Inhibition of Human Alveolar Macrophage Production of Leukotriene B4 by Acute In Vitro and In Vivo Exposure to Tobacco Smoke Johanne Tardif, Pierre Borgeat, and Michel Laviolette Unite de Recherche, Centre de Pneumologie, Hapital, Ste-Foy, and Unite de Recherche, Inflammation et ImmunologieRhumatologie, Centre Hospitalier de l'Universite Laval, Quebec, Canada
The number of neutrophils in the lungs of cigarette smokers is increased. This could be a consequence of the chemotactic mediator synthesis by alveolar macrophages (AM). In order to evaluate the possible role of leukotriene B4 (LTB4) in this condition, we studied the formation of LTB4 by nonsmokers' AM exposed in vitro and in vivo to cigarette smoke and by smokers' AM. In the absence of stimulus or upon stimulation, nonsmokers' AM exposed in vitro to tobacco smoke formed less LTB4 than did nonexposed AM, e.g., the cells incubated with arachidonic acid and ionophore produced, respectively, 241 ± 132 and 425 ± 106 pmol LTB4 !lQ6 cells (mean ± SEM) (P < 0.01). In other experiments, smokers' AM were incubated in absence of stimulus and produced less LTB4 than did nonsmokers' AM; during a 3-h incubation, smokers' and nonsmokers' adherent AM released, respectively, 3 ± 2 and 40 ± 28 pmol LTB4/1Q6 cells (P < 0.05). Similarly stimulated smokers' AM produced less LTB4 than did nonsmokers' AM, e.g., the cells incubated with arachidonic acid and ionophore formed, respectively, 225 ± 41 and 573 ± 150 pmol LTBJIQ6 cells (P < 0.05). In a group including mild smokers and nonsmokers, in vivo exposure to the smoke of 4 cigarettes produced a decrease in the release of LTB4 by AM, e.g., in the presence of arachidonic acid and ionophore, nonexposed and exposed AM produced, respectively, 198 ± 38 and 143 ± 38 pmol LTB./IQ6 cells (P < 0.05). In all those conditions, AM exposed to cigarette smoke exhibited an inhibition of 5-hydroxy-eicosatetraenoic acid. These results show that acute in vitro or in vivo exposures to tobacco smoke and chronic smoking inhibit the spontaneous and stimulated AM production of arachidonic acid metabolites. Consequently, it is very unlikely that LTB4 could account for the neutrophil recruitment into the lungs of smokers.
Human alveolar macrophages (AM) can produce important amounts of leukotriene (LT) B4 , a biologically active metabolite of arachidonic acid (AA) (1-4). Stimuli such as phagocytosis or calcium ionophore induce the synthesis of this mediator by AM. LTB4 acts on lung smooth muscles, vascular permeability, and lymphocyte functions (5-7) but most importantly is a potent neutrophil chemotactic factor. It is as effective as the Sa complement fragment in this function (8-10). The neutrophils carry a large amount of hydrolytic enzymes capable of destroying the lung structure (11-14). An increased number of neutrophils is found in smokers' bronchoalveolar lavage (BAL). It has been postulated that these Key IJbrds: leukotrienes, alveolar macrophages, smoking, arachidonic acid (Received in original form June 30, 1989 and in revised form September 25, 1989) Address correspondence to: Michel Laviolette, M.D., Centre de Pneumologie, Hopital Laval, Tl25, Chemin Ste-Foy, Ste-Foy, Quebec GlY 4G5, Canada. Abbreviations: alveolar macrophages, AM; bronchoalveolar lavage, BAL; carboxyhemoglobin, COHb; 5S-hydroxy-6,8,II,14-(E,Z,Z,Z)-eicosatetraenoic acid, 5-HETE; leukotriene B4 , LTB4 • Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 155-161, 1990
cells might be at the origin of chronic inflammatory process leading to the development of emphysema (11-16). Smoking provokes many morphologic and functional alterations in AM (17-20), and tobacco smoke induces AM to release many mediators, including chemotactic factors (13, 21). Consequently, AM could be involved in the pathogenesis of emphysema via a chemotactic factor release and neutrophil recruitment to the lungs. Hunninghake and Crystal (13) previously demonstrated that nonsmokers' AM acutely exposed in vitro to tobacco products release a chemotactic factor for neutrophils. They also found that smokers' AM spontaneously release this chemotactic factor. Because this mediator had a small molecular weight and was lipidic in nature, they postulated that it could be LTB4 • However, chronic smoking induces a significant inhibition of AM LTB4 and prostaglandin synthesis (22-26). Moreover, acute in vivo exposure of smokers' AM to tobacco smoke does not induce LTB4 production (27). These results suggest that the mediator described by Hunninghake and Crystal may not be LTB4 • The purpose of this study was to evaluate the release of LTB4 by nonsmokers' AM acutely exposed in vitro or in vivo to tobacco smoke and also by chronic smokers' AM. We found that AM acutely or
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chronically exposed to tobacco smoke released less LTB4 than did the nonexposed cells.
Materials and Methods Subjects The study involved 58 healthy volunteers, 31 males and T1 females, with a mean ageof27.6yr (range, 19t042yr), who all gave informed consent: 34 were current nonsmokers, 33 never smoked, and one had stopped smoking 6 yr previously. Twelve were current heavy smokers (>20 cigarettes/d for more than 2 yr), and twelve were occasional or light smokers, of whom eight smoked 1 or 2 cigarettes per month and four smoked less than 4 cigarettes/d. None of the subjects had taken any medication for 1 mo prior to the study nor had a common cold or pulmonary symptoms, and none had a history suggestive of lung disease. BAL and Cell Processing All lavages were performed by fiberoptic bronchoscopy as previously described (28) using 240 or 300 ml of warm (J"l0 C) saline (0.9% NaCl, wt/vol) in 60-ml aliquots. The cells were separated from the lavage fluid by centrifugation (250 g, 10 min, 4°C) and resuspended in tissue culture medium RPMI 1640 (GIBCO, Grand Island, NY) (pH 7.4) or in Dulbecco's PBS (without Ca2+ and Mg2+). A portion of those cell suspensions was used for the determination of total cell number (hemocytometer), differential counts (Wright-Giemsa or Diff-Quik stain), and viability (trypan blue exclusion). Cell viability was always greater than 90%.
In Vitro Exposure to Tobacco Smoke The lavage cells obtained from 18 nonsmokers were resuspended in culture medium RPMI 1640 containing MgC12 and CaC12 (final concentration, 0.5 mM and 2 mM, respectively) to a concentration of 1.0 X 1()6 cells/0.8 ml and placed for adherence in 35-mm culture dishes (Falcon Plastics, Los Angeles, CA). The incubation volume was completed by adding 0.2 ml of medium containing the smoke extracts, or 0.2 ml of medium bubbled with air. Those media were prepared according to the method of Hunninghake and Crystal (13). Briefly, the smoke from 1 cigarette (Winston®, unfiltered; 1. R. Reynolds Tobacco Co.) or the equivalent amount of air was bubbled through 24 ml of RPMI 1640, providing, respectively, the smoke medium and the air medium used in those experiments. The cells were incubated with or without stimulus. The adherent AM were incubated with air medium or smoke medium at J"l0 C under an atmosphere of 5 % CO2 and 95 % air. After 1 h, the cellular monolayers were washed 3 times with warm (J"l0 C) HBSS, then 1 ml of fresh medium (RPMI 1640) was added to the dishes and adherent AM were incubated for an additional 3 h. The incubations were stopped by adding 0.5 ml methanol/acetonitrile. This solution also contained 50 ng of prostaglandin (PG) B2 per ml as an internal standard. The denatured media were harvested and analyzed to seek the presence of 5-lipoxygenase products by reverse-phase HPLC. The cellular viability was estimated in control culture dishes using trypan blue dye exclusion. We found no difference between the viability of AM exposed to
air medium compared to those exposed to smoke medium (97 ± 0.2% and 96 ± 0.6%, respectively; P = 0.21). In other experiments, adherent cells were washed as described above, preincubated for 10 min with fresh HBSS, and then stimulated for 10 min with 30 J,tM AA and/or 2 J,tM calcium ionophore A23187. For the control experiments, AM were incubated with 0.2 % of ethanol (final concentration). At the end of the incubations, the reactions were stopped as described above and the denatured media were kept for HPLC analysis. Comparison between Heavy Smokers' and Nonsmokers' Cells In 12 experiments, lavage cells were obtained from 1 heavy smoker and 1 nonsmoker on the same morning and were processed in parallel. The cell pellets were resuspended in RPMI 1640. One-milliliter aliquots of cellular suspension (1.5 X 1()6 cells/ml) were added to 35-mm culture dishes. In some experiments, the cells were incubated for 3 h in culture medium RPMI 1640 at J"l0 C, according to the method described by Hunninghake and Crystal (13). This was done to evaluate whether smokers' AM could spontaneously release LTB4 • The culture fluids were harvested for HPLC analysis. At the end of the incubation, there was no significant difference in cellular viability between the nonsmokers' and smokers' cells (93 ± 1% and 95 ± 1%, respectively; P = 0.11). In other experiments, the cells were incubated for 1 h at J"l0 C on culture dishes in RPMI 1640 with 10% FBS. The cell monolayers were then washed 3 times with warm HBSS to remove nonadherent cells. One milliliter of fresh HBSS with MgCl 2 and CaC12 (final concentration, 0.5 mM and 2 mM, respectively) was added to the cultures, and the cells were preincubated for 10 min. The adherent cells were then stimulated with 2 J,tM ionophore A23187 and/or 30 J,tM AA for 10 min. In the control experiments, AM were incubated with ethanol (0.2%). The reactions were stopped, and the denatured media kept for HPLC analysis.
In Vivo Exposure to Tobacco Smoke For these experiments, a first BAL (four 60-ml aliquots) was performed in the lingula. The subjects then smoked 4 cigarettes within 1 h, and a second BAL (also using 240 ml) was done this time in the right middle lobe. Eight light or occasional smokers and four nonsmokers volunteered for this experiment. The smokers had not smoked for more than 1 wk. Briefly, the subjects smoked 1 cigarette (Export 'W'®, unfiltered; MacDonald Inc.) every 15 min, taking 1 puff with a deep inhalation every 30 s. Approximately 10 min were required to smoke 1 cigarette, leaving a rest interval of about 5 min between each cigarette. Blood samples for carboxyhemoglobin (COHb) measurement were withdrawn before and after smoking (29). The cellular viability was similar before and after smoking (91 ± 2 % and 85 ± 2 %, respectively; P = 0.12). The cells from these 2 consecutive lavages were compared to evaluate the effect of an acute in vivo tobacco smoke exposure on AM LTB4 production. A I-ml aliquot of cell suspension (1.0 X 1()6 cells/ml PBS) was added to polystyrene tubes and preincubated in motion at J"l0 C for 10 min.
Tardif, Borgeat, and Laviolette: Acute Smoking and Leukotriene Synthesis by Alveolar Macrophages
The AM were then stimulated for 10 min with 30 /-tM exogenous AA and/or 2/-tM calcium ionophore A23187. The controls contained the same final concentrations of CaCl h MgClz, and ethanol as described above. The reactions were stopped, and the denatured cell suspensions kept for HPLC analysis. Four light or occasional smokers and three nonsmokers, who participated to the first part of this in vivo experiment, also volunteered to form a control group. These subjects were submitted to the same procedures except that they had to inhale air through a straw (sham smoking) rather than smoke a cigarette. No blood samples were withdrawn in this case. The lavage cells were processed according to the same protocol. Reverse-phase HPLC Analysis of Lipoxygenase Products 5-Lipoxygenase products were measured by HPLC as previously described (30). The metabolites were identified by their co-migration with authentic standards and the specificity of UV absorption. Their quantitation was done by measuring the peak height and comparing with calibrated standards of lipoxygenase products, after correction for recoveries using the internal standard PGB2 • Supernatants were centrifuged to eliminate precipitated material and injected into a cartridge (Radial Pak Cs, 100 X 5 mm, 10/-tm particle size; Waters Millipore Corp., Milford, MA). All the different metabolites were eluted at a solvent flow of 1.5 ml/min using a 3-s01vent mixture system, following a predetermined program. LTB4 and 5S-hydroxy-6,8,1l,14(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE) were measured with a detection limit of "-'5 pmol. Statistical Analysis Data were expressed as the mean ± SEM. The comparisons between the BAL results of the 3 groups of subjects were done using a Kruskal-Wallis test (multiple comparison) for nonparametric values. The productions of lipoxygenase products were compared using either a Wilcoxon-Mann-Whitney two-sample test for unpaired nonparametric values or a Wilcoxon's signed rank test for paired nonparametric values. A value of P < 0.05 was considered significant.
Results Bronchoalveolar Lavage Lavage parameters are presented in Table 1. The lavage return was similar in the 3 groups. Heavy smokers had a
157
greater total number of cells, more macrophages, but less lymphocytes than did nonsmokers (P < 0.05). However, there was no significant difference in the percentage and in the absolute number (data not shown) ofneutrophils between heavy smokers and nonsmokers. The number of cells recovered from occasional and light smokers was lower than that from heavy smokers (135.2 ± 22 X 103 versus 205.3 ± 21.6 X 103 cells/ml [P < 0.05]) but similar to that obtained from nonsmokers (93 ± 13.6 X 103 cells/ml). The occasional and light smokers' BAL had similar differential cell counts as nonsmokers' BAL but contained fewer macrophages and more lymphocytes than heavy smokers' BAL (P < 0.05). However, in light smokers' BAL, about 5% of the AM had some morphologic characteristics of heavy smokers' AM (17-20). These AM had lipidic inclusions and were somewhat bigger than nonsmokers' AM. Occasional smokers' AM were similar to those of nonsmokers. In the experiments on in vivo exposure to cigarette smoke, the subjects had 2 BAL. The mean recovered volumes of lavage fluid before and after smoking 4 cigarettes were similar: 59.6 ± 2.3% versus 61.1 ± 1.4%, respectively (P > 0.05). The total number of recovered cells and the cellular differentials were also similar: total cell numbers, 147.9 ± 23.9 X 103 and 141.6 ± 28.3 X 103 cells/ml; macrophages, 83.1 ± 4.8% and 81.1 ± 4.9%; lymphocytes, 14.7 ± 4.8% and 17.6 ± 5%; and neutrophils, 1.8 ± 0.5% and 1 ± 0.3%. Acute In Vitro Exposure to Tobacco Smoke Unstimulated AM exposed in vitro to smoke medium and incubated for 3 h did not synthesize detectable amounts of LTB4 nor 5-HETE, whereas AM exposed to air medium produced small amounts of these products: LTB4 , 1 ± 1 pmol/106 cells; 5-HETE, 5 ± 5 pmol/106 cells. Upon stimulation, AM exposed to smoke medium produced less LTB4 and 5-HETE than did AM exposed to air medium (Figure 1). Incubated with ionophore A23187, AM exposed to tobacco smoke produced about 6 times less LTB4 and 8 times less 5-HETE than did those exposed to the air medium: respectively, LTB4 , 38 ± 16 and 254 ± 85 pmol/106 cells; 5-HETE,29 ± 15 and 239 ± 80 pmol/106 cells (P < 0.01). A similar decrease in LTB4 and 5-HETE production by AM exposed to smoke medium was observed in cultures stimulated with both AA (30 /-tM) and calcium ionophore A23187: respectively LTB4 , 241 ± 132 and 425 ± 106 pmol/lQ6 cells in smoke medium and air medium; 5-HETE, 156 ± 91 and 429 ± 100 pmol/1Q6 cells (P < 0.01). In the control incubations, the smoke medium-incubated cells synthesized less 5-HETE: 8 ± 4 and 48 ± 14 pmol/1Q6 cells,
TABLE 1
Subjects' characteristics and lavage data* BAL Return
n
Nonsmokers 34 Occasional and light smokers 12 Smokers 12
* Values
are ll\ean
Sex (M/F)
Age (yr)
(% of infused
Cell Yield
Macrophages
Lymphocytes
Neutrophils
Eosinophils
volume)
(X llYlml)
(%)
(%)
(%)
(%)
16/18
25.6
± 0.9
65.7
±
1.3
93.0
7/5 8/4
30.7 29.3
± 2.1 ± 1.6
60.6 63.3
± ±
1.4 1.5
135.2 205.3
± SEM.
t p < 0.05 compared to smokers.
±
13.6t
84.0
± 2.2t
14.4
± 2.2t
1.2
± 0.2
0.3
± 0.1
± 22.0t ± 21.6
81.5 94.6
± 5.0t ± 1.0
17.0 3.7
± 5.4t ± 0.9
1.2 1.1
± 0.3 ± 0.2
0.4 0.6
± 0.2 ± 0.2
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
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AIR-MEDIUM
~
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Figure 1. Effects of acute in vitro exposure to tobacco smoke on lipxygenase product synthesis by nonsmokers' alveolar macrophages (AM). Adherent bronchoalveolar lavage (BAL) cells were incubated in the presence of medium bubbled with tobacco smoke or control medium bubbled with air and were stimulated 10 min with 2 JLM ionophore A23187 (IONO) alone (n = 13) or with 30 JLM arachidonic acid and ionophore A23l87 (AA + IONO) (n = 12). *P < 0.05, tp < 0.01; Wilcoxon's signed rank test.
400
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t
t
O
.... .... o
I:
200
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o 1""--" C
I
WPM
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I
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IONO
C
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respectively (P = 0.01). The decrease in LTB4 production, however, was not significant: 7 ± 2 and 13 ± 5 pmol/106 cells. Comparison between Heavy Smokers' and Nonsmokers' Cells Unstimulated adherent AM from smokers failed to produce a greater amount of LTB4 than did cells from nonsmokers. In fact, their production of LTB4 was significantly less than that of the nonsmokers' AM: 3 ± 2 and 40 ± 28 pmol/lQ6 cells, respectively (P < 0.05) (Figure 2). The production of 5-HETE by cells of smokers was also lower: 4 ± 4 and 69 ± 32 pmol/lQ6 cells, respectively; this difference did not reach statistical significance (P = 0.06). Adherent smokers' AM, incubated with exogenous AA, produced, respectively, about 8 and 3 times less LTB4 and 5-HETE than did AM of nonsmokers; LTB4 , 9 ± 3 and 69 ± 23 pmol/l06 cells (P < 0.01); 5-HETE, 68 ± 20 and 230 ± 59 pmol/lQ6 cells (P = 0.01) (Figure 3). The smokers' AM incubated with calcium ionophore A23187 produced approximately 2.8 times less metabolites than did nonsmokers' AM: respectively, LTB4 , 191 ± 36 and 531 ± 140 pmol/ 106 cells (P = 0.02); 5-HETE, 202 ± 41 and 745 ± 204 pmol/lQ6 cells (P < 0.01). When smokers' AM were stimulated with both exogenous AA and ionophore A23187, the production of both metabolites was 2.5 times less than that of the nonsmokers' cells: respectively, LTB4 , 225 ± 41 and 573 ± 150 pmol/106 cells (P = 0.03); 5-HETE, 544 ± 173 and 1,185 ± 288 pmol/lQ6 cells (P < 0.01). In the control incubations, LTB 4 synthesis by smokers' AM was significantly less than that of nonsmokers' AM: undetectable «5 pmol) and 26 ± 22 pmol/lQ6 cells in smokers and nonsmokers, respectively (P = 0.01). Acute In Vivo Exposure to Tobacco Smoke After smoking 4 cigarettes, the COHb concentration increased from 0.5 ± 0.2 to 2.7 ± 0.8 mol/mol (COHb/Hb) (P < 0.01). In the group undergoing sham smoking, there was no observable difference in LTB4 or 5-HETE production between the first and second lavage, both under control and stimulated conditions (P > 0.33) (Figure 4, upper panel). Af-
AA+IONO
ter an acute in vivo exposure to the smoke of 4 cigarettes, AM released less lipoxygenase products (Figure 4, lower panel). Upon stimulation, AM not exposed to cigarette smoke produced more lipoxygenase products than did tobacco smoke-exposed cells: with AA, LTB4 , 15 ± 4 and 7 ± 2 pmol/106 cells (P < 0.01) and 5-HETE, 90 ± 25 and 48 ± 17 pmol/lQ6 cells (P = 0.01); with AA and ionophore, LTB4 , 198 ± 38 and 143 ± 38 pmol/lQ6 cells (P < 0.05) and 5-HETE, 756 ± 146 and 558 ± 133 pmol/106 cells (P < 0.05), respectively. Upon stimulation with ionophore A23187, the LTB4 and 5-HETE synthesis by tobacco smoke-exposed AM did not decrease significantly compared to the exposed cells (P = 0.18). No differences were found in the control incubations. In these experiments, LTB4 and
150
(f)
...."".... U
-........
o
NONSMOKERS
~
SMOKERS -r-
100
\Do
o
~ Cl.
*
-r-
50
o
~
LTB4
~
S-HETE
Figure 2. LTB 4 and 5-HETE production by unstimulated AM of nonsmokers and heavy smokers. Adherent lavage cells were incubated for 3 h in RPMI 1640 to evaluate the spontaneous production of LTB4 and 5-HETE (n = 6). *P < 0.05; Wilcoxon-MannWhitney two-sample test.
Tardif, Borgeat, and Laviolette: Acute Smoking and Leukotriene Synthesis by Alveolar Macrophages
159
Figure 3. Lipoxygenase product synthesis by smokers' and nonsmokers' AM incubated in adherence. Adherent cells were incubated for 10 min with 2 JLM ionophore A23187 (lONO) or/ and 30 JLM AA (n = 11). 'p < 0.05, tp < 0.01; Wilcoxon-MannWhitney two-sample test.
5-HETE productions by AM of occasional and light smokers (n = 8) were similar to those of nonsmokers (n = 4), e.g., in the presence of AA and ionophore, light smokers' cells produced 200 ± 50 pmol LTB.fl()6 cells and 598 ± 155 pmol 5-HETE/IQ6 cells, and nonsmokers' cells produced
Figure 4. Effect of inhaling air (sham smoking) or smoking 4 cigarettes on AM's AA metabolism. Upper panel: Production of LTB4 and 5-HETE after sham smoking (n = 7). Lower panel: Production of LTB 4 and 5-HETE after smoking 4 cigarettes (n = 12). Cell suspensions were incubated for 10 min with 2 JLM ionophore (IONO) orland 30 JLM AA. 'p < 0.05, tp < 0.01; Wilcoxon's signed rank test.
224 ± 95 pmol LTBJIQ6 cells (P = 0.32) and 936 ± 361 pmol 5-HETE/106 cells (P = 0.27). The difference in 5HETE production before smoking seen between the upper and lower panels of Figure 4 could be explained by the variability of 5-HETE production from one subject to another
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
and by the fact that data presented in the upper panel were obtained from seven out ofthe 12 subjects shown in the lower panel.
Discussion The present study demonstrates that acute in vitro (Figure 1) and in vivo (Figure 4) exposures to tobacco smoke inhibit the release of lipoxygenase products by AM obtained from nonsmokers or light and occasional smokers. Moreover, neither acute in vivo and in vitro exposures to tobacco smoke nor chronic smoking induce spontaneous production of LTB. (Figures 2 and 4). As previously reported (22-26), chronic cigarette smoking reduces the metabolism of AA by smokers' AM, leading to a decrease in the production ofleukotrienes (Figure 3). Rios-Mollineda and colleagues ('lJ) suggested that an acute in vivo exposure to cigarette smoke inhibits the production of LTB. by smokers' AM. Because they studied smokers, one cannot differentiate the effect of chronic smoking from that of acute smoke exposure from their study. The acute in vitro exposure to tobacco smoke used in this study induced a significant inhibition of the smokers' AM 5lipoxygen;l~e activity. A smaller but similar inhibition was obtained by smoking4 cigarettes. The difference in the magnitude of the effects produced by cigarette smoke between in vivo and in vitro exposures could be explained by the difference in the exposure levels. In vitro, adherent cells were incubated in the presence of cigarette smoke extracts. This exposure may be quite different than the in vivo exposure of AM to tobacco smoke during smoking. In vivo, the smoke particles are submitted to the respiratory system's various mechanisms of particle elimination (31) and the smoke is dispersed throughout lungs. The in vitro exposure, used in this study, could be much greater than the exposure in in vivo smoking; however, our results show that short acute smoking of 4 cigarettes is sufficient to cause a significant impairment in the AM metabolism of AA. This effect was not due to chronic smoking because in our experiments, 4 subjects were nonsmokers. Also, the occasional and light smokers' cells showed none, or only few, of the morphologic alterations seen in cells recovered from chronic smokers. Moreover, the light and occasional smokers' and nonsmokers' cells produced similar amounts of LTB. and 5-HETE. The effect of acute smoking oil lipoxygenase product synthesis is further supported by the fact that sham smoking did not inhibit the production of LTB. by AM. It has been also shown that acute smoking provokes other alterations of AM functions; these include an increase in the superoxide anion production by unstimulated AM of nonsmokers and an increased production of O2 radicals by unstimulated and stimulated AM of smokers (32). However, even though short acute smoking inhibits the LTB. production by AM, the duration of its action and its long-term effects on the lung homeostasis remain unknown. One purpose of this study was to evaluate a possible LTB. production by AM in acute exposure to tobacco smoke. According to Hunninghake and Crystal (13), smokers' and nonsmokers' AM exposed to tobacco smoke release a chemotactic factor for neutrophils. Our results show that, in similar experimental conditions as those of Hunninghake and Crystal, no LTB. was produced spontaneously by smokers' AM
or by nonsmokers' AM exposed to tobacco smoke (Figure 2). Previous studies have shown that smokers' AM do not spontaneously release LTB. (24, 'lJ). This study also demonstrates that, in another experimental condition, acute in vivo smoking does not cause a spontaneous release of LTB. (Figure 4). Those previous experiments did not specifically evaluate the mechanism of the inhibition of AM AA metabolism by tobacco smoke. With smokers' cells, the addition of AA to the incubations did not correct the LTB. production decrease. This suggests that the defect in the AA metabolism is not at the level of the substrate release but rather at the level of lipoxygenase enzymatic pathways. However, because we did not measure the total release of AA and its metabolites, we can not eliminate the possibility of a defect at the phospholipase level. In conclusion, this study confirms the previously reported findings of the LTB. synthesis inhibition in smokers' and nonsmokers' AM exposed to tobacco smoke. It also shows that acute in vitro and in vivo exposures to cigarette smoke inhibit the LTB. production. Finally, our findings suggest that the chemotactic factor released by. AM in the presence of the tobacco smoke is not LTB•. Acknowledgments: The writers are grateful to Aline Huard and Lise Petitclerc for fibroscopy assistance and to Francine Gauvin and Fran90ise Maher for typing the manuscript. This study was supported by a grant to M. Laviolette from the Medical Research Council of Canada. This is part of the work submitted for M.Sc. thesis by J. Tardif (Laval University, Quebec, Canada). J. Tardif is supported by the "Fonds de la recherche en sante du Quebec."
References 1. Fels, A. O. S., N. A. Pawlowski, E. B. Cramer, T. K. C. King, Z. A. Cohn, and W. A. Scott. 1982. Human alveolar macrophages produce leukotriene B•. Proc. Natl. Acad. Sci. USA 79:7866-7870. 2. Martin, T. p.., L. C. Altman, R. K. Albert, and W. R. Henderson. 1984. Leukotriene B. production by the human alveolar macrophage: a potential mechanism for amplifying inflammation in the lung. Am. Rev. Respir. Dis. 129: 106-111. 3. Godard, P., M. Damon, F. B. Michel, E. J. Corey, and K. F. Austen. 1983. Leukotriene B. production from human alveolar macrophages (ab7 stract). Clin. Res. 31:548. 4. Laviolette, M., R. Coulombe, E. Braquet, S. Picard, aIidP. Borgeat. 1987. Lipoxygenase-mediated metabolism of arachidonic acid in human alveolar macrophages: a study with cell suspensions and adherent cells. Collogue Inserm: Institut National de la Sante et de la Recherche Medicale 152: 109-118. 5. Sirois, P., S. Roy, P. Borgeat, S. Picard, and E. J. Corey. 1981. Structural requirement for the action of leukotriene B. on the guinea-pig lung: importance of double bond geometry in the 6, 8, IO-triene unit. Biochem. Biophys. Res. Commun. 99:385-390. 6. Sirois, P., and P. Borgeat. 1982. Mediators of immediate hypersensitivity. In Immunopharmacology. P. Sirois, M. Rola-Pleszczynski, editors. Elsevier, North Holland. 201-222. 7. Rola-Pleszczynski, M., P. Borgeat, and P. Sirois. 1982. Leokotriene B. induces human suppressor lymphocytes. Biochem. Biophys. Res. Commun. 108:1531-1537. 8. Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, and M. J. H. Smith. 1980. Leukotriene B., a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264-265. 9. Bray, M. A., A. W. Ford-Hutchinson, and M. J. H. Smith. 1981. Leukotriene B.: an inflammatory mediator in vivo. Prostaglandins 22:213223. 10. Palmblad, J.,C. L. Malmsten, A. M. Uden, O. Radmark, L. Engstedt, and B. Samuelsson. 1981. Leukotriene B. is a potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 58:658-661. 11. Hunninghake, G. W., J. Gadek, and R. G. Crystal. 1980. Mechanism by which cigarette smoke attracts polymorphonuclear leukocytes to the lung. Chest 77(2 Suppl.):273. 12. Cohen A. B., and M. Rossi. 1983. Neutrophils in normal lungs. Am. Rev. Respir. Dis. 127:S3-S9.
Tardif, Borgeat, and Laviolette: Acute Smoking and Leukotriene Synthesis by Alveolar Macrophages
13. Hunninghake, G. W., and R. G. Crystal. 1983. Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers. Am. Rev. Respir. Dis. 128:833-838. 14. Tharwat, F., R. T. Abboud, A. Richter, and S. S. Johal. 1986. Acute effect of smoking on elastaselike esterase activity and immunologic neutrophil elastase levels in bronchoalveolar lavage fluid. Am. Rev. Respir. Dis. 133:568-573. 15. Blue, M. L. and A. Janoff. 1978. Possible mechanisms of emphysema in cigarette smokers: release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensate in vitro. Am. Rev. Respir. Dis. 117:317-325. 16. Niewoehner, D. E., J. Kleinerman, and D. B. Rice. 1974. Pathologic changes in the peripheral airways of young cigarette smokers. N. Eng/. J. Med. 291:755-758. 17. Pratt, S. A., M. H. Smith, A. J. Ladman, and T. N. Finley. 1971. The ultrastructure of alveolar macrophages from human cigarette smokers and non-smokers. Lab. Invest. 24:331-338. 18. Harris, J. 0., G. N. Olsen, J. R. Castle, and A. S. Maloney. 1975. Comparison of proteolytic enzyme activity in pulmonary alveolar macrophages and blood leukocytes in smokers and non-smokers. Am. Rev. Respir. Dis. 111 :579-586. 19. Brody, A. B., andJ. E. Craighead. 1975. Cytoplasmic inclusions in pulmonary macrophages of cigarette smokers. Lab. Invest. 32: 125-132. 20. Davis, G. S., A. R. Brody, and K. B. Adler. 1979. Functional and physiologic correlates of human alveolar macrophage cell shape and surface morphology. Chest 75(2 Suppl.):280-282. 21. Hoidal, J. R., and D. E. Niewoehner. 1982. Lung phagocyte recruitment and metabolic alterations induced by cigarette smoke in humans and in hamsters. Am. Rev. Respir. Dis. 126:548-552. 22. Laviolette, M., J. Chang, and D. S. Newcombe. 1981. Human alveolar macrophages: a lesion in arachidonic acid metabolism in cigarette smokers. Am. Rev. Respir. Dis. 124:397-401.
161
23. Wolter, N. J., S. L. Kunkel, J. P. Lynch III, and P. A. Ward. 1983. Production of cyclooxygenase products by alveolar macrophages in pulmonary sarcoidosis. Chest 83(5 Suppl.):79-81. 24. Laviolette, M., R. Coulombe, S. Picard, P. Braquet, and P. Borgeat. 1986. Decreased leukotriene B, synthesis in smoker's alveolar macrophages in vitro. J. Clin. Invest. 77:54-60. 25. Wieslander, E., M. Linden, L. Hakansson et a/. 1987. Human alveolar macrophages from smokers have an impaired capacity to secrete LTB, but not other chemotactic factors. Eur. J. Respir. Dis. 71:263-272. 26. Linden, M., E. Wieslander, A. Eklund, K. Larsson, and R. Brattsand. 1988. Effects of oral N-acetylcysteine on cell content and macrophage function in bronchoalveolar lavage from healthy smokers. Eur. Respir. J. 1:645-650. 27. Rios-Mollineda, A., R. M. Santiago, S. K. Endicott, W. F. Stenson, and E. J. Campbell. 1986. Transient suppression of human alveolar macrophage secretion of leukotriene B, by in vivo cigarette smoke exposure (abstract). Am Rev. Respir. Dis. 133(Suppl.):AI40. 28. Laviolette, M. 1985. Lymphocyte fluctuation in bronchoalveolar lavage fluid in normal volunteers. Thorax 40:651-656. 29. Norgaard-Pedersen, B., O. Siggaard-Andersen, and J. Rem. 1972. Spectrophotometric determination of oxy-, carboxy-, met-, and sulph-hemoglobin in capillary blood. Clin. Chim. Acta 42:85-100. 30. Borgeat, P., and S. Picard. 1988. 19-Hydroxyprostaglandin B2 as an internal standard for on-line extraction-high-perforrnance liquid chromatography analysis of lipoxygenase products. Anal. Biochem. 171 :283289. 31. Brain, J. D., D. F. Proctor, and L. M. Reid. 1977. Respiratory Defense Mechanisms. Parts I and II. Marcel Dekker, New York. 32. Richter, A. M., R. T. Abboud, S. S. Johal, and T. A. Fera. 1986. Acute effect of smoking on superoxide production by pulmonary alveolar macrophages. LUng 164:233-242.
The number of neutrophils in the lungs of cigarette smokers is increased. This could be a consequence of the chemotactic mediator synthesis by alveolar macrophages (AM). In order to evaluate the possible role of leukotriene B4 (LTB4) in this condition, we studied the formation of LTB4 by nonsmokers' AM exposed in vitro and in vivo to cigarette smoke and by smokers' AM. In the absence of stimulus or upon stimulation, nonsmokers' AM exposed in vitro to tobacco smoke formed less LTB4 than did nonexposed AM, e.g., the cells incubated with arachidonic acid and ionophore produced, respectively, 241 ± 132 and 425 ± 106 pmol LTB4 !lQ6 cells (mean ± SEM) (P < 0.01). In other experiments, smokers' AM were incubated in absence of stimulus and produced less LTB4 than did nonsmokers' AM; during a 3-h incubation, smokers' and nonsmokers' adherent AM released, respectively, 3 ± 2 and 40 ± 28 pmol LTB4/1Q6 cells (P < 0.05). Similarly stimulated smokers' AM produced less LTB4 than did nonsmokers' AM, e.g., the cells incubated with arachidonic acid and ionophore formed, respectively, 225 ± 41 and 573 ± 150 pmol LTBJIQ6 cells (P < 0.05). In a group including mild smokers and nonsmokers, in vivo exposure to the smoke of 4 cigarettes produced a decrease in the release of LTB4 by AM, e.g., in the presence of arachidonic acid and ionophore, nonexposed and exposed AM produced, respectively, 198 ± 38 and 143 ± 38 pmol LTB./IQ6 cells (P < 0.05). In all those conditions, AM exposed to cigarette smoke exhibited an inhibition of 5-hydroxy-eicosatetraenoic acid. These results show that acute in vitro or in vivo exposures to tobacco smoke and chronic smoking inhibit the spontaneous and stimulated AM production of arachidonic acid metabolites. Consequently, it is very unlikely that LTB4 could account for the neutrophil recruitment into the lungs of smokers.
Human alveolar macrophages (AM) can produce important amounts of leukotriene (LT) B4 , a biologically active metabolite of arachidonic acid (AA) (1-4). Stimuli such as phagocytosis or calcium ionophore induce the synthesis of this mediator by AM. LTB4 acts on lung smooth muscles, vascular permeability, and lymphocyte functions (5-7) but most importantly is a potent neutrophil chemotactic factor. It is as effective as the Sa complement fragment in this function (8-10). The neutrophils carry a large amount of hydrolytic enzymes capable of destroying the lung structure (11-14). An increased number of neutrophils is found in smokers' bronchoalveolar lavage (BAL). It has been postulated that these Key IJbrds: leukotrienes, alveolar macrophages, smoking, arachidonic acid (Received in original form June 30, 1989 and in revised form September 25, 1989) Address correspondence to: Michel Laviolette, M.D., Centre de Pneumologie, Hopital Laval, Tl25, Chemin Ste-Foy, Ste-Foy, Quebec GlY 4G5, Canada. Abbreviations: alveolar macrophages, AM; bronchoalveolar lavage, BAL; carboxyhemoglobin, COHb; 5S-hydroxy-6,8,II,14-(E,Z,Z,Z)-eicosatetraenoic acid, 5-HETE; leukotriene B4 , LTB4 • Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 155-161, 1990
cells might be at the origin of chronic inflammatory process leading to the development of emphysema (11-16). Smoking provokes many morphologic and functional alterations in AM (17-20), and tobacco smoke induces AM to release many mediators, including chemotactic factors (13, 21). Consequently, AM could be involved in the pathogenesis of emphysema via a chemotactic factor release and neutrophil recruitment to the lungs. Hunninghake and Crystal (13) previously demonstrated that nonsmokers' AM acutely exposed in vitro to tobacco products release a chemotactic factor for neutrophils. They also found that smokers' AM spontaneously release this chemotactic factor. Because this mediator had a small molecular weight and was lipidic in nature, they postulated that it could be LTB4 • However, chronic smoking induces a significant inhibition of AM LTB4 and prostaglandin synthesis (22-26). Moreover, acute in vivo exposure of smokers' AM to tobacco smoke does not induce LTB4 production (27). These results suggest that the mediator described by Hunninghake and Crystal may not be LTB4 • The purpose of this study was to evaluate the release of LTB4 by nonsmokers' AM acutely exposed in vitro or in vivo to tobacco smoke and also by chronic smokers' AM. We found that AM acutely or
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chronically exposed to tobacco smoke released less LTB4 than did the nonexposed cells.
Materials and Methods Subjects The study involved 58 healthy volunteers, 31 males and T1 females, with a mean ageof27.6yr (range, 19t042yr), who all gave informed consent: 34 were current nonsmokers, 33 never smoked, and one had stopped smoking 6 yr previously. Twelve were current heavy smokers (>20 cigarettes/d for more than 2 yr), and twelve were occasional or light smokers, of whom eight smoked 1 or 2 cigarettes per month and four smoked less than 4 cigarettes/d. None of the subjects had taken any medication for 1 mo prior to the study nor had a common cold or pulmonary symptoms, and none had a history suggestive of lung disease. BAL and Cell Processing All lavages were performed by fiberoptic bronchoscopy as previously described (28) using 240 or 300 ml of warm (J"l0 C) saline (0.9% NaCl, wt/vol) in 60-ml aliquots. The cells were separated from the lavage fluid by centrifugation (250 g, 10 min, 4°C) and resuspended in tissue culture medium RPMI 1640 (GIBCO, Grand Island, NY) (pH 7.4) or in Dulbecco's PBS (without Ca2+ and Mg2+). A portion of those cell suspensions was used for the determination of total cell number (hemocytometer), differential counts (Wright-Giemsa or Diff-Quik stain), and viability (trypan blue exclusion). Cell viability was always greater than 90%.
In Vitro Exposure to Tobacco Smoke The lavage cells obtained from 18 nonsmokers were resuspended in culture medium RPMI 1640 containing MgC12 and CaC12 (final concentration, 0.5 mM and 2 mM, respectively) to a concentration of 1.0 X 1()6 cells/0.8 ml and placed for adherence in 35-mm culture dishes (Falcon Plastics, Los Angeles, CA). The incubation volume was completed by adding 0.2 ml of medium containing the smoke extracts, or 0.2 ml of medium bubbled with air. Those media were prepared according to the method of Hunninghake and Crystal (13). Briefly, the smoke from 1 cigarette (Winston®, unfiltered; 1. R. Reynolds Tobacco Co.) or the equivalent amount of air was bubbled through 24 ml of RPMI 1640, providing, respectively, the smoke medium and the air medium used in those experiments. The cells were incubated with or without stimulus. The adherent AM were incubated with air medium or smoke medium at J"l0 C under an atmosphere of 5 % CO2 and 95 % air. After 1 h, the cellular monolayers were washed 3 times with warm (J"l0 C) HBSS, then 1 ml of fresh medium (RPMI 1640) was added to the dishes and adherent AM were incubated for an additional 3 h. The incubations were stopped by adding 0.5 ml methanol/acetonitrile. This solution also contained 50 ng of prostaglandin (PG) B2 per ml as an internal standard. The denatured media were harvested and analyzed to seek the presence of 5-lipoxygenase products by reverse-phase HPLC. The cellular viability was estimated in control culture dishes using trypan blue dye exclusion. We found no difference between the viability of AM exposed to
air medium compared to those exposed to smoke medium (97 ± 0.2% and 96 ± 0.6%, respectively; P = 0.21). In other experiments, adherent cells were washed as described above, preincubated for 10 min with fresh HBSS, and then stimulated for 10 min with 30 J,tM AA and/or 2 J,tM calcium ionophore A23187. For the control experiments, AM were incubated with 0.2 % of ethanol (final concentration). At the end of the incubations, the reactions were stopped as described above and the denatured media were kept for HPLC analysis. Comparison between Heavy Smokers' and Nonsmokers' Cells In 12 experiments, lavage cells were obtained from 1 heavy smoker and 1 nonsmoker on the same morning and were processed in parallel. The cell pellets were resuspended in RPMI 1640. One-milliliter aliquots of cellular suspension (1.5 X 1()6 cells/ml) were added to 35-mm culture dishes. In some experiments, the cells were incubated for 3 h in culture medium RPMI 1640 at J"l0 C, according to the method described by Hunninghake and Crystal (13). This was done to evaluate whether smokers' AM could spontaneously release LTB4 • The culture fluids were harvested for HPLC analysis. At the end of the incubation, there was no significant difference in cellular viability between the nonsmokers' and smokers' cells (93 ± 1% and 95 ± 1%, respectively; P = 0.11). In other experiments, the cells were incubated for 1 h at J"l0 C on culture dishes in RPMI 1640 with 10% FBS. The cell monolayers were then washed 3 times with warm HBSS to remove nonadherent cells. One milliliter of fresh HBSS with MgCl 2 and CaC12 (final concentration, 0.5 mM and 2 mM, respectively) was added to the cultures, and the cells were preincubated for 10 min. The adherent cells were then stimulated with 2 J,tM ionophore A23187 and/or 30 J,tM AA for 10 min. In the control experiments, AM were incubated with ethanol (0.2%). The reactions were stopped, and the denatured media kept for HPLC analysis.
In Vivo Exposure to Tobacco Smoke For these experiments, a first BAL (four 60-ml aliquots) was performed in the lingula. The subjects then smoked 4 cigarettes within 1 h, and a second BAL (also using 240 ml) was done this time in the right middle lobe. Eight light or occasional smokers and four nonsmokers volunteered for this experiment. The smokers had not smoked for more than 1 wk. Briefly, the subjects smoked 1 cigarette (Export 'W'®, unfiltered; MacDonald Inc.) every 15 min, taking 1 puff with a deep inhalation every 30 s. Approximately 10 min were required to smoke 1 cigarette, leaving a rest interval of about 5 min between each cigarette. Blood samples for carboxyhemoglobin (COHb) measurement were withdrawn before and after smoking (29). The cellular viability was similar before and after smoking (91 ± 2 % and 85 ± 2 %, respectively; P = 0.12). The cells from these 2 consecutive lavages were compared to evaluate the effect of an acute in vivo tobacco smoke exposure on AM LTB4 production. A I-ml aliquot of cell suspension (1.0 X 1()6 cells/ml PBS) was added to polystyrene tubes and preincubated in motion at J"l0 C for 10 min.
Tardif, Borgeat, and Laviolette: Acute Smoking and Leukotriene Synthesis by Alveolar Macrophages
The AM were then stimulated for 10 min with 30 /-tM exogenous AA and/or 2/-tM calcium ionophore A23187. The controls contained the same final concentrations of CaCl h MgClz, and ethanol as described above. The reactions were stopped, and the denatured cell suspensions kept for HPLC analysis. Four light or occasional smokers and three nonsmokers, who participated to the first part of this in vivo experiment, also volunteered to form a control group. These subjects were submitted to the same procedures except that they had to inhale air through a straw (sham smoking) rather than smoke a cigarette. No blood samples were withdrawn in this case. The lavage cells were processed according to the same protocol. Reverse-phase HPLC Analysis of Lipoxygenase Products 5-Lipoxygenase products were measured by HPLC as previously described (30). The metabolites were identified by their co-migration with authentic standards and the specificity of UV absorption. Their quantitation was done by measuring the peak height and comparing with calibrated standards of lipoxygenase products, after correction for recoveries using the internal standard PGB2 • Supernatants were centrifuged to eliminate precipitated material and injected into a cartridge (Radial Pak Cs, 100 X 5 mm, 10/-tm particle size; Waters Millipore Corp., Milford, MA). All the different metabolites were eluted at a solvent flow of 1.5 ml/min using a 3-s01vent mixture system, following a predetermined program. LTB4 and 5S-hydroxy-6,8,1l,14(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE) were measured with a detection limit of "-'5 pmol. Statistical Analysis Data were expressed as the mean ± SEM. The comparisons between the BAL results of the 3 groups of subjects were done using a Kruskal-Wallis test (multiple comparison) for nonparametric values. The productions of lipoxygenase products were compared using either a Wilcoxon-Mann-Whitney two-sample test for unpaired nonparametric values or a Wilcoxon's signed rank test for paired nonparametric values. A value of P < 0.05 was considered significant.
Results Bronchoalveolar Lavage Lavage parameters are presented in Table 1. The lavage return was similar in the 3 groups. Heavy smokers had a
157
greater total number of cells, more macrophages, but less lymphocytes than did nonsmokers (P < 0.05). However, there was no significant difference in the percentage and in the absolute number (data not shown) ofneutrophils between heavy smokers and nonsmokers. The number of cells recovered from occasional and light smokers was lower than that from heavy smokers (135.2 ± 22 X 103 versus 205.3 ± 21.6 X 103 cells/ml [P < 0.05]) but similar to that obtained from nonsmokers (93 ± 13.6 X 103 cells/ml). The occasional and light smokers' BAL had similar differential cell counts as nonsmokers' BAL but contained fewer macrophages and more lymphocytes than heavy smokers' BAL (P < 0.05). However, in light smokers' BAL, about 5% of the AM had some morphologic characteristics of heavy smokers' AM (17-20). These AM had lipidic inclusions and were somewhat bigger than nonsmokers' AM. Occasional smokers' AM were similar to those of nonsmokers. In the experiments on in vivo exposure to cigarette smoke, the subjects had 2 BAL. The mean recovered volumes of lavage fluid before and after smoking 4 cigarettes were similar: 59.6 ± 2.3% versus 61.1 ± 1.4%, respectively (P > 0.05). The total number of recovered cells and the cellular differentials were also similar: total cell numbers, 147.9 ± 23.9 X 103 and 141.6 ± 28.3 X 103 cells/ml; macrophages, 83.1 ± 4.8% and 81.1 ± 4.9%; lymphocytes, 14.7 ± 4.8% and 17.6 ± 5%; and neutrophils, 1.8 ± 0.5% and 1 ± 0.3%. Acute In Vitro Exposure to Tobacco Smoke Unstimulated AM exposed in vitro to smoke medium and incubated for 3 h did not synthesize detectable amounts of LTB4 nor 5-HETE, whereas AM exposed to air medium produced small amounts of these products: LTB4 , 1 ± 1 pmol/106 cells; 5-HETE, 5 ± 5 pmol/106 cells. Upon stimulation, AM exposed to smoke medium produced less LTB4 and 5-HETE than did AM exposed to air medium (Figure 1). Incubated with ionophore A23187, AM exposed to tobacco smoke produced about 6 times less LTB4 and 8 times less 5-HETE than did those exposed to the air medium: respectively, LTB4 , 38 ± 16 and 254 ± 85 pmol/106 cells; 5-HETE,29 ± 15 and 239 ± 80 pmol/106 cells (P < 0.01). A similar decrease in LTB4 and 5-HETE production by AM exposed to smoke medium was observed in cultures stimulated with both AA (30 /-tM) and calcium ionophore A23187: respectively LTB4 , 241 ± 132 and 425 ± 106 pmol/lQ6 cells in smoke medium and air medium; 5-HETE, 156 ± 91 and 429 ± 100 pmol/1Q6 cells (P < 0.01). In the control incubations, the smoke medium-incubated cells synthesized less 5-HETE: 8 ± 4 and 48 ± 14 pmol/1Q6 cells,
TABLE 1
Subjects' characteristics and lavage data* BAL Return
n
Nonsmokers 34 Occasional and light smokers 12 Smokers 12
* Values
are ll\ean
Sex (M/F)
Age (yr)
(% of infused
Cell Yield
Macrophages
Lymphocytes
Neutrophils
Eosinophils
volume)
(X llYlml)
(%)
(%)
(%)
(%)
16/18
25.6
± 0.9
65.7
±
1.3
93.0
7/5 8/4
30.7 29.3
± 2.1 ± 1.6
60.6 63.3
± ±
1.4 1.5
135.2 205.3
± SEM.
t p < 0.05 compared to smokers.
±
13.6t
84.0
± 2.2t
14.4
± 2.2t
1.2
± 0.2
0.3
± 0.1
± 22.0t ± 21.6
81.5 94.6
± 5.0t ± 1.0
17.0 3.7
± 5.4t ± 0.9
1.2 1.1
± 0.3 ± 0.2
0.4 0.6
± 0.2 ± 0.2
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
600
U'l .... .... IO.l
t
o
AIR-MEDIUM
~
SMOKE-MED IUM
Figure 1. Effects of acute in vitro exposure to tobacco smoke on lipxygenase product synthesis by nonsmokers' alveolar macrophages (AM). Adherent bronchoalveolar lavage (BAL) cells were incubated in the presence of medium bubbled with tobacco smoke or control medium bubbled with air and were stimulated 10 min with 2 JLM ionophore A23187 (IONO) alone (n = 13) or with 30 JLM arachidonic acid and ionophore A23l87 (AA + IONO) (n = 12). *P < 0.05, tp < 0.01; Wilcoxon's signed rank test.
400
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ID
t
t
O
.... .... o
I:
200
Cl.
o 1""--" C
I
WPM
10NO
I
AA+IONO
IONO
C
5-HETE
LTB4
respectively (P = 0.01). The decrease in LTB4 production, however, was not significant: 7 ± 2 and 13 ± 5 pmol/106 cells. Comparison between Heavy Smokers' and Nonsmokers' Cells Unstimulated adherent AM from smokers failed to produce a greater amount of LTB4 than did cells from nonsmokers. In fact, their production of LTB4 was significantly less than that of the nonsmokers' AM: 3 ± 2 and 40 ± 28 pmol/lQ6 cells, respectively (P < 0.05) (Figure 2). The production of 5-HETE by cells of smokers was also lower: 4 ± 4 and 69 ± 32 pmol/lQ6 cells, respectively; this difference did not reach statistical significance (P = 0.06). Adherent smokers' AM, incubated with exogenous AA, produced, respectively, about 8 and 3 times less LTB4 and 5-HETE than did AM of nonsmokers; LTB4 , 9 ± 3 and 69 ± 23 pmol/l06 cells (P < 0.01); 5-HETE, 68 ± 20 and 230 ± 59 pmol/lQ6 cells (P = 0.01) (Figure 3). The smokers' AM incubated with calcium ionophore A23187 produced approximately 2.8 times less metabolites than did nonsmokers' AM: respectively, LTB4 , 191 ± 36 and 531 ± 140 pmol/ 106 cells (P = 0.02); 5-HETE, 202 ± 41 and 745 ± 204 pmol/lQ6 cells (P < 0.01). When smokers' AM were stimulated with both exogenous AA and ionophore A23187, the production of both metabolites was 2.5 times less than that of the nonsmokers' cells: respectively, LTB4 , 225 ± 41 and 573 ± 150 pmol/106 cells (P = 0.03); 5-HETE, 544 ± 173 and 1,185 ± 288 pmol/lQ6 cells (P < 0.01). In the control incubations, LTB 4 synthesis by smokers' AM was significantly less than that of nonsmokers' AM: undetectable «5 pmol) and 26 ± 22 pmol/lQ6 cells in smokers and nonsmokers, respectively (P = 0.01). Acute In Vivo Exposure to Tobacco Smoke After smoking 4 cigarettes, the COHb concentration increased from 0.5 ± 0.2 to 2.7 ± 0.8 mol/mol (COHb/Hb) (P < 0.01). In the group undergoing sham smoking, there was no observable difference in LTB4 or 5-HETE production between the first and second lavage, both under control and stimulated conditions (P > 0.33) (Figure 4, upper panel). Af-
AA+IONO
ter an acute in vivo exposure to the smoke of 4 cigarettes, AM released less lipoxygenase products (Figure 4, lower panel). Upon stimulation, AM not exposed to cigarette smoke produced more lipoxygenase products than did tobacco smoke-exposed cells: with AA, LTB4 , 15 ± 4 and 7 ± 2 pmol/106 cells (P < 0.01) and 5-HETE, 90 ± 25 and 48 ± 17 pmol/lQ6 cells (P = 0.01); with AA and ionophore, LTB4 , 198 ± 38 and 143 ± 38 pmol/lQ6 cells (P < 0.05) and 5-HETE, 756 ± 146 and 558 ± 133 pmol/106 cells (P < 0.05), respectively. Upon stimulation with ionophore A23187, the LTB4 and 5-HETE synthesis by tobacco smoke-exposed AM did not decrease significantly compared to the exposed cells (P = 0.18). No differences were found in the control incubations. In these experiments, LTB4 and
150
(f)
...."".... U
-........
o
NONSMOKERS
~
SMOKERS -r-
100
\Do
o
~ Cl.
*
-r-
50
o
~
LTB4
~
S-HETE
Figure 2. LTB 4 and 5-HETE production by unstimulated AM of nonsmokers and heavy smokers. Adherent lavage cells were incubated for 3 h in RPMI 1640 to evaluate the spontaneous production of LTB4 and 5-HETE (n = 6). *P < 0.05; Wilcoxon-MannWhitney two-sample test.
Tardif, Borgeat, and Laviolette: Acute Smoking and Leukotriene Synthesis by Alveolar Macrophages
159
Figure 3. Lipoxygenase product synthesis by smokers' and nonsmokers' AM incubated in adherence. Adherent cells were incubated for 10 min with 2 JLM ionophore A23187 (lONO) or/ and 30 JLM AA (n = 11). 'p < 0.05, tp < 0.01; Wilcoxon-MannWhitney two-sample test.
5-HETE productions by AM of occasional and light smokers (n = 8) were similar to those of nonsmokers (n = 4), e.g., in the presence of AA and ionophore, light smokers' cells produced 200 ± 50 pmol LTB.fl()6 cells and 598 ± 155 pmol 5-HETE/IQ6 cells, and nonsmokers' cells produced
Figure 4. Effect of inhaling air (sham smoking) or smoking 4 cigarettes on AM's AA metabolism. Upper panel: Production of LTB4 and 5-HETE after sham smoking (n = 7). Lower panel: Production of LTB 4 and 5-HETE after smoking 4 cigarettes (n = 12). Cell suspensions were incubated for 10 min with 2 JLM ionophore (IONO) orland 30 JLM AA. 'p < 0.05, tp < 0.01; Wilcoxon's signed rank test.
224 ± 95 pmol LTBJIQ6 cells (P = 0.32) and 936 ± 361 pmol 5-HETE/106 cells (P = 0.27). The difference in 5HETE production before smoking seen between the upper and lower panels of Figure 4 could be explained by the variability of 5-HETE production from one subject to another
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
and by the fact that data presented in the upper panel were obtained from seven out ofthe 12 subjects shown in the lower panel.
Discussion The present study demonstrates that acute in vitro (Figure 1) and in vivo (Figure 4) exposures to tobacco smoke inhibit the release of lipoxygenase products by AM obtained from nonsmokers or light and occasional smokers. Moreover, neither acute in vivo and in vitro exposures to tobacco smoke nor chronic smoking induce spontaneous production of LTB. (Figures 2 and 4). As previously reported (22-26), chronic cigarette smoking reduces the metabolism of AA by smokers' AM, leading to a decrease in the production ofleukotrienes (Figure 3). Rios-Mollineda and colleagues ('lJ) suggested that an acute in vivo exposure to cigarette smoke inhibits the production of LTB. by smokers' AM. Because they studied smokers, one cannot differentiate the effect of chronic smoking from that of acute smoke exposure from their study. The acute in vitro exposure to tobacco smoke used in this study induced a significant inhibition of the smokers' AM 5lipoxygen;l~e activity. A smaller but similar inhibition was obtained by smoking4 cigarettes. The difference in the magnitude of the effects produced by cigarette smoke between in vivo and in vitro exposures could be explained by the difference in the exposure levels. In vitro, adherent cells were incubated in the presence of cigarette smoke extracts. This exposure may be quite different than the in vivo exposure of AM to tobacco smoke during smoking. In vivo, the smoke particles are submitted to the respiratory system's various mechanisms of particle elimination (31) and the smoke is dispersed throughout lungs. The in vitro exposure, used in this study, could be much greater than the exposure in in vivo smoking; however, our results show that short acute smoking of 4 cigarettes is sufficient to cause a significant impairment in the AM metabolism of AA. This effect was not due to chronic smoking because in our experiments, 4 subjects were nonsmokers. Also, the occasional and light smokers' cells showed none, or only few, of the morphologic alterations seen in cells recovered from chronic smokers. Moreover, the light and occasional smokers' and nonsmokers' cells produced similar amounts of LTB. and 5-HETE. The effect of acute smoking oil lipoxygenase product synthesis is further supported by the fact that sham smoking did not inhibit the production of LTB. by AM. It has been also shown that acute smoking provokes other alterations of AM functions; these include an increase in the superoxide anion production by unstimulated AM of nonsmokers and an increased production of O2 radicals by unstimulated and stimulated AM of smokers (32). However, even though short acute smoking inhibits the LTB. production by AM, the duration of its action and its long-term effects on the lung homeostasis remain unknown. One purpose of this study was to evaluate a possible LTB. production by AM in acute exposure to tobacco smoke. According to Hunninghake and Crystal (13), smokers' and nonsmokers' AM exposed to tobacco smoke release a chemotactic factor for neutrophils. Our results show that, in similar experimental conditions as those of Hunninghake and Crystal, no LTB. was produced spontaneously by smokers' AM
or by nonsmokers' AM exposed to tobacco smoke (Figure 2). Previous studies have shown that smokers' AM do not spontaneously release LTB. (24, 'lJ). This study also demonstrates that, in another experimental condition, acute in vivo smoking does not cause a spontaneous release of LTB. (Figure 4). Those previous experiments did not specifically evaluate the mechanism of the inhibition of AM AA metabolism by tobacco smoke. With smokers' cells, the addition of AA to the incubations did not correct the LTB. production decrease. This suggests that the defect in the AA metabolism is not at the level of the substrate release but rather at the level of lipoxygenase enzymatic pathways. However, because we did not measure the total release of AA and its metabolites, we can not eliminate the possibility of a defect at the phospholipase level. In conclusion, this study confirms the previously reported findings of the LTB. synthesis inhibition in smokers' and nonsmokers' AM exposed to tobacco smoke. It also shows that acute in vitro and in vivo exposures to cigarette smoke inhibit the LTB. production. Finally, our findings suggest that the chemotactic factor released by. AM in the presence of the tobacco smoke is not LTB•. Acknowledgments: The writers are grateful to Aline Huard and Lise Petitclerc for fibroscopy assistance and to Francine Gauvin and Fran90ise Maher for typing the manuscript. This study was supported by a grant to M. Laviolette from the Medical Research Council of Canada. This is part of the work submitted for M.Sc. thesis by J. Tardif (Laval University, Quebec, Canada). J. Tardif is supported by the "Fonds de la recherche en sante du Quebec."
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