Previous reports suggested that cigarettesmoke had a protective effect of on the develop-ment of hypersensitivity pneumonitis (HP).However, smoking rate in chronic pigeon breeder’’slung (PBL) seemed to be high in our clinical expe-riences. We developed a murine model of PBL byintranasal instillation with pigeon droppingextracts (PDE) for 4 weeks (short-term exposure)and 17 weeks (long-term exposure) to investigatethe effect of cigarette smoke on disease processes.In this model, lung inflammation associated withthe production of anti-PDE antibodies and antigendependent lymphocyte proliferation was induced.Long-term exposure to PDE without cigarettesmoking resulted in an increase in lung weight /body weight ratio, total cell number in bron-choalveolar lavage (BAL) fluid, and content ofhydroxyproline in the lung compared to short-term exposure. After a short-term exposure, ciga-rette smoke lessened the lymphocytosis in BALfluid, and lymphocyte proliferation. On the otherhand, after a long-term exposure cigarette smokeincreased lung hydroxyproline. These results sug-gest that a short-term cigarette smoking attenuateslung inflammation, but a long-term cigarette

smoking enhances lung inflammation with fibrosis.

Key words: pigeon breeder’s lung, cigarettesmoke, lung inflammation, lunghydroxyproline

Introduction

Hypersensitivity pneumonitis (HP) is an immuno-logically induced lung disease caused by repeatedinhalation of various environmental antigens in sus-ceptible individuals. Pigeon breeder’s lung (PBL) is oneof the common HP caused by inhalation of pigeon-related antigens which are included in pigeon droppingsand in other pigeon materials1-4. PBL tends to developchronic disease with few acute symptoms includingfever and chills (insidious onset) as compared to sum-mer type HP5.

The clinical outcome of PBL is variable and theresponsible factors for its variation remain undeter-mined5,6. Several articles have reported protectiveeffects of cigarette smoking in developing acute HP7-9.Smokers in bird fanciers produced lower concentrationsof antibody to bird-related antigens in serum andbronchoalveolar lavage fluid than non-smokers10-12.Several proinflammatory cytokines such as TNF-Ïand IFN-Î are crucial in the development of HP13-17, butcigarette smoke is known to downregulate the produc-tion of these cytokines18,19. In line with these observa-tions, cigarette smoke decreased pulmonary dendriticcells and suppressed lipopolysaccharide-induced pro-

Original Article

Effect of cigarette smoking on the development of murine chronic pigeon breed-er’s lung The difference between a short-term and a long-term exposure

Masashi Furuiye, Shuji Miyake, Yasunari Miyazaki, Yoshio Ohtani, Naohiko Inase, Takeshi Umino,and Yasuyuki Yoshizawa.

Department of Integrated Pulmonology, Graduate School, Faculty of Medicine, Tokyo Medical and DentalUniversity

J Med Dent Sci 2007; 54: 1–149

Corresponding Author: Yasuyuki Yoshizawa, M.D., Ph.DProfessor and ChairmanDepartment of Integrated Pulmonology, Tokyo Medical and DentalUniversity, 1-5-45, Bunkyo-ku, Tokyo, 113-8519, JapanTel: 81-35803-5950 Fax: 81-35803-0167E-mail: yoshizawa.pulm@tmd.ac.jp Received October 16; Accepted December 1, 2006

duction of inflammatory cytokines by inhibiting theactivation of activator protein-1 in bronchial epithelialcells20,21. In contrast, short-term cigarette smokingenhanced allergic airway inflammation in mice associ-ated with eosinophilic inflammation, an increaseddendritic cells in bronchoalveolar lavage fluid, andincreased serum antigen-specific IgE22. Cigarettesmoke has been reported to directly stimulate alveolarmacrophages to produce proinflammatory cytokinessuch as IL-1 and IL-623. Furthermore, pulmonaryinflammation was induced accompanied with anincreased amount of infiltration of the dendritic cells inthe airways and the lung parenchyma in a mousemodel exposed with cigarette smoke for 24 weeks24.The effects of cigarette smoke on immune responsesare contrasting in the development of chronic diseaseand it was reported more than 50% of chronic PBLpatients were smokers25.

We hypothesized that cigarette smoke may modifythe disease process of PBL leading to chronic disease.In the present study, we have evaluated the effect ofcigarette smoke on the development of chronic PBL ina murine model by studying the difference between ashort-term and a long-term exposure. Lung weight /body weight ratio, serum antibodies and lymphocyteproliferation in response to pigeon-related antigen,BAL profiles and the expressions of INF-Î, TNF-Ï andIL-10 mRNA in the lung were evaluated to analyze theinflammatory process. The content of hydroxyproline inthe lung and histological findings were also examined toevaluate the fibrotic process.

Materials and Methods

AnimalsSpecific pathogen-free C57BL/6J mice were pur-

chased from Sankyo Medical animal supply (SankyoLabo. Co., Tokyo Japan). Female mice, weighting 18-24g, were housed in plastic cages and were fed stan-dard mouse chow and water at Tokyo Medical andDental University. Animal protocols were reviewedand approved by the Institutional Animal Care and UseCommittee.

Instillation with pigeon dropping extracts (PDE)PDE was obtained according to the previously

described method26. Briefly, fresh pigeon droppingwere stirred with a 20 volume of phosphate bufferedsaline solution (pH 7.4) for 24 h followed by dialysisagainst distilled water and lyophilized. PDE contains

large amounts of endtoxin, whose biological activitiesmight play a role in the initiation of PBL. Freeze-driedPDE was dissolved with sterile 0.9% saline andadjusted to 0.2 mg/ml. Under light anesthesia withinhalation of diethyl ether, PDE dissolved in 40 ml of0.9% saline (8 mg) was applied at the tip of the nose ofmice and they inhaled PDE involuntarily. Instillation wasconducted 3 days per week for an indicated period oftime. Control mice were administrated 40 ml of physio-logic saline solution in the same manner includinganesthesia.

Cigarette smokingMice were put into the capsules toward the cylinder

with Hamburg type (Borgwalt Co.) filled with cigarettesmoke of 10 filtered commercial cigarettes (Marlboro,Philip Morris Products, SA) once a day. Exposure wasdone 5 days per week from 1 week before the initiationof instillation with PDE.

Experimental designThree groups of mice were studied. (1) control mice

instilled with saline solution (group C). (2) miceinstilled with PDE (group P). (3) mice instilled with PDEand exposed to cigarette smoke (group P+S). Eachgroup was divided into two subgroups; instilled foreither 4 weeks (short-term exposure) and for 17weeks (long-term exposure).

Bronchoalveolar lavage (BAL)Plastic cannula was inserted into the trachea. BAL

samples were obtained by washing the whole lungswith 1.0 ml aliquots of 0.9% saline three times. Aftercentrifugation, BAL cell pellets were washed andresuspended in RPMI and total cell counts wereexamined. Cytospin preparations were fixed andstained using Diff-Quick staining, and differential cellcounts were made.

ELISA assay for anti-PDE IgG antibodyAntibody against PDE in sera was measured by an

enzyme-linked immunosorbent assay6. Each well ofpolystyrene plates (Immulon 2; DynatechLaboratories; Alex-andria, VA) was coated with a 100ml of 1mg/ml PDE in carbonate buffer (pH 9.6) at 4°Covernight. After washing the wells three times withphosphate buffered saline solution (pH 7.4) containing0.05% polysorbate-20 (Tween-20) (PBST), each wellwas treated with 0.05% bovine serum albumin-PBST toblock the free binding surface of the wells. Our previousstudy showed that the optical dilution to evaluate anti-

M. FURUIYE et al. J Med Dent Sci2

bodies was 1: 400 for the sera. Samples in 100 Òl quan-tities were added to each well and incubated at 37 °Cfor 1h. After washing the wells with PBST, 100 Òl of a1:1000 diluted solution of rabbit anti-mouse IgG cou-pled to horseradish peroxidase was added and incu-bated at room temperature for 1h. After washing thewells again, the substrate was added, and color wasdeveloped and measured at 490-nm and 620-nm.

Quantification of cytokine mRNA expression inthe lung

RNA was extracted from the left lung and INF-γ,TNF-α and IL-10 mRNA were quantified by a real timequantitative reverse transcriptase-polymerase chainreaction (RT-PCR)27. Amplification of cDNA was per-formed by ABI PRISM 7700 Sequence DetectionSystem using Taqman real time RT-PCR kit (AppliedBiosystems) with specific probe and primers accordingto the manufacture’s instructions. The results wereexpressed as the ratio of the experimental mice to thecontrol mice.

Lymphocyte proliferationBAL cells were resuspended in RPMI and 1x105 cells

were plated into wells of a 96-well plate in the presenceor in the absence of pigeon sera. We have notemployed PDE as the antigen for lymphocytes prolifer-ation test because crude PDE contains cytotoxic com-ponents to lymphocytes, but we have successfullyapplied pigeon sera to human BAL cells. After 5 day-culture with or without pigeon sera, [3H] thymidine wasadded to the culture for the last 24 h. Cell proliferationwas measured by an automated liquid scintillationcounter. The results were expressed as stimulationindex, which is the geometric mean counts per minuteof stimulated cultures with pigeon sera divided by thegeometric mean counts per minute of unstimulated cul-tures as control (without pigeon sera).

Quantification of hydroxyprolineContent of hydroxyproline in the right lung was

determined as described in detail elsewhere28. Briefly,lungs were homogenized with cold TCA followed byhydrochloric acid. Chloramine-T was added, followed byperchloric acid and p-demethyl-aminobenzaldehyde.Color change was assessed by spectrophotometryand estimated on a standard curve of hydroxyproline.

Histological evaluationIn the mice which had not been lavaged, the left lung

was perfused with 2% paraformaldehyde through the

left main bronchus to be fixed in 2% paraformaldehyde-PBS. The sections were embedded in paraffin, cut in5 Òm-thick sections, and stained with hematoxylin andeosin. The sections were evaluated under lightmicroscopy.

StatisticsStatistical analysis was done with post-hoc test

using Scheffe’s F test or Mann-Whitney nonparametricunpaired analysis using a statistical software (Statcel2). Values are expressed as the mean ±SEM. Valuesof p < 0.05 were considered as significant.

Results

Lung weight / body weight ratioLung weight / body weight ratio was estimated

before BAL. A short- term exposure to PDE induced anincrease in lung weight / body weight ratio, and a long-term exposure to PDE caused a more prominentincrease (Figure 1). There were no significantchanges of lung weight / body weight ratio between themice instilled with saline solution (0.0078±0.0009) andexposed to cigarette smoke alone (0.0071±0.0006)after a short-term exposure.

BALThe recovery rate of BAL fluid in all groups was more

than 80%. Total cells in BAL fluid were significantly (p <0.0001) increased after a long-term exposure com-

3CIGARETTE SMOKING IN PIGEON BREEDER’S LUNG MODEL

Fig. 1. Lung / body weight ratio. Open bar: control mice instilled with saline solution (group C);closed bar: mice instilled with PDE (group P); hatched bar: miceinstilled with PDE and exposed to cigarette smoke (group P+S); 4W:after 4 weeks; 17W: after 17 weeks. Data are expressed as themean±SEM for 4 (4W-P and 4W-P+S), 5 (4W-C), and 6 (17W) ani-mals. ** p<0.01 (Scheffe’s F test).

pared to those after a short-term exposure (Figure 2).There was no significant difference in the total cells inBAL fluid between group P and group P+S both in ashort-term and a long-term exposure.

Instillation with PDE also resulted in an increase inthe percentage of lymphocytes both in a short-term anda long-term exposure. The percentage of lympho-cytes in group P+S was significantly less than group Pin a short-term exposure, whereas no significant differ-ence was detected between them in a long-termexposure (Figure 3).

Production of anti-PDE IgG antibodySignificantly higher titer of anti-PDE IgG antibody

was detected in group P than in group C both in ashort-term and a long-term exposure. Group P+Sseemed to produce less titer of the antibody than groupP both in a short-term and a long-term exposure, how-ever no significant difference was demonstrated(Figure 4).

Cytokine mRNA expression in the lung The ratio of mRNA expression of IFN-γ, TNF-α and

IL-10 in the lung compared with the controls after along-term exposure was demonstrated in Figure 5.Instillation of PDE upregulated the expression ofthese cytokines more than 100 times than controls.Cigarette smoke attenuated the increased expressionof these three cytokines.

Lymphocyte proliferation Proliferation of lymphocyte in BAL fluid induced by

pigeon sera was shown in Figure 6. The antigen-dependent lymphocyte proliferation was not demon-strated in group C with a stimulation index of 1.06(mean, n=2). In group P after a short-term and a long-term exposure, higher lymphocyte proliferation wasinduced with a stimulation index of more than 2.0.Cigarette smoke inhibited the induction of lymphocyteproliferation in a short-term exposure. However, in a

M. FURUIYE et al. J Med Dent Sci4

Fig. 2. Total cells in BAL fluid . Open bar: control mice instilled with saline solution (group C);closed bar: mice instilled with PDE (group P); hatched bar: miceinstilled with PDE and exposed to cigarette smoke (group P+S); 4W:after 4 weeks; 17W: after 17 weeks. Data are expressed as themean±SEM for 4 (4W-P and 4W-P+S), 5 (4W-C), and 6 (17W) ani-mals. ** p<0.01 (Scheffe’s F test).

Fig. 3. Subpopulation in BAL cells. Closed bar: neutrophils; hatched: lymphocytes. C: control miceinstilled with saline solution; P: mice instilled with PDE; P+S: miceinstilled with PDE and exposed to cigarette smoke; 4W: after 4weeks; 17W: after 17 weeks. Data are expressed as the mean±SEMfor 4 (4W-C, 4W-P+S, 17W-P, and 17W-P+S), 5 (4W-P), and 6 (17W-C) animals. *p<0.05; ** p<0.01 (Scheffe’s F test).

Fig. 4. Anti-PDE IgG antibody in sera.O.D.: optical density; open bar: control mice instilled with saline solu-tion (group C); closed bar: mice instilled with PDE (group P);hatched bar: mice instilled with PDE and exposed to cigarettesmoke (group P+S); 4W: after 4 weeks; 17W: after 17 weeks. Dataare expressed as the mean±SEM for 4 (4W-P and 17W-C), 5 (4W-C), and 6 (4W-P+S, 17W-P, and 17W-P+S) animals.** p<0.01(Scheffe’s F test).

long-term exposure, cigarette smoke did not alter theinduction, showing no significant difference betweengroup P and group P+S (Figure 6).

Lung hydroxyproline contentLung collagen was quantified by measuring the

content of hydroxyproline in the lung. In a short-termexposure, instillation with PDE with or without cigarettesmoke did not alter the lung hydroxyproline contentcompared with the controls (group P: 1.10±0.08;group P+S : 1 .10±0.05). On the other hand, group Pin a long-term exposure showed an increase in

hydroxyproline compared to a short-term exposure(Figure 7). In addition, a significant increase wasfound in group P+S compared to group P after a long-term exposure, showing that cigarette smokeenhanced the fibrotic process in the lung.

Histological findingsRepetitive intranasal instillation with PDE resulted in

inflammatory changes in the lung (Figure 8). A short-term exposure caused inflammatory changes in pre-dominantly peribronchiolar and perivascular regioncharacterized by infiltration with lymphocytes.Inflammatory changes in group P+S were less severecompared with group P. After a long-term exposure,inflammatory changes in peribronchiolar and perivas-cular region were less prominent compared to ashort-term exposure and alveolar macrophages infil-trating into alveolar space were observed. In group P+Safter a long-term exposure, increased numbers ofalveolar macrophages in the alveolar space wereobserved compared to group P.

Discussion

PBL is a form of HP caused by hypersensitivity reac-tions to inhaled pigeon-related antigens and the clinicaloutcome of PBL is variable1-4. Although many factorssuch as cigarette smoking, as well as the duration ofinhaled antigen exposure, the dosage of exposedantigens, and individual susceptibility and responsive-ness to the inhaled antigen may contribute to the out-

5CIGARETTE SMOKING IN PIGEON BREEDER’S LUNG MODEL

Fig. 5. Cytokine mRNA expression in the lung compared with con-trols after a long-term exposure.Vertical line represents the ratio of mRNA expression compared withcontrols. Closed bar: mice instilled with PDE (group P); hatched bar:mice instilled with PDE and exposed to cigarette smoke (group P+S).Data are expressed as the mean±SEM for 4 animals in each group.* p<0.05 (Mann-Whitney test).

Fig. 6. Lymphocyte proliferation in BAL fluid induced by pigeon sera. S.I.: stimulation index, mean counts in cells stimulated with pigeonsera divided by those unstimulated (without pigeon sera). Closed bar:mice instilled with PDE (group P); hatched bar: mice instilled withPDE and exposed to cigarette smoke (group P+S); 4W: after 4weeks; 17W: after 17 weeks. Data are expressed as the mean±SEMfor 4 animals in each group. * p<0.05 (Scheffe’s F test).

Fig. 7. Content of hydroxyproline in the lung. Vertical line represents the ratio of hydroxyproline compared with con-trols. Closed bar: mice instilled with PDE (group P); hatched bar:mice instilled with PDE and exposed to cigarette smoke (group P+S);4W: after 4 weeks; 17W: after 17 weeks. Data are expressed as themean±SEM for 4 (4W) and 6 (17W) animals. * p<0.05 (Scheffe’s Ftest).

M. FURUIYE et al. J Med Dent Sci6

Fig. 8. Microscopic photograph of the lung (Hematoxylin-eosin stain).Upper left: mice instilled with PDE (group P) after a short-term exposure (original magnification x 4); upper right: mice instilled with PDE andexposed to cigarette smoke (group P+S) after a short-term exposure (original magnification x4); middle left: group P after a long-term expo-sure (original magnification x4); middle right: group P after a long-term exposure (original magnification x 10); bottom left: group P+S after along-term exposure (original magnification x 4); bottom right: group P+S after a long-term exposure (original magnification x10).

come of the clinical course5,6, each factor has not beenfully analyzed because of the limitations of the retro-spective studies in humans. To better understand thedetailed pathogenesis of PBL, we developed an animalmodel of PBL using mice. In the present study wedemonstrated that repetitive intranasal instillation ofPDE induced lung inflammation associated with lym-phocytosis in BAL fluid, the production of anti-PDE anti-body, and a positive lymphocyte proliferation in thepresence of pigeon sera. Long-term exposure to PDEresulted in an increase in lung weight / body weightratio and in the total cell number in BAL fluids com-pared to short-term exposure. In addition, increasedamounts of hydroxyproline after a long-term exposuresuggested the development of fibrotic changes.These findings are similar to the pathological featuresof PBL in humans.

It has been shown that HP occurs more frequently innonsmokers than smokers, and several investigationshave demonstrated that cigarette smoke had a protec-tive effect on the development of HP both in human andanimal models7-9. Previous studies reported thatsmokers in bird fanciers produced lower concentrationsof IgG antibody against bird related antigens in seraand BAL fluids than non-smokers10-12. In contrast,short-term cigarette smoking was reported toenhance allergic airway inflammation in mice associat-ed with eosinophilic inflammation, increase the dendriticcells in BAL fluid, and increase serum antigen-specificIgE22. The present study using mice showed thatexposure to cigarette smoke had a tendency toreduce the production of anti-PDE IgG antibody.Cigarette smoke also lessened lymphocytosis in BALfluid and histological inflammatory changes after ashort-term exposure. The molecular mechanismsinvolved in the above result of our study are not clear. Ithas been reported that several proinflammatorycytokines such as TNF-α 13-15 and IFN-γ16, 17 play crucialroles in the development of HP and cigarette smokedownregulate the production of these cytokines18,19. Inthis study, exposure to cigarette smoke resulted in adecrease in mRNA expression of these proinflamma-tory cytokines in a short-term and a long-term expo-sure. Our results are consistent with the attenuation oflung inflammation induced by cigarette smoke in theearly phase of PBL. However, the enhanced lunginflammation by cigarette smoking after a long-termexposure is inconsistent with the reduced mRNAexpression of these cytokines. Cigarette smoking wasreported to directly stimulate alveolar macrophages toproduce other proinflammatory cytokines such as IL-1

and IL-623. Further studies will be necessary to eluci-date the molecular mechanism of the effect of cigarettesmoke on the pathogenesis of PBL.

The expression of CD80 and CD86 on alveolarmacrophages has been shown to increase in HPpatients compared with normal subjects as a result ofactive immune reactions, and to decrease in smokerscompared to nonsmokers leading to the less immunereactivity29,30. The low level of the expression of co-stim-ulatory molecules on alveolar macrophages of smokersmay be one reason for the protective effect of cigarettesmoke in developing HP30,31. In line with these obser-vations, cigarette smoke decreased pulmonary den-dritic cells and suppressed lipopolysaccharide-induced production of inflammatory cytokines byinhibiting the activation of activator protein-1 inbronchial epithelial cells20,21. In contrast, pulmonaryinflammation was induced accompanied with anincreased amount of infiltration of the dendritic cells inthe airways and the lung parenchyma in a mousemodel exposed with cigarette smoke for 24 weeks24.We have examined the expression of co-stimulatorymolecules on alveolar macrophages by flow cytometryand showed no significant decrease in the expressionof CD80 and CD86 on alveolar macrophages of miceexposed to cigarette smoke for 4 weeks in a preliminarystudy. These results suggest that the effect of cigarettesmoke on decreased inflammation in the lungs is notdue to the decreased expression of co-stimulatorymolecules on antigen presenting cells in the airways.However, since we have not examined the expressionof co-stimulatory molecules on dendritic cells in the tho-racic lymph nodes, our results are not conclusive32.

The protective effects of cigarette smoke seen in ashort-term exposure were no longer observed in a long-term exposure. The lung weight / body weight ratio andthe total cell numbers of the BAL fluid tended to beincreased compared to unexposed mice, and a signifi-cant increase in lung hydroxyproline was induced bycigarette smoke. These observations are consistentwith our previous study of 32 chronic PBL patientsshowing the high rate of smokers, accounting for62.5% at the time when the sensitization begun25. Toour knowledge, there had been no reported animalmodel of chronic HP showing the effect of cigarettesmoke exposure for the extended period of experi-mental time (long-term exposure). Our study is likely tobe more closely related to chronic PBL in humans thanprevious studies in sense of long-term sensitization33. Ina clinical study of chronic farmer’s lung, Ohtsuka et al.reported that cigarette smoke might promote an insid-

7CIGARETTE SMOKING IN PIGEON BREEDER’S LUNG MODEL

ious form of the disease that led to deterioration of theclinical outcome34. Our results also suggest that ciga-rette smoke attenuates the inflammation in the lung atthe early phase of PBL but enhances lung inflammationand lung fibrosis at the later phase. Cigarette smokemay induce the production of some profibroticcytokines such as TGF-β after a long-term exposure,and may upregulate the expression of matrix metallo-proteinase, such as MMP-9, observed in COPDpatients35. However, in this study, direct effect of ciga-rette smoke on the process of lung inflammation andfibrosis has insufficiently been examined, because wehave not prepared smoking alone group without instil-lation of PDE in the experimental design.

In conclusion, this study has demonstrated that along term repetitive intranasal instillation of PDEinduced lung inflammation associated with anincrease of lung hydroxyproline. Although a short-term cigarette smoke attenuated lung inflammation, along-term cigarette smoke enhanced lung inflammationwith more lung hydroxyproline. This is the first report inwhich an increase in lung hydroxyproline by a long termcigarette smoke was documented in an animal model ofPBL. Further studies are required to unveil the mecha-nisms involved in an enhancement of lung fibrosis bycigarette smoke.

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9CIGARETTE SMOKING IN PIGEON BREEDER’S LUNG MODEL