Exp. Pathol. 1990; 39: 37-43 VEB Gustav Fischer Verlag lena
Department of Pneumonology and Allergology, Medical Academy, L6di, Poland
Nicotine increases human polymorphonuclear leukocytes chemotactic response a possible additional mechanism of lung injury in cigarette smokers By D. NOWAK, U. RUTA and G.
PIAS~CKA
With 4 figures
Address for correspondence: DARIUSZ NOWAK, M. D., Department of Pneumonology and Allergology, Medical Academy, Kopcinskiego st. 22,90-153 L6di, Poland Key words: nicotine; leukocytes; granulocytes ; chemotoxin; smoking, cigarette ; cigarette smoking; polymorphonuclear leukocytes; lung
Summary Human polymorphonuclear leukocytes (PMNL) which are a potential source of proteolytic enzymes and reactive oxidant species contribute to the development of pulmonary emphysema in cigarette smokers. We found that nicotine at concentrations that occur in smokers' plasma enhances human PMNL chemotactic response to zymosan-activated serum (ZAS) and n-formylmethionyl-leucyl-phenylalanine (FMLP). Maximal increase in chemotactic migration was at nicotine concentration 1 f.lmolll. Higher concentrations, above 0.1 mmol!l inhibited PMNL chemotactic response and spontaneous migration. Nicotine also enhanced PMNL influx to the place of inflammation developed in the mouse pleural cavity after injection of ZAS . The number of PMNL found in the pleural cavity was I. 9-fold higher (p < 0.00 1, n = 5) when animals were pretreated with 0.15 mg of nicotine . However, this drug itself (concentrations of 0.1 f.lmolll to 10 mmolll) had weak chemotactic activity for PMNL. It seems that the stimulatory action of nicotine on PMNL chemotaxis may be partly responsible for increased PMNL numbers in the lower airways of cigarette smokers and following formation of the elastase!antielastase imbalance in lung tissue.
Introduction The central assumption of the proteolytic theory of premature pulmonary emphysema is the increased elastin degradation resulting from the elastase/antielastase imbalance in the lower airways . Cigarette smoking is the major exogenic factor in the epidemiology of pulmonary emphysema. One of several links between cigarette smoking and the development of emphysema is the recruitment of mononuclear phagocytes and polymorphonuclear leukocytes (PMNL) to the lower airways (8, 17). This process may be an important first step in the formation of elastase/ antielastase imbalance because these cells release proteases including elastases as well as increased amounts of reactive oxygen species (7, 13) which may inactivate 0.01) for FMLP, respectively. The higher nicotine concentrations, above 0.1 mmolll diminished cell migration to chemoattractants. The mean decrease in the chemotactic index was 1.04 ± 0.79 (not significant), 1.38 ± 0.59 (p < 0.01) at nicotine concentration 1 and 10 mmol/l, for assay with FMLP and 0.54 ± 0.16 (p < 0.01), 0.74 ± 0.33 (p < 0.01) for assay with ZAS, respectively. These concentrations also inhibited PMNL spontaneous migration under agarose, fig. 3. However, concentration of nicotine of 0.1 f,lmolll enhanced PMNL migration as expressed by the migration index. The PMNL viability after incubation with nicotine in RPMI-1640 medium for 90 min at 37°C was lower than in cells incubated without nicotine. At nicotine concentrations of 0, 0.1, 1, 10 f,lmolll and 0.1, 1, 10 mmol/l viability was 96 ± 3, 93 ± 3, 92 ± 3, 89 ± 6, 86 ± 6, 84 ± 5, 69 ± 10%, respectively; results were obtained from 5 separate experiments. Table 1 shows the total number of cells and PMNL and lymphocytes present in the mouse pleural cavity at 3 h after injection of various agents. Injection of PBS alone increased the total number of cells and PMNL recovered from the pleural cavity. ZAS caused influx of inflammatory cells, predominantly PMNL to the pleural cavity. The total number of cells, number of PMNL and lymphocytes was about 2-,2.6-, 1.7-fold higher than in animals injected with PBS, respectively. The previous intraperitoneal nicotine administration enhanced the total number of cells (l.4-fold) and the number of PMNL (l.9-fold) Exp. Pathol. 39 (1990) 1
39
Table 1.
The effect of nicotine on the total number of cells and number of PMNL and lymphocytes found in the pleural cavity after injection of ZAS. No.
Total cells/mouse (x 106)
Agent
a 1 2 3 4
PBS PBS Ni
b
+ PBS + ZAS + ZAS
0.44 0.56 1.1
1.5
PMNL (x 106)
± 0.05 ± 0.03 & ± 0.10 ± 0.15 #
0.11 0.19 0.50 0.96
± 0.02 ± 0.02 ! ± 0 .05 ± 0 .08 *
Lymphocytes (X
106)
0.32 0.36 0.60 0 .53
± 0.07 ± 0.06 ± 0.16 ± 0 . 12
PBS: phosphate buffered saline pH 7.4; ZAS: zymosan-activated serum; Ni : nicotine. Animals were injected intraperitoneally (a) with PBS or Ni and then after 2 h intrapleuraUy (b) with PBS or ZAS. Results (mean values and standard deviation) were obtained from 5 separate experiments. &: significantly different (p < 0.02) from no 1; !: significantly different (p < 0.01) from no 1; #: significantly different (p < 0.01) from no 3; *: significantly different (p < 0.001) from no 3. CHEt10TACTlC INDEX CHEt10TACTlC INDEX
~r----r-----r-----r---.
Sr---~----~----~----~
3
o
10:' 10-6 10-5 10-'1 10-3 10-2
NICOTINE CONCENTRATION [motif]
o~~~
o
__~~__~~__~~
10-7 10-6 10-5 10-* 10-3 10-2
NICOTINE CONCENTRATION [mot/{]
Fig.l. The effect of nicotine on PMNL chemotactic response to FMLP. PMNL were incubated with appropriate nicotine solutions in RPMI-1640 medium for 90 min at 37°C and then their chemotactic response to FMLP was measured under agarose. Each curve represents results of one experiment with PMNL obtained from separate donors .
Fig. 2. The effect of nicotine on PMNL chemotactic response to ZAS. PMNL were incubated with appropriate nicotine concentration in RPMI-I640 medium for 90 min at 37°C and then their chemotactic response to ZAS was measured under agarose. Each curve represents results of one experiment with PMNL obtained from separate donors .
but not that of lymphocytes found in the pleural cavity after injection of ZAS. Results of assays of nicotine chemotactic activity for PMNL are shown in fig . 4. Strong PMNL migration to nicotine was observed when it was used in concentrations of 1 and 10 mmoUI but only in 2 of 9 assays. 40
Exp. Patho!' 39 (1990) I
CHEMOTACTIC INDEl
MIGRATION INDEX
1\",
1,1t r - - - - - - - - - - - - - - ,
1,2
1.0
2.5
r1\0.92 !:o,1 Io.8!:0, 09
O.8f----
!isz;;
a76!o,06
1.8 1.6
0.61-----
1.'-
O'~f---------
1.2
0.21----------
1.0
10- 7 10- 6 10-$ 'O-~ 10-3 10-2
NICOTINE CONCENTRATION [moll/]
...
0
0
(>
..
"
10- 7 10-6 10-5 IO- It 10-3 10-2
NICOTINE CONCENTRATION [mollf)
Fig.3. The effect of nicotine on PMNL spontaneous migration under agarose. PMNL were incubated with appropriate nicotine concentration in RPMI 1640 medium for 90 min at 37°C. Values of migration index below 0.8-significant migration inhibition (22).
Fig. 4. The chemotactic activity of nicotine for PMNL. Nicotine was dissolved in RPMI 1640 medium to various final concentrations and chemotactic response of PMNL to 10 ",,1 of these solutions was measured under agarose technique. Results are obtained from 9 series of separate experiments with PMNL from 9 various donors. These cells were able to migrate to ZAS and FMLP, part of them were used in experiments showed in figs. 1 and 2. However, the weak nicotine chemotactic activity (values of chemotactic index below 1.5) was found for all concentrations of this alkaloid used. The number of the weak positive PMNL chemotactic responses to nicotine was dependent on its concentration and ranged from 3 to 6 of 9 assays.
Discussion Our results of the in vitro nicotine influence on PMNL chemotactic response are in agreement with opposed data presented in two previous works (2, 26). BRIDGES et ai. (2) found that nicotine concentrations of 6 to 10 mmolll inhibited PMNL chemotactic response to endotoxin-activated human serum. TOTTI et ai. (26) observed stimulation of PMNL migration to FMLP and C5fr by nicotine when it was used in concentrations of 0.31 to 30.8 ""molli. We found that the effect of nicotine on PMNL chemotactic response depends on its concentration: concentrations of 0.1 to 10 ""molll enhance and of 1 to 10 mmolll inhibit PMNL migration to chemoattractants. This inhibition is probably due to the cytotoxic action of high nicotine concentrations. However, CORBERAND et ai. (3) reported no influence of nicotine at concentrations of 1.85 to 185 ""molll on PMNL chemotaxis to caseine under agarose. It may be explained by differences between our experimental protocol and that of CORBERAND et ai. who incubated PMNL with nicotine only for 30 min. Our in vitro results are supported by in vivo nicotine enhancement of the PMNL influx to the place of inflammation developed in the pleural cavity after injection of ZAS. The nicotine dose of 0.15 mg per mouse was very high and represents the action of toxic nicotine concentrations on PMNL inflammatory influx (all animals had convulsions during the first 20 min after nicotine injection). In mice, nicotine after intravenous injection is rapidly distributed throughout the tissues and body fluids (23). Also nicotine is relatively rapidly excreted from the mouse body. Exp. Pathol. 39 (1990) 1
41
Approximately 50 % of the single nicotine dose was excreted with urine within 6 h after injection (14). In our experiments the drug administered intraperitoneally passed through the liver before it was able to enter the general circulation. And in addition the mouse liver can uptake and metabolize nicotine mostly to cotinine (6). Therefore, for these reasons we used such high single nicotine doses in this experiment. In contrast to data presented by TOTTI et al. (26) we failed to find significant chemotactic activity of nicotine for PMNL. They measured PMNL chemotaxis to nicotine using modified Boyden chambers and observed nicotine-dependent migration at its concentration of 0.5 [tmol/l to 0.5 mmol/l with the maximum at 30.8 [tmoVI. In our study nicotine as a low molecular weight compound could relatively rapidly diffuse around well in agarose layer and thus induce weak PMNL chemotactic migration. Probably, for this reason only high nicotine concentrations induced sometimes strong PMNL migration. Human PMNL have a noncholinergic nicotine receptor on their surface which can be occupied by this alkaloid at concentrations present in the blood of smokers (4). Also the average blood nicotine concentration in smokers (21) is approximate to its concentrations used in our experiments which enhanced PMNL chemotactic response to ZAS and FMLP. The direct action of nicotine via its receptor may be responsible for this enhancement. PMNL are a rich potential source of elastase (10) which is released from these cells after phagocytosis or their chemotactic stimulation (19, 25). Increased PMNL inflammatory recruitment to the lung and proteases release may lead to the lung injury resulting in emphysema. It is possible that enhancement of PMNL chemotactic response to chemoattractants created in the lung in the course of smoking by nicotine may contribute to the increase in number of PMNL in the lower airways of cigarette smokers and following lung injury. However, the chemotactic responsiveness of PMNL has been shown to be depressed in cigarette smokers as compared to nonsmokers (18). Some unsaturated aldehydes present in cigarette smoke such as acrolein, crotonaldehyde may be responsible for it (2). On the other hand, there are no differences in monocyte chemotaxis between smokers and nonsmokers (16), and human alveolar macrophages from smokers have shown an enhancement of chemotactic responsiveness (27).
Acknowledgements This work was supported partly by the Department of Lung Medicine, University of Lund, Sweden. We thank Prof. B. G. SIMONSSON for this support.
References \. BOYUM, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 1968; 97 (Suppl.): 77-89. 2. BRIDGES, R. B., KRAAL, J. H., HUANG, 1. J. T., CHANCELLOR, M. B.: Effects of cigarette smoke components on in vitro chemotaxis of human polymorphonuclear leukocytes. Infect. Immun. 1977; 16:
240-248. 3. CORBERAND, 1., LAHARRAGUE, P., NGUEN, F., DUTAU, G., FONTANILLES, A. M. , GLEIZES, B., GYRARD, E.: In vitro effect of tobacco smoke components on the functions of normal human polymorphonuclear leukocytes. Infect. Immun. 1980; 30: 649-655. 4. DAVIES, B. D., Hoss, W., LIN, J. P., LIONETTI, F.: Evidence for a noncholinergic nicotine receptor on human phagocytic leukocytes. Mol. Cell. Biochem. 1982; 44: 23-3\. 5. GADEK, J. E., HUNNINGHAKE, G. W., FELLS, G. A., ZIMMERMANN, R. 1., KEBYH, B. A., CRYSTAL, R. G.: Evaluation of the protease-antiprotease theory of human destructive lung disease. Bull. Eur. Physio-Pathol. Respir. 1980; 16 (Suppl.): 27-34. 6. HANSSON, E., SCHMITERLOW, C. G.: Metabolism of nicotine in various tissues. In: VON EULER, U. S. (ed.): Tobacco alkaloids and related compounds. Pergamon Press 1964; 87-99. 7. HOIDAL, J., Fox, R. B., LEMARBE, P. A., PERRI, R., REPINE, 1. E.: Altered oxidative metabolic response in vitro of alveolar macrophages from asymptomatic cigarette smokers. Am. Rev. Respir. Dis.
1981; 123: 85-89. 8. HUNNINGHAKE, G. W., CRYSTAL, R. G.: Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am. Rev. Respir. Dis. 1983; 128: 833-836.
42
Exp. Pathol. 39 (1990) 1
9. - GADEK, J. E., FALES, H. W., CRYSTAL, R. G.: Human alveolar macrophage-derived chemotactic factor for neutrophils. J. Clin. Invest. 1980; 66: 473-483. 10. JANOFF, A.: Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis. Am. Rev. Respir. Dis. 1985; 132: 417-433. 11. KEW, R. R., GHEBREHIWET, B., JANOFF, A.: Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement. 1. Clin. Invest. 1985; 75: 1000-1007. 12. - - - The role of complement in cigarette smoke-induced chemotactic activity of lung fluids. Am. Rev. Respir. Dis. 1986; 133: 478-481. 13. LUDWIG, P. W., HOIDAL, J. R.: Alterations in leukocyte oxidative metabolism in cigarette smokers. Am. Rev. Respir. Dis. 1982; 126: 977-980. 14. McKENNIS, H.: Disposition and fate of nicotine in animals. In: VON EULER, U. S. (ed.): Tobacco alkaloids and related compounds. Pergamon Press 1964; 53-74. 15. NELSON, R. D., QUIE, P. G., SIMMONS, R. L.: Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immuno!. 1975; 115: 1650-1656. 16. NIELSEN, H.: A quantitative and qualitative study of blood monocytes in smokers. Eur. J. Respir. Dis. 1985;
66: 327 - 332. 17. NIEWOEHNER, D. E., KLEINERMAN, 1. , RICE, D. P.: Pathologic changes in the peripheral airways of young cigarette smokers. N. Eng!. J. Med. 1974; 291: 755-758. 18. NOBLE, R. c., PENNY, B. B.: Comparison of leukocyte count and function in smoking and nonsmoking young men. Infect. Immun. 1975; 12: 550-555. 19. OHLSSON, K., OLSSON, J.: The extracellular release of granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Scand. J. Haemato!. 1977; 19: 145-152. 20. POSTLEWAITE, A. E., KANG, A. H.: Collagen and collagen peptide-induced chemotaxis of human blood monocytes. J. Exp. Med. 1976; 143: 1299-1307. 21. RUSSEL, M. A. H., JARVIS, M., IYER, R., FEYERABEND, c.: Relation of nicotine yield of cigarettes to blood nicotine concentrations in smokers. Br. Med. 1. 1980; 280: 972-976. 22. SAURAT,1. W., PONVERT, c., HERVE, M., SCHEINMAN, P., PAUPE, J.: Criteres d'interpretation du test de migration leucocytaire applique au depistage des sensibilisationis medicamenteuses. Rev. Franc. Allergo!.
1974; 14: 169-174. 23. SCHMITERLDW, C. G., HANSSON, E.: Tissue distribution ofC14-nicotine.ln: VON EULER, U. S. (ed.): Tobacco alkaloids and related compounds. Pergamon Press 1964; 75-86. 24. SENIOR, R. M., GRIFFIN, G. L., MECHAM, R. P.: Chemotactic activity of elastin-derived peptides. J. Clin. Invest. 1980; 66: 859-862. 25. SHOWELL, H. 1., FREER, R. T., ZIGMOND, S. H., SCHIFTMAN, E.: The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J. Exp. Med. 1976; 143: 1155-1169. 26. TOTTI, N., MCCUSKER, K. T., CAMPBELL, E. J., GRIFFIN, G. L., SENIOR, R. M.: Nicotine is chemotactic for neutrophils and enhances neutrophil responsiveness to chemotactic peptides. Science 1984; 223: 169-171. 27. WARR, G. A., MARTIN, R. R.: Chemotactic responsiveness of human alveolar macrophages: effects of cigarette smoking. Infect. Immun. 1974; 9: 769-771. (Received July 14, 1988; Accepted July 18, 1988)
Exp. Pathol. 39 (1990) I
43
Department of Pneumonology and Allergology, Medical Academy, L6di, Poland
Nicotine increases human polymorphonuclear leukocytes chemotactic response a possible additional mechanism of lung injury in cigarette smokers By D. NOWAK, U. RUTA and G.
PIAS~CKA
With 4 figures
Address for correspondence: DARIUSZ NOWAK, M. D., Department of Pneumonology and Allergology, Medical Academy, Kopcinskiego st. 22,90-153 L6di, Poland Key words: nicotine; leukocytes; granulocytes ; chemotoxin; smoking, cigarette ; cigarette smoking; polymorphonuclear leukocytes; lung
Summary Human polymorphonuclear leukocytes (PMNL) which are a potential source of proteolytic enzymes and reactive oxidant species contribute to the development of pulmonary emphysema in cigarette smokers. We found that nicotine at concentrations that occur in smokers' plasma enhances human PMNL chemotactic response to zymosan-activated serum (ZAS) and n-formylmethionyl-leucyl-phenylalanine (FMLP). Maximal increase in chemotactic migration was at nicotine concentration 1 f.lmolll. Higher concentrations, above 0.1 mmol!l inhibited PMNL chemotactic response and spontaneous migration. Nicotine also enhanced PMNL influx to the place of inflammation developed in the mouse pleural cavity after injection of ZAS . The number of PMNL found in the pleural cavity was I. 9-fold higher (p < 0.00 1, n = 5) when animals were pretreated with 0.15 mg of nicotine . However, this drug itself (concentrations of 0.1 f.lmolll to 10 mmolll) had weak chemotactic activity for PMNL. It seems that the stimulatory action of nicotine on PMNL chemotaxis may be partly responsible for increased PMNL numbers in the lower airways of cigarette smokers and following formation of the elastase!antielastase imbalance in lung tissue.
Introduction The central assumption of the proteolytic theory of premature pulmonary emphysema is the increased elastin degradation resulting from the elastase/antielastase imbalance in the lower airways . Cigarette smoking is the major exogenic factor in the epidemiology of pulmonary emphysema. One of several links between cigarette smoking and the development of emphysema is the recruitment of mononuclear phagocytes and polymorphonuclear leukocytes (PMNL) to the lower airways (8, 17). This process may be an important first step in the formation of elastase/ antielastase imbalance because these cells release proteases including elastases as well as increased amounts of reactive oxygen species (7, 13) which may inactivate 0.01) for FMLP, respectively. The higher nicotine concentrations, above 0.1 mmolll diminished cell migration to chemoattractants. The mean decrease in the chemotactic index was 1.04 ± 0.79 (not significant), 1.38 ± 0.59 (p < 0.01) at nicotine concentration 1 and 10 mmol/l, for assay with FMLP and 0.54 ± 0.16 (p < 0.01), 0.74 ± 0.33 (p < 0.01) for assay with ZAS, respectively. These concentrations also inhibited PMNL spontaneous migration under agarose, fig. 3. However, concentration of nicotine of 0.1 f,lmolll enhanced PMNL migration as expressed by the migration index. The PMNL viability after incubation with nicotine in RPMI-1640 medium for 90 min at 37°C was lower than in cells incubated without nicotine. At nicotine concentrations of 0, 0.1, 1, 10 f,lmolll and 0.1, 1, 10 mmol/l viability was 96 ± 3, 93 ± 3, 92 ± 3, 89 ± 6, 86 ± 6, 84 ± 5, 69 ± 10%, respectively; results were obtained from 5 separate experiments. Table 1 shows the total number of cells and PMNL and lymphocytes present in the mouse pleural cavity at 3 h after injection of various agents. Injection of PBS alone increased the total number of cells and PMNL recovered from the pleural cavity. ZAS caused influx of inflammatory cells, predominantly PMNL to the pleural cavity. The total number of cells, number of PMNL and lymphocytes was about 2-,2.6-, 1.7-fold higher than in animals injected with PBS, respectively. The previous intraperitoneal nicotine administration enhanced the total number of cells (l.4-fold) and the number of PMNL (l.9-fold) Exp. Pathol. 39 (1990) 1
39
Table 1.
The effect of nicotine on the total number of cells and number of PMNL and lymphocytes found in the pleural cavity after injection of ZAS. No.
Total cells/mouse (x 106)
Agent
a 1 2 3 4
PBS PBS Ni
b
+ PBS + ZAS + ZAS
0.44 0.56 1.1
1.5
PMNL (x 106)
± 0.05 ± 0.03 & ± 0.10 ± 0.15 #
0.11 0.19 0.50 0.96
± 0.02 ± 0.02 ! ± 0 .05 ± 0 .08 *
Lymphocytes (X
106)
0.32 0.36 0.60 0 .53
± 0.07 ± 0.06 ± 0.16 ± 0 . 12
PBS: phosphate buffered saline pH 7.4; ZAS: zymosan-activated serum; Ni : nicotine. Animals were injected intraperitoneally (a) with PBS or Ni and then after 2 h intrapleuraUy (b) with PBS or ZAS. Results (mean values and standard deviation) were obtained from 5 separate experiments. &: significantly different (p < 0.02) from no 1; !: significantly different (p < 0.01) from no 1; #: significantly different (p < 0.01) from no 3; *: significantly different (p < 0.001) from no 3. CHEt10TACTlC INDEX CHEt10TACTlC INDEX
~r----r-----r-----r---.
Sr---~----~----~----~
3
o
10:' 10-6 10-5 10-'1 10-3 10-2
NICOTINE CONCENTRATION [motif]
o~~~
o
__~~__~~__~~
10-7 10-6 10-5 10-* 10-3 10-2
NICOTINE CONCENTRATION [mot/{]
Fig.l. The effect of nicotine on PMNL chemotactic response to FMLP. PMNL were incubated with appropriate nicotine solutions in RPMI-1640 medium for 90 min at 37°C and then their chemotactic response to FMLP was measured under agarose. Each curve represents results of one experiment with PMNL obtained from separate donors .
Fig. 2. The effect of nicotine on PMNL chemotactic response to ZAS. PMNL were incubated with appropriate nicotine concentration in RPMI-I640 medium for 90 min at 37°C and then their chemotactic response to ZAS was measured under agarose. Each curve represents results of one experiment with PMNL obtained from separate donors .
but not that of lymphocytes found in the pleural cavity after injection of ZAS. Results of assays of nicotine chemotactic activity for PMNL are shown in fig . 4. Strong PMNL migration to nicotine was observed when it was used in concentrations of 1 and 10 mmoUI but only in 2 of 9 assays. 40
Exp. Patho!' 39 (1990) I
CHEMOTACTIC INDEl
MIGRATION INDEX
1\",
1,1t r - - - - - - - - - - - - - - ,
1,2
1.0
2.5
r1\0.92 !:o,1 Io.8!:0, 09
O.8f----
!isz;;
a76!o,06
1.8 1.6
0.61-----
1.'-
O'~f---------
1.2
0.21----------
1.0
10- 7 10- 6 10-$ 'O-~ 10-3 10-2
NICOTINE CONCENTRATION [moll/]
...
0
0
(>
..
"
10- 7 10-6 10-5 IO- It 10-3 10-2
NICOTINE CONCENTRATION [mollf)
Fig.3. The effect of nicotine on PMNL spontaneous migration under agarose. PMNL were incubated with appropriate nicotine concentration in RPMI 1640 medium for 90 min at 37°C. Values of migration index below 0.8-significant migration inhibition (22).
Fig. 4. The chemotactic activity of nicotine for PMNL. Nicotine was dissolved in RPMI 1640 medium to various final concentrations and chemotactic response of PMNL to 10 ",,1 of these solutions was measured under agarose technique. Results are obtained from 9 series of separate experiments with PMNL from 9 various donors. These cells were able to migrate to ZAS and FMLP, part of them were used in experiments showed in figs. 1 and 2. However, the weak nicotine chemotactic activity (values of chemotactic index below 1.5) was found for all concentrations of this alkaloid used. The number of the weak positive PMNL chemotactic responses to nicotine was dependent on its concentration and ranged from 3 to 6 of 9 assays.
Discussion Our results of the in vitro nicotine influence on PMNL chemotactic response are in agreement with opposed data presented in two previous works (2, 26). BRIDGES et ai. (2) found that nicotine concentrations of 6 to 10 mmolll inhibited PMNL chemotactic response to endotoxin-activated human serum. TOTTI et ai. (26) observed stimulation of PMNL migration to FMLP and C5fr by nicotine when it was used in concentrations of 0.31 to 30.8 ""molli. We found that the effect of nicotine on PMNL chemotactic response depends on its concentration: concentrations of 0.1 to 10 ""molll enhance and of 1 to 10 mmolll inhibit PMNL migration to chemoattractants. This inhibition is probably due to the cytotoxic action of high nicotine concentrations. However, CORBERAND et ai. (3) reported no influence of nicotine at concentrations of 1.85 to 185 ""molll on PMNL chemotaxis to caseine under agarose. It may be explained by differences between our experimental protocol and that of CORBERAND et ai. who incubated PMNL with nicotine only for 30 min. Our in vitro results are supported by in vivo nicotine enhancement of the PMNL influx to the place of inflammation developed in the pleural cavity after injection of ZAS. The nicotine dose of 0.15 mg per mouse was very high and represents the action of toxic nicotine concentrations on PMNL inflammatory influx (all animals had convulsions during the first 20 min after nicotine injection). In mice, nicotine after intravenous injection is rapidly distributed throughout the tissues and body fluids (23). Also nicotine is relatively rapidly excreted from the mouse body. Exp. Pathol. 39 (1990) 1
41
Approximately 50 % of the single nicotine dose was excreted with urine within 6 h after injection (14). In our experiments the drug administered intraperitoneally passed through the liver before it was able to enter the general circulation. And in addition the mouse liver can uptake and metabolize nicotine mostly to cotinine (6). Therefore, for these reasons we used such high single nicotine doses in this experiment. In contrast to data presented by TOTTI et al. (26) we failed to find significant chemotactic activity of nicotine for PMNL. They measured PMNL chemotaxis to nicotine using modified Boyden chambers and observed nicotine-dependent migration at its concentration of 0.5 [tmol/l to 0.5 mmol/l with the maximum at 30.8 [tmoVI. In our study nicotine as a low molecular weight compound could relatively rapidly diffuse around well in agarose layer and thus induce weak PMNL chemotactic migration. Probably, for this reason only high nicotine concentrations induced sometimes strong PMNL migration. Human PMNL have a noncholinergic nicotine receptor on their surface which can be occupied by this alkaloid at concentrations present in the blood of smokers (4). Also the average blood nicotine concentration in smokers (21) is approximate to its concentrations used in our experiments which enhanced PMNL chemotactic response to ZAS and FMLP. The direct action of nicotine via its receptor may be responsible for this enhancement. PMNL are a rich potential source of elastase (10) which is released from these cells after phagocytosis or their chemotactic stimulation (19, 25). Increased PMNL inflammatory recruitment to the lung and proteases release may lead to the lung injury resulting in emphysema. It is possible that enhancement of PMNL chemotactic response to chemoattractants created in the lung in the course of smoking by nicotine may contribute to the increase in number of PMNL in the lower airways of cigarette smokers and following lung injury. However, the chemotactic responsiveness of PMNL has been shown to be depressed in cigarette smokers as compared to nonsmokers (18). Some unsaturated aldehydes present in cigarette smoke such as acrolein, crotonaldehyde may be responsible for it (2). On the other hand, there are no differences in monocyte chemotaxis between smokers and nonsmokers (16), and human alveolar macrophages from smokers have shown an enhancement of chemotactic responsiveness (27).
Acknowledgements This work was supported partly by the Department of Lung Medicine, University of Lund, Sweden. We thank Prof. B. G. SIMONSSON for this support.
References \. BOYUM, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 1968; 97 (Suppl.): 77-89. 2. BRIDGES, R. B., KRAAL, J. H., HUANG, 1. J. T., CHANCELLOR, M. B.: Effects of cigarette smoke components on in vitro chemotaxis of human polymorphonuclear leukocytes. Infect. Immun. 1977; 16:
240-248. 3. CORBERAND, 1., LAHARRAGUE, P., NGUEN, F., DUTAU, G., FONTANILLES, A. M. , GLEIZES, B., GYRARD, E.: In vitro effect of tobacco smoke components on the functions of normal human polymorphonuclear leukocytes. Infect. Immun. 1980; 30: 649-655. 4. DAVIES, B. D., Hoss, W., LIN, J. P., LIONETTI, F.: Evidence for a noncholinergic nicotine receptor on human phagocytic leukocytes. Mol. Cell. Biochem. 1982; 44: 23-3\. 5. GADEK, J. E., HUNNINGHAKE, G. W., FELLS, G. A., ZIMMERMANN, R. 1., KEBYH, B. A., CRYSTAL, R. G.: Evaluation of the protease-antiprotease theory of human destructive lung disease. Bull. Eur. Physio-Pathol. Respir. 1980; 16 (Suppl.): 27-34. 6. HANSSON, E., SCHMITERLOW, C. G.: Metabolism of nicotine in various tissues. In: VON EULER, U. S. (ed.): Tobacco alkaloids and related compounds. Pergamon Press 1964; 87-99. 7. HOIDAL, J., Fox, R. B., LEMARBE, P. A., PERRI, R., REPINE, 1. E.: Altered oxidative metabolic response in vitro of alveolar macrophages from asymptomatic cigarette smokers. Am. Rev. Respir. Dis.
1981; 123: 85-89. 8. HUNNINGHAKE, G. W., CRYSTAL, R. G.: Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am. Rev. Respir. Dis. 1983; 128: 833-836.
42
Exp. Pathol. 39 (1990) 1
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