Published ahead of print on December 30, 2003, doi:10.1164/rccm.200210-1154OC
© 2004 American Thoracic Society
Inhibitory Effect of Nicotine on Experimental Hypersensitivity Pneumonitis In Vivo and In VitroUnité de Recherche, Centre de Pneumologie, Institut Universitaire de Cardiologie et de Pneumologie de l'Université Laval, Hôpital Laval, Quebec City, Québec, Canada Correspondence and requests for reprints should be addressed to Yvon Cormier, M.D., Hôpital Laval, 2725 Chemin Ste-Foy, Quebec City, PQ, G1V 4G5 Canada. E-mail: yvon.cormier{at}med.ulaval.ca
The incidence of hypersensitivity pneumonitis (HP) is lower in smokers than in nonsmokers. Because nicotine is immunosuppressive, we hypothesized that it could have a protective effect on HP induction in vivo. HP was induced in mice that were treated with nicotine either intraperitoneally (IP) (0.5 to 2.0 mg/kg/day) or intranasally (IN) (0.025 to 2.0 mg/kg/day). Both IP- and IN-treated animals had fewer bronchoalveolar lavage total cells and lymphocytes and a decreased lung tissue inflammation. IFN-  but not interleukin-10 mRNA expression was reduced in lung tissue of 2.0-mg/kg IN-treated animals. To test the effect of nicotine on alveolar macrophages, AMJ2-C11 cells were treated with nicotine and stimulated with lipopolysaccharide or Saccharopolyspora rectivirgula, a causative agent of HP. Nicotine reduced tumor necrosis factor release and tumor necrosis factor, interleukin-10, and IFN- mRNA expression after stimulation and decreased CD80 expression by 55% in lipopolysaccharide-stimulated cells and by 41% in S. rectivirgula–stimulated cells. We conclude that nicotine could be, at least in part, responsible for the protection observed in smokers against HP. The inhibitory effect of nicotine on alveolar macrophages could be one of the mechanisms involved.
Key Words: alveolar macrophages • nicotine • hypersensitivity pneumonitis • cytokines Nicotine, a tertiary cholinomimetic alkaloid, is a major component of cigarette smoke. This molecule is highly lipophilic and thus has the capacity to cross the blood/brain barrier and cause addiction (1). This nicotinic agonist has some immunomodulatory and antifibrotic effects. In fact, it inhibits lymphocyte proliferation (2), interleukin (IL)-1ß, tumor necrosis factor (TNF), IL-6 and IL-12 production by macrophages (3, 4), the expression of costimulatory molecules such as CD28 and CTLA-4 on T cells (5), and fibroblast proliferation (6). Nicotine is effective in the treatment of ulcerative colitis, an intestinal inflammatory disease (7), and has been shown to have beneficial effects in a type 1 diabetes mouse model (8). Interestingly, certain inflammatory diseases, such as sarcoidosis and ulcerative colitis are less frequent in smokers than in nonsmokers, and cigarette smoking protects against radiation pneumonitis (9, 10). When exposed to an environment that can cause hypersensitivity pneumonitis (HP), smokers have lower levels of specific antibodies to the causative antigen (11). On the other hand, when HP does occur in smokers, it promotes an insidious and more chronic form of the disease and worsens the clinical outcome (12).
HP is a pulmonary inflammatory disease characterized by the accumulation and proliferation of lymphocytes in the lung (13). This disease is caused by an immune reaction to inhaled antigens such as Saccharopolyspora rectivirgula, which is responsible for the induction of farmer's lung, a form of HP (14). The costimulation between T lymphocytes (CD28 and CTLA-4 molecules) and alveolar macrophages (AMs) (B7 molecules or CD80-86) plays a major role in the development of this disease: blockade of this pathway inhibits the inflammatory response to S. rectivirgula antigen in mice (15). Cytokines such as TNF, IFN- Generally, smokers have lung inflammation characterized by an increased number of AMs (up to 99% of total cells), as demonstrated by bronchoalveolar lavage (BAL) (19). The aim of this study was to verify whether nicotine could, at least in part, be responsible for the protection against the development of HP observed in smokers and to look at possible mechanisms of action. For this, we used a previously described mouse model of HP that has been used extensively in our laboratory (15) and treated mice with nicotine, either intraperitoneally (IP) or intranasally (IN).
In addition, an AM cell line stimulated with S. rectivirgula antigen or Escherichia Coli lipopolysaccharide (LPS), a potent activator of AM (3), was used to test the effect of nicotine on TNF, IFN-
Induction of HP and BAL C57Bl/6 female mice (Charles River, St-Constant, PQ, Canada) received 50 µl of IN S. rectivirgula antigen 3 consecutive days a week for 3 weeks. Mice were simultaneously treated with either 100-µl IP or 50-µl IN nicotine tartrate salt (Sigma, St. Louis, MO) daily or twice a day (Table 1) . Four days after the last S. rectivirgula instillation, mice were sacrificed by overexposure to isoflurane and tracheotomized, and a BAL was performed using three aliquots of 1 ml phosphate-buffered saline. Total cells were counted. Cytospin preparations were stained with Hemacolor Stain Set (EM Diagnostic Systems, Middletown, VA), and differential counts were obtained.
Histopathologic Studies A section of the left lung from the highest nicotine dose (2.0 mg/kg) IN-treated mice from a separate group that did not undergo BAL was collected on the day of the sacrifice and stained with hematoxylin/eosin. Inflammatory parameters of the lung tissue were evaluated blindly by a pathologist. The histology score was graded from 0 to 4.
Semiquantitative Cytokine mRNA Expression The PCR reaction was run on a 1.5% agarose gel, stained with ethidium bromide, and exposed to ultraviolet light. Densitometric analyses of the bands were performed (ScionImage; Scion Corporation, Frederick, MD).
In Vitro Studies
Semiquantitative RT-PCR for In Vitro Studies
Flow Cytometry Analysis for CD80/86 Expression
Statistical Analysis
Total Cell and Differential Counts in BAL Results of BAL total cell counts and differential counts are presented in Figures 1–3 . A significant reduction of total cell counts was observed in mice treated with 0.5 mg/kg of nicotine given IP daily and twice a day and 1.0 mg/kg given twice a day (p = 0.02, p = 0.02, and p = 0.03; n = 8 mice per group), whereas the number of lymphocytes decreased in all the IP-treated groups (p = 0.003, p = 0.007, and p = 0.04) (Figure 1). IN administration (Figure 2) first resulted in a significant decrease in total cells at a very low dose of nicotine (0.025 mg/kg, p = 0.04, n = 16) and at the 0.25-, 0.5-, and 2.0-mg/kg dose (p = 0.01 for all groups, n = 16 mice per group). The lymphocyte population was significantly decreased in all IN-treated groups except for the 1.0-mg/kg group (p < 0.0001 for all groups). Because the 1.0-mg/kg IN-treated group did not first respond to the treatment, a separate experiment was performed with this particular dose to verify whether this result was due to experimental variations. The results (Figure 3) show that this dose did indeed reduce both total cells (p = 0.01) and lymphocytes (p = 0.002) in the BAL (n = 8 mice per group).
Histopathologic Studies Results of the histopathologic studies are presented in Figure 4 and show a marked peribronchial, perivascular, and parenchymal infiltration of inflammatory cells in the S. rectivirgula group (Figure 4B) compared with control mice (Figure 4A). Mice treated with 2.0 mg/kg nicotine (Figure 4C) showed a decreased tissue infiltration of mononuclear cells in lung tissue compared with nontreated mice. This result was confirmed by a reduction of total histologic score (p = 0.01, n = 9 mice per group) (Figure 4D).
RT-PCR for In Vivo Studies The results for RT-PCR for detection of IFN- and IL-10 mRNA expression in mice lung sections from the highest dose–treated mice (2.0 mg/kg) are presented in Figure 5
. The ratio between the intensity of the ß-actin band and that of the cytokine band was calculated. Results are expressed as a percentage of expression, with 100% being attributed to the S. rectivirgula alone ratio. Each band represents a different animal. The 2.0-mg/kg treatment significantly reduced IFN- mRNA expression (p = 0.01, n = 4 mice per group). The expression of IL-10 was not affected by the nicotine treatment at this dose.
TNF Concentration in AMJ2-C11 Cell Line Supernatants (In Vitro Studies) Results on the effect of nicotine on TNF levels in LPS- and S. rectivirgula–stimulated AMJ2-C11 cells supernatants are presented in Figure 6 . Results are expressed as a percentage of release, with 100% being attributed to LPS- or S. rectivirgula–stimulated and untreated cells. The TNF release was significantly reduced to 84% for 160-µM nicotine-treated and LPS-stimulated cells (Figure 6A) (n = 4, p = 0.004). Similarly, TNF release was significantly reduced to 76% for 160-µM nicotine-treated and S. rectivirgula–stimulated cells (Figure 6B) (n = 4, p = 0.02). AMJ2-C11 cells failed to release IL-10 and IFN- (data not shown).
Flow Cytometry Analysis In the AMJ2-C11 cell line, CD86 was expressed in 100% of the cells; no stimulation of expression was therefore achievable by LPS or S. rectivirgula. Nicotine had no effect on CD86 expression. CD80 was expressed in 5% of unstimulated cells. Nicotine alone increased its expression to 20% of the cells, whereas LPS stimulated the expression to 38.1% and S. rectivirgula to 34.6% of cells. Despite the fact that nicotine alone stimulated CD80 expression, nicotine treatment on LPS- or S. rectivirgula–stimulated cells lowered CD80 expression to 19.8% (p = 0.0015) and 26.1% (p = 0.01), respectively (n = 4) (Figure 7) .
RT-PCR for In Vitro Studies (AMJ2-C11 Cell Line) Results are presented in Figure 8 (TNF), Figure 9 (IL-10), and Figure 10 (IFN- ) and are expressed as a percentage of expression, with 100% being attributed to the LPS- or S. rectivirgula–alone stimulated cells. The results are representative of four different experiments. Nicotine treatment had an inhibitory effect on TNF mRNA expression that was reduced by 98% (p < 0.0001), with the 40-µM dose in LPS-treated cells and 34% (p = 0.03) with the 160-µM dose in the S. rectivirgula–stimulated cells. A similar effect was observed with the IL-10 mRNA expression. In fact, nicotine treatment reduced IL-10 mRNA expression by 88% (p < 0.0001) in LPS-stimulated cells (40-µM nicotine) and 62% (p = 0.01) in S. rectivirgula–stimulated cells (160 µM). AMJ2-C11 cells failed to express IFN- mRNA with LPS stimulation, but it was detected with the S. rectivirgula antigen stimulation. Nicotine treatment reduced IFN- mRNA expression by 80% (p < 0.0001) in 40-µM treated cells.
This study was performed to verify the effect of nicotine on lung inflammation in an in vivo mouse model of HP and in vitro using a mouse AM cell line. Nicotine treatment, either IP or IN, had a significant antiinflammatory effect in the mouse model. Total lung cells as well as tissue inflammation were significantly decreased in nicotine-treated animals. The BAL cell population that was the most affected by the nicotine treatment was the lymphocyte population. These cells were significantly decreased in BAL, either in IP- or IN-treated animals. Nicotine treatment also had an inhibitory effect on IFN- mRNA expression but had no effect on IL-10 mRNA expression by lung tissue. The effect of nicotine in the IP-treated mouse model did not follow a positive dose response pattern. For the IP administration, the most effective dose on total cell accumulation in BAL was 0.5 mg/kg administered twice a day. This is a relatively high dose, corresponding to that of people smoking an average of one pack a day (21). The decrease of the antiinflammatory effect seen with increasing nicotine doses could be attributed to nicotinic acetylcholine receptor desensitization at high doses or to toxic effects of nicotine at these very high doses. The IN administration first dose response also showed an unusual pattern. The 0.025-, 0.25-, 0.5-, and 2.0-mg/kg doses had significant antiinflammatory effects on total cells, whereas the 1.0-mg/kg dose did not. Of interest is the significant effect of the lowest dose of 0.025 mg/kg. This dose corresponds to a single administration of NT nasal spray in humans (22). The results obtained with the 1.0-mg/kg group were surprising. Such results could be explained by a double dose response curve. However, because in this group half of the animals seemed to have a response and half did not (data not shown), we questioned whether this was due to a technical error. To verify this, an additional group of mice was treated at this IN dose. This second trial showed that a 1.0-mg/kg IN administration significantly reduces both total cells and lymphocytes in the BAL; we can conclude that the previous result for that dose was due to experimental variability and that nicotine did not have a double dose response curve, as it was suggested by the first results. Nicotine had a striking inhibitory effect on lymphocyte accumulation in the lung, as demonstrated by BAL. This effect is in agreement with other studies showing an inhibitory effect of nicotine on lymphocyte proliferation (23). The CD4/CD8 subtype of lymphocytes found in the BAL was not affected by the nicotine treatment (result not shown). Because significant effects were obtained at low doses in both IP and IN protocols, further studies are needed to identify the optimal antiinflammatory dose. Similar results with low doses of nicotine being more effective to reduce inflammation than high concentrations have also been reported in treatment of ulcerative colitis (24). The effect of nicotine on tissue infiltration of inflammatory cells further supports the antiinflammatory effects of nicotine. One of the reasons that we used IN administration was to avoid the liver first pass effect and to achieve an antiinflammatory effect at lower doses than with IP administration. When administered IN, nicotine is rapidly absorbed by the vessels surrounding the nasal sinuses. These vessels drain into the superior vena cava, and blood passes into pulmonary circulation before entering the peripheral circulation (22). Our research supports the better effect of direct administration into the lungs compared with systemic administration; the 0.025-mg/kg dose administered IN had a significant antiinflammatory effect, whereas the 0.25-mg/kg dose administered IP did not.
The downregulating effects of nicotine on IFN- The inhibitory effect of nicotine on CD80 expression on the mouse AM cell line suggests that AMs could be at least partially responsible for the observed lymphocyte suppression. Because blockade of the CD80/86-CTLA-4/CD28 pathway inhibits lymphocyte response to S. rectivirgula antigen (15), the decrease in expression of CD80 that we observed could be another mechanism by which cigarette smoking could decrease the risk for HP.
TNF is a cytokine that is involved in the pathology of HP (16). The fact that TNF production by the AMJ2-C11 cell line as well as TNF mRNA expression was reduced by the nicotine treatment could explain in part the immunosuppressive effect of nicotine in vivo. Nicotine also downregulated IL-10 and IFN-
Once again, the dose response curve of nicotine showed that the lowest dose (40 µM) had the best inhibitory effect on LPS-stimulated cells and that the highest dose (160 µM) had the best effect on S. rectivirgula–stimulated cells. This could be explained by a difference in the level of activation of the cells when stimulated with 0.1-µg/ml LPS compared with 50 µg/ml for S. rectivirgula antigen stimulation and by the different mechanisms of action of the two antigens. S. rectivirgula is phagocytosed by AMs, whereas LPS acts through its own receptor, CD14 (26). This hypothesis is further supported by the fact that all the LPS-treated cells showed a better response with the lowest dose of nicotine compared with S. rectivirgula–stimulated cells. We also believe that a shorter time of incubation could prevent receptor desensitization and have a better effect on cytokine mRNA inhibition. Finally, the fact that AM produced IFN- Because nicotine did reduce IL-10 mRNA production by AM in vitro, the expression found in lung tissue from the mouse lungs could come from other inflammatory cells or from structural cells. The inhibitory effect of nicotine could not only explain the lower prevalence of HP in smokers but also the different outcome of HP that does occur in some smokers (12). The downregulating effect could be sufficient to prevent acute HP. Subjects could thus develop a more insidious form that could progress to irreversible lung damage before the diagnosis is made, explaining the poorer outcome in these subjects. Lymphocytes are highly activated and recruited by the cytokines released by AMs. The inhibitory effect of nicotine on cytokines mRNA production in AMs could, together with inhibition of the costimulatory pathway, explain the decrease in the lymphocyte population in the BAL of nicotine-treated animals. We are aware that inhibition of mRNA expression does not always transfer to a protein production inhibition, but however, there was concordance between TNF mRNA levels and protein release in AMJ2-C11 cells. Because in our study the decrease in mRNA and protein expression corresponds to cellular findings, we believe that the overall inhibitory effect of nicotine could explain, at least to some extent, the protection that smokers have against the development of this disease.
Conclusions
The authors thank Jocelyne Simard and Alina Milahia for their help with the mRNA extraction and RT-PCRs and Dr. Marcien Fournier for the histopathologic studies. They also thank the Canadian Institutes of Health Research, the J-D. Bégin foundation, and Institut Robert-Sauvé en Santé et Sécurité au Travail for financial support.
Conflict of Interest Statement: M-R.B. has no conflict of interest and the work in this paper is part of the patent number PCT/CA02/00412 [March 2002] and is the property of Laval University; E.I-A. has no conflict of interest and the work in this paper is part of the patent number PCT/CA02/00412 [March 2002] and is the property of Laval University; Y.C. has no conflict of interest and the work in this paper is part of the patent number PCT/CA02/00412 [March 2002] and is the property of Laval University. Received in original form October 8, 2002; accepted in final form December 30, 2003
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