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Biochemical and Cellular Interrelationships in the Development of Ozone Tnclucec! Pulmonary Fibrosis JEROLD A. LAST University of California, Davis Difficulties in Relating Ozone Exposure to Lung Disease / 416 Response to Fibrogenic Insult / 417 Acute Lung Injury and Edema / 417 Transition from Pulmonary Edema to Cellular Inflammation / 418 The Middle Phase / 420 Lung Fibrosis / 425 Models of Exposure / 427 Experimental Approaches / 427 Chronic Exposure / 429 Progression of Lung Injury After Cessation of Exposure / 430 Synergistic Interactions / 430 Determination of Human Risk / 431 Identification of Susceptible Populations / 432 Summary 1 433 Summary of Research Recommendations / 434 Air Pollution, the Automobile' and Public Health. @) 1988 by the Health Effects Institute. National Academy Press, Washington, D.C. 415

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416 Biochemical and Cellular Interrelationships Difficulties in Relating Ozone Exposure to Lung Disease The focus of this chapter is on the evidence that exposure to ozone (O3) can cause pul- monary fibrosis, generally considered to be a chronic lung disease. A description of the underlying mechanisms of lung injury that might be predictive of other adverse health effects provides a background to the discus- sion of O3-induced pulmonary fibrosis. True chronic experiments have rarely been performed with O3. Examinations of the development of lung fibrosis, a disease widely perceived as chronic, have been made primarily on the basis of data from experiments performed in an acute time frame, usually days or weeks. Long-term data are generally products of earlier exper- iments not performed under present-day standards of care and precision. Animal hygiene in those experiments was not al- ways adequate to prevent outbreaks of pneumonia and other confounding disease. It may be that the human disease analo- gous to that caused by O3 is not chronic lung fibrosis but acute forms of pulmonary fibrosis such as the adult respiratory dis- tress syndrome. The many similarities be- tween the acute disease in humans and the responses of animals to O3 are also dis- cussed in this chapter. The misconception that O3 can cause emphysema deserves special note. Early work of Stokinger and coworkers (1957) suggested this to be the case. However, in those studies O3 was routinely generated from air rather than oxygen, and there was concurrent exposure to nitrogen dioxide (arising from oxidation of atmospheric ni- trogen), a known emphysema-provoking agent. Intercurrent animal illness might also have influenced those results. Perhaps the frequency of occurrence of chronic obstructive pulmonary disease (COPD) vis-a-vis fibrosis tempted investi- gators to find a solution to this much more prevalent disease in humans. Although data are lacking that suggest that exposure to O3, by itself, can cause emphysema, with- out large-scale, careful, lifetime exposures of animals to O3, the possibility that expo- sure to O3 may contribute to the develop ment of COPD in humans cannot be to- tally ruled out. Another difficulty lies in trying to distin- guish between an "effect" and an "adverse health effect." Acute exposure to O3, espe- cially at higher-than-ambient concentra- tions, causes changes in a wide variety of measurable biological parameters. Some of these changes are probably relevant to the mechanisms of action of O3. However, some are not they are more likely to be epiphenomena (for example, changes in barbiturate-induced sleeping time) or arti- facts (for example, sleeplessness and lack of appetite in rats for 1-2 days after the start of exposures to levels of O3 that presumably are irritating enough to cause "sore throats". Interpretation of whether mea- sured changes, even statistically significant changes, are "adverse health effects" may be a particularly difficult problem when human pulmonary function tests are per- formed on volunteers and one or two inter- dependent parameters out of 20 are shown to change slightly. Such "experiments of nature" as epide- miologic studies of the risk of breathing polluted atmospheres in, for example, the Los Angeles air basin, have essentially nothing to tell us thus far. This is true for several reasons. Most important, from the perspective of this chapter, the clinical rec- ognition of pulmonary fibrosis is difficult, except for the most severe manifestations of this disease. Routine spirometry (simple pulmonary function testing), such as may be performed on large populations, is a very insensitive technique for scoring lung fibrosis. There have been very few studies (and no large-scale ones) on autopsies of people routinely exposed to O3 and dying of random causes. Costs of such a large- scale study might be prohibitive. Confounding effects of cigarette smok- ing further complicate large-scale epidemi- ologic studies, as do definitions of appro- priate control groups. The relationship between outcome in an individual and ac- tual dose of O3 to that individual is never known; personal monitoring of individuals in a large-scale study probably has not been performed for O3. Finally, epidemiologic studies are most powerful when the disease

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Jerold A. Last 417 at risk is a rare one, and fibrosis is not rare. All of these limitations also pertain to de- termination of whether O3 exposure in- creases the risk of cancer. Finally, the characteristics of particularly susceptible populations at risk for pulmo- nary fibrosis are unknown. Intuitively- and limited animal data support this intu- ition it is hypothesized that neonates and youngsters whose lungs are still developing might be one such sensitive population. Populations receiving higher-than-ordi nary doses of 03, such as joggers and other ~ ' outdoor exercisers and athletes, and obli gate mouth breathers, might constitute an other. Definition of such potentially sus ceptible populations has profound impact on the proper design and interpretation of animal exposure studies, but this is an area where little has been done experimentally. Some may argue that mathematical modeling of calculated dose delivered to tissue sites can replace such actual experi- mentation, but this is not yet possible. Not enough is known about lung structure, target cells and molecules, thickness and reactivity of protective layers at different sites in the lung, or the role of secondary reactions in amplification of injury, to in- terpret such modeling exercises, either bi- ologically or for regulatory purposes. The requisite data to refine such models to the point where they may replace animal ex- periments will most probably not be avail- able in the near future. In the meantime, measurement of equivalence of biological responses may be the best indicator for interpretation of exposure/dose relation of fibroblasts to these sites (Reiser and Last 1979~. These fibroblasts would then syn- thesize the collagen that constitutes the fi- brotic "scar." Macrophages may release substances that stimulate fibroblasts to pro- duce collagen, or to produce more collagen than their baseline synthesis levels (Reiser and Last 1979~. Other cells, including lymphocytes, granulocytes, and eosinophils, also seem able to release substances chemotactic for one another (Sober and Gallin 1979~. Some or these same inflammatory cell types also seem to be able to release factors that stim- ulate fibroblast mitogenesis (Nathan et al. 1980), which in turn increases the capacity for collagen synthesis at such sites. The interplay of chemotactic, mitogenic, and collagen synthesis-stimulating factors in the etiology of organ fibrosis and the reg- ulation of cell infiltration and turnover in the damaged lung is poorly understood but presently under very active investigation. It will be discussed more thoroughly be- low. Acute Lung Injury and Edema The earliest quantifiable response of the lung to many types of injury, including exposure to 03, is pulmonary edema. At high levels of injury, this response is easy to quantify; the animal is sacrificed and the lung weighed. An increase in lung wet weight over weight of lungs from matched control animals exposed to filtered air can be equated with the increase of fluid that r-~ r- ~constitutes edema. To control for differ ships in humans vis-a-vis animal experi ments. Response to Fibrogenic Insult When the lung is exposed to O3 or other fibrogenic agents administered systemically or by inhalation, the critical response seems to be an inflammatory influx of macro- phages to sites of injury, with accompany- ing edema. This influx may be responsible for the release of chemotactic factors that, in turn, are responsible for the recruitment ences in animal size, data are expressed as the ratio of lung weight to body weight. However, the best way to quantify edema in most animal models that is, the ratio of wet weight to dry weight of the lung is not appropriate with O3 (Cross et al. 1981~. The edema fluid accumulating in lungs of rats exposed to less than 1 part per million (ppm) of O3 has a wet weight to dry weight ratio similar to lung and to blood serum; therefore, pulmonary edema in- duced by O3 has little or no effect on the lung wet weight: dry weight ratio, even in lungs whose wet weight has doubled. Mea

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418 Biochemical and Cellular Interrelationships surement of wet weight (either directly or normalized to body weight) alone, how- ever, can be used to quantify O3-induced edema. Such measurements show concen- tration/response behavior between about 0.5 and 1.0 ppm O3, but these measure- ments are insensitive compared to quanti- fication by other methods. Two sensitive methods deserve special mention: accumulation of serum proteins in lung ravage fluid (Hu et al. 1982; Guth et al. 1986) and tracer transport from blood to ravage fluid (and vice versa) (Alpert et al. 1971; Guth et al. 1986~. Both techniques have been used to examine effects of expo- sure of laboratory animals to O3 at low concentrations. Under the assay conditions used in those studies, the increased protein content of ravage fluid from lungs of rats exposed to O3 was due almost completely to movement of serum albumin from blood to broncho-alveolar and pulmonary interstitial fluid, as determined by electro- phoretic analysis of proteins in the ravage fluid. More recent studies (Warren et al. 1986; Warren and Last 1987) document a positive response by this assay at 0.12 ppm O3, the current peak hourly National Am- bient Air Quality Standard. Increased permeability of the lung to serum proteins can be equated with pulmo- nary edema, a recognized adverse health effect. Increased permeability has been ob- served in animal experiments at O3 concen- trations routinely encountered, or even routinely exceeded, as peak hourly values in southern California smog episodes. At a given concentration of O3, rats as obligate nose breathers probably receive a lower total dose to the peripheral lung than do humans. Further work is necessary to as- certain the reversibility of these changes suggestive of pulmonary edema and the long-term significance of a succession of short-term episodes of pulmonary edema. Current experiments in humans, using la- beled aerosols of diethylenetriaminepenta- acetic acid (DTPA) to probe for permeabil- ity changes of the lung after exposure to O3 in humans (see, for example, Gellert et al. 1985) and rats (for example, Minty and Royston 1985) should be correlated with similar animal work. Recommendation 1. Using experimen- tal animals, anatomic regions in the lung associated with tracer transport from the blood to ravage fluid and vice versa after O3 exposure should be localized and correlated with the bulk movement of albumin into the airspaces. ~ Recommendation 2. To facilitate inter- pretation of human studies, sensitive lav- age-based assays of protein content need to be correlated with DTPA aerosol transport . . assays in anlma s. ~ Recommendation 3. Human studies us- ing DTPA aerosols or other tracers in conjunction with concentration/response assays for edema should be undertaken. There are two important barriers to the accumulation of fluid in the lung in pulmo- nary edema. The first, the endothelial cell permeability barrier, is thought to be in- trinsically leaky. Fluid is constantly cross- ing this barrier and constantly being pumped from the interstitial region back to the vasculature to maintain homeostasis. O3 does not appear to damage endothelial cells directly since it is too chemically reac- tive to traverse the distance from the air- ways to the vasculature and thereby to reach these cells. However, products of O3 reaction with epithelial cells or mucous constituents might well have long enough lifetimes to affect cells deeper in the lung or elsewhere in the body. The other barrier the epithelial cell layer with its tight junctions between cells is thought to prevent fluid move- ment between the vasculature (and intersti- tial space) and the airways. It is thus easy to visualize the relationship between damage to epithelial cells in the small airways and centriacinar regions of the lung and pulmo- nary edema. Transition from Pulmonary Edema to Cellular Inflammation The first lung cells to encounter inhaled or intratracheally instilled fibrogenic agents are the epithelial cells lining the respiratory tract. Oxidant gases such as nitrogen diox

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.lerold A. Last 419 ide (NO2) and O3 have long been known to damage epithelial cells directly; much has been published `,n the mechanisms bv which such oxidant gases may exert their cytotoxic effects (see, for example, Mustafa and Tierney 1978~. Systemically adminis- tered fibrogenic agents such as paraquat, radiation, and possibly bleomycin may also exert a direct cytotoxic effect on lung pa- renchymal cells through generation of free radicals in vitro, and thus these agents may share common pathways of injury and fi- brogenesis with the oxidant gases. There appears to be a wide range of re- sponse of the type I and II pneumonocytes to such agents. In general, type I pneumo- nocytes appear to be more sensitive to damage than type II pneumonocytes, al- though the much greater surface area of type I cells (about 95 percent of normal alveolar epithelium) may merely present a correspondingly larger target for injury. Exposure of an animal to a pneumotoxic agent often results in a characteristic pattern of injury and repair in which type II cells rapidly begin proliferating to repair the epithelial lesions resulting from injury and death of type I cells. Despite their greater cytoplasmic differentiation, type II cells apparently serve as progenitor cells of type I pneumonocytes (Haschek and ~Vitschi 1979). Cellular damage may also result in the local release of soluble mediators thought to be chemotactic for phagocytotic cells. Such mediators could include products from fibrin, fibronectin, albumin, prosta- glandins, leukotrienes, and a vast array of poorly characterized chemoattractants and other factors. Present concepts of the critical sequence of events in lung injury suggest that the response of the lung to cellular damage and/or to pulmonary edema is the move- ment of alveolar macrophages and (per- haps) the recruitment of leukocytes to sites of damage within or near the lung epithelial surface. This process may also contribute to increased airway reactivity. For most models of lung damage, the neutrophil is a ubiquitous participant in this inflammatory response. O3 iS unusual as a pneumotoxicant; the inflammatory response of the lung to O3 seems to be predominantly macrophagic, and there is as yet no evidence for a role of the neutrophil in this process, especially in the critical early stages. It is tempting to speculate that the lack of a neutrophilic response to O3 may have mechanistic sig- nificance, especially with regard to lung fibrosis and, perhaps, to the failure to ob- serve an emphysematous response to O3. This would be consistent with the pre- sumed role of neutrophil elastase in the pathogenesis of emphysema, as discussed by Wright (this volume). Accumulation of alveolar macrophages at sites of lung damage can be beneficial or detrimental. If the response is limited, dogma suggests that the lung repairs itself. However, the long-term consequences to the lung of multiple episodes of this type and whether the lung maintains its putative ability for self-repair in the face of repeated limited insults are unknown. If the macro- phagic response is more exuberant, then the alveolar macrophages elaborate factors that stimulate the proliferation of lung fi- broblasts. Such fibroblast proliferation gives rise to increased numbers of intersti- tial lung cells producing collagen, the hall- mark of pulmonary fibrosis. This process is not well understood. Specifically, the ex- tent of macrophage accumulation at sites of lung damage cannot be used to determine whether lung repair or progression to fi- brosis occurs. However, macrophage accu- mulation elicited by "inert" particles (for example, fly ash) does not result in lung fibrosis. The concept of cellular damage is often invoked in discussions of toxicity but sel- dom defined in biochemical terms. There seems to be a general consensus as to the mechanism of action of 03, which is as- sumed to initiate lipid peroxidation in lungs by reacting with unsaturated fatty acids of cell membranes. This putative mechanism of action, although widely accepted, has not been proven. Neither is the chemical basis for generation of free radicals by these reactions completely straightforward. It is likely that if free radicals and active oxygen species do play a role in cellular damage elicited by 03, macrophages and leuko

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420 Biochemical and Cellular Interrelationships cytes participating in the inflammatory re- sponse might well be the source of such active oxygen species. The initiation of lipid peroxidation in the lung is a possible mechanism for oxidant damage by gases such as O3 and NO2 (Fridovich 1976; Waling 1963~. In recent years a considerable body of evidence has been amassed in support of the hypothesis that lung damage may be mediated in large part by reactions involving free radicals. Such radicals may be directly generated by reaction of toxicants with target molecules (for example, O3 or NO2 reaction with polyunsaturated fatty acids), or indirectly as a result of phagocytotic activity by alve- olar macrophages or neutrophils. These cells release free radicals such as superoxide and hydroxyl during increased metabolism which is often termed "oxidative burst." Evidence for the role of free radicals in lung damage includes a wide variety of observations. Uncontrolled clinical trials have shown increased survival of patients suffering from adult respiratory distress syndrome after they are treated with vita- min E (Wolf and Seeger 1982~. Treatment with various hydroxyl radical scavengers protected rats from pulmonary edema in- duced by high doses of thiourea or from otherwise lethal levels of gamma irradia- tion (Fox et al. 1983~. Recent studies have shown protection against hyperopia by su- peroxide dismutase or catalase stabilized by encapsulation in liposomes (Turrens et al. 1984~. Production of oxygen radicals in rat lungs during hyperoxia has been directly demonstrated by Freeman and Crapo (1981), and in vitro studies by Martin (1984) support this observation. Numerous studies, among them Crapo et al. (1978) and Mustafa and Tierney (1978), have re- ported increases in the activity of free rad- ical-scavenging enzymes in lungs of ani- mals surviving insult with 03, NO2, or other toxicants, thereby indirectly support- ing this hypothesis. In vitro experiments also support the concept that O3 can di- rectly injure cells by a mechanism involv- ing free radicals (see, for example, Morgan and Wenzel 1985~. This is a field of intense investigation at present, with new observations reported in practically every issue of the relevant jour- nals. The linkages by which early damage are connected to "late" pulmonary fibrosis have not been defined and remain an area for future studies. Recommendation 4. Animal studies should be done to determine the role of free radicals and active oxygen species in lung . . 1nJury. The Middle Phase The middle phase, or the events that occur between injury to cells and deposition of excess collagen, is the component of the lung's response to injury that we under- stand the least. The recruitment of alveolar macrophages to sites of injury and the subsequent proliferation of fibroblasts are key cellular events in the pathogenesis of pulmonary fibrosis. In addition, there are published studies (reviewed by Haschek and Wits chi 1979) suggesting an important role for the alveolar type II cells in the determination of the balance between cell repair and fibrogenesis in the damaged lung. Epithelial Cells. Alveolar type II cell pro- liferation may represent a critical period in terms of repair (Haschek and Witschi 1979~. Disruption of the normal sequence of events during this period may favor the development of pulmonary fibrosis. For example, systemic administration of buty- lated hydroxytoluene to mice results in widespread necrosis of type I cells within 24 hr. On days 2 and 3, most dividing cells are type II pneumonocytes. If mice are exposed to high (70 percent) concentrations of oxygen (02) during this period, type II cells are severely affected, whereas dividing interstitial cells are not. Inhibition of the epithelial cell proliferation allows the inter- stitial cells to proliferate relatively un- checked, resulting in increased collagen pro- duction and pulmonary fibrosis. Animals exposed to x rays instead of O2 showed similar results (Witschi et al. 1980~. These experiments also showed that the timing of the second insult was critical. If it

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Jerold A. Last 421 did not coincide with the period of reepi- thelialization, then the enhanced fibrosis was not observed. Other investigators have found a similar enhancement of fibrosis in rats exposed to bleomycin followed by high levels of O2 (Rinaldo et al. 1982; Tryka et al. 1982~. Interestingly, paraquat, which by itself can induce irreversible fi- brosis, has been observed to destroy type I as well as type II cells (Vejeyaratnam and Corrin 1971; Smith and Heath 1973; Skill- rud and Martin 1984~. These data suggest that epithelial cell control of fibroblast pro- liferation may be an important early mech- anism in pulmonary fibrosis of various etiologies (Bowden 1984~. Recommendation 5. Studies of the link- ages between epithelial cell damage and repair and changes in populations of pul- monary macrophages and interstitial cells should be undertaken in whole animals. Pulmonary Alveolar Macrophages. Many investigators have allocated a central role to the pulmonary alveolar macrophage in the initiation of fibrosis. The role of the mac- rophage in inflammatory and immunologic processes is enormously complex and the topic is reviewed periodically (Hocking and Golde 1979; Nathan et al. 1980~. A related topic is the effects of fibrogenic agents on pulmonary alveolar macrophages in terms of their recruitment and functional response. The time course of macrophage accumulation in relation to a fibrogenic stimulus appears to vary with the insult and, to some extent, with the experimental design. In many models of fibrosis, macro- phages begin accumulating extremely rap- idly after exposure to the fibrogenic agent. Brody and coworkers (1985) hypothe- sized that complement may play a key role in particle-induced macrophage migration. They found that rats depleted of comple- ment by cobra venom and mice genetically deficient in complement had far less mac- rophage accumulation following asbestos exposure. In addition, the molecular weight of the chemotactic factor from lav- age fluid was consistent with its being C5a. These researchers did not speculate on the source of the complement component, ex cept to point out that edema fluid (serum transudate) is an important potential source of lower-molecular-weight components of complement in the damaged lung. Other investigators have shown that macrophages themselves may participate in macrophage recruitment. Dauber and Da- niele (1980) found that guinea pig lung macrophages secreted a chemoattractant for macrophages as well as for neutrophils and lymphocytes. Kagan and coworkers (1983b) found that alveolar macrophages from rats exposed to asbestos appear to secrete a chemoattractant for macrophages. That study did not, however, examine the effects of this chemoattractant on other effecter cells. Partial characterization sug- gested it to be a protein and heterogeneous with regard to molecular weight. Circulat- ing immune complexes, whose etiology is unknown, are also believed to stimulate macrophage accumulation (Hunninghake et al. 1981~. The direct effects of fibrogenic agents on macrophages also seem to vary consider- ably, depending on the specific agent and the experimental design. The role of the macrophage in fibrogenesis has often been envisioned as passive; that is, the macro- phage may release various soluble media- tors that play a role in fibrogenesis. More recent studies suggest that macrophages may respond to a fibrogenic agent in vari- ous ways. Silica-exposed rat lungs showed numer- ous silica-containing macrophages in lav- age fluid as well as in situ immediately after exposure, with high percentages of silica . . . . . contaln1ng macrop aages remalmng SIX weeks later (Brody et al. 1982; Warheit et al. 1984). Ultrastructural examination re- vealed that the silica-containing macro- phages appeared normal, and functional studies (measurement of oxygen consump- tion and phagocytosis) of ravaged macro- phages revealed no abnormalities. A decrease in phagocytic capacity is not necessarily synonymous with functional impairment. For example, Tryka and co- workers (1984a) found that alveolar macro- phages ravaged from lungs of hamsters exposed to bleomycin and O2 at time points ranging up to 120 days increased in

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422 Biochemical and Cellular Interrelationships number but had a decreased amount of cell surface antigen, indicating that they were relatively immature. Those researchers at- tribute the markedly decreased phagocytic capability at least partly to the decrease in surface antigen. Other data from that study suggest that fibrosis may be associated with increased macrophage turnover. Once macrophages have been recruited, they are capable of releasing many media- tors that modulate the behavior of other effecter cells in the lung. Kazmierowski et al. (1977) observed that macrophages ob- tained from normal primate lungs secreted at least two chemotactic factors. One factor had properties consistent with its identity as the complement component Csa, and was chemotactic for neutrophils, mononu- clear cells, and eosinophils. The other fac- tor had an apparent molecular weight of about 5000, did not appear to be a comple- ment component, and was a specific che- moattractant for neutrophils. Similarly, Merrill and coworkers (1980) found that human alveolar macrophages secreted two chemoattractants for neutro- phils, and Dauber and Daniele (1980) found that macrophages from guinea pig lung secreted chemoattractants for macro- phages, neutrophils, and lymphocytes. A phagocytic stimulus increased the release of the neutrophil chemoattractant. Recommendation 6. Basic research in cell culture systems should be performed to examine the biochemical basis of cell-cell communication and the molecular nature of various mediators, released by leuko- cytes and macrophages from damaged lungs, that enhance or prolong the cellular inflammatory response. Macrophages may also modulate effecter cells in another way. Stimulated macro- phages are capable of releasing arachidonic acid from cell phospholipid pools. Further metabolism through the cyclooxygenase pathway results in the production of the prostaglandins PGE~ and PGI~. among other products. In vitro data show that PGE2 and PGI2 suppress a variety ot neu- trophil, macrophage, and lymphocyte functional responses. They may also partic ~ ~O mate in the induction of suppressor T cells. In marked contrast, metabolic products of arachidonic acid resulting from the li- pooxygenase pathway appear to enhance the inflammatory response (Hunninghake et al. 1984~. Recommendation 7. Mediators possi- bly released by damaged lung epithelial cells or derived from damaged matrix com- ponents, which maintain and amplify lung injury after acute cellular or organ damage, should be characterized. Macrophage secretion of effecter cell chemoattractants appears to be increased in fibrosis. Schoenberger and coworkers (1982) observed that asbestos stimulates alveolar macrophages to increase neutro- phil migration to the lung. Wesselius and coworkers (1984) found that macrophages ravaged from exposed rat lungs between 5 and 20 days after bleomycin instillation secreted increased amounts of neutrophil chemoattractant as compared with macro- phages from controls. Since bleomycin did not directly stimulate macrophages, the mechanism for the increased macrophage secretion is unclear. The authors postulate that the stimulus may result from cell in- jury caused by bleomycin. In addition, data from a study of macrophage-derived che- moattractants from hamsters instilled with bleomycin suggest that macrophages may be regulating the sequence of effecter cell mi- gration following injury (Kaelin et al. 1983~. The interaction between macrophages and fibroblasts is particularly complex since macrophages appear to have the capacity to stimulate fibroblasts as well as to suppress them. Stimulation of fibroblast prolifera- tion alone is also complex. Stiles and co- workers (1979) proposed dividing growth factors into competence factors (which pro- vide a signal early in the G. phase of the cell cycle) and progression factors (which pro- vide a signal later in Gil, stimulating the cell to replicate. Bitterman and coworkers (1982) showed that alveolar macrophages secrete a growth factor (alveolar macrophage-derived growth factor, mol. wt. = 18,000) for fibroblasts that is distinct from other described growth fac

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lerold A. Last 423 tars. The alveolar macrophage-derived growth factor is believed to function as a progression factor and to stimulate fibro blasts to produce their own progression factor. Macrophages secrete fibronectin, a large glycoprotein known to mediate cell/matrix interactions through a variety of functions including its chemotactic properties (Hun ninghake et al. 1984~. Fibronectin is be lieved to act as a competence factor for fibroblast growth (Bitterman et al. 1983~. In addition, fibronectin is chemotactic for fibroblasts. Indeed, macrophage-derived fi bronectin is 1,000-fold more potent as a chemoattractant than is plasma fibronectin (Rennard et al. 1982~. Macrophages also appear to be capable of suppressing fibroblast growth. Elias and coworkers (1985) found that supernatants from normal human alveolar macrophages inhibit growth of log-phase fibroblasts. Their study showed that the inhibitory capacity of the supernatant was directly related to its capacity to stimulate fibroblast prostaglandin production. They separated macrophage subpopulations by density gradients and found that the factor(s) an peared to be preferentially elaborated by smaller and denser macrophages. In some respects this factor resembles those de- r -a scribed by Korn et al. (1980) and Clark et al. (1982~. Investigation of the effects of specific fibrogenic agents on macrophage regula tion of fibroblast growth illustrates the complexities involved. For example, Lu gano and coworkers (1984) found that 2 and 14 days after silica exposure, ravaged macrophages depressed fibroblast prolifer ation, whereas at 42 days macrophages stimulated fibroblast proliferation. Clark and others (1982) discovered that macro phages from hamsters instilled with bleomycin had a greater suppressive ettect on fibroblast proliferation and collagen synthesis compared with control macro phages. They found that suppression was associated with increased PGE2 and intra cellular cAMP, and that fibroblast suppres sive activity decreased in the first days after bleomycin instillation, and then increased after 8 days. This suppressive activity may ~ . . be a mechanism for modulating fibroblast proliferation and fibrosis following fibro- gen~c exposure. Recently published data of Schmidt et al. (1984) suggest that interleukin-1 may play a role in fibroblast proliferation. Although other researchers have not found that inter- leukin-1 has fibroblast-stimulating proper- ties, Schmidt and coworkers point out dif- ferences in experimental design that might account for this discrepancy. Peripheral blood monocytes were used in that partic- ular study, and it should be noted that alveolar macrophages are also capable of secreting interleukin-1. No detailed studies have been reported with macrophages or effecter cells from animals exposed to O3. Such work should be done to test whether (and which of) these pathways might be operative in the lungs of animals exposed to o3. Broncho-alveolar ravage was used to ob- tain macrophages in many of the studies discussed above. Given the heterogeneity of macrophages, it is unclear if the popu- lations present in ravage fluid accurately reflect the populations actually present in lung tissue, particularly in disease states. The problem is compounded by the fact that there is no consensus as to the ap- oronriate functional and/or structural def- ~n~t~ons of such macrophage subpopu- lations. Brain and coworkers (1977), Mason (1977), and Lum and coworkers (1983) have reviewed some of the potential problems in studying ravaged macro- phages. In detailed morphometric analyses of centriacinar macrophages in situ and pul- monary macrophages ravaged from control and O3-exposed rats, Lum and coworkers (1983) observed significant differences in most parameters studied between the la- vaged and in situ macrophages in the con- trol as well as the O3-exposed rats. These data suggest that, at least in this model of fibrosis, ravaged macrophages are not rep- resentative of macrophages present at the sites of greatest lung damage. Alterna- tively, it is questionable whether interstitial macrophages, and those resident in airways and therefore accessible by ravage, are a homogeneous population in equilibrium or

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424 Biochemical and Cellular Interrelationships are somehow "different." Clearly, similar problems may exist in studying any of the effecter cells obtained by ravage. Fibroblasts. Presumably the cells respon- sible for synthesizing the "abnormal" col- lagen of pulmonary fibrosis whether it be abnormal in amount, location, or type are the fibroblasts. Although they often are perceived as passive target cells for the effecter cells and their mediators, fibro- blasts may on occasion play a more active role in directing the course of fibrosis. In some cases fibroblasts may directly interact with the fibrogenic agent. Several workers have examined the effects of bleomycin on fibroblasts. Sterling and co- workers (1982) found that collagen synthe- sis increased in human fetal lung fibroblasts exposed to bleomycin for 48 hr. but deg- radation also increased. They also found that polysomes from bleomycin-treated fi- broblasts synthesized twice as much colla- gen as control polysomes, but noncollagen protein synthesis was not affected. Similar results have been reported by Clark et al. (1980) and Phan et al. (1985~. In an examination of the effects of bleomycin, hyperopia, and the presence of lung macrophages on collagen synthesis by human WI-38 fibroblasts, Robin and Juhos (1983) found that bleomycin alone directly stimulated collagen synthesis, as measured by hydroxyproline in the culture dishes. The addition of hyperoxia and/or the pres- ence of lung macrophages did not further increase collagen synthesis, and hyperopia alone significantly decreased collagen syn- thesis. However, hyperopia in the presence of lung macrophages increased collagen synthesis about the same extent as did bleomycin. There is also evidence that bleomycin affects fibroblast proliferation. In a study of the effects of in vitro and in viva exposure to bleomycin on growth characteristics of fibroblasts, Absher and coworkers (1984) observed that both types of bleomycin ex- posure appeared to decrease growth of fi- broblasts in comparison with controls. In a similar system of in vitro exposure, Phan and coworkers (1985) found that bleomy- cin exposure had no effect on growth. However, since that study examined iso- lated fibroblasts 14 days after bleomycin instillation, its authors suggest that the difference in timing may account for the apparently discrepant findings. That is, during the first week after instillation, bleomycin toxicity may impair fibroblast growth, whereas during the second week, recovery may be occurring. Furthermore, this recovery may involve recruitment or selection of a population of fibroblasts with different growth characteristics. Whether these provocative findings in the bleomycin system accurately model events in lungs of animals exposed to O3 remains to be tested. Fibroblasts are believed to play a role in epithelial cell growth and function. Fibro- blast pneumonocyte factor has been iso- lated from glucocorticoid-treated fibro- blasts and has been shown to stimulate disaturated phosphatidylcholine synthesis in whole lung in vitro (Smith 1979) and in isolated type II pneumonocytes (Smith 1978~. A small somatomedin-like growth factor specific for pneumonocytes is be- lieved to be secreted by fibroblasts after pneumonectomy (Smith et al. 1980~. Pul- monary fibroblasts exposed to hyperoxia in vitro secrete an epithelial cell growth factor as well as a lipid-synthesis-stimulating fac- tor (Tanswell 1983~. These particular fac- tors appear to differ from any of the others previously described. Fibroblasts may also affect effecter cells. Although their predominant function is apparently production of collagen, other matrix components, and mucopolysaccha- rides, they also secrete biologically active products such as Cal and interferon (Al- Adnani and McGee 1976~. Cultured fibro- blasts produce a factor chemotactic for mo- nocytes as well as for neutrophils (Sober and Gallin 1979~. In addition, fibroblast culture fluid is capable of generating chemotactic activity from human serum, probably by cleaving C5a from C5. Fibro- blasts are capable of producing macrophage migration inhibition factor (Tubergen et al. 1972~. Whether such "fibrokines" play a role in pulmonary fibrosis has yet to be demonstrated. Collagen itself is another fibroblast prod- uct with chemotactic properties. Type I

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.lerold A. Last collagen and its isolated chains are chemo- tactic for monocytes but not neutrophils (Stecher 1975; Postlethwaite and Kang 1976~. In contrast, rat collagen is chemo- tactic for rat neutrophils in viva (Chang and Houck 1970~. ~ Recommendation 8. Examination of factors released by inflammatory cells that modulate collagen synthesis or fibroblast proliferation, especially in response to 03, might help to define the mechanisms un- derlying the transition from the damaged, inflamed lung to the fibrotic lung. Lung Fibrosis Lung fibrosis, as defined clinically, refers to interstitial fibrosis as is seen in the later stages of idiopathic pulmonary fibrosis (also called cryptogenic fibrosing alveolitis in the literature of the United Kingdom). In this disease the hallmark of pulmonary fibrosis as seen by the pathologist is in- creased focal staining of collagen fibers in the alveolar interstitium. Despite earlier misconceptions, it seems clear that fibrotic lungs from humans with either acute or chronic pulmonary fibrosis contain in- creased amounts of collagen as evaluated biochemically, in agreement with the his- tological findings. In the normal lung, interstitial collagen fibers are thought to provide structural matrix or scaffolding upon which the lung cells are assembled. These fibers are also thought to be responsible for the limits to which the alveoli can be stretched during inhalation or to which they can relax dur- ing expiration. The deposition of additional collagen in the fibrotic lung is presumably responsible for the increased stiffness of these lungs, whereby the volume to which they can expand at a given distending pres- sure is decreased as compared with normal values. Unfortunately, pure interstitial fibrosis does not generally occur in lungs damaged by toxicants. In many ways such toxicants cause disease that more closely resembles adult or infant respiratory distress syn- drome than chronic fibrosis. Excess lung collagen is usually observed not only in the 425 alveolar ~nterst~t~um, but also throughout the centriacinar region, including the alve- olar ducts and respiratory bronchioles. The relationship between increased collagen deposition around small airways and lung mechanics is not understood, either theo- retically or empirically. Recommendation 9. The reversibility of excess collagen deposition in the fibrotic lung, as reflected by increased hydroxy- proline content of the lung, should be determined. There are at least 13 genetically distinct collagen types known to occur in all mam- mals, of which.at least seven have been found in normal lungs or synthesized by isolated lung cells. Two types predominate in the lung, representing about 90 percent or more of the total lung collagen. Type I and III collagens are the major interstitial components and are found in the normal lungs of all mammals in an approximate ratio of 2:1. This ratio is altered in fibrotic lungs. It is not known whether shifts in collagen types, as compared with absolute increases in collagen content, account for the in- creased stiffness of fibrotic lungs. Type III collagen is much more compliant than is type I; thus, an increasing proportion of type I relative to type III collagen might result in a stiffer lung as is observed in pulmonary fibrosis. Changes in collagen cross-linking in fibrotic lungs may also contribute to the increased stiffness ob- served. In addition, because type I collagen is the material that stains histologically as "collagen," whereas type III collagen does not, an increased proportion of type I rel- ative to type III collagen would be appre- ciated histologically as an "increased amount of lung collagen." Therefore, it is unclear whether the observed increase in stainable collagen is due solely to the in- crease in collagen content of the lungs observed biochemically, or whether altered collagen types or cross-linking might also contribute to the histological changes seen. Some types of pulmonary fibrosis, in- cluding that induced by 03, involve abnor- malities in the type of collagen being made.

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430 Biochemical and Cellular Interrelationships posed and control rats reached the same endpoint collagen content after six months, with the exposed animals reaching this value more rapidly (at three months). It seems likely that the changes in hydroxy- proline content of the lungs from exposed rats observed over the first three months of the study were related to excessive deposi- tion of collagen (fibrosis) in their lungs (as observed morphologically), but a role for accelerated aging (or growth) of the lungs in O3-exposed rats cannot be ruled out as an alternative explanation of these observa- tions. One disadvantage of rats as an ex~oer- imental animal for chronic studies Is that rats continue to grow throughout their en- tire lifetime, and lung growth is not "com- plete" at maturity as in humans. Recommendation 12. The response of the developing lung versus the mature lung in animals exposed to O3 should be studied to ascertain whether the developing lung is more susceptible to damage, fibrosis, or change. Progression of Lung Injury After Cessation of Exposure What are the consequences of allowing animals to "recover" during a postexpo- sure period? In another set of experiments, intermittent exposure to high levels of O3 (0.64 or 0.96 ppm) for 8 hr/day elicited the same increases in lung collagen content of adult rats as did a continuous exposure protocol of 23.5 hr/day (Last et al. 1984b). This finding emphasizes the importance of not assuming that effects of O3 exposure may be estimated by a simple concentration x time relationship. A six-week postexpo- sure "recovery period" breathing filtered air exacerbated the increase in lung collagen content appreciated immediately after ces- sation of exposure. This result suggests that not only are these effects irreversible, at least in this time frame, but that they are also progressive. The mechanism for exacerbating dam- age, or repairing such damage, to the lung by a postexposure period breathing filtered air is not obvious. Before any meaningful O ~ hypothesis can be presented, further exper- iments examining cellular changes within the lung during such a postexposure period are necessary. Examination of the time course and cellular components of reepithe- lialization of the alveolar ducts and walls during the postexposure period would be especially important in this regard. Haschek and Witschi (1979) stressed the importance of this component of lung re- pair in potentiation by O2 of pulmonary fibrosis after lung damage with butylated hydroxytoluene. Further, to avoid misin- terpretation of data because of altered growth rates in exposed and control ani- mals, use of appropriate (pair-fed) controls in experiments with growing animals ex- posed to high levels of O3 iS obligatory. This specific area of research on postexpo- sure effects of O3 iS an important one for further study. Recommendation 13. Lung structure and biochemistry over long postexposure periods should be studied in detail. Synergistic Interactions The design of experiments to assess fibro- genicity of a given agent becomes much more complex when the possibility of syn- ergistic (or antagonistic) actions between agents is considered. For example, Haschek and coworkers found that exposure of mice to 70 percent O2 enhances pulmonary fi- brosis previously induced by intraperito- neal injection of butylated hydroxytoluene (Haschek and Wits chi 1979; Has chek et al. 1983~. They concluded that the severe fi- brosis seen after the combination of buty- lated hydroxytoluene and O2 is the result of synergism, and proposed the following mechanism: when butylated hydroxytol- uene causes lung damage, there is an initial phase of epithelial proliferation. If mice are exposed to 70 percent O2 during this pe- riod, the epithelial type II cells are either inhibited from dividing or killed. Damage to the epithelial cells may then lead to unin- hibited interstitial cell growth. This specific interaction between butylated hydroxytol- uene and O2 seems to occur only in mice.

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Jerold A. Last 431 A similar synergism apparently occurs with bleomycin and 70 percent O2 in rats, hamsters, and other laboratory rodents (Tryka et al. 1984b) and is not specific to the mouse. Other such two-agent models also seem to work in multiple species (Has- chek et al. 1983~; such combinations in- clude drugs and 02, radiation and 02, and cytotoxic agents and bleomycin. In another study of synergism between particulates and gases, McTilton and Charles (1976) examined the effects on guinea pigs of exposure to sodium chloride aerosols and SO2. When the mixture was at a high humidity, those researchers found decreases in airway flow resistance. Nor- mally, SO2 does not penetrate deep into the lung, and McTilton and Charles proposed that the highly soluble SO2 dissolved in droplets and thus was able to "piggyback" into the lower respiratory tract. Ellison and Wailer (1978) have reviewed this topic. In a study of oxidant gases plus SO2, Gardner and coworkers (1977) found that a protocol of O3 and sulfuric acid (H2SO4) aerosol presented sequentially was more toxic than either agent alone, based on mortality rates of mice exposed to Strepto- coccus pyogenes. Juhos and coworkers (1978) found evidence for synergism between O3 and H2SO4, based on very limited his- tologic evaluations of rat lungs. Last and Cross (1978), using several biochemical cri- teria and studying the effects upon tracheal mucous-producing cells, reported a syner- gism between O3 and H2SO4 aerosols at relatively low concentrations of each. Ha- zuka and Bates (1975) observed synergism between O3 and sulfate aerosols and sug- gested that it was responsible for decreased maximal flow rates observed during human exposure to O3 and SO2 or H2SO4 at near-ambient levels. In a recent study of the effects of ammo- nium sulfate aerosols in combination with O3 or NO2 on collagen metabolism in rat lungs, Last and coworkers (1983a) found that ammonium sulfate aerosols alone had no effect on collagen synthesis rates; how- ever, they significantly potentiated the effects of the oxidant gases. Guth and coworkers (1986) found that lavagable pro tein content increased significantly in lungs of rats continuously exposed for 1-2 days to 0.12 ppm O3, and, more important, Warren and Last (1987) saw elevated colla- gen synthesis when rats were exposed to 0.12 ppm O3 plus H2SO4 aerosol. The latter researchers obtained similar results with rats exposed to 0.2 ppm O3 + 40 ,ug/m3 of acid aerosol. We interpret these results as being indicative of a prefibrotic response of the lung to injury. Furthermore, responses in this and other assays in rats exposed to 0.2 ppm O3 for 8 hr/night and in rats exposed continuously have been similar. That is, the increase in lung collagen synthesis rate is not contin- gent upon continuous exposure of rats to O3, but may occur under an intermittent exposure regimen that models human diur- nal exposures as well. These results must be confirmed, especially in another species. Last and coworkers (1984a) suggested a hypothetical mechanism for this synergy involving increases in the stability of free radicals generated in situ. Since the mech- anisms of injury elicited by individual pneumotoxins are so poorly understood, it is hardly surprising that mechanisms un- derlying synergistic interactions remain highly speculative. Recommendation 14. A rational basis should be developed for prediction of syn- ergistic or antagonistic interactions of pol- lutant mixtures by systematically examin- ing binary and ternary combinations of the pollutants. The possibility that agents known to affect epithelial cell turnover might interact synergistically with O3 de- serves special attention. Determination of Human Risk What are the implications of these experi- ments for those charged with setting ambi- ent air quality standards? Clearly, the tra- ditional data base used for setting such standards is heavily skewed in the direction of detecting acute, short-term effects such as reflex bronchoconstriction in human

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432 Biochemical and Cellular Interrelationships subjects undergoing controlled experimen- tal exposures. However, increased airway resistance may occur in response to release of stored histamine from airway mast cells in response to signals from irritant receptors (Dixon and Mountain 1965) and may have little or no long-term conse- quences to health. The inflammatory re- sponse of the lung to O3 may also contrib- ute to increased airway reactivity after exposure. The potential adverse health effects of air pollution that constitute the major concern, however, are chronic effects from intermit- tent, long-term, low-level exposures: can- cer, emphysema, pulmonary fibrosis, and chronic obstructive lung disease. Con- trolled human exposures are of absolutely no value for assessing these types of risks. To date, epidemiologic studies have also been of little value in assessing these risks (Committee on Medical and Biological Ef- fects of Environmental Pollutants 1979), partly because of the overwhelming im- pacts of smoking and occupational expo- sures on the incidence of these diseases in our population, and the uncertainty about individual doses as compared with expo- sures of entire populations. It is not practical to look at each pollutant and every possible combination of pollut- ants in long-term dose/response experi- ments that may require inhalation expo- sures for six months, or a year, or an entire lifetime (three years or more in rats or mice). Thus the need is for short-term assays that probe for potential structural changes in the lung, such as those for collagen and elastin metabolism described above. Such short-term assays may detect potential adverse health effects of air pol- lutants that cannot be ascertained by con- trolled human exposures or other currently used methodology for risk assessment. However, the regulatory climate until now has tended to ignore animal inhalation tox- icology experiments in favor of data from controlled human exposures (Whitten- berger 1985~. ~ Recommendation 15. Greater use should be made of animal models of sus- ceptible populations. We also seem to be allowing ourselves to be lulled into a sense of security with the current ambient air quality standards, on the basis of the belief that we adapt to pollutants upon continued exposure. The concept of adaptation comes from the at- tenuation of reflex bronchoconstriction in controlled human exposures to, for exam- ple, O3 upon continued exposure. There are no data suggesting that the lung can adapt to continued exposure to O3 when the assay for effects is based upon structural changes in the lung rather than upon tran- sient responses such as reflex bronchocon- striction or localized inflammation. It is only by designing experiments that correlate data between short-term assays for structural change (to evaluate dose/re- sponse characteristics of the lung) with selected long-term chronic exposures that include detailed examination of lung struc- ture that we will be able to evaluate prop- erly the true risks of exposure to ambient air pollutants and their mixtures. Recommendation 16. Long-term (life- time) exposures of rodents should be per- formed under realistic protocols with so- phisticated assays at termination. Such studies should also examine tumor inci- dence and, to be meaningful, would require very large groups of animals. Identification of Susceptible Populations The wording of the 1977 version of the Clean Air Act raises the question of poten- tial susceptible populations in relation to any attempt to evaluate actual or hypothet- ical human risks of exposure to O3. Are there identifiable susceptible populations from the standpoint of potential lung fi- brosis? First, and most important, are very young children and infants, whose lungs are still developing and who would seem from animal experiments to be at risk of their lungs developing differently in atmo- spheres containing O3. "Differently" in this context relates to the higher collagen con- tent found in lungs of young rats than in

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herald A. Last 433 lungs of matched, pair-fed controls (Last et al. 1984b) and the elongated alveolar ducts or respiratory bronchioles observed in rats or monkeys after exposure to O3 (Tyler et al. 1985~. It is not known if these observa- tions constitute adverse health effects, hence, use of the word "differently." Second, and obvious, is the population that breathes polluted outdoor air rather than conditioned indoor air by the nature of their professions, by choice, or by necessity during episodes of pollution, especially in conjunction with strenuous exercise (which increases the inhaled dose to the lungs the prototypical "freeway joggers. Since fibrosis is an insidious disease and the lung has a substantial reserve capacity to protect itself from the physiological con- sequences of fibrosis, a third population at risk includes people whose lungs are al- ready severely compromised by chronic obstructive pulmonary disease, emphy- sema, congenital disease, or fibrosis. A fourth group at risk would be those with diseases of the small airways, the focal site of O3-induced fibrosis. The largest group with small airways pathology would be cigarette smokers. If the cellular mech- anisms of lung inflammation and injury discussed earlier pertain in human popula- tions, subjects with chronic lung inflamma- tion (alveolitis) should be at increased risk. This group would include workers and others with allergic alveolitis and patients (such as those with idiopathic pulmonary fibrosis) who are subject to recurrent epi- sodes of inflammatory infiltration of the distal lung with leukocytes and macro- phages. Summary Inhalation of O3 by experimental animals causes pulmonary edema after exposures of short duration and results in peribronchio- lar and centriacinar pulmonary fibrosis af- ter longer exposures. This discussion has stressed possible linkages between these outcomes, including the probable central role of the alveolar macrophage as an effec- tor cell. The phenomenon and the under lying mechanisms of pathogenesis of pul- monary edema are probably well enough known for these observations to be extrap- olated to assessment of human risks upon exposure to a given concentration of 03, and sensitive clinical methods applicable to hu- mans are available to compare directly with such estimates. However, the reversibility of such changes and whether increased fluid in the bronchial and alveolar airspaces is "damage" in the regulatory sense (that is, "an adverse health effect") are unknown. At the other end of the process, much is being learned about the biochemistry of collagen in the fibrotic lung. Little or noth- ing is known of the cellular mechanisms responsible for excessive synthesis and dep- osition of fibrotic collagen, or whether such changes are reversible. As far as O3 is concerned, nothing is known of the corre- sponding cellular and biochemical mecha . . nlsms in Humans. The events linking acute and chronic responses of the lung to O3 are poorly understood and should be a primary focus of future research, in cell biology as well as in biochemistry. Most of the methodology necessary for such studies in animals is probably available. Other topics of interest are progressive versus self-limited injury to the lung, re- versibility of damage, and the potential for synergism between mixtures of air pollut- ants, especially O3 and respirable aerosols or particulates. Recent studies in various animal models have suggested that certain combinations of agents, which alone cause limited or undetectable lung injury, can cause progressive pulmonary fibrosis. These models have certain features in common with the continuing changes ob- served in lungs of rats and monkeys ex- posed to O3 followed by periods where they breathe only filtered pure air. Rats exposed to O3 in combination with various acidic aerosols, which by themselves appar- ently cause no lung damage at the concen- trations tested, have suffered enhanced acute lung damage. The significance of these observations to potential chronic, ir- reversible changes in rat lungs, or to poten- tial human health effects, remains to be determined.

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434 Biochemical and Cellular Interrelationships Summary of Research Recommendations Acute Phase of Injury In the acute injury phase, the greatest need is for a better understanding of the pathophysiological significance of apparent alterations in broncho-alveolar epithelial permeability in animals and humans acutely exposed to O3. HIGH PRIORITY Recommendlation 2 To facilitate interpretation of human studies, sensitive ravage-based assays of protein content need to be correlated with diethylenetri aminepentaacetic (DTPA) acid aerosol transport assays in animals. MEDIUM PRIORITY Recommendlation 1 Using experimental animals, anatomic regions in the lung asso ciated with tracer transport from blood to ravage fluid, and vice versa, after O3 exposure, should be localized and correlated with bulk movement of albumin into airspaces. Recommendation 3 Human studies using DTPA aerosols or other tracers in conjunction with concentration/response assays for edema should be undertaken. LOW PRIORITY Recommendation4 Animal studies should be done to determine the role of free radicals and active oxygen species in lung injury. Middle Phase of Injury The greatest needs for research in the middle phase of injury/ fibrosis are for a better understanding of the basis of cellular changes that perpetuate and amplify inflammation and cellular damage in lungs of animals exposed to O3. HIGH PRIORITY Recommendation 5 Studies of the linkages between epithelial cell damage and repair and changes in populations of pulmonary macrophages and inter stitial cells should be undertaken in whole animals. Recommendlation 6 Basic research in cell culture systems should be performed to examine the biochemical basis of cell-cell communication and the molecular nature of various mediators, released by leukocytes and macrophages from damaged lungs, that enhance or prolong the cellular inflammatory response. MEDIUM PRIORITY Recommendation 7 Mediators possibly released by damaged lung epithelial cells or derived from damaged matrix components (arachidonic acid

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Jerold A. Last 435 metabolites, chemotactic factors, cytokines), which maintain and amplify lung injury after acute cellular or organ damage should be characterized. Recommendation 8 Examination of factors released by inflammatory cells that modulate collagen synthesis or fibroblast proliferation, especially in response to 03, might help to define the mechanisms under lying the transition from the damaged, inflamed lung to the fibrotic lung. Late Phase of Injury In the fibrotic phase, or late stages of injury, the greatest need is to understand whether the increased collagen synthesis observed in lungs of rats and monkeys acutely exposed to O3 results in pulmonary fibrosis. HIGH PRIORITY Recommendation9 The reversibility of excess collagen deposition in the fibrotic lung, as reflected by increased hydroxyproline content of the lung, should be determined. Recommendation 11 The relationship between the presence of specific abnormal collagen cross-links in the lung and the reversibility of fibrosis should be examined. . Recommendation 12 The response of the developing lung versus the mature lung in animals exposed to O3 should be studied to ascertain whether the developing lung is more susceptible to damage, fibrosis, or change. MEDIUM PRIORITY Recommendation 10 In addition to collagen, the content and structure of other extracellular matrix components in the fibrotic lung should be examined. Other Considerations H I G H P R I O R I T Y Recommendation 13 Detailed study of lung structure and biochemistry over long postexposure periods should be made to allow better understand ing of progression of injury after exposure to O3 is terminated. Recommendation 14 A rational basis should be developed for prediction of synergistic or antagonistic interactions of pollutant mixtures by systematically examining binary and ternary combinations of the pollutants. The possibility that agents known to affect epithelial cell turnover might interact synergistically with O3 deserves special attention. Recommendation 15 Greater use should be made of animal models of susceptible populations.

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436 Biochemical and Cellular Interrelationships Recommendation 16 Descriptive, subjective pathology is of little or no value. Long term (lifetime) exposures of rodents under realistic protocols with sophisticated assays at termination are required, with morphomet ric and detailed biochemical examination of structural components at the least. Such studies should also examine tumor incidence and, to be meaningful, would require very large groups of animals. References Absher, M., Hildebran, J., Trombley, L., Woodcock- Mitchell, J., and Marsh, J. 1984. Characteristics of cultured lung fibroblasts from bleomycin-treated rats: comparisons with in vitro exposed normal fibroblasts, Am. Rev. Respir. Dis. 129:12~129. Al-Adnani, M. S., and McGee, J. O'D. 1976. Clq production and secretion by f~broblasts, Nature 263:14~146. Alpert, S. M., Schwartz, B. B., Lee, S. D., and Lewis. T. R. 1971. Alveolar protein accumulatir~n __ 7 - r A sensitive indicator of low level oxidant toxicity, Arch. Intern. Med. 128:69. Bartlett, J. D., Faulkner, C. S., and Cook, K. 1974. Effects of chronic ozone exposure on lung elasticity in young rats, J. Appl. Physiol. 37:92-96. Beck, B. D., Gerson, B., Feldman, H. A., and Brian, J. D. 1983. Lactate dehydrogenase isoenzymes in hamster lung ravage fluid after lung injury, Toxicol. Appl. Pharmacol. 71:59-71. Bitterman, P. B., Rennard, S. I., Hunninghake, G. W., and Crystal, R. G. 1982. Human alveolar macrophage growth factor for fibroblasts. Regulation and partial characterization, J. Clin. Invest. 70:806-822. Bitterman, P. B., Rennard, S. I., Adelberg, S., and Crystal, R. G. 1983. Role of fibronectin as a growth factor for fibroblasts, J. Cell Biol. 97: 192~1932. Bowden, D. H. 1984. Unraveling pulmonary fibrosis: the bleomycin model, Lab. Invest. 50(5):487088. Brain, J. D., Godleski, J. J., and Sorokin, S. P. 1977. Quantification, origin and fate of pulmonary mac- rophages, In: Respiratory Defense Mechanisms, Part II a D. Brain, D. F. Proctor, and L. 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