Inflammation, Vol. 3, No. 3, 1979
TOBACCO SMOKE Effects on Pulmonary Host Defense DAVID B. DRATH, MANFRED L. KARNOVSKY, and GARY L. HUBER Department of Biological Chemistry and Department of Medicine Thorndike Laboratory, Harvard Medical School, Boston, Massachusetts
Abstract--Tobacco smoke affected both the metabolism and function of pulmonary alveolar macrophages (PAM), Phagocytosis of viable Staphylococcus aureus and inert starch particles was minimally but consistently depressed in PAM from rats exposed to tobacco smoke for six months. Oxygen consumption, superoxide and hydrogen peroxide release, and hexose monophosphate shunt activity were elevated in cells from smokers. Oxidation of glucose, labelled in the carbon-six position, remained unchanged. All observed effects of tobacco smoke on oxygen metabolism occurred during phagocytosis and did not affect the basal metabolism of the nonstimulated cell.
INTRODUCTION Despite the very high prevalence of cigarette consumption and the importance of the alveolar macrophage to the defenses of the lung, very little is known about the effect of long-term exposure to tobacco smoke, under controlled conditions, on the function and metabolism of this important cell. In this communication we report that alveolar macrophages isolated from the lungs of rats exposed chronically to tobacco smoke consistently phagocytize viable and inert particles to a lesser extent than macrophages from control animals. These cells, which are thought to be responsible for maintaining the sterility of the lower respiratory tract (1, 2), are similar to other phagocytic cells in that they exhibit several metabolic perturbations during phagocytosis (3-5). Therefore, we also have examined the effect of carefully controlled experimental exposure to tobacco smoke on several aspects of the metabolic response to phagocytosis, including oxygen consumption, superoxide and hydrogen peroxide release, and oxidation of specifically labeled glucose. 281
0360-3997/79/0700-0281503.00/09 1979PlenumPublishingCorporation
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MATERIALS AND METHODS Tobacco Smoke Delivery. Pathogen-free male rats of the CD strain, weighing 125 g at the onset of the experiment, were placed in conical animal holding tubes with openings for their snouts at the narrow end. Exposure racks holding 50 such tubes were connected to 30-port smoking machines which delivered fresh whole smoke from 10 2R1 reference cigarettes (University of Kentucky, Lexington, Kentucky). The performance and characteristics of this smokegenerating machine, as well as the details and monitoring of our smoke delivery system, have been published previously (6). The smoke was stabilized by dilution with 10 volumes of air and delivered to the animals three times a day. This regimen was followed for 180 consecutive days, during which time the machines were intermittently monitored to assure consistency of the delivery system. The amount of fresh smoke inhaled by individual animals, determined by labeling the cigarettes with an inert tracer of the particulate matter, decachlorobiphenyl, and quantifying the retention of these tracers of smoke particulate matter in the lungs of experimental animals (7), was equivalent to approximately 189packs per day consumption of unfiltered cigarettes by man (8). Cell Isolation. On the day following the last exposure, the animals were sacrificed and macrophages recovered by bronchopulmonary lavage with isotonic saline at room temperature (9). Monolayers containing more than 95% viable alveolar macrophages, as determined by differential cell counts and trypan blue staining, were prepared according to the technique of Michell and coworkers (10). Cells were recovered from controls and smoke-exposed rats in similar numbers, and contained approximately the same protein content per l0 6 cells. The plating efficiency of both groups was similar as determined by the protein content of the monolayer. Uptake of Labeled Particles. In order to assess effects of tobacco on phagocytosis, separate ~4C-radiolabeled challenges of viable Staphylococcus aureus or inert starch granules were used. Consistent with desiderata for measurement of phagocytosis stated by Michell et al. (10), starch particles were present in considerable excess and uptake at the various times monitored. Thus, both the initial rate of phagocytosis and the total capacity of the cells for ingestion could be determined. The live bacterial particle was used at levels more consistent with biological reality (10 bacteria/macrophage). Monolayers consisting of 3 X 10 6 cells (when added) were formed at 37~ as previously described. After one hour, nonadherent ceils were washed off (approX. 35%). Fresh Hanks' balanced salt solution containing glucose (mg/ml) was added to the monolayers followed by the appropriate particle, 14C-labeled Staphylococcus aureus (strain 209P) at a multiplicity of 10 particles/macrophage and ~4C-labeled starch (5 mg/ml). At the indicated times, particles were removed, the monolayers were vigorously washed in saline and dried in preparation for counting in a gas-flow counter. Zero time counts were subtracted from subsequent measurements. Protein determinations were performed on the monolayers as well as on the bacteria. Metabolic Assays. Oxygen consumption was measured polargraphicaUy with an oxygen electrode (Yellow Spring Instrument Co., Yellow Springs, Ohio) on alveolar macrophage samples consisting of 4 X 106 ceils. Superoxide-dependent cytochrome c reduction was determined spectrophotometrically at 550 nm in the presence or absence of superoxide dismutase, as previously described (11). Hydrogen peroxide was measured by the fluorometric technique of Root et al. (12), where a decrease in the fluorescence of scopoletin (ICN, Cleveland, Ohio) in the presence of horseradish peroxidase (Sigma Chemical Co., St. Louis, Missouri) is proportional to the amount of H202 in the medium. Oxidation of specifically labeled glucose was determined by the monolayer technique of Miehell et al. (10), where 4/~mol (0.5/.t Ci) of [ 1-14C]glucose (for hexose monophosphate shunt determination) or 4 #mol (2.5 #Ci) of [6-~4C] glucose (New England Nuclear, Boston, Massachusetts) were used. Opsonized zymosan at a multiplicity of
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Smoke
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283
10:1 served as the phagocytic stimulus in the metabolic studies. All values were expressed as nanomoles per 30 minutes per milligram of protein and are shown as the arithmetic mean plus or minus one standard error of the mean of three experiments each consisting of multiple determinations.
RESULTS
The uptake of Staphylococcus aureus was depressed in macrophages recovered from smoke-exposed animals, as the three independent experiments in Figure la all show. Statistical significance was determined by the use of the paired t test over the three separate experiments at each time point studied. The depression, at least through 60 min, appears to affect both the capacity of the cell for total ingestion as well as the rate at which bac-
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Fig. 1. Uptake of viable particles (a, left panels) and inert particles (b, right panels) by alveolar maerophages lavaged from the lungs of control and chronically smoke-exposed rats (6 months). Filled circles indicate smokers and empty circles are controls. Final results were expressed as micrograms of particle phagocytized per milligram of macrophage protein. The figures represent results obtained in three individual experiments. The mean + S E of triplicate determinations are shown.
284
Drath, Karnovsky, and Huber
teria may be ingested (P < 0.05 at each time point). The "initial rate" was measured with the limitation that the earliest practical time point in these experiments was 15 min. After 60 min, phagocytosis apparently levels off in the cells recovered from smoke-exposed animals, while continuing in control cells. The response to a challenge of nonviable 14C-labeled starch was slightly different (Figure lb). In this case, macrophages from both groups phagocytized at rates that were not significantly different during the first 30 min. After this time, phagocytosis by control cells clearly exceeded that by cells from smoke-exposed animals (P < 0.02 for 45 and 60 min). An approximate 30% inhibition in particle uptake was evident at the end of 60 min, reflecting a diminished capacity. Although phagocytosis appears to be adversely affected by long-term experimental exposure to tobacco smoke, the metabolism of the macrophage, which is normally stimulated during the process of ingestion, was further stimulated following chronic exposure to smoke. The change in metabolism due solely to particle ingestion (Ap) may be calculated from the information presented in Table 1. The Ap for oxygen consumption and hydrogen peroxide release were approximately doubled when compared to controls, while AP for superoxide release was increased sixfold in macrophages from smoke-exposed rats. It is interesting to note that the observed effects of tobacco smoke on oxygen metabolism occurred only during phagocytosis and did not affect the basal metabolism of the resting cell (P < 0.01 for all measures except glucose C-6 oxidation, which was not affected). Oxidation of glucose by the direct oxidative pathway (hexose monophosphate shunt) under resting conditions, while apparently increased in response to tobacco smoke, was in fact not significantly altered. During phagocytosis, however, shunt activity was significantly elevated (P < 0.01). No effect was demonstrated on oxidation of [6--14C]glucose to 14CO2. Table 1. Effects of Chronic Cigarette Smoke Exposure (6 months) on Metabolism of Rat Alveolar Macrophages at Rest and During Phagocytosis a Control Measure Oxygen consumption Superoxide release Hydrogen peroxide release Glucose-C-1 "*CO2 Glucose-C-6 -~CO2
Rest 486.7 1.2 1.9 17.5 2.0
+ + + + _
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Phagocytosis 8.0 0.96 0.18 1.1 0.30
662.9 5.4 16.2 14.2 1.8
+ + + + +
20.5 2.0 2.9 2.5 0.26
Rest 508.9 1.8 1.9 23.6 2.4
+ + + _ +
Phagocytosis 20.1 0.84 0.15 4.7 0.51
915.2 26.9 25.5 29.5 1.9
+ + + _+ +
6.7 5.8 3.2 6.2 0.43
aOpsonized zymosan at a multiplicity of 10:1 served as the phagocytic stimulus. All values are expressed as nanomoles per 30 minutes per milligram of protein and are shown as the m e a n + S E of three experiments each consisting of multiple determinations.
285
Tobacco Smoke and Lung Defenses
DISCUSSION Our results are consistent with a state of metabolic activation in the macrophage population recovered from smoke-exposed animals. Agents such as Listeria monocytogenes and sodium caseinate are known to cause activation in peritoneal macrophages. The former does so through a sequence of reactions involving the immune system (13), while the latter appears to be nonspecific in nature (14). We would postulate at present that prolonged exposure to tobacco smoke nonspecifically activates the metabolism of phagocytizing alveolar macrophages, although no information is available from our studies regarding the specific direct effect of tobacco on the immune system. A nonspecific activation of this nature would be different, of course, from other types of activation reported for macrophages in that no direct effect was seen on resting cells. Perhaps what we observed is the tobacco smoke-induced uncovering of a site on the membrane responsible for the initiation of the metabolic alterations normally seen during phagocytosis. It is difficult to compare this study with others in the literature because of the lack of conformity of experimental smoking conditions in animals, as well as the variability in quantity and type of smoking products consumed by man. All too often, individual studies have been compared in which similarities between cigarettes smoked with respect to type (i.e., filtered or unfiltered), tar and nicotine content, quantity smoked, duration of the smoking habit, as well as mode of smoking (e.g., by man or through machine intervention), type of machine used, and exposure and dose regimen, did not exist. Additionally, comparisons have been made between the effects of whole smoke and isolated smoke components. In some cases, macrophages have been removed from either man or experimental animal after smoking, while in other studies smoke or its components were added to monolayers of normal macrophages in vitro. In previous experiments using identical smoking conditions, except for the duration of the smoke exposures, we were able to show that tobacco smoke apparently produces an "activated" macrophage after 30 days (15). Cells isolated from animals following 30 consecutive days of smoke exposure, in contrast to cells recovered after six months of experimental smoking, phagocytized inert starch more avidly than control cells and handled viable Staphylococcus aureus with equal efficiency. The metabolism during phagocytosis in the short-term exposures, however, was more variable than was shown in our studies on rats subjected to six months of exposure to tobacco smoke. Oxygen consumption and hydrogen peroxide release were elevated; the increase of the latter was more than threefold greater than in the six-month smokers. In contrast, superoxide release was not increased
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in the 30-day smoke-treated animals, whereas it was at six months. Oxidation of glucose via the direct oxidative pathway, while unremarkable in the 30-day exposed group, was definitely elevated in rats exposed to smoke for longer periods. In comparing the two series of experiments, it was apparent that prolonged smoking for six months exerted a depressant effect upon phagocytosis of viable and inert particles, under the conditions of these experiments. Since this effect was not seen at 30 days, the results of exposure to tobacco smoke on phagocytosis appear to be cumulative in nature. Whether the target site for tobacco smoke is an energy-yielding metabolic reaction or a structural component of the cellular phagocytic apparatus (i.e., contractile proteins) is not presently known. Our earlier studies of 30-day smoke-treated animals indicated that energy-yielding metabolic pathways were not affected by tobacco smoke (15). Low and colleagues have recently suggested that smoke components, particularly acrolein, inhibit phagocytosis by altering the cell's contractile apparatus through an action on calcium-dependent adenosine triphosphatase (16). This is an attractive hypothesis. However, since their results were obtained not under in vivo smoking conditions, but by adding smoke or smoke components to control macrophages in vitro, they could not explain why under in vivo smoking conditions tobacco apparently has little or no effect on phagocytosis at least up to 30 days (15, 17, 18). Our results suggest that prolonged exposure to tobacco smoke impairs the crucial process of particulate and bacterial uptake by alveolar macrophages, The functional impairment in phagocytosis of bacteria may be an important pathophysiologic mechanism rendering the host susceptible to pulmonary infection, as has been reported for human smokers (19). In addition, a reduced capacity to phagocytize inert particles may delay their clearance from the lung and thus enhance, through a longer particle-tissue contact time, their potential to cause parenchymal injury directly. These metabolically altered cells release considerably more superoxide and hydrogen peroxide than cells from control animals. Furthermore, superoxide and hydrogen peroxide may react and form even more reactive substances, such as singlet oxygen and hydroxyl radicals (20). Evidence from our laboratory indicates that alveolar macrophages do indeed release hydroxyl radicals during phagocytosis (21). Therefore it is possible that the long-term effects of the metabolic activation observed in our experiments may lead to pulmonary parenchymal damage through the release of such reactive entities. In this regard, we also note the recent finding by Rodriguez and colleagues of increased release of elatase by alveolar macrophages harvested from smokers (22). Although not directly applicable to our results, their results do indicate that tobacco smoke may enhance parenchymal damage through the mediation of alveolar macrophages.
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REFERENCES 1. GREEN, G. M. 1968. Pulmonary clearance of infectious agents. Annu. Rev. Med. 19:315336. 2. HUMOR, G. L., W. G. JOHANSON,and F. M. LAFORCE. 1977. Experimental models and pulmonary antimicrobial defenses. In Lung Biology in Health and Disease. C. LENFANT,editor. Marcel Dekker, New York. 983-1022. 3. OREN, R., A. E. FARNHAM,K. SAITO, R. MILoVSKY,and M. L. KARNOVS~CY.1963. Metabolic patterns in three types of phagocytizing cells. J. Cell Biol. 17:487-501. 4. DRATH, D.B.0 A.H. HARPER, and M.L. KARNOVSr:Y. 1976. Biochemical characteristics of alveolar macrophages. In Lung Cells in Disease, A. Bouhuys, editor. North-Holland, Amsterdam. 153-161. 5. KARNOVSKY, M. L. 1974. The biological basis of the functions of polymorphonuclear leukocytes and macrophages. In Progress in Immunology, L. Brent and J. Holborrow, editors. North-Holland, Amsterdam. 83-93. 6. HUBER, G. L., V. E. POCHAY, J. W. SnEA, W. E. HINDS, R. R. WEKER, M. W. FIRST, and G.C. SORNBERGER. 1979. An experimental animal model for quantifying the biologic effects of marijuana on the defense system of the lung. In Marijuana and Membranes: Quantitation, Metabolism, Cellular Response, Reproduction and Brain. G. G. Nahas and W. D. M. Paton, editors. Pergamon Press, Oxford (in press). 7. BINNS, R., J. L. BEVEN, L. V. WILTON, and W. G. LtJYTON. 1976. Inhalation toxicity studies on cigarette smoke. IV. Expression of the dose of smoke particulate material applied to the lungs of experimental animals. Toxicology 6:207-211. 8. HUaER, G. L., V. E. POCHAY, V. K. MAHAJAN, C. R. MCCARTHY, W. C. HINDS, P. DAVIES, D.B. DRATH, and G.C. SORNBERGER. 1977. The effect of chronic exposure to tobacco smoke on the antibacterial defenses of the lung. Cur. J. Resp. Physiopathol. 13:145-156. 9. MYRVIK,Q. M., E. S. LEAKE,and B. J. FARISS. 1961. Studies on pulmonary alveolar macrophages from the normal rabbit: A technique to procure them in a high state of purity. Immunology 86:128-132. 10. MICHELL, R., S. J. PANCAKE,J. NOSEWORTHY,and M. L. KARNOVSKY. 1970. Measurements of rates of phagocytosis. The use of cellular monotayers. J. CeIL Biol. 40:216-224. 11. DRATH, D. B., and M. L. KARNOVSKY. 1975. Superoxide production by phagocytic leukocytes. J. Exp. Med. 141:257-262. 12. RooT, R., J. MELTCALF, N. OSHINO, and B. CHANCE. 1974. H202 release from human granulocytes during phagocytosis. I. Documentation, quantitation and some regulating factors. J. Clin. Invest. 55:944-955. 13. MACKANESS, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105-114. 14. KARNOVSKY, M. L., J. LAZDINS, and S. SIMMONS. 1975. Metabolism of activated mononuclear phagocytes at rest and during phagocytosis. /n Mononuclear Phagocytes in Immunity Infection and Pathology. R. Van Furth, editor. Blackwell Scientific Publications, Oxford. 423-439. 15. DRATH, D., A. HARPER, J. GHARIBIAN, M. L. KARNOVSKY, and G. L. HUBER. 1978. The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages. J. Cell. Physiol. 95:105-114. 16. Low, E., R. Low, and G. M. GREEN. 1977. Correlated effects of cigarette smoke components on alveolar macrophages adenosine triphosphatase activity and phagocytosis. Am. Rev. Resp. Dis. 115:963. 17. HARRIS,J., E. SWENSON,and J. E. JOHNSON, III. 1970. Human alveolar macrophages: Comparison of phagocytic ability, glucose utilization and ultra structure in smokers and nonsmokers. J. Clin. Invest. 49:2086-2097.
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18. REYNOLDS, H. Y., J. KAZMIEROWSKI,and H. NEWBALL 1975. Specificity of opsonic antibodies to enhance phagocytosis of Pseudomonas aeruginosa by human alveolar macrophages. J. Clin. Invest. 56:376-385. 19. HAYES,W. F., JR., V. KRSTULOVIC,and A. L. BELL, JR. 1966. Smoking habit and incidence of respiratory tract infections in a group of adolescent males. Am. Rev. Resp. Dis. 93:730734. 20. BEAUCHAMP,C., and I. FRIDOVICH. 1970. A mechanism for the production of ethylene from methional. The generation of hydroxyl radical by xanthine oxidase. J. Biol. Chem. 245: 4641-4646. 21. DRATH, D. B., M.L. KARNOVSKY,and G. L. HUBER. 1979. Hydroxyl radical formation in phagocytic cells of the rat. J. Appl. Physiol. 46:136-140. 22. RODRIGUEZ, R.J., R. R. WHITE, R. M. SENIOR, and E. A. LEVINE. 1977. Elastase release from human alveolar macrophages: Comparison between smokers and non-smokers. Science 198:313-314.
TOBACCO SMOKE Effects on Pulmonary Host Defense DAVID B. DRATH, MANFRED L. KARNOVSKY, and GARY L. HUBER Department of Biological Chemistry and Department of Medicine Thorndike Laboratory, Harvard Medical School, Boston, Massachusetts
Abstract--Tobacco smoke affected both the metabolism and function of pulmonary alveolar macrophages (PAM), Phagocytosis of viable Staphylococcus aureus and inert starch particles was minimally but consistently depressed in PAM from rats exposed to tobacco smoke for six months. Oxygen consumption, superoxide and hydrogen peroxide release, and hexose monophosphate shunt activity were elevated in cells from smokers. Oxidation of glucose, labelled in the carbon-six position, remained unchanged. All observed effects of tobacco smoke on oxygen metabolism occurred during phagocytosis and did not affect the basal metabolism of the nonstimulated cell.
INTRODUCTION Despite the very high prevalence of cigarette consumption and the importance of the alveolar macrophage to the defenses of the lung, very little is known about the effect of long-term exposure to tobacco smoke, under controlled conditions, on the function and metabolism of this important cell. In this communication we report that alveolar macrophages isolated from the lungs of rats exposed chronically to tobacco smoke consistently phagocytize viable and inert particles to a lesser extent than macrophages from control animals. These cells, which are thought to be responsible for maintaining the sterility of the lower respiratory tract (1, 2), are similar to other phagocytic cells in that they exhibit several metabolic perturbations during phagocytosis (3-5). Therefore, we also have examined the effect of carefully controlled experimental exposure to tobacco smoke on several aspects of the metabolic response to phagocytosis, including oxygen consumption, superoxide and hydrogen peroxide release, and oxidation of specifically labeled glucose. 281
0360-3997/79/0700-0281503.00/09 1979PlenumPublishingCorporation
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MATERIALS AND METHODS Tobacco Smoke Delivery. Pathogen-free male rats of the CD strain, weighing 125 g at the onset of the experiment, were placed in conical animal holding tubes with openings for their snouts at the narrow end. Exposure racks holding 50 such tubes were connected to 30-port smoking machines which delivered fresh whole smoke from 10 2R1 reference cigarettes (University of Kentucky, Lexington, Kentucky). The performance and characteristics of this smokegenerating machine, as well as the details and monitoring of our smoke delivery system, have been published previously (6). The smoke was stabilized by dilution with 10 volumes of air and delivered to the animals three times a day. This regimen was followed for 180 consecutive days, during which time the machines were intermittently monitored to assure consistency of the delivery system. The amount of fresh smoke inhaled by individual animals, determined by labeling the cigarettes with an inert tracer of the particulate matter, decachlorobiphenyl, and quantifying the retention of these tracers of smoke particulate matter in the lungs of experimental animals (7), was equivalent to approximately 189packs per day consumption of unfiltered cigarettes by man (8). Cell Isolation. On the day following the last exposure, the animals were sacrificed and macrophages recovered by bronchopulmonary lavage with isotonic saline at room temperature (9). Monolayers containing more than 95% viable alveolar macrophages, as determined by differential cell counts and trypan blue staining, were prepared according to the technique of Michell and coworkers (10). Cells were recovered from controls and smoke-exposed rats in similar numbers, and contained approximately the same protein content per l0 6 cells. The plating efficiency of both groups was similar as determined by the protein content of the monolayer. Uptake of Labeled Particles. In order to assess effects of tobacco on phagocytosis, separate ~4C-radiolabeled challenges of viable Staphylococcus aureus or inert starch granules were used. Consistent with desiderata for measurement of phagocytosis stated by Michell et al. (10), starch particles were present in considerable excess and uptake at the various times monitored. Thus, both the initial rate of phagocytosis and the total capacity of the cells for ingestion could be determined. The live bacterial particle was used at levels more consistent with biological reality (10 bacteria/macrophage). Monolayers consisting of 3 X 10 6 cells (when added) were formed at 37~ as previously described. After one hour, nonadherent ceils were washed off (approX. 35%). Fresh Hanks' balanced salt solution containing glucose (mg/ml) was added to the monolayers followed by the appropriate particle, 14C-labeled Staphylococcus aureus (strain 209P) at a multiplicity of 10 particles/macrophage and ~4C-labeled starch (5 mg/ml). At the indicated times, particles were removed, the monolayers were vigorously washed in saline and dried in preparation for counting in a gas-flow counter. Zero time counts were subtracted from subsequent measurements. Protein determinations were performed on the monolayers as well as on the bacteria. Metabolic Assays. Oxygen consumption was measured polargraphicaUy with an oxygen electrode (Yellow Spring Instrument Co., Yellow Springs, Ohio) on alveolar macrophage samples consisting of 4 X 106 ceils. Superoxide-dependent cytochrome c reduction was determined spectrophotometrically at 550 nm in the presence or absence of superoxide dismutase, as previously described (11). Hydrogen peroxide was measured by the fluorometric technique of Root et al. (12), where a decrease in the fluorescence of scopoletin (ICN, Cleveland, Ohio) in the presence of horseradish peroxidase (Sigma Chemical Co., St. Louis, Missouri) is proportional to the amount of H202 in the medium. Oxidation of specifically labeled glucose was determined by the monolayer technique of Miehell et al. (10), where 4/~mol (0.5/.t Ci) of [ 1-14C]glucose (for hexose monophosphate shunt determination) or 4 #mol (2.5 #Ci) of [6-~4C] glucose (New England Nuclear, Boston, Massachusetts) were used. Opsonized zymosan at a multiplicity of
Tobacco
Smoke
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283
10:1 served as the phagocytic stimulus in the metabolic studies. All values were expressed as nanomoles per 30 minutes per milligram of protein and are shown as the arithmetic mean plus or minus one standard error of the mean of three experiments each consisting of multiple determinations.
RESULTS
The uptake of Staphylococcus aureus was depressed in macrophages recovered from smoke-exposed animals, as the three independent experiments in Figure la all show. Statistical significance was determined by the use of the paired t test over the three separate experiments at each time point studied. The depression, at least through 60 min, appears to affect both the capacity of the cell for total ingestion as well as the rate at which bac-
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Fig. 1. Uptake of viable particles (a, left panels) and inert particles (b, right panels) by alveolar maerophages lavaged from the lungs of control and chronically smoke-exposed rats (6 months). Filled circles indicate smokers and empty circles are controls. Final results were expressed as micrograms of particle phagocytized per milligram of macrophage protein. The figures represent results obtained in three individual experiments. The mean + S E of triplicate determinations are shown.
284
Drath, Karnovsky, and Huber
teria may be ingested (P < 0.05 at each time point). The "initial rate" was measured with the limitation that the earliest practical time point in these experiments was 15 min. After 60 min, phagocytosis apparently levels off in the cells recovered from smoke-exposed animals, while continuing in control cells. The response to a challenge of nonviable 14C-labeled starch was slightly different (Figure lb). In this case, macrophages from both groups phagocytized at rates that were not significantly different during the first 30 min. After this time, phagocytosis by control cells clearly exceeded that by cells from smoke-exposed animals (P < 0.02 for 45 and 60 min). An approximate 30% inhibition in particle uptake was evident at the end of 60 min, reflecting a diminished capacity. Although phagocytosis appears to be adversely affected by long-term experimental exposure to tobacco smoke, the metabolism of the macrophage, which is normally stimulated during the process of ingestion, was further stimulated following chronic exposure to smoke. The change in metabolism due solely to particle ingestion (Ap) may be calculated from the information presented in Table 1. The Ap for oxygen consumption and hydrogen peroxide release were approximately doubled when compared to controls, while AP for superoxide release was increased sixfold in macrophages from smoke-exposed rats. It is interesting to note that the observed effects of tobacco smoke on oxygen metabolism occurred only during phagocytosis and did not affect the basal metabolism of the resting cell (P < 0.01 for all measures except glucose C-6 oxidation, which was not affected). Oxidation of glucose by the direct oxidative pathway (hexose monophosphate shunt) under resting conditions, while apparently increased in response to tobacco smoke, was in fact not significantly altered. During phagocytosis, however, shunt activity was significantly elevated (P < 0.01). No effect was demonstrated on oxidation of [6--14C]glucose to 14CO2. Table 1. Effects of Chronic Cigarette Smoke Exposure (6 months) on Metabolism of Rat Alveolar Macrophages at Rest and During Phagocytosis a Control Measure Oxygen consumption Superoxide release Hydrogen peroxide release Glucose-C-1 "*CO2 Glucose-C-6 -~CO2
Rest 486.7 1.2 1.9 17.5 2.0
+ + + + _
Smoker
Phagocytosis 8.0 0.96 0.18 1.1 0.30
662.9 5.4 16.2 14.2 1.8
+ + + + +
20.5 2.0 2.9 2.5 0.26
Rest 508.9 1.8 1.9 23.6 2.4
+ + + _ +
Phagocytosis 20.1 0.84 0.15 4.7 0.51
915.2 26.9 25.5 29.5 1.9
+ + + _+ +
6.7 5.8 3.2 6.2 0.43
aOpsonized zymosan at a multiplicity of 10:1 served as the phagocytic stimulus. All values are expressed as nanomoles per 30 minutes per milligram of protein and are shown as the m e a n + S E of three experiments each consisting of multiple determinations.
285
Tobacco Smoke and Lung Defenses
DISCUSSION Our results are consistent with a state of metabolic activation in the macrophage population recovered from smoke-exposed animals. Agents such as Listeria monocytogenes and sodium caseinate are known to cause activation in peritoneal macrophages. The former does so through a sequence of reactions involving the immune system (13), while the latter appears to be nonspecific in nature (14). We would postulate at present that prolonged exposure to tobacco smoke nonspecifically activates the metabolism of phagocytizing alveolar macrophages, although no information is available from our studies regarding the specific direct effect of tobacco on the immune system. A nonspecific activation of this nature would be different, of course, from other types of activation reported for macrophages in that no direct effect was seen on resting cells. Perhaps what we observed is the tobacco smoke-induced uncovering of a site on the membrane responsible for the initiation of the metabolic alterations normally seen during phagocytosis. It is difficult to compare this study with others in the literature because of the lack of conformity of experimental smoking conditions in animals, as well as the variability in quantity and type of smoking products consumed by man. All too often, individual studies have been compared in which similarities between cigarettes smoked with respect to type (i.e., filtered or unfiltered), tar and nicotine content, quantity smoked, duration of the smoking habit, as well as mode of smoking (e.g., by man or through machine intervention), type of machine used, and exposure and dose regimen, did not exist. Additionally, comparisons have been made between the effects of whole smoke and isolated smoke components. In some cases, macrophages have been removed from either man or experimental animal after smoking, while in other studies smoke or its components were added to monolayers of normal macrophages in vitro. In previous experiments using identical smoking conditions, except for the duration of the smoke exposures, we were able to show that tobacco smoke apparently produces an "activated" macrophage after 30 days (15). Cells isolated from animals following 30 consecutive days of smoke exposure, in contrast to cells recovered after six months of experimental smoking, phagocytized inert starch more avidly than control cells and handled viable Staphylococcus aureus with equal efficiency. The metabolism during phagocytosis in the short-term exposures, however, was more variable than was shown in our studies on rats subjected to six months of exposure to tobacco smoke. Oxygen consumption and hydrogen peroxide release were elevated; the increase of the latter was more than threefold greater than in the six-month smokers. In contrast, superoxide release was not increased
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in the 30-day smoke-treated animals, whereas it was at six months. Oxidation of glucose via the direct oxidative pathway, while unremarkable in the 30-day exposed group, was definitely elevated in rats exposed to smoke for longer periods. In comparing the two series of experiments, it was apparent that prolonged smoking for six months exerted a depressant effect upon phagocytosis of viable and inert particles, under the conditions of these experiments. Since this effect was not seen at 30 days, the results of exposure to tobacco smoke on phagocytosis appear to be cumulative in nature. Whether the target site for tobacco smoke is an energy-yielding metabolic reaction or a structural component of the cellular phagocytic apparatus (i.e., contractile proteins) is not presently known. Our earlier studies of 30-day smoke-treated animals indicated that energy-yielding metabolic pathways were not affected by tobacco smoke (15). Low and colleagues have recently suggested that smoke components, particularly acrolein, inhibit phagocytosis by altering the cell's contractile apparatus through an action on calcium-dependent adenosine triphosphatase (16). This is an attractive hypothesis. However, since their results were obtained not under in vivo smoking conditions, but by adding smoke or smoke components to control macrophages in vitro, they could not explain why under in vivo smoking conditions tobacco apparently has little or no effect on phagocytosis at least up to 30 days (15, 17, 18). Our results suggest that prolonged exposure to tobacco smoke impairs the crucial process of particulate and bacterial uptake by alveolar macrophages, The functional impairment in phagocytosis of bacteria may be an important pathophysiologic mechanism rendering the host susceptible to pulmonary infection, as has been reported for human smokers (19). In addition, a reduced capacity to phagocytize inert particles may delay their clearance from the lung and thus enhance, through a longer particle-tissue contact time, their potential to cause parenchymal injury directly. These metabolically altered cells release considerably more superoxide and hydrogen peroxide than cells from control animals. Furthermore, superoxide and hydrogen peroxide may react and form even more reactive substances, such as singlet oxygen and hydroxyl radicals (20). Evidence from our laboratory indicates that alveolar macrophages do indeed release hydroxyl radicals during phagocytosis (21). Therefore it is possible that the long-term effects of the metabolic activation observed in our experiments may lead to pulmonary parenchymal damage through the release of such reactive entities. In this regard, we also note the recent finding by Rodriguez and colleagues of increased release of elatase by alveolar macrophages harvested from smokers (22). Although not directly applicable to our results, their results do indicate that tobacco smoke may enhance parenchymal damage through the mediation of alveolar macrophages.
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