In Vitro Study of Gas Effects 151
IN
VITRO
STUDY OF GAS EFFECTS MACROPHAGES B. W A L L A E R T
ON ALVEOLAR
and C. V O I S I N
Laboratoire de Pollution Atmosph6rique et de Pathologie Respiratoire Exp6rimentale and CJF INSERM, Institut Pasteur, Lille, France
INTRODUCTION The lung is the target of multiple aggressions due to occupational hazards, environmental noxious substances and personal risks such as tobacco smoke. In all of these situations, alveolar macrophages (AM) located on the respiratory membrane, are in close, permanent and direct contact with the airborne pollutants and are directly exposed to particles and toxic gases. Thus, taking into account the multiple functional activities of this cell population (Fels and Cohn, 1986), it seems reasonable to hypothezise that alveolar macrophages play a pivotal role in the responses of the respiratory tract to the inhaled materials. During the past 25 years, extensive investigations have been devoted to alveolar macrophage response to silica, coal dust and asbestos (Parazzi et al., 1968; Scheule and Holian, 1991; Brody, 1986). The presence of the dust inside the cells, either obtained ex vivo from dusted animals or humans, or in vitro submitted to selected particles under strictly controlled conditions, allows a qualitative and quantitative evaluation of cell disturbances. In marked contrast, AM responses to toxic gases are difficult to approach. However the understanding of the mechanisms of the functional and pathological disorders resulting from the inhalation of toxic gases in the respiratory tract requires the study of the direct effects of pollutants on the state and the activity of AM. Up to now, three series of methods were used: the first ones consist in animal exposure to the toxic gas followed by bacterial challenge against infectious agents, in order to evaluate the possible alterations in the phagocytic and bactericidal activity of AM (Ehrlich, 1966; Henry et al., 1969); the second technic is an "in vitro" study of AM harvested by bronchoalveolar lavage either from animals or from human volunteers exposed to controlled inhalation of toxic gases (Denicola et al., 1981; Sone et al., 1983). However, in such experiments, cell disturbances are not well correlated to the gas exposure; the third one deals with in vitro systems for exposure of alveolar macrophages to gases in controlled conditions. I N V I T R O SYSTEMS FOR EXPOSURE OF ALVEOLAR MACROPHAGES TO GASES
The description of various in vitro system for exposure of lung cells to oxidant gases have been recently reviewed by Rasmussen (Rasmussen, 1984). Exposure to gases may be Corresponding Author: B. WALLAERTMD, Laboratoire de Pollution atmosph6rique, Institut Pasteur de Lille, 1, rue Calmette, 59800 Lille, FRANCE. Abbreviations: Alveolar macrophages: AM, Chemiluminescence:CL. Key Words: alveolar macrophages, gases, in vitro systems, oxidants. Supported by Universit6 de Lille II and INSERM CJF 90-06. Cell Biology end Toxicology, Vol. 8, No. 3, pp. Copyright © 1992 Princeton Scientific Publishing ISSN: 0742-2091
151-156 Co., Inc.
152
Wallert and Voisin
performed using roller bottles fitted with modified rotating caps with tubing connections (Bolton et al., 1982) or dishes on rocker platforms tilting back and forth to periodically expose the cell culture to gases (Guerbero et al., 1979a; Guerboro et al., 1979b). Lastly, exposure of cells may be obtained using gas-permeable membranes on which cells grow on very thin membrane allowing gas to permeate through the film (Mink et al., 1979). However, it is clear that using these systems, culture medium constitutes a barrier between the gas and the target cell and does not permit a physiological approach of the toxic effects of gases. This is the reason why we developed an experimental model, using a cell culture technique in gas phase (Voisin et al., 1974; Voisin et al, 1977a; Voisin et al, 1977b). Herein we reported the value and the limits of this original method using the model of alveolar macrophage responses to NO2. BIPHASIC SYSTEM CULTURE OF ALVEOLAR MACROPHAGES AM were harvested by broncho alveolar lavage from healthy subjects according to the method previously described (Voison et al., 1977a). AM were layered on a Gelman membrane, which was applied to the surface of a reservoir filled up with culture medium, so as to be saturated by capillarity. In such experimental conditions, in air enriched with CO2 5% and saturated with water at 37°C, it was possible to maintain the cells alive, with normal metabolic and functional activities up to two weeks. I N V I T R O EXPOSURE OF ALVEOLAR MACROPHAGES TO NO 2
Exposure to NO2 was carried out using an original device consisting in a special exposure chamber, in which air with 5% CO2 enriched with the toxic gas was pulsed in continuous flow with continuous monitoring. Long term (24 h) and short term exposure (30 min) of human AM to various low concentrations NO2 were performed. The effects of gases on AM were studied by examination of theft morphological appearance under optical and electronic microscopy and by the determination of the ATP content of the cells. The cell injury index reported in this paper corresponds to the percent decrease in cellular ATP content of gas-exposed cells when compared with the ATP content of aft-exposed cells, measured at the same time. Long term exposure (24 h) at low concentration NO2 (0.2 ppm) resulted in a significant decrease in cell ATP content. Cell injury index clearly depended on NO2 concentration. Experiments done with 2 and 5 ppm NO2 resulted in a complete destruction of the cellular preparation. In marked contrast, short term exposure (30 min) at low concentration NO2 (0.2 ppm) did not induce cytotoxic effects of NO2 as assessed by the absence of modification of beta-glu-
In Vitro Study of Gas Effects 153
curonidase cell content and of ATP cell content after exposure. Conversely, NO2 short term exposure was responsible for cell membrane activation as demonstrated by the increased generation of both superoxide anion and neutrophil chemotactic activity by alveolar macrophages.
NEUTROPHIL CHEMOTACTIC ACTIVITY
SUPEROXIDE ANION GENERATION
PMN/HPF
RLU
p < 0,05 20000
P < 0,05
T
10000
i
AIR
20
10
fliiiiJ I?1 NO2
AIR
NO2
FIGURE 1. Effects of short term (30 min) exposure to low concentration NO 2 (0.2 ppm) on alveolar macrophages. Left panel: Spontaneous superoxide anion generation by alveolar macrophages. Generation of superoxide anion by alveolar macrophages was assayed using a lucigenin-dependent chemiluminescence (CL) method in which lucigenin served as a chemilucigenic probe. The results were expressed as relative luminescent units (RLU) per 500,000 viable alveolar macrophages. Right panel: Spontaneous neutrophil chemotactic activity generation by alveolar macrophages. Results are expressed as neutrophils (PMN) per high power field (HPF). Spontaneous chemiluminescence of the AM exposed to NO2 was significantly higher than spontaneous CL of control cells (Figure 1). This was largely due to the release of superoxide anion, as demonstrated by the inhibition of CL in presence of SOD (-77.4% for AM exposed to air and -90% for AM exposed to NO2). The neutrophil chemotactic activity generated by the cells exposed to NO2 was significantly higher than by the control cells of the same batches (8.82 PMN/HPF for cells exposed to air and 14.28 PMN/HPF for cells exposed to NO2, Figure 1). DISCUSSION The experimental model we described is in agreement with the objectives that we determined to approach as nearly as possible the exposure conditions that obtain in vivo: 1) culture of
154
Wallert and Voisin
alveolar macrophages in aerobiosis reproducing the physiological conditions of the bronchoalveolar spaces, 2) precise control of the experimented toxic gases, and 3) available biological techniques to characterize the cell alterations. Our results clearly demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either a cell injury or a cell activation associated with secretion of various bioactive products and mediators. Stimulation of various AM activities was observed with short term exposure to low concentrate NO2 (0.2 ppm), particularly superoxide anion release and neutrophil chemotactic activity generation. These results are in agreement with recent studies demonstrating that acute smoking resulted in increased superoxide production by unstimuiated AM recovered from smokers 1 h after smoking (Richter et al., 1986). These data are consistent with a stimulating effect of smoking or of NO2 on superoxide production by AM. Although precise mechanism remains unclear (Patel and Block, 1986), increased superoxide production by AM may have pathogenic significance leading to cellular and bronchioloalveolar injury (Murlas and Roum, 1985; Mohsenin and Gee, 1987). In addition, the finding that NO2 exposed AM released neutrophil chemotactic activity might explain in part the effects of short term, low level nitrogen dioxide exposure on bronchial hypersensitivity of asthmatic patients (Orehek et al, 1976). Experimental studies clearly indicated that airway hyper responsiveness after ozone exposure was associated with an influx of neutrophils into the distal airways (O'Byrne et al., 1984). Although there is no direct evidence that airway inflammation causes the hyperractivity of asthma, our data suggest a possible role for alveolar macrophages in the development of neutrophil inflammation in the distal airways in NO2 exposed subjects. Our experimental model of cell culture in aerobiosis is of particular interest to determine the biological effects of toxic gases at ambient concentrations on target cells. Additional studies demonstrated that this model offers new ways of research with various cell types (alveolar macrophages as well a type II pneumocytes) either for the analysis of biological mechanisms involved in the cell response to gas pollutants and or for evaluation of cell protective methods. SUMMARY
To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems have been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO2 on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.
In Vitro Study o f Gas Effects
155
REFERENCES ALINK, G.M., VAN DER HOEVEN, J.C.M., DEBETS, F.M.H., VAN DE VEN, W.S.M., and KOEMAN, J.H. (1979). A new exposure model for in vitro testing of effects of gaseous pollutants on mammalian cells by mean of gas diffusion through plastic films. Chemosphere 2:63-73. BOLTON, D.C., TARKINGTON, B.K., ZEE, Y.C., and OSEBOLD, J.W. (1982). An in vitro system for studying the effects of ozone on mammalian cell cultures and viruses. Environ Res. 27:466475. BRODY, A.R. (1986). Pulmonary cell interactions with asbestos fibers in vivo and in vitro. Chest. 89:1555-1595. DENICOLA, D.B., REBAR, A.H., and HENDERSON, R.F. (1981). Early damage Indicators in the lung. V. Biochemical and cytological response to NO2 inhalation. Toxicol Appl Pharmacol 60:301-312. EHRLICH, R. (1966). Effect of nitrogen dioxide on resistance to respiratory infection. Bact Rev. 30:604-614. FELS, A.O.S. and COHN, Z.A. (1986). The alveolar macrophage. J Appl Physiol. 60: 353-369. GUERBERO, R.R., ROUNDS, D.E., BOOHER, J., OLSON, R.S., and MACKNEY, T.D. (1979a). Ozone sensitivity in against WII-38 cells based on acid phosphatase content. Arch Environm Health 34:407-412. GUERBORO, R.R., ROUNDS, D.E., OLSON, R.S,, and MAKNEY, J.D. (1979b). Mutagenic effects of ozone on human cells exposed in vivo and in vitro based in sister chromatid exchange analysis. Environ Res. 18:336-346. HENRY, M.C., EHRLICH, R., and BLAIR, W.H. (1969). Effect of nitrogen dioxide on resistance of squirrel monkeys to Klebsiella pneumonia infection. Arch Environm Health 18:580-587. MOHSENIN, V. and GEE, B.L. (1987). Acute effect of nitrogen dioxide exposure on the functional activity of Alpha-l-protease inhibitor in bronchoalveolar lavage fluid or normal subjects. Am Rev Respir Dis. 136:646-650. MURLAS, C.G. and ROUM, J.H. (1985). Sequence of pathologic changes in the airway mucosa of guinea pigs during ozone-induced bronchial hyperreactivity. Am Rev Respir Dis. 131:314320. O'BYRNE, P.M., WALTERS, E.H., GOLD, B.D., AIZAWA, H.A., FABBRI, L.M., ALPERT, S.E., NADEL, J.A., and HOLTZMAN, M.J. (1984). Neutrophil depletion inhibits airway hyper responsiveness induced by ozone exposure. Am Rev Respir Dis. 130:214-219. OREHEK, J., MASSARI, J.P., GAYRARD, P., GRIMAUD, C., and CHARPIN, J. (1976). Effects of short-term, low levels nitrogen dioxide exposure on bronchial sensitivity of asthmatic patients. J Clin Invest 57:301-307. PARAZZI, E., SECCHI, G.C., PERNIS, B., and VIGLIANI, E. (1968). Studies on the cytotoxic action of silica dusts on macrophages "in vitro". Arch Environ Health 17: 850-855. PATEL, J.M. and BLOCK, E.R. (1986). Nitrogen Dioxide-induced changes in cell membrane fluidity and function. Am Rev Respir Dis. 134:1196-1202. RASMUSSEN, R.E. In vitro systems for exposure of lung cells to NO2 and 03 (1984). J Toxicol Environm Health 13:397-411. RICHTER, A.M., ABBOUD, R.T., JOHAL, S.S., and FERA, T.A. (1986). Acute effect of smoking on superoxide production by pulmonary alveolar macrophages. Lung 1164:233-242. SCHEULE, R.K. and HOLIAN, A. (1991). Immunologic aspects of pneumoconiosis. Exp Lung Res. 17:661-685. SONE, S., BRENNAN, L.M., and CREASIA, D.A. (1983). In vitro and in vitro NO2 exposures enhance phagocytic and tumoricidal activities of rat alveolar macrophages. J Toxicol Environm Health 11:151-161. VOISIN, C., AERTS, C., and HOUDRET, J.L. (1974). M6thode d'6tude des effets du NO2 sur les macrophages alvdolaires de cobaye en survie in vitro. Rev Fr Mal Resp 2 (suppl. 1):93-97.
156
Wallert and Voisin
VOISIN, C., AERTS, C., JAKUBSCZAK, E., and TONNEL, A.B. (1977). La culture cellulaire en phase gazeuse. Un nouveau module exp6rimental d'6tude in vitro des activit6s des macrophages • Bull Europ Physiopathol Respir 13:69-82. VOISIN, C., ALERTS,C., JAKUBSCZAK, E., HOUDRET, J.L., and TONNEL, A.B. (1977). Effects du bioxyde d'azote sur les macrophages alv~olaires en survie en phase gazeuse. Un nouveau module exp6rimental pour l'6tude in vitro de la cytotoxicit6 des gaz nocifs. Bull Eur Physiopathol Respir 13:137-144.
IN
VITRO
STUDY OF GAS EFFECTS MACROPHAGES B. W A L L A E R T
ON ALVEOLAR
and C. V O I S I N
Laboratoire de Pollution Atmosph6rique et de Pathologie Respiratoire Exp6rimentale and CJF INSERM, Institut Pasteur, Lille, France
INTRODUCTION The lung is the target of multiple aggressions due to occupational hazards, environmental noxious substances and personal risks such as tobacco smoke. In all of these situations, alveolar macrophages (AM) located on the respiratory membrane, are in close, permanent and direct contact with the airborne pollutants and are directly exposed to particles and toxic gases. Thus, taking into account the multiple functional activities of this cell population (Fels and Cohn, 1986), it seems reasonable to hypothezise that alveolar macrophages play a pivotal role in the responses of the respiratory tract to the inhaled materials. During the past 25 years, extensive investigations have been devoted to alveolar macrophage response to silica, coal dust and asbestos (Parazzi et al., 1968; Scheule and Holian, 1991; Brody, 1986). The presence of the dust inside the cells, either obtained ex vivo from dusted animals or humans, or in vitro submitted to selected particles under strictly controlled conditions, allows a qualitative and quantitative evaluation of cell disturbances. In marked contrast, AM responses to toxic gases are difficult to approach. However the understanding of the mechanisms of the functional and pathological disorders resulting from the inhalation of toxic gases in the respiratory tract requires the study of the direct effects of pollutants on the state and the activity of AM. Up to now, three series of methods were used: the first ones consist in animal exposure to the toxic gas followed by bacterial challenge against infectious agents, in order to evaluate the possible alterations in the phagocytic and bactericidal activity of AM (Ehrlich, 1966; Henry et al., 1969); the second technic is an "in vitro" study of AM harvested by bronchoalveolar lavage either from animals or from human volunteers exposed to controlled inhalation of toxic gases (Denicola et al., 1981; Sone et al., 1983). However, in such experiments, cell disturbances are not well correlated to the gas exposure; the third one deals with in vitro systems for exposure of alveolar macrophages to gases in controlled conditions. I N V I T R O SYSTEMS FOR EXPOSURE OF ALVEOLAR MACROPHAGES TO GASES
The description of various in vitro system for exposure of lung cells to oxidant gases have been recently reviewed by Rasmussen (Rasmussen, 1984). Exposure to gases may be Corresponding Author: B. WALLAERTMD, Laboratoire de Pollution atmosph6rique, Institut Pasteur de Lille, 1, rue Calmette, 59800 Lille, FRANCE. Abbreviations: Alveolar macrophages: AM, Chemiluminescence:CL. Key Words: alveolar macrophages, gases, in vitro systems, oxidants. Supported by Universit6 de Lille II and INSERM CJF 90-06. Cell Biology end Toxicology, Vol. 8, No. 3, pp. Copyright © 1992 Princeton Scientific Publishing ISSN: 0742-2091
151-156 Co., Inc.
152
Wallert and Voisin
performed using roller bottles fitted with modified rotating caps with tubing connections (Bolton et al., 1982) or dishes on rocker platforms tilting back and forth to periodically expose the cell culture to gases (Guerbero et al., 1979a; Guerboro et al., 1979b). Lastly, exposure of cells may be obtained using gas-permeable membranes on which cells grow on very thin membrane allowing gas to permeate through the film (Mink et al., 1979). However, it is clear that using these systems, culture medium constitutes a barrier between the gas and the target cell and does not permit a physiological approach of the toxic effects of gases. This is the reason why we developed an experimental model, using a cell culture technique in gas phase (Voisin et al., 1974; Voisin et al, 1977a; Voisin et al, 1977b). Herein we reported the value and the limits of this original method using the model of alveolar macrophage responses to NO2. BIPHASIC SYSTEM CULTURE OF ALVEOLAR MACROPHAGES AM were harvested by broncho alveolar lavage from healthy subjects according to the method previously described (Voison et al., 1977a). AM were layered on a Gelman membrane, which was applied to the surface of a reservoir filled up with culture medium, so as to be saturated by capillarity. In such experimental conditions, in air enriched with CO2 5% and saturated with water at 37°C, it was possible to maintain the cells alive, with normal metabolic and functional activities up to two weeks. I N V I T R O EXPOSURE OF ALVEOLAR MACROPHAGES TO NO 2
Exposure to NO2 was carried out using an original device consisting in a special exposure chamber, in which air with 5% CO2 enriched with the toxic gas was pulsed in continuous flow with continuous monitoring. Long term (24 h) and short term exposure (30 min) of human AM to various low concentrations NO2 were performed. The effects of gases on AM were studied by examination of theft morphological appearance under optical and electronic microscopy and by the determination of the ATP content of the cells. The cell injury index reported in this paper corresponds to the percent decrease in cellular ATP content of gas-exposed cells when compared with the ATP content of aft-exposed cells, measured at the same time. Long term exposure (24 h) at low concentration NO2 (0.2 ppm) resulted in a significant decrease in cell ATP content. Cell injury index clearly depended on NO2 concentration. Experiments done with 2 and 5 ppm NO2 resulted in a complete destruction of the cellular preparation. In marked contrast, short term exposure (30 min) at low concentration NO2 (0.2 ppm) did not induce cytotoxic effects of NO2 as assessed by the absence of modification of beta-glu-
In Vitro Study of Gas Effects 153
curonidase cell content and of ATP cell content after exposure. Conversely, NO2 short term exposure was responsible for cell membrane activation as demonstrated by the increased generation of both superoxide anion and neutrophil chemotactic activity by alveolar macrophages.
NEUTROPHIL CHEMOTACTIC ACTIVITY
SUPEROXIDE ANION GENERATION
PMN/HPF
RLU
p < 0,05 20000
P < 0,05
T
10000
i
AIR
20
10
fliiiiJ I?1 NO2
AIR
NO2
FIGURE 1. Effects of short term (30 min) exposure to low concentration NO 2 (0.2 ppm) on alveolar macrophages. Left panel: Spontaneous superoxide anion generation by alveolar macrophages. Generation of superoxide anion by alveolar macrophages was assayed using a lucigenin-dependent chemiluminescence (CL) method in which lucigenin served as a chemilucigenic probe. The results were expressed as relative luminescent units (RLU) per 500,000 viable alveolar macrophages. Right panel: Spontaneous neutrophil chemotactic activity generation by alveolar macrophages. Results are expressed as neutrophils (PMN) per high power field (HPF). Spontaneous chemiluminescence of the AM exposed to NO2 was significantly higher than spontaneous CL of control cells (Figure 1). This was largely due to the release of superoxide anion, as demonstrated by the inhibition of CL in presence of SOD (-77.4% for AM exposed to air and -90% for AM exposed to NO2). The neutrophil chemotactic activity generated by the cells exposed to NO2 was significantly higher than by the control cells of the same batches (8.82 PMN/HPF for cells exposed to air and 14.28 PMN/HPF for cells exposed to NO2, Figure 1). DISCUSSION The experimental model we described is in agreement with the objectives that we determined to approach as nearly as possible the exposure conditions that obtain in vivo: 1) culture of
154
Wallert and Voisin
alveolar macrophages in aerobiosis reproducing the physiological conditions of the bronchoalveolar spaces, 2) precise control of the experimented toxic gases, and 3) available biological techniques to characterize the cell alterations. Our results clearly demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either a cell injury or a cell activation associated with secretion of various bioactive products and mediators. Stimulation of various AM activities was observed with short term exposure to low concentrate NO2 (0.2 ppm), particularly superoxide anion release and neutrophil chemotactic activity generation. These results are in agreement with recent studies demonstrating that acute smoking resulted in increased superoxide production by unstimuiated AM recovered from smokers 1 h after smoking (Richter et al., 1986). These data are consistent with a stimulating effect of smoking or of NO2 on superoxide production by AM. Although precise mechanism remains unclear (Patel and Block, 1986), increased superoxide production by AM may have pathogenic significance leading to cellular and bronchioloalveolar injury (Murlas and Roum, 1985; Mohsenin and Gee, 1987). In addition, the finding that NO2 exposed AM released neutrophil chemotactic activity might explain in part the effects of short term, low level nitrogen dioxide exposure on bronchial hypersensitivity of asthmatic patients (Orehek et al, 1976). Experimental studies clearly indicated that airway hyper responsiveness after ozone exposure was associated with an influx of neutrophils into the distal airways (O'Byrne et al., 1984). Although there is no direct evidence that airway inflammation causes the hyperractivity of asthma, our data suggest a possible role for alveolar macrophages in the development of neutrophil inflammation in the distal airways in NO2 exposed subjects. Our experimental model of cell culture in aerobiosis is of particular interest to determine the biological effects of toxic gases at ambient concentrations on target cells. Additional studies demonstrated that this model offers new ways of research with various cell types (alveolar macrophages as well a type II pneumocytes) either for the analysis of biological mechanisms involved in the cell response to gas pollutants and or for evaluation of cell protective methods. SUMMARY
To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems have been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO2 on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.
In Vitro Study o f Gas Effects
155
REFERENCES ALINK, G.M., VAN DER HOEVEN, J.C.M., DEBETS, F.M.H., VAN DE VEN, W.S.M., and KOEMAN, J.H. (1979). A new exposure model for in vitro testing of effects of gaseous pollutants on mammalian cells by mean of gas diffusion through plastic films. Chemosphere 2:63-73. BOLTON, D.C., TARKINGTON, B.K., ZEE, Y.C., and OSEBOLD, J.W. (1982). An in vitro system for studying the effects of ozone on mammalian cell cultures and viruses. Environ Res. 27:466475. BRODY, A.R. (1986). Pulmonary cell interactions with asbestos fibers in vivo and in vitro. Chest. 89:1555-1595. DENICOLA, D.B., REBAR, A.H., and HENDERSON, R.F. (1981). Early damage Indicators in the lung. V. Biochemical and cytological response to NO2 inhalation. Toxicol Appl Pharmacol 60:301-312. EHRLICH, R. (1966). Effect of nitrogen dioxide on resistance to respiratory infection. Bact Rev. 30:604-614. FELS, A.O.S. and COHN, Z.A. (1986). The alveolar macrophage. J Appl Physiol. 60: 353-369. GUERBERO, R.R., ROUNDS, D.E., BOOHER, J., OLSON, R.S., and MACKNEY, T.D. (1979a). Ozone sensitivity in against WII-38 cells based on acid phosphatase content. Arch Environm Health 34:407-412. GUERBORO, R.R., ROUNDS, D.E., OLSON, R.S,, and MAKNEY, J.D. (1979b). Mutagenic effects of ozone on human cells exposed in vivo and in vitro based in sister chromatid exchange analysis. Environ Res. 18:336-346. HENRY, M.C., EHRLICH, R., and BLAIR, W.H. (1969). Effect of nitrogen dioxide on resistance of squirrel monkeys to Klebsiella pneumonia infection. Arch Environm Health 18:580-587. MOHSENIN, V. and GEE, B.L. (1987). Acute effect of nitrogen dioxide exposure on the functional activity of Alpha-l-protease inhibitor in bronchoalveolar lavage fluid or normal subjects. Am Rev Respir Dis. 136:646-650. MURLAS, C.G. and ROUM, J.H. (1985). Sequence of pathologic changes in the airway mucosa of guinea pigs during ozone-induced bronchial hyperreactivity. Am Rev Respir Dis. 131:314320. O'BYRNE, P.M., WALTERS, E.H., GOLD, B.D., AIZAWA, H.A., FABBRI, L.M., ALPERT, S.E., NADEL, J.A., and HOLTZMAN, M.J. (1984). Neutrophil depletion inhibits airway hyper responsiveness induced by ozone exposure. Am Rev Respir Dis. 130:214-219. OREHEK, J., MASSARI, J.P., GAYRARD, P., GRIMAUD, C., and CHARPIN, J. (1976). Effects of short-term, low levels nitrogen dioxide exposure on bronchial sensitivity of asthmatic patients. J Clin Invest 57:301-307. PARAZZI, E., SECCHI, G.C., PERNIS, B., and VIGLIANI, E. (1968). Studies on the cytotoxic action of silica dusts on macrophages "in vitro". Arch Environ Health 17: 850-855. PATEL, J.M. and BLOCK, E.R. (1986). Nitrogen Dioxide-induced changes in cell membrane fluidity and function. Am Rev Respir Dis. 134:1196-1202. RASMUSSEN, R.E. In vitro systems for exposure of lung cells to NO2 and 03 (1984). J Toxicol Environm Health 13:397-411. RICHTER, A.M., ABBOUD, R.T., JOHAL, S.S., and FERA, T.A. (1986). Acute effect of smoking on superoxide production by pulmonary alveolar macrophages. Lung 1164:233-242. SCHEULE, R.K. and HOLIAN, A. (1991). Immunologic aspects of pneumoconiosis. Exp Lung Res. 17:661-685. SONE, S., BRENNAN, L.M., and CREASIA, D.A. (1983). In vitro and in vitro NO2 exposures enhance phagocytic and tumoricidal activities of rat alveolar macrophages. J Toxicol Environm Health 11:151-161. VOISIN, C., AERTS, C., and HOUDRET, J.L. (1974). M6thode d'6tude des effets du NO2 sur les macrophages alvdolaires de cobaye en survie in vitro. Rev Fr Mal Resp 2 (suppl. 1):93-97.
156
Wallert and Voisin
VOISIN, C., AERTS, C., JAKUBSCZAK, E., and TONNEL, A.B. (1977). La culture cellulaire en phase gazeuse. Un nouveau module exp6rimental d'6tude in vitro des activit6s des macrophages • Bull Europ Physiopathol Respir 13:69-82. VOISIN, C., ALERTS,C., JAKUBSCZAK, E., HOUDRET, J.L., and TONNEL, A.B. (1977). Effects du bioxyde d'azote sur les macrophages alv~olaires en survie en phase gazeuse. Un nouveau module exp6rimental pour l'6tude in vitro de la cytotoxicit6 des gaz nocifs. Bull Eur Physiopathol Respir 13:137-144.
