Abstract
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-γ 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-γ 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.
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)-1β, 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-γ, and IL-10 are involved in HP. Increased TNF serum bioactivity was reported in farmer's lung patients (16). Animal models have also shown that IL-10 modulates inflammation and that IFN-γ is necessary for development of the disease (17, 18).
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-γ, and IL-10 mRNA expression and B7 costimulatory molecule expression in vitro. Some of the results in these studies have been previously published in the form of an abstract (20).
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)
Nicotine Administration | Group Name | Instillation | Treatment (mg/kg) | Frequency of Treatment (times per day) |
|---|---|---|---|---|
| IP | Sal | Sal | Sal | 2 |
| Nic | Sal | Nic | 2 | |
| 0 | S. rectivirgula | Sal | 2 | |
| 0.5 (1×) | S. rectivirgula | Nic, 0.5 | 1 | |
| 0.5 (2×) | S. rectivirgula | Nic, 0.5 | 2 | |
| 1.0 (2×) | S. rectivirgula | Nic, 1.0 | 2 | |
| IN | Sal | Sal | Saline | 1 |
| Nic | Sal | Nic | 1 | |
| 0 | S. rectivirgula | Sal | 1 | |
| 0.025 | S. rectivirgula | Nic, 0.025 | 1 | |
| 0.25 | S. rectivirgula | Nic, 0.25 | 1 | |
| 0.5 | S. rectivirgula | Nic, 0.5 | 1 | |
| 1.0 | S. rectivirgula | Nic, 1.0 | 1 | |
| 2.0 | S. rectivirgula | Nic, 2.0 | 1 |
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.
The reverse transcription-polymerase chain (RT-PCR) reaction experiments were done on lung sections (from the 2.0-mg/kg IN-treated group) lysed in TRIZOL reagent (GIBCO BRL, Grand Island, NY). Total RNA was extracted and quantified with Ribo-Green reagent (Molecular Probes, Eugene, OR). One microgram of RNA was reverse transcribed with MMLV-Reverse Transcriptase (GIBCO BRL), and a PCR was performed with a Peltier Thermal Cycler 200 for detection of IFN-γ and IL-10 mRNA expression using Taq DNA polymerase (Promega, Madison, WI). Primers used for IFN-γ and IL-10 were from Biosource International (PQ, Canada).
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).
A mouse AM cell line, AMJ2-C11 (ATCC, Manassas, VA), was used. Cells (150,000) were plated in a 24-well plate, stimulated with S. rectivirgula (50 μg/ml) or LPS (0.1 μg/ml) for 24 hours, and treated with 0, 40, 80, or 160 μM of nicotine. Supernatant TNF levels were measured by ELISA (RD Diagnostics, Minneapolis, MN), and cells were collected for analysis of the B-7 (CD80/86) costimulatory molecule expression or lysed using TRIZOL reagent (GIBCO BRL) for RNA extraction.
Cell lysis, RNA extraction, quantification, and RT-PCR were performed as for the in vivo studies. Primers used for TNF were 3′-CCT GGC TAG TGG GGC TTC AAG TCA TCT GTC TT-5′ and 5′-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3′. Primers used for IFN-γ and IL-10 were the same as for the in vivo studies.
Cells were stimulated with LPS or S. rectivirgula, treated with 40 μM of nicotine for 24 hours, and incubated for 45 minutes with a mouse anti-CD80 antibody coupled to fluorescein isothiocyanate fluorochrome or a mouse anti-CD86 coupled to phycoerythrin fluorochrome or their isotype control. The percentage of cells positive for CD80/86 was analyzed in an Epics ELITE flow cytometer.
Statistical analysis was made using an analysis of variance table followed by a Fisher's post hoc test.
Results of BAL total cell counts and differential counts are presented in Figures 1–3
Figure 1. Bronchoalveolar lavage (BAL) total cell and differential counts of intraperitoneally (IP) nicotine-treated mice. Nicotine (nic) administration at doses of 0.5 mg/kg treated once or twice daily and 1.0 mg/kg treated twice daily decreased the total cell accumulation compared with the Saccharopolyspora rectivirgula (SR)-alone group. All nicotine-treated groups had significantly fewer BAL lymphocytes than the nontreated mice. *p < 0.05 compared with the SR-alone group.
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Figure 2. BAL total cell and differential counts of intranasally (IN) nicotine-treated mice. All nicotine-treated groups had fewer total BAL cells and BAL lymphocytes, except for the 1.0-mg/kg group. *p < 0.05 compared with the SR-alone group.
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Figure 3. BAL total cell and differential counts of 1.0-mg/kg IN nicotine-treated mice. Nicotine administration significantly reduced both total cells and lymphocytes in the BAL. *p < 0.05 compared with the SR-alone group.
[More] [Minimize]Results of the histopathologic studies are presented in Figure 4
Figure 4. Histopathology results (×10) of control (SAL; A), nontreated (SR; B), and 2.0-mg/kg treated (SR + nicotine; C) mice. SR treatment induced a marked peribronchial, perivascular, and parenchymal infiltration of inflammatory cells compared with saline-treated mice, whereas nicotine treatment reduced this infiltration. Results are expressed in D as the total histologic score (graded on an arbitrary scale, from 0 to 4: 0, no inflammatory cells; 1, less than 10%; 2, 10–25%; 3, 25–50%; and 4, more than 50%), which was significantly reduced in nicotine-treated mice (total score of 1.6 ± 0.6) compared with nontreated mice (total score of 2.46 ± 0.68) (*p = 0.01; n = 9).
The results for RT-PCR for detection of IFN-γ and IL-10 mRNA expression in mice lung sections from the highest dose–treated mice (2.0 mg/kg) are presented in Figure 5
Figure 5. IFN-γ and interleukin (IL)-10 mRNA expression in lung sections of untreated (SR) and 2.0-mg/kg IN nicotine-treated (NIC) mice (n = 4). (A and C) Lung mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). β-Actin primers were used as a positive control. Each lane represents a different animal. (B and D) Semiquantification of mRNA expression was done by densitometry. Nicotine treatment had no effect on IL-10 mRNA expression but significantly reduced IFN-γ mRNA expression in the 2.0-mg/kg group (SR + NIC). *p < 0.05 compared with SR-alone group.
Results on the effect of nicotine on TNF levels in LPS- and S. rectivirgula–stimulated AMJ2-C11 cells supernatants are presented in Figure 6
Figure 6. Percentage of tumor necrosis factor (TNF) release by lipopolysaccharide (LPS) (A)- and SR (B)-stimulated cells; 100% is attributed to the LPS- or SR-stimulated and untreated cells. Nicotine treatment significantly reduced TNF release by 160-μM treated cells for both types of stimulations (84% in LPS-stimulated and nicotine-treated cells and 76% in SR-stimulated and nicotine-treated cells). *p < 0.05; n = 4.
[More] [Minimize]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)
Figure 7. Expression of CD80 costimulatory molecule in AMJ2-C11 LPS- or SR-stimulated cells. Nicotine treatment (40 μM) reduced CD80 expression by 51% in LPS-stimulated cells (LPS + Nic) and by 25% in SR-stimulated cells (SR + Nic). *p < 0.01, n = 4.
[More] [Minimize]Results are presented in Figure 8
Figure 8. TNF mRNA expression in AMJ2-C11 cells. (A) Expression was determined by RT-PCR after LPS or SR stimulation, and β-actin primers were used as positive control subjects. The first band represents untreated cells, and the second, third, and fourth bands represent, respectively, 40-, 80-, and 160-μM nicotine treatments. This is a representative result of four different experiments. (B and C) Quantification of the bands was achieved by densitometry. Nicotine treatment reduced TNF mRNA expression by 98% in LPS-stimulated and 40-μM–treated cells and by 34% in SR-stimulated and 160-μM–treated cells. (*p < 0.05 compared with the nontreated group, n = 4).
Figure 9. IL-10 mRNA expression in AMJ2-C11 cells. (A) mRNA expression was determined by RT-PCR, and β-actin primers were used as a positive control. The first band represents untreated cells, and the second, third, and fourth bands represent, respectively, 40-, 80-, and 160-μM nicotine treatments. This is a representative result of four different experiments. (B and C) Nicotine treatment reduced IL-10 mRNA expression by 88% in LPS-stimulated and 40-μM–treated cells and by 62% in SR-stimulated and 160-μM–treated cells (*p < 0.05 compared with the nontreated group, n = 4).
Figure 10. IFN-γ mRNA expression in AMJ2-C11 SR-stimulated cells. (A) Expression was determined by RT-PCR, and β-actin primers were used as a positive control. The first band represents untreated cells, and the second, third and fourth bands represent, respectively, 40-, 80-, and 160-μM nicotine treatments. This is a representative result of four different experiments. (B) Densitometric quantification of the bands showed a 80% reduction of expression in 40-μM SR-stimulated and nicotine-treated cells. *p < 0.05 compared with the nontreated group, n = 4).
[More] [Minimize]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-γ 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-γ mRNA expression by lung tissue from the highest dose of nicotine-treated mice could be one of the intracellular mechanisms involved in the protection effect of cigarette smoking on the development of HP. In fact, IFN-γ expression was reported to be essential in the development of this disease (18). The lack of effect of nicotine on IL-10 mRNA expression in lung tissue is not surprising. Nicotine-treated mice had lung tissue inflammation, and IL-10 is an antiinflammatory cytokine induced by proinflammatory cytokines. The preservation of high levels of IL-10 mRNA could also be a positive effect added to other nicotine effects in controlling the inflammation response to the S. rectivirgula antigen.
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-γ mRNA expression by AMJ2-C11 cells. These results are similar to those previously published on nicotine inhibitory effect on cytokine production by peripheral blood monocytes (3, 25). AMJ2-C11 cells failed to release IL-10 and IFN in vitro. This is consistent with the ATCC technical data on this cell line, which states that AMJ2-C11 cells preferably produce IL-6 and a small amount of TNF on stimulation with LPS.
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-γ mRNA expression on S. rectivirgula stimulation but did not with LPS stimulation is understandable because IFN-γ is a TH1-type cytokine and S. rectivirgula is known to induce HP, a TH1-polarized disease (27).
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.
Results of this study show that nicotine reduces the alveolar inflammatory response to S. rectivirgula antigen and affects some AM (stimulated with LPS or S. rectivirgula) functions in vitro. This influence could be, at least in part, responsible for the protection that smokers have against development of HP. Because nicotine is effective in the treatment of ulcerative colitis, it could also be of interest in the treatment of HP and other pulmonary inflammatory diseases.
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.
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