs are gratefully acknowledged.

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CARP, H., and JANOFF, A. (1977). Possible mechanisms of emphysema in smokers: In vitro suppression of serum elastase-inhibitor capacity by fresh cigarette smoke and its prevention by antioxidants. Amer. Rev. Respir. Dis. 118, 617-621. CARP, H., and JANOFF, A. (1979). In vitro suppression of serum elastase-inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes. .I. Clin. Invest. 63, 793-797.

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JOHNSON, D., and TRAVIS, J. (1978). Structural evidence for methionine at the reactive site of human a,-proteinase inhibitor. J. Biol. Chem. 253, 7142-7144. JOHNSON, D., and TRAVIS, J. (1979). The oxidative interaction of human a,-proteinase inhibitor: Further evidence for methionine at the reactive site. J. Biol. Chem. 254, 4022-4026. KLEINERMAN, J., IP, M. P. C., and GORDON, R. (1985). The reaction of the respiratory tract to chronic NO, exposure. In “The Pathologist and the Environment-International Academy of Pathology Monograph” (D. G. Scarpelli, J. E. Craighead, and N. Kaufman, Eds.), pp. 200-210. Williams & Wilkins, Baltimore. KUEPPERS, F., ABRAMS, W. R., WEINBAUM, G., and ROSENBLOOM, J. (1981). Resistance of tropoelastin and elastin peptides to degradation by a,-macroglobulin-protease complexes. Arch. Bio&em. Biophys, 211, 143-150. LASKOWSKI, M., and KATO, I. (1980). Protein inhibitors of proteinases. Annu. Rev. Biochem. 49, 593 -626.

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MUDD, J. B., and FREEMAN, B. A. (1977). Reaction of ozone with biological membranes. In “Biochemical Effects of Environmental Pollutants” (S. D. Lee, Ed.), pp. 97- 133. Ann Arbor Science Pub., Ann Arbor, MI. MUDD, J., LEAV~~T, R., ORGON, A., and MCMANUS T. (1969). Reaction of ozone with amino acids and proteins. Atmos. Environ. 3, 669-682. REMINGTON, R. D., and SCHORTZ, M. A. (1970). “Statistics with Applications to Biological and Health Sciences.” Prentice-Hall, Englewood Cliffs, NJ.

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ROBERTS, R. C. (1982). Alpha-2-macroglobulin. In “Reviews of Hematology, Proteinase Inhibitors of Human Plasma” (G. Murano, Ed.). P. J. D. Pub. STARKEY, P M. (1977). Elastase and catheporn G, the serine proteinases of human neutrophil leucocytes and spleen. In “Proteinases in Mammalian Cells and Tissues” (A. J. Barrett, Ed.), Chap. 1, pp. 181-207. Elsevier/North-Holland, Amsterdam. STARKEY, P. M., and BARREN, A. J. (1977). a,-Macroglobulin, a physiological regulator of proteinase activity. In “Proteinases in Mammalian Cells and Tissues” (A. J. Barrett, Ed.), pp. 663-696. Elsevier/North-Holland, Amsterdam. STONE, P. J., CALORE, J. D., SNIDER, G. L., and FRANZBLAU, C. (1982). Role of a,-macroglobulin complexes in the pathogenesis of elastase induced emphysema in the hamster. J. C/in. Invest. 69, 920-93 1. SWITZER, M. E., GORDON, H. J., and MCKEE, P A. (1983). Proteolytic activity of a,-macroglobulin-enzyme complexes toward human factor VIII/van Willebrand factor. Biochemistry 22, 1437-1444. TRAVIS, J., and SALVESEN, G. S. (1983). Human plasma proteinase inhibitors. Annu. Rev. Biochem. 55, 655-709. WERB, Z. (1986). Multiple pathways for regulating the expression of metalloproteinases. .Z. Cell. Biothem. 10A (suppl.), 258.