Cigarette Smoke Decreases Pulmonary Dendritic Cells ... - ATS Journals

Cigarette Smoke Decreases Pulmonary Dendritic Cells and Impacts Antiviral Immune Responsiveness Clinton S. Robbins, David E. Dawe, Susanna I. Goncharova, Mahmoud A. Pouladi, Anna G. Drannik, Filip K. Swirski, Gerard Cox, and Martin R. Sta¨mpfli Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, and Department of Medicine, McMaster University, Hamilton, Ontario, Canada

We investigated the impact of cigarette smoke exposure on respiratory immune defense mechanisms. Mice were exposed to two cigarettes daily, 5 d/wk, for 2–4 mo. Tobacco smoke decreased the number of dendritic cells (DCs) in the lung tissue. Furthermore, smoke exposure dramatically reduced the percentage of B7.1-expressing DCs. Because DCs are believed to be indispensable to the initiation of adaptive immune responses, we investigated the impact of cigarette smoke on immune responsiveness toward adenovirus. Mice were exposed to two cigarettes for 2–4 mo and inoculated with 2 ⫻ 108 pfu of a replication-deficient adenovirus on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Smoke exposure specifically prevented the expansion and maximal activation of CD4 T cells and reduced the number of both activated CD4 and CD8 T cells. Consequently, smoke exposure shifted the activated CD4:CD8 T cell ratio from 3 to 1.5 when compared with sham exposure. Significant decreases were also observed in serum adenovirus-specific pan IgG, IgG1, and IgG2a immunoglobulin levels, which was associated with diminished viral neutralization capacity. We demonstrate that chronic tobacco smoke exposure impairs the immune response against adenovirus. This may, in part, explain the increased prevalence of viral infections in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality in the adult population (1–3). WHO projections indicate that by 2020 COPD will be the fourth leading cause of death worldwide (4). COPD is characterized by airway obstruction that is not fully reversible and typically progressive (1–3). Although COPD is almost exclusively associated with cigarette smoking, only ⵑ 10–15% of smokers develop clinically manifest airway obstruction (1–3). This suggests that although smoking is the major risk factor, it is not sufficient for disease elicitation and progression. Additional risk factors for COPD include socioeconomic status (5), airway hyperresponsiveness and atopy (6, 7), childhood respiratory infections (8–10), air pollution (11), occupational hazard (12), and a rare heredi-

(Received in original form July 10, 2003 and in revised form August 7, 2003) Address correspondence to: Martin R. Sta¨mpfli, Ph.D., McMaster University, Department of Pathology and Molecular Medicine, Health Sciences Centre, Room 4H21A, 1200 Main Street West, Hamilton, ON, L8N 3Z5 Canada. E-mail: [email protected] Abbreviations: adenovirus, Ad; antigen-presenting cells, APCs; chronic obstructive pulmonary disease, COPD; cytotoxic lymphocytes, CTL; dendritic cells, DCs; enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC; Hanks’ balanced salt solution, HBSS; interferon-␥, IFN-␥; interleukin, IL; phycoerythrin, PE. Am. J. Respir. Cell Mol. Biol. Vol. 30, pp. 202–211, 2004 Originally Published in Press as DOI: 10.1165/rcmb.2003-0259OC on August 14, 2003 Internet address: www.atsjournals.org

tary deficiency of ␣1-antitrypsin (13). The clinical course of COPD is often associated with acute exacerbations of symptoms (14–17). Although exacerbations may be initiated by multiple factors, the most common identifiable associations are with bacterial and viral infections (18–22). This may suggest that cigarette smoke impacts respiratory defense mechanisms. Tobacco smoke impacts both innate and adaptive immunity (23), and it has long been proposed that human diseases associated with cigarette smoke may, in part, reflect an effect of smoking on the immune system (24). Cigarette smoke has been shown to decrease serum levels of all immunoglobulin classes (25–29) except for IgE (30). In a series of studies in smoking and nonsmoking pigeon breeders, lower levels of precipitating antibodies were detected in smokers (25, 26). Similar differences were observed between smoking and nonsmoking workers in poultry plants (27), cotton mills (28), and cigar plants (29), suggesting that cigarette smoke impacts the humoral arm of adaptive immunity. In contrast, conflicting results have been reported with respect to the effects of cigarette smoke on T cell responses. Although some studies suggest decreased proliferative responsiveness of lymphocytes from the lung-associated lymph nodes in cigarette smoke–exposed mice (31, 32), other studies do not report such differences (33). Similarly, Hughes and colleagues showed no change in the proliferative responses of peripheral blood T cells to PHA in smokers compared with nonsmokers (34). Antigen-presenting cells (APCs) are an important component in the initiation and maintenance of adaptive immune responses. Interestingly, there is a paucity of data regarding the impact of cigarette smoke on antigen presentation. Conflicting results have been reported with respect to levels of HLA-DR in the lungs of smokers (35–38). Moreover, Chang and coworkers have reported no change in the ability of APCs from lungassociated lymph nodes of smoke-exposed mice to stimulate T cell proliferation (32). Overall, whether or not cigarette smoke impacts APCs remains an unresolved issue. The objective of this study was to investigate the impact of chronic cigarette smoke exposure on respiratory immune defense mechanisms. Using a murine model of mainstream cigarette smoke exposure, we demonstrate that tobacco smoke decreases the number and alters the costimulatory molecule expression profile of dendritic cells in the lung. Following administration of a replication-deficient adenovirus, we observed decreased expansion of activated CD4 and CD8 T cells in the lung and altered antigen-specific immunoglobulin production. Given the association of tobacco smoke related disease with bacterial and viral infection,

Robbins, Dawe, Goncharova, et al.: Cigarette Smoke Alters Antiviral Immune Responsiveness

that cigarette smoke impairs lung adaptive immune responses to pathogens may have important implications for the pathogenesis of COPD.

Materials and Methods Animals Female C57BL/6 mice (6–8 wk old) were purchased from Charles River Laboratories (Montreal, PQ, Canada). All mice were maintained in Level B housing conditions in a 12-h light-dark cycle. Level B is an access-restricted area; cages, food, and bedding are autoclaved. All of the experiments described in this study were approved by the Animal Research Ethics Board of McMaster University.

Smoke Exposure Protocol Mice were exposed to mainstream tobacco smoke in a smoke exposure system that was initially developed for guinea pigs (39) and has since been adapted for mice (40). Mice were exposed to two cigarettes daily (1R1 or 1R3 reference cigarettes, Tobacco and Health Research Institute, University of Kentucky), 5 d/wk. To control for handling, groups of mice were placed into restrainers only (sham-exposure).

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Splenocyte and Lung Cell Isolation Spleens were removed and immediately placed on ice in Hanks’ balanced salt solution (HBSS; Gibco BRL, Grand Island, NY). The tissue was triturated between the ends of sterile frosted slides and the resulting suspension was filtered through nylon mesh (BSH Thompson, Scarborough, ON, Canada). The cell suspension was centrifuged at 250 ⫻ g for 10 min at 4⬚C. Red blood cells were lysed with ACK lysis buffer (0.5M NH4Cl, 10 mM KHCO3, and 0.1nM Na2EDTA at pH 7.2–7.4), and the splenocytes were washed twice with HBSS and resuspended in RPMI (Gibco BRL) supplemented with 10% fetal bovine serum, 1% L-glutamine (Sigma Chemicals Co., Oakville, ON, Canada) and 1% penicillin/streptomycin. For isolation of lung cells, lungs were flushed via the right ventricle with 10 ml of warm (37⬚C) HBSS (calcium- and magnesium-free) containing 5% fetal bovine serum (Sigma Chemicals Co.), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco BRL). The lungs were then cut into small pieces (ⵑ 2 mm in diameter) and shaken at 37⬚C for 1 h in 15 ml of 150 U/ml collagenase III (Worthington Biochemical, Freehold, NJ) in HBSS. Using a plunger from a 5-ml syringe, the lung pieces were triturated through a metal screen into HBSS, and the resulting cell suspension was filtered through nylon mesh. Cells were washed twice and mononuclear cells were isolated by density centrifugation in 30% Percoll (Pharmacia, Uppsala, Sweden).

Splenocyte and Lung Cell Culture Adenovirus Infection Mice were either sham-exposed or exposed to cigarette smoke for 2–4 mo. A quantity of 2 ⫻ 108 pfu of an E1/E3-deleted, replicationdeficient human type 5 adenovirus (Ad) (41) was administered intranasally on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Ad was delivered in a total volume of 20 ␮l of phosphate-buffered saline vehicle (two 10-␮l administrations 5 min apart) into anaesthetized animals.

Measurements of Cytokines Enzyme-linked immunosorbent assay (ELISA) kits for murine interleukin (IL)-4, IL-13, and interferon (IFN)-␥ were purchased from R&D Systems (Minneapolis, MN). The threshold of detection for each of these systems was ⬍ 2pg/ml.

Measurement of Immunoglobulins in Serum Adenovirus (Ad)-specific immunoglobulins were detected in serum by ELISA. Blood was collected by retro-orbital bleeding and serum obtained by centrifugation after incubating whole blood for 30 min at 37⬚C. Samples were stored at ⫺20⬚C until assayed. MaxiSorb plates (NUNC Brand Products, Roskilde, Denmark) were coated overnight at 4⬚C with a cell lysate from adenoviral infected HELA cells. Subsequently, coated wells were blocked with 1% bovine serum albumin in phosphate-buffered saline for 2 h at room temperature. After washing, serum samples were incubated overnight at 4⬚C, washed, and developed with biotin-labeled, antimouse panIgG, IgG1, and IgA (Southern Biotechnology Associates, Birmingham, AL) and IgG2a (Pharmingen, Mississauga, Ontario, Canada). Plates were washed and incubated with alkalinephosphatase streptavidin for 1 h at room temperature. The color reaction was developed with p-Nitrophenyl phosphate tablets. Units were calculated based on serial dilutions from each sample as previously described (42).

Splenocytes and lung cells were cultured in RPMI (Gibco BRL) alone or with 109 pfu ultraviolet-inactivated, E1/E3-deleted, replication-deficient adenovirus at 8 ⫻ 105 cells/well in a 96-well flatbottom plate (Becton Dickinson, Lincoln Park, NJ). After 5 d of culture, supernatants were harvested for cytokine measurement.

Flow Cytometry The following monoclonal antibodies were selected to study the phenotype of lung mononuclear cells: anti-CD3 (biotin-conjugated 145–2C11); anti-CD4 (fluorescein isothiocyanate [FITC]-conjugated L3T4); anti-CD8 (FITC-conjugated Ly-2); anti-CD69 (phycoerythrin [PE]-conjugated H1 2F3); anti-B7.1 (biotin-conjugated 16–10AI); anti-B7.2 (biotin-conjugated GLI); anti-CD11b (Mac-1) (PE-conjugated MI/70); anti-CD11c (PE-conjugated HL3); and Streptavidin PerCP (Becton Dickinson, San Jose, CA) was used as a second-step reagent for detection of biotin-labeled antibodies. Titration was performed to determine the optimal concentration of each antibody. All antibodies were purchased from BD PharMingen (San Diego, CA). To minimize nonspecific binding, 106 cells were incubated with 0.5 ␮g Fc Block (CD16/CD32; Pharmingen) on ice for 15 min. Cells were then stained with first-stage monoclonal antibodies on ice for 30 min, washed, and then treated with second-stage reagents. Cells were fixed in 1% paraformaldehyde and counted on a FACScan (Becton Dickinson, Sunnyvale, CA). Analyses were performed using WinMDI software (Scripps Research Institute, La Jolla, CA). A total of 50,000–150,000 events were acquired and gated appropriately based on forward and side scatter plots.

Cytotoxic Lymphocyte Assay Cytotoxic lymphocyte (CTL) assays were performed on splenocytes isolated from smoke- or sham-exposed mice inoculated with Ad. Spleens were removed 5 d following last Ad administration, and the splenocytes isolated and stimulated with C57SV cells infected with Addl70–3 (E1/E3-deleted adenovirus) at an MOI of

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 30 2004

50. Target cells for the 51Cr-release assay were prepared by infecting C57SV cells for 24 h with an MOI of 50 of Addl70–4 (E1/E3 competent adenovirus). The effector cells (i.e., co-cultured splenocytes) were harvested, counted, and mixed in V-bottom microtiter dishes with target cells at various E:T ratios. After 4 h incubation at 37⬚C, 51Cr-release was measured by a ␥ counter, and the specific release was calculated as follows: [(experimental release ⫺ spontaneous release)/(maximum release ⫺ spontaneous release)] ⫻ 100%.

Ad Neutralization Assay The neutralizing capacity of sera was determined using a protocol described in detail elsewhere (43). Briefly, serial dilutions of serum (1:125–1:3,125) were incubated with 5 ⫻ 106 pfu of an adenoviral vector expressing the ␤-galactosidase gene of Escherichia coli (AdLacZ) (41) at 37⬚C for 60 min. Subsequently, the AdLacZ/serum mixtures were placed on confluent Hela cells for 60 min at 37⬚C and then washed. 1 ml of MEM F11 was added and cells were left for 16 h at 37⬚C. Cells were then lysed and ␤-galactosidase activity was developed with ␱-nitrophenol-␤-D-galactopyranoside (4 g/liter; Sigma). The reaction was terminated and the OD was read at 420 nm. Number of infectious particles was calculated according to a standard curve and percent neutralization was expressed relative to serum from naive animals.

Data Analysis Data are expressed as means ⫾ SEM. Statistical interpretation of results is indicated in figure legends. Differences were considered statistically significant when P ⬍ 0.05.

Results Impact of Cigarette Smoke Exposure on the Cellular Profile in the Lung Tissue Mice were exposed to two cigarettes/d for 5 d/wk. At 3, 6, and 10 mo, lung histology was assessed. In agreement with previous observations (40), we observed emphysematous lesions at 6 mo that were more pronounced after 10 mo (data not shown). Interestingly, emphysematous changes were not associated with marked tissue inflammation at any of the time points investigated. To assess the effect of cigarette smoke exposure on the cellular profile in the lung, mice were exposed to cigarette smoke for 2–4 mo. Lung mononuclear cells were isolated and CD4 T cells (CD3⫹/ CD4⫹), CD8 T cells (CD3⫹/CD8⫹), dendritic cells (MHCII⫹/CD11chigh), B cells (MHCII⫹/B220⫹), and macrophages (MHCII⫹/CD11b⫹) were assessed by flow cytometry. The total number of cells isolated from lungs of either naive or smoke-exposed mice was similar (1.54 ⫾ 0.32 ⫻ 106 versus 1.79 ⫾ 0.76 ⫻ 106/lung, respectively; mean ⫾ SEM, n ⫽ 6, Student’s t test P ⫽ 0.62). This cell population included both intra-alveolar and interstitial mononuclear cells. T cells were analyzed within the lymphocyte gate based on forward and side scatter patterns. No differences were observed in the percentage of CD4 and CD8 T cells in the lungs of smoke-exposed mice compared with naive animals (Figures 1A and 1B). Furthermore, the activation status of T cells was unchanged by tobacco smoke exposure as assessed by

Figure 1. Flow cytometric analysis of T cell populations in the lung tissue. Lung mononuclear cells were isolated from either naive mice or animals exposed to two cigarettes daily, 5 d/wk for 2–4 mo. (A ) Activated CD4 T cells (CD3, CD4, and CD69). (B ) Activated CD8 T cells (CD3, CD8 and CD69); 50,000 events were collected within the lymphocyte gate. Data show one representative experiment of two. Cells were pooled from five mice in each experimental group.


expression of the early activation marker, CD69. Although cigarette smoke did not impact the number of macrophages (MHCII⫹/CD11b⫹) and B cells (MHCII⫹/B220⫹) (data not shown), we observed a significant decrease in the number of DCs (MHCII⫹/CD11chigh) in the lungs of smokeexposed mice compared with naive animals (Figure 2). The decrease was specific to the lung, because we did not observe similar changes in the draining lymph nodes (data not shown). Because expression of costimulatory molecules is required for effective antigen presentation by APC and we observed decreased numbers of DCs in the lungs of smokeexposed animals, we next assessed expression of CD80 (B7.1) and CD86 (B7.2) on pulmonary DCs by flow cytometry. Table 1 indicates that smoke exposure markedly decreased the percentage of DCs expressing CD80 compared with naive animals. Smoke exposure had much less of an effect on the percentage of pulmonary DCs expressing CD86. Cigarette Smoke Exposure Decreases CD4 T Cells in the Lung following Ad Infection Next, we investigated whether the impact of cigarette smoke exposure on DCs was associated with altered cellular responses to adenovirus. Mice were exposed to cigarette smoke for 2–4 mo and 2 ⫻ 108 pfu of replication-deficient Ad was delivered on three occasions, 2 wk apart, during the

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TABLE 1

B7.1 and B7.2 expression on DCs isolated from the lung tissue

Naive Smoke

B7.1 B7.2 B7.1 B7.2

Experiment #1

Experiment #2

42.8 21.3 9.3 18.4

22.5 28.4 10.7 21.6

Lung mononuclear cells were isolated from either naive mice or animals exposed to two cigarettes daily, 5 d/wk for 2–4 mo. Cells were gated on the DC population (MHCII⫹/CD11chigh) and then evaluated for the distribution of B7 markers on the cell surface. Data shown represent percentage of cells from two independent experiments. Cells were pooled from five mice in each experimental group.

last month of tobacco smoke exposure. Qualitative histologic assessment of the lung tissue revealed a mild pulmonary inflammatory infiltrate in both sham- and smoke-exposed animals following Ad delivery (data not shown). Quantification of the mononuclear cell compartment, however, showed a significant increase in the number of cells isolated from the lungs of sham-exposed, but not smoke-exposed mice following administration of Ad when compared with naive controls (Figure 3). We next characterized specific subsets of lymphocytes within the lung mononuclear cell compartment

Figure 2. Flow cytometric analysis of DCs in the lung tissue. Lung mononuclear cells were isolated from either naive mice or animals exposed to two cigarettes daily, 5 d/ wk for 2–4 mo. (A ) Gated mononuclear cells were assessed for cell surface expression of MHCII/CD11c. Data show one representative experiment of six. Cells were pooled from five mice in each experimental group. (B ) Total number of MHCII⫹/CD11chigh cells isolated per lung. Data were obtained from six independent experiments in which cells were pooled from five animals per group (mean ⫾ SEM; statistical analysis was performed using Student’s t test; P ⬍ 0.013).

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Figure 3. Total mononuclear cell number following adenovirus infection. Mice were either sham- or cigarette smoke–exposed for 2–4 mo and inoculated with 2 ⫻ 108 pfu replication-deficient Ad on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Animals were killed 5 d following the last Ad exposure. Data depict total number of mononuclear cells isolated per lung. Shown is the average of three independent experiments in which cells were pooled from five animals per group (mean ⫾ SEM; statistical analysis was performed on one-way ANOVA with Fisher LSD; P ⬍ 0.05).

using flow cytometry, namely CD4 (CD4⫹/CD3⫹) and CD8 (CD8⫹/CD3⫹) T cells. We observed a significant increase in the percentage of CD4 T cells in sham-exposed mice following Ad delivery (Table 2 and Figure 4A) compared with naive animals. Interestingly, the percentage of CD4 T cells in smoke-exposed mice inoculated with Ad was significantly reduced compared with sham-exposed animals following viral delivery and similar to the naive control. To investigate the activation status of the CD4 T cells, we assessed cell surface expression of the early activation marker, CD69. We observed an increase in the percentage of activated CD4 T cells in sham-exposed mice following Ad administration when compared with naive animals (Table 2 and Figure 4A). Smoke exposure led to a significant reduction in the percentage of CD69 expressing CD4 T cells

TABLE 2

T cell expansion and activation in the lung tissue

CD4 T cells CD3⫹CD4⫹ CD69⫹ CD8 T cells CD3⫹CD8⫹ CD69⫹

Naive

Sham/Ad

Smoke/Ad

13.7 ⫾ 0.9 10.8 ⫾ 1.3

19.7 ⫾ 1.5* 46.6 ⫾ 8.2*

13.9 ⫾ 0.8† 26.3 ⫾ 2.6†

7.7 ⫾ 0.4 10.4 ⫾ 1.2

10.3 ⫾ 0.9 27.9 ⫾ 8.3

9.7 ⫾ 0.2 26.0 ⫾ 5.0

Mice were exposed to cigarette smoke for 2–4 mo and administered 2 ⫻ 108 pfu Ad on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Lung mononuclear cells pooled from five animals per treatment group were isolated 5 d after the last Ad exposure and stained for flow cytometric analysis of CD3, CD4, CD8, and CD69 expression; 50,000 events were collected within the lymphocyte gate as assessed by forward and side scatter. Data shown represent percentage of cells within the lymphocyte gate (mean ⫾ SEM; n ⫽ 3). Statistical analysis was performed by one-way ANOVA with Fisher LSD; P ⬍ 0.05. * Statistically different from naive. † Statistically different from sham/Ad.

following Ad delivery. When factoring in the total cell number, Figure 4C shows that smoke exposure decreased the number of activated CD4 T cells in smoke-exposed mice by ⵑ 80% when compared with sham-exposed animals following Ad delivery. Because the number of activated CD4 T cells was similar between naive mice and animals exposed to tobacco smoke only (data not shown), the differences that we observed between sham- and smoke-exposed mice receiving Ad reflected an altered responsiveness to the virus and not an effect of smoke alone. We observed similar percentages of CD8 T cells expressing CD69 in the lungs of both sham- and cigarette smoke– exposed animals following Ad administration (Table 2 and Figure 4B). Taking the total cell number into consideration however, smoke-exposed mice receiving Ad had, on average (based on three independent experiments), ⵑ 60% fewer activated CD8 T cells than sham-exposed mice inoculated with Ad. Figure 4C shows one representative experiment. Overall, we observed a shift in the ratio of activated CD4:CD8 T cells following Ad delivery from 3 in shamexposed mice, to 1.5 in cigarette smoke–exposed animals. In naive mice this ratio was ⵑ 2.0. Impact of Cigarette Smoke Exposure on In Vitro Ad-Specific Cytokine Production and CTL Responsiveness Given the decrease in activated CD4 T cells in smokeexposed mice, we investigated in vitro Ad-specific cytokine production by lung and spleen mononuclear cells. Mice were exposed to cigarette smoke for 2–4 mo, and 2 ⫻ 108 pfu of a replication-deficient Ad was delivered on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Lung mononuclear cells and splenocytes were isolated 5 d after the last viral delivery and placed in culture. Ad-specific production of IFN-␥, IL-4, and IL-13 was subsequently assessed in cell culture supernatants. Sham- and smoke-exposed animals yielded significant Adspecific production of both IFN-␥ and IL-13 in the lung and the spleen (Table 3). Levels of IFN-␥ production were similar in both groups. In contrast, we observed a 30% decrease in IL-13 production in the spleens of smokeexposed animals following administration of Ad compared with sham-exposed mice that had received Ad. IL-4 production was not detected in any of the culture conditions (data not shown). Furthermore, we did not detect virus-specific cytokine production in the lungs and spleens of naive animals (data not shown). Next we investigated the impact of cigarette smoke exposure on Ad-specific CTL responses. Mice were exposed to cigarette smoke for 2–4 mo, and 2 ⫻ 108 pfu Ad was delivered on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Splenocytes were harvested from mice 5 d after the last viral administration. Though we observed Ad-specific CTL activity in both sham- and smoke-exposed mice compared with uninfected controls (Figure 5), there were no differences in CTL activity between the two groups. Altered Ad-Specific Immunoglobulin Production in Cigarette Smoke–Exposed Mice An important function of CD4 T cells in viral immune responses is to provide B cell help. Therefore, that the


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Figure 4. Expansion and activation of T cell subsets in the lung. Mice were either sham- or cigarette smoke– exposed for 2–4 mo and inoculated with 2 ⫻ 108 pfu replication-deficient adenovirus on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Lung mononuclear cells were isolated and pooled from five animals per treatment group, 5 d after the last Ad delivery, and stained for flow cytometric analysis of (A ) activated CD4 T cells (CD3, CD4, and CD69) and (B ) activated CD8 T cells (CD3, CD8, and CD69); 50,000 events were collected within the lymphocyte gate as assessed by forward and side scatter. (C ) Total number of activated CD4 and CD8 T cells isolated per lung. Data show one representative experiment of three.

number of activated CD4 T cells was decreased in smokeexposed mice receiving Ad led us to investigate the impact of cigarette smoke exposure on Ad-specific immunoglobulin production. Mice were exposed to cigarette smoke for 2–4 mo, and 2 ⫻ 108 pfu Ad was delivered on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Ad-specific panIgG, IgG1, IgG2a, and IgA were measured in serum 5 d after the last viral administration. In sham-exposed mice, Ad delivery induced a robust antigenspecific immunoglobulin response (Figure 6). Cigarette smoke exposure significantly inhibited the production of panIgG, IgG1, and IgG2a by 60, 90, and 40%, respectively. Similar levels of IgA were detected in both sham- and smoke-exposed mice inoculated with Ad. We did not observe Ad-specific immunoglobulins in naive mice. To elucidate whether decreased titres of Ad-specific IgG in smoke-exposed mice were associated with altered neutralization of virus, we assayed the neutralizing activity of

the serum. We observed a decreased neutralizing capacity of serum from smoke-exposed mice that had received Ad compared with sham-exposed animals inoculated with Ad (Figure 7).

Discussion The objective of this study was to investigate the impact of chronic tobacco smoke exposure on respiratory immune defense mechanisms. To this end, we used a mainstream cigarette smoke exposure system that is widely used as an experimental murine model of emphysema (39, 40). In agreement with these previous studies, we observed emphysematous lesions following 6 mo of cigarette smoke exposure that were even more pronounced by 10 mo (44). Despite reports of increased inflammation in the lungs of smokers (45–48), emphysema formation in our model was not associated with tissue inflammation or an expansion of CD8 T

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TABLE 3

In vitro Ad-specific cytokine production Lung Sham/Ad

IL-13 IFN-␥

Medium Ad Medium Ad

5 22 1 1,793

⫾ ⫾ ⫾ ⫾

5 9* 0 471*

Spleen Smoke/Ad

15 24 0 2,134

⫾ ⫾ ⫾ ⫾

2 4* 0 286*

Sham/Ad

10 125 894 17,468

⫾ ⫾ ⫾ ⫾

2 23* 394 2,384

Smoke/Ad

2 86 370 20,581

⫾ ⫾ ⫾ ⫾

2 11*† 86 4,364*

Mice were exposed to cigarette smoke for 2–4 mo and infected with 2 ⫻ 108 pfu Ad on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Sham mice were infected with Ad, but were not exposed to cigarette smoke. Lung mononuclear cells and splenocytes were harvested 5 d after the last Ad exposure and placed in culture for 5 d in either medium or ultraviolet-inactivated Ad. Cytokine production was assessed in cell culture supernatants by ELISA (n ⫽ 3 for naive; n ⫽ 5 for all other groups). Data are expressed as means ⫾ SEM (pg/ml). Statistical analysis was performed by one-way ANOVA with Fisher’s LSD; P ⬍ 0.05. * Statistically different than medium. † Statistically different than sham/Ad.

cells. This may underscore the importance of other factors in the induction of inflammatory responses associated with cigarette smoking status (5–12). Cigarette smoke exposure dramatically reduced the number of dendritic cells in the lung. These changes were observed following 2 mo of tobacco smoke exposure and preceded the formation of emphysematous lesions. Furthermore, cigarette smoke exposure resulted in fewer DCs expressing the costimulatory molecule, B7.1. That smoke exposure impacts the DC compartment may have profound effects on immune responsiveness. Specifically, antigens are captured at the mucosal surface by APCs and subsequently processed and presented to lymphocytes in the draining lymphoid structures (49, 50). DCs are the most potent APCs and are likely indispensable to the initiation of T cell immunity. To assess the effect of cigarette smoke on adaptive immune responses, mice were inoculated with a replicationdeficient Ad. Ad is clinically relevant, because an increased

Figure 5. Ad-specific CTL responsiveness in the spleen. Mice were either sham- or cigarette smoke–exposed for 2–4 mo and inoculated with 2 ⫻ 108 pfu replicationdeficient adenovirus on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Splenocytes were harvested 5 d following the last viral delivery and stimulated with Ad-infected C57/SV cells for 5 d. Splenocytes were assessed by 51Chromium release assay for their ability to lyse either uninfected or Ad-infected C57/SV cells at effector:target ratios of 10, 30, and 90. Data represent three independent experiments in which cells were pooled from five animals per group (mean ⫾ SEM; statistical analysis was performed using one-way ANOVA with Fisher LSD; P ⬍ 0.05. *Statistically different from uninfected controls). Filled circles, smoke-infected; open circles, sham-infected; filled triangles, smoke-uninfected; open triangles, sham-uninfected.

incidence of infection has been reported in both stable COPD and during acute exacerbation (51). Intranasal delivery of Ad resulted in a mild lung inflammatory infiltrate in both sham- and smoke-exposed animals. Quantification of the mononuclear cell population, however, revealed a significant increase in the number of cells in sham-exposed mice receiving Ad only. Interestingly, tobacco smoke exposure led to a specific decrease in the percentage of activated CD4, but not activated CD8 T cells in the lung following Ad administration when compared with sham-exposed animals inoculated with Ad. Furthermore, upon taking the total cell number into effect, tobacco smoke decreased the numbers of both activated CD4 and CD8 T cells by approximately

Figure 6. In vivo Ad-specific immunoglobulin production. Mice were either sham- or cigarette smoke–exposed for 2–4 mo and inoculated with 2 ⫻ 108 pfu replication-deficient Ad on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Lung mononuclear cells were isolated 5 d after the last Ad exposure. Levels of Ad-specific pan-IgG, IgG1, IgG2a, and IgA were measured in the serum by ELISA. Data shown represent mean ⫾ SEM; n ⫽ 3 for naive, n ⫽ 7 for sham, n ⫽ 8 for smoke; except for IgG2a where n ⫽ 17 for sham, n ⫽ 18 for smoke. Statistical analysis was performed using one-way ANOVA with Tukey Test; P ⬍ 0.05.


Figure 7. Ad neutralization capacity. Mice were either sham- or cigarette smoke–exposed for 2–4 mo and inoculated with 2 ⫻ 108 pfu replication-deficient adenovirus on three occasions, 2 wk apart, during the last month of tobacco smoke exposure. Serum was collected 5 d after the last viral delivery and adenovirus neutralization was determined in vitro. Data show serum samples from ten animals/ group (mean ⫾ SEM; statistical analysis was performed using Student’s t test; P ⬍ 0.05; *statistically different from Sham/Ad).

80% and 60%, respectively. Consequently, the ratio of activated CD4:CD8 T cells decreased from 3 to 1.5. It is noteworthy that similar observations have been reported in the peripheral blood and bronchoalveolar lavage fluid of smokers (52, 53). Our data also suggest that the increased levels of CD8 T cells observed in COPD may not necessarily be due to the selective recruitment/expansion of CD8 T cells, but rather a failure in the CD4 T cell population to respond to virus. The aforementioned changes were specific to an effect of tobacco smoke on the antiviral immune response, because cigarette smoke on its own did not impact the CD4 and CD8 T cell compartments. Also, the inhibitory effect of cigarette smoke on T cell populations was associated with prolonged exposure to cigarette smoke, because only 2 wk of smoke exposure before Ad delivery did not induce similar changes (data not shown). The impact of cigarette smoke on T cell responsiveness to Ad may be related to the decreased number and altered costimulatory molecule profile of lung DCs observed in tobacco smoke–exposed mice. DCs have multiple functions, including antigen presentation and production of proinflammatory and immune regulatory cytokines, all of which are important to antiviral immunity. Although we have not determined the impact of tobacco smoke on in vivo DC function, Nouri-Shirazi and coworkers recently demonstrated that in vitro exposure of bone marrow–derived DCs to nicotine, one of the main components of tobacco smoke, inhibited their ability to produce cytokines and stimulate allogeneic T cells (54). Furthermore, that we still observe markedly decreased DC numbers following viral infection (data not shown) suggests that even in the event that DC function is not impaired, there are fewer cells in the lungs to perform these tasks. Alternatively, it may be argued that cigarette smoke impairs T cell function directly as suggested by Kalra and colleagues, who demonstrated impaired antigen-mediated T cell signaling in tobacco smoke–exposed rats (55). To our knowledge, our data demonstrate for the first time that chronic cigarette

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smoke exposure directly impacts lung T cell responses toward Ad in vivo. That smoke exposure did not affect in vitro levels of IFN-␥ and IL-13 produced by lung mononuclear cells or CTL activity in the spleen was unexpected, given the changes observed in T cell phenotype. However, we did observe a significant reduction in IL-13 production in the spleens of smoke-exposed animals, suggesting an impact of tobacco smoke on T cell effector function. Furthermore, because Ad-specific immunoglobulin production is dependent upon CD4 T cell help (56), the observed reduction in antibody production and decreased antiviral neutralizing activity following cigarette smoke exposure may be a consequence of alterations in the lung CD4 T cell compartment. Although T and B cell interactions are generally thought to occur in the draining lymph nodes, Bachmann and colleagues have shown that the maturation of memory B cells into antibody-producing plasma cells is induced at the site of antigen persistence (57). Because Ad was delivered to the airway, tobacco smoke may impact this process. Our observation that tobacco smoke inhibits immunoglobulin production is consistent with previous reports (58, 59). Interestingly, this inhibition was more pronounced on IgG1 production, a Th2 immunoglobulin phenotype, than IgG2a. This finding is consistent with the specific reduction observed in Ad-specific IL-13 in splenocyte cultures, suggesting a potential role for tobacco smoke exposure in the polarization of T helper responses as proposed by Majori and colleagues (60). Adaptive immune responses are important in both the initial clearance of viral pathogens and protection from recurring infection (61). Here we present data that indicates cigarette smoke exposure impacts both cellular and humoral immune responses against viral antigens. It could therefore be argued that the increased prevalence of viral infections as well as the persistence of cells expressing viral proteins in patients with COPD (62) may be due to tobacco smoke associated impairment of antiviral immunity. Speculatively then, early treatment of patients with COPD should include strategies to combat infection and/or to stimulate the immune system. This notion is supported by a study demonstrating an effect of an immunostimulating agent in reducing the likelihood of severe respiratory events in COPD leading to hospitalization (63). In summary, chronic tobacco smoke exposure reduced the percentage of pulmonary DCs in the lung and altered their costimulatory molecule expression profile. In addition, smoke exposure prevented the specific expansion and maximal activation of CD4 T cells and reduced the number of both activated CD4 and CD8 T cells in response to Ad. Finally, marked reductions were observed in serum levels of Ad-specific immunoglobulins which were associated with diminished viral neutralizing capacity. Because both cellular and humoral immunity provide protection against viral and bacterial pathogens, our findings may, in part, explain the increased prevalence of infections in COPD. Acknowledgments: The secretarial assistance of Mary Kiriakopoulos is gratefully acknowledged. The authors also thank Christine Moore, Mark Jordana, Sussan Kianpour, and Tina Walker for their expert technical assistance in exposing mice to cigarette smoke. And finally, a special thank you to Greg Harder and Dr. Jonathan Bramson for their help with the CTL assays. This study was supported

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by AstraZeneca Canada, and the Hamilton Health Sciences Corporation and St. Joseph’s Hospital. F.K.S. is holder of a K. M. Hunter/CIHR Doctoral Research Award, A.G.D. holds a CIHR R&D fellowship, and M.R.S. is holder of a Fellowship from the Parker B. Francis Foundation.

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