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Apr 24, 2006 - Am J Respir Crit Care Med Vol 174. pp 1342–1351, 2006. Originally Published in ...... kine regulation in the lungs of cigarette smokers. Am J Respir Crit ... Natural killer cell activity in cigarette smokers and asbestos workers. Am Rev Respir Dis .... Lymph node dendritic cells control CD8 T cell responses ...
Cigarette Smoke Impacts Immune Inflammatory Responses to Influenza in Mice Clinton S. Robbins, Carla M. T. Bauer, Neda Vujicic, Gordon J. Gaschler, Brian D. Lichty, Earl G. Brown, and Martin R. Sta¨mpfli Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, Department of Biology, and Department of Medicine, McMaster University, Hamilton; and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada

Rationale: Studies have shown that cigarette smoke impacts respiratory host defense mechanisms; however, it is poorly understood how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents. Objective: The objective of this study was to investigate the impact of mainstream cigarette smoke exposure on immune inflammatory responses and viral burden after respiratory infection with influenza A. Methods: C57BL/6 mice were sham- or smoke-exposed for 3 to 5 mo and infected with either 2.5 ⫻ 103 pfu (low dose) or 2.5 ⫻ 105 pfu (high dose) influenza virus. Measurements and Main Results: Although smoke exposure attenuated the airway’s inflammatory response to low-dose infection, we observed increased inflammation in smoke-exposed compared with sham-exposed mice after infection with high-dose influenza, despite a similar rate of viral clearance. The heightened inflammatory response was associated with increased expression of tumor necrosis factor-␣, interleukin-6, and type 1 IFN in the airway, and increased mortality. Importantly, smoke exposure did not interfere with the development of influenza-specific memory responses; sham- and smoke-exposed animals were equally protected upon viral rechallenge. Conclusion: Our study suggests that, in mice, cigarette smoke affects primary antiviral immune-inflammatory responses, whereas secondary immune protection remains intact. Keywords: chronic obstructive pulmonary disease; inflammation; immunity; neutrophils

Epidemiologic studies clearly show that smoking is associated with an increased incidence of both upper and lower respiratory tract infections (1). For example, Aronson and colleagues reported that young smokers were at significantly greater risk of acquiring lower respiratory tract illness, with a longer duration of cough than nonsmokers (2). Furthermore, chronic obstructive pulmonary disease (COPD), a condition largely associated with cigarette smoking, is associated with periods of acute exacerbation of symptoms largely due to bacterial and viral infections (3–7). Together, these findings suggest that cigarette smoking may alter the way respiratory pathogenic microorganisms are handled.

(Received in original form April 24, 2006; accepted in final form October 5, 2006 ) Supported by the Canadian Institutes of Health Research. M.R.S. is a holder of a Canadian Institutes of Health Research New Investigator award. Correspondence and requests for reprints should be addressed to Martin R. Sta¨mpfli, Ph.D., McMaster University, Department of Pathology and Molecular Medicine, MDCL, Room 4011, 1200 Main Street West, Hamilton, ON, L8N 3Z5 Canada. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 174. pp 1342–1351, 2006 Originally Published in Press as DOI: 10.1164/rccm.200604-561OC on October 5, 2006 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Epidemiological studies show that smoking is associated with an increased incidence of respiratory tract infections. These findings suggest that cigarette smoking may alter the way respiratory pathogenic microorganisms are handled. What This Study Adds to the Field

Cigarette smoke affects primary antiviral responses, yet secondary immune protection remains intact. Exaggerated inflammatory responses to viral agents likely contribute to the decline in clinical status associated with COPD exacerbations.

Multiple host defense mechanisms are involved in protecting the lung from the potentially harmful effects of infection. These include physical barriers as well as a complex network of innate and adaptive immune mechanisms (8–10). That exposure to tobacco smoke impacts a number of these processes is suggested by studies demonstrating that cigarette smoke impairs mucociliary clearance and damages epithelial cell tight junctions (11, 12) (reviewed in Reference 13). Other studies have shown that cigarette smoke impacts numerous cell types of the immune system, including bronchial epithelial cells (14), alveolar macrophages (15–18), natural killer cells (19–21), dendritic cells (22, 23), and B and T lymphocytes (24–26). It is poorly understood, however, how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents. In the present study, we assessed the impact of mainstream tobacco smoke (MTS) on the course of viral infection with a replication-competent, mouse-adapted influenza A virus. Influenza infection is a significant concern for smoking populations. Nicholson and colleagues reported influenza infection in 23% of smokers compared with 6% in nonsmokers in a nonimmunized population between the ages of 60 and 90 yr (27). In another 3-yr study, 20% of reported hospital admissions for acute exacerbation of COPD presented with positive serology for influenza A (28). In addition to smoking cessation, annual influenza vaccination is an important measure for preventing COPD exacerbation. We demonstrate that MTS differentially affects airway inflammatory responses depending on the initial infectious dose. Specifically, we observed decreased inflammation in the lungs of mice infected with 2.5 ⫻ 103 pfu of influenza virus. In contrast, MTS exposure exacerbated airways inflammation and induced death after infection with a 2-log higher infectious dose (2.5 ⫻ 105 pfu), despite a similar lung viral burden as in sham-exposed mice. Importantly, MTS exposure did not interfere with the

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induction of protective antiviral immunity. Our study suggests that inappropriate inflammatory responses to viral agents may in part contribute to the inflammation observed in smokers and the decline in health status associated with COPD exacerbations. Some of the results in this study were previously reported in abstract form (29).

Measurement of Viral Titers in the Lung

METHODS

Flow cytometric analysis was used to study the phenotype of lung cells and is described in detail in the online supplement. Data were collected using a FACScan (Becton Dickinson, Sunnyvale, CA) and analyzed using WIN-MDI software (Scripps Research Institute, La Jolla, CA). A total of 5 ⫻ 104 to 1 ⫻ 105 events were acquired.

Animals Female C57BL/6 mice (6–8-wk-old) were purchased from Charles River Laboratories (Montreal, PQ, Canada). All experiments described in this study were approved by the McMaster University (Hamilton, ON, Canada) Animal Research Ethics Board.

Cigarette Smoke Exposure Mice were exposed to MTS in a smoke exposure system, as previously described (30). Mice were exposed to two cigarettes daily (1R3 reference cigarettes, Tobacco and Health Research Institute, University of Kentucky), 5 d/wk, for 3–5 mo. Sham-exposed animals were placed in restrainers only. Further details are provided in the online supplement.

Influenza Infection Isoflurane-anesthetized mice were infected intranasally with either 2.5 ⫻ 103 pfu (low-infectious dose) or 2.5 ⫻ 105 pfu (high infectious dose) of a mouse-adapted H1N1 influenza A (A/FM/1/47) virus in 35 ␮l of phosphate-buffered saline (PBS) vehicle. A/FM/1/47 is a fully sequenced, plaque-purified preparation that is biologically characterized with respect to mouse lung infections (31). Animals were not exposed to cigarette smoke on the day of viral delivery. After low-dose viral infection, animals were exposed to cigarette smoke until the day before they were killed. After high-dose viral infection, animals were exposed to cigarette smoke until 3 d after infection. After this time point, infected animals did not tolerate smoke exposure well.

Collection and Measurement of Specimens Bronchoalveolar lavage (BAL) fluid and peripheral blood were collected as described in the online supplement. The left lung lobe was removed before collecting the BAL fluid for assessment of viral burden. For histologic evaluation, lungs were inflated with 10% formalin at 25 cm H2O pressure and embedded in paraffin; 3-␮m-thick sections were stained with hematoxylin and eosin. A detailed description of all the methods used can be found in the online supplement.

Viral titers were determined from lung homogenates on Madin-Darby canine kidney monolayers, as outlined in the online supplement. Viral titers were expressed as the number of infectious viral particles per gram of lung tissue.

Flow Cytometry

Innate Immunostimulation with Polyinosine-Polycytidylic Acid At 24 h before influenza infection, animals were anesthetized and 100 ␮g polyinosine-polycytidylic acid (poly[I:C]; Sigma, Oakville, ON, Canada) was delivered intranasally in 30 ␮l PBS vehicle. Mice were killed 5 d after infection with either 2.5 ⫻ 103 pfu or 2.5 ⫻ 105 pfu influenza virus.

Statistical Analysis Data are expressed as means (⫾ either SD or SEM). Statistical interpretation of results is indicated in figure legends. All statistical analysis was performed using SigmaStat statistical software, version 2.0 (SPSS Inc., Chicago, IL). Differences were considered statistically significant when the p value was less than 0.05.

RESULTS Clinical Outcome

To assess the impact of MTS on the clinical course of influenza infection, sham- and MTS-exposed mice were infected with either low- (2.5 ⫻ 103 pfu) or high- (2.5 ⫻ 105 pfu) dose influenza A virus. Neither sham- nor MTS-exposed mice displayed overt clinical symptoms after infection with low-dose virus. On the other hand, infection with high-dose influenza resulted in a progressive worsening of clinical symptoms in both sham- and MTSexposed animals (data not shown). Animals became ruffled, hypomobile, and displayed evidence of labored breathing. Importantly, three of eight MTS-exposed animals died between 6 and 7 d after infection (Table 1). None of the sham-exposed mice died.

Myeloperoxidase Activity

BAL and Peripheral Blood Inflammatory Profile

Myeloperoxidase (MPO) activity was assessed in lung homogenates as described in the online supplement. MPO activity was expressed as units per gram lung tissue, where 1 U MPO was defined as the amount of enzyme capable of degrading 1 ␮mol H2O2/min at room temperature.

Infection of sham-exposed mice with either low- or high-dose influenza virus resulted in the induction of significant airways inflammation (Figure 1). Unlike the lower infectious dose, infection with 2.5 ⫻ 105 pfu influenza was associated with a more pronounced BAL neutrophilia. Whereas MTS attenuated the inflammatory response after low-dose viral challenge, we observed increased inflammation in MTS- compared with shamexposed mice after high-dose infection. Specifically, 7 d after

Measurements of Cytokines and Immunoglobulins Tumor necrosis factor (TNF)-␣, IL-6, and IFN-␥ levels were determined using Beadlyte mouse multicytokine fluorescent bead–based FLEX assays (Upstate Biotechnology, Charlottesville, VA) and quantified using a Luminex100 instrument (Upstate Biotechnology), according to the manufacturer’s instructions. The limit of detection for TNF-␣, interleukin (IL)-6, and IFN-␥ was 0.3, 1.2, and 6.8 pg/ml, respectively. Macrophage inflammatory protein (MIP)-2 was measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN). The limit of detection for MIP-2 was less than 1.5 pg/ml. Influenza-specific immunoglobulins were measured as described in the online supplement.

TABLE 1. SURVIVAL PROFILE Dose

Experimental Condition*

No. of Survivors/Total No.†

Low

Sham/influenza MTS/influenza Sham/influenza MTS/influenza

10/10 10/10 8/8 5/8

Plaque Reduction Assay

High

BAL samples were assessed for their ability to protect primary IFN regulatory factor-3–deficient mouse embryonic fibroblasts (kindly provided by Dr. Karen Mossman, McMaster University) from infection with a vesicular stomatitis virus expressing a green fluorescence protein under an endogenous viral promoter (VSV-GFP). Additional details of this protocol are provided in the online supplement.

Definition of abbreviation: MTS ⫽ mainstream tobacco smoke. * MTS- and sham-exposed mice were infected with either low- (2.5 ⫻ 103 pfu) or high- (2.5 ⫻ 105 pfu) dose influenza A. † Survival was assessed 7 d (low dose) and between 6 and 7 d (high dose) after infection.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006

Figure 1. Inflammatory profile in the bronchoalveolar lavage (BAL) fluid. Sham- (closed bars) and mainstream tobacco smoke (MTS)–exposed (open bars) mice were infected with either low-dose (upper panels) or high-dose (lower panels) influenza virus. BAL was performed at the indicated time points after infection, and the number of mononuclear cells and neutrophils was determined. Data shown represent means ⫾ SEM; n ⫽ 6–10. Statistical analysis was performed using one-way analysis of variance (ANOVA) with the Student-Newman-Keuls method; p ⬍ 0.05. *Statistically significant compared with uninfected mice; †statistically significant compared with sham-exposed, influenza-infected mice.

low-dose infection, we detected significantly fewer mononuclear cells and a trend toward decreased numbers of neutrophils in MTS- compared with sham-exposed mice. In contrast, infection with high-dose influenza A virus resulted in increased numbers of inflammatory cells in the BAL of MTS- compared with shamexposed animals; we observed more mononuclear cells and neutrophils in the airways of MTS-exposed mice compared with those of sham-exposed animals at 3 and 5 d after infection. Compared with uninfected animals, we observed significantly fewer lymphocytes in the peripheral blood of both sham- and MTS-exposed mice after high-dose influenza infection (Figure 2). Furthermore, MTS exposure was associated with elevated numbers of peripheral blood neutrophils compared with sham

exposure at 3 and 5 d after infection. In contrast, we did not observe any significant differences in the number of leukocytes isolated from the peripheral blood of sham- and MTS-exposed animals after infection with low-dose influenza virus (data not shown). Histologic Assessment of the Lung

Next, we investigated the impact of cigarette smoke exposure on lung pathology. We demonstrate that 3–5 mo of cigarette smoke exposure alone does not lead to overt tissue pathology (Figure 3B). Low-dose influenza infection was mainly associated with peribronchiolar and perivascular inflammation in both sham- and MTS-exposed mice (Figures 3C and 3D). In addition to peribronchiolar and perivascular inflammation, high-dose influenza infection resulted in the accumulation of inflammatory cells within the lung parenchyma of both sham- and MTSexposed mice, suggestive of greater alveolar involvement (Figures 3E–3H). Consistent with these observations, we isolated fewer cells from the lungs of sham- and MTS-exposed animals infected with low-dose (9.07 ⫾ 2.50 ⫻ 106 and 9.16 ⫾ 2.94 ⫻ 106 cells/lung, respectively; mean ⫾ SEM; n ⫽ 10) compared with high-dose (20.24 ⫾ 3.65 ⫻ 106 and 22.79 ⫾ 4.93 ⫻ 106 cells/lung, respectively; mean ⫾ SEM; n ⫽ 9) influenza virus. To determine whether MTS exposure was associated with increased tissue inflammation in animals infected with high-dose influenza, we assessed MPO activity in lung tissue homogenates. Although MPO activity was increased in the lungs of animals infected with influenza compared with uninfected mice (Table 2), we did not observe any significant differences in MPO levels between sham- and MTS-exposed, influenza-infected animals. Cytokine Levels in the Airway

Figure 2. Inflammatory profile in the peripheral blood. Sham- (closed bars) and MTS-exposed (open bars) exposed mice were infected with high-dose influenza virus. At the indicated time points after infection, peripheral blood was obtained, and the number of monocytes, lymphocytes, and neutrophils determined. Data shown represent means ⫾ SEM; n ⫽ 5–6. Statistical analysis was performed using one-way ANOVA with the Student-Newman-Keuls method; p ⬍ 0.05. *Statistically significant compared with uninfected mice; †statistically significant compared with sham-exposed, influenza-infected mice.

The result that MTS exposure altered inflammatory cell responses associated with influenza infection led us to investigate whether the expression of inflammatory cytokines were similarly affected. Levels of MIP-2, TNF-␣, IL-6, and IFN-␥ were assessed in the BAL fluid of MTS- and sham-exposed mice infected with either low- or high-dose influenza A virus. We observed increased expression of MIP-2, TNF-␣, and IL-6 after infection with either low- or high-dose influenza A compared with uninfected animals (Table 3). We detected similar levels of MIP-2, TNF-␣, and IL-6 in sham- and MTS-exposed animals 3 and 7 d after infection with low-dose influenza. Similarly, infection with

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Figure 3. Light photomicrograph of paraffin-embedded lung tissue sections. Sham- and MTS-exposed mice were infected with either low- or high-dose influenza virus, and lung tissues were collected and stained with hematoxylin and eosin. (A ) Sham-only exposure. (B ) MTS-only exposure. Sham- and MTS-exposed mice (C and D, respectively) infected with low-dose influenza were killed 7 d after infection. Sham- and MTS-exposed mice infected with highdose influenza virus were killed either 5 (E and F, respectively) or 7 (G and H, respectively) d after infection. Original magnification: ⫻50 of representative sections.

high-dose influenza was associated with comparable levels of MIP-2 in the airways of sham- and MTS-exposed animals. However, MTS exposure led to significantly increased levels of IL-6 and TNF-␣ compared with sham-exposed animals 3 and 5 d after infection. Plaque reduction assays further demonstrated that MTS exposure induced heightened type-1 IFN production compared with sham exposure after infection with high-dose influenza (Figure 4).

TABLE 2. MYELOPEROXIDASE ACTIVITY IN THE LUNG MPO (U/g Tissue) d.a.i. 0 3 5

Sham

MTS

240 ⫾ 72 1,181 ⫾ 213* 662 ⫾ 118

215 ⫾ 47 1,212 ⫾ 137* 852 ⫾ 133*

Definition of abbreviations: d.a.i. ⫽ days after infection; MPO ⫽ myeloperoxidase; MTS ⫽ mainstream tobacco smoke. MTS- and sham-exposed mice were infected with high-dose (2.5 ⫻ 105 pfu) influenza virus. MPO activity was assessed in lung tissue homogenates at the indicated time points. Data shown represent mean ⫾ SEM; n ⫽ 3 for sham and MTS (0 d.a.i.); n ⫽ 9 for sham and MTS (3 and 5 d.a.i). Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls method; p ⬍ 0.05. * Statistically significant compared with uninfected mice.

Compared with uninfected mice, IFN-␥ levels in the BAL fluid were elevated to a similar extent in both sham- and MTSexposed animals 1 wk after low-dose infection (data not shown). We did not observe increased levels of IFN-␥ in either sham- or MTS-exposed animals after high-dose infection (measurements were taken 5 d after infection, which is likely too early a time point to detect this cytokine). Viral Burden in the Lung

Next we assessed the effect of MTS on viral burden in the lungs. No virus was isolated from the lungs of uninfected animals (data not shown). MTS exposure was associated with a decreased viral burden compared with sham exposure 7 d after infection with low-dose influenza (Figure 5). MTS exposure had no impact on viral burden in the lungs either 3 or 5 d after infection with highdose influenza (Figure 5). T-Cell Profile of the Lung

The involvement of T-cell responses in the resolution of influenza infection is well established. To determine if MTS impacted the expansion and activation of CD4 and CD8 T cells after infection with influenza, sham- and MTS-exposed mice were infected with either low- or high-dose influenza virus. We assessed the percentage of CD4 (CD3⫹/CD4⫹) and CD8 (CD3⫹/ CD8⫹) T cells in the lung, as well as expression of the early activation marker CD69. Infection with both infectious doses of influenza led to significant and comparable increases in the

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006 TABLE 3. BRONCHOALVEOLAR LAVAGE CYTOKINES

Dose Low High

d.a.i. 0 3 7 3 5

MIP2

TNF-␣

IL-6

(pg/ml)

(pg/ml)

(pg/ml)

Sham 0 63 32 119 75

⫾ ⫾ ⫾ ⫾ ⫾

0 9* 6* 23* 11*

MTS 0 72 19 134 67

⫾ ⫾ ⫾ ⫾ ⫾

0 21* 4 12* 8*

Sham 5 48 55 64 74

⫾ ⫾ ⫾ ⫾ ⫾

2 14* 11* 6* 8*

MTS 5 42 43 90 98

⫾ ⫾ ⫾ ⫾ ⫾

2 9* 10* 13*,† 12*,†

Sham 2 331 487 272 400

⫾ ⫾ ⫾ ⫾ ⫾

2 83* 104* 78* 58

MTS 0 303 389 583 896

⫾ ⫾ ⫾ ⫾ ⫾

0 98* 106* 113*† 295*†

Definition of abbreviations: d.a.i. ⫽ days after infection; MIP ⫽ macrophage inflammatory protein; MPO ⫽ myeloperoxidase; MTS ⫽ mainstream tobacco smoke; TNF ⫽ tumor necrosis factor. MTS- and sham-exposed mice were infected with either low- (2.5 ⫻ 103 pfu) or high- (2.5 ⫻ 105 pfu) dose influenza A virus. Lungs were lavaged and cytokine production was assessed at the indicated time points. Data shown represent means ⫾ SEM; n ⫽ 3–10. Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls method; p ⬍ 0.05. * Statistically significant compared with uninfected mice. † Statistically significant compared with sham-exposed, influenza-infected mice.

percentage of activated CD4 and CD8 T cells in the lungs of sham- and MTS-exposed mice compared with uninfected animals (Table 4). MTS exposure resulted in a modest reduction in the percentage of CD69-expressing CD4 T cells in animals infected with high-dose influenza. We also determined the impact of MTS on intracellular expression of granzyme B expression in CD8 T cells, a previously described surrogate for antigenspecific T-cell activation (32). Table 4 demonstrates that, compared with sham exposure, MTS attenuated the percentage of granzyme B⫹ CD8 T cells in mice infected with low-dose influenza. Unexpectedly, we did not observe an increase in the percentage of granzyme B⫹ CD8 T cells in high-dose–infected animals compared with uninfected mice. Secondary Responses to Influenza A

Because MTS exposure was associated with an attenuated airways inflammatory response and accelerated clearance of influenza from the lung after infection with low-dose influenza, we investigated whether MTS affected the establishment of protective antiviral immunity. To this end, serum (IgG1 and IgG2a) and BAL fluid (IgA) levels of influenza-specific immunoglobulins were assessed 6 wk after infection. Similar levels of influenzaspecific IgG1, IgG2a, and IgA were detected in sham- and MTS-

exposed animals (Figure 6A). We did not detect influenzaspecific immunoglobulins in uninfected mice (data not shown). To assess whether MTS impacted immune protection, shamand MTS-exposed mice infected with 2.5 ⫻ 103 pfu influenza virus were rechallenged with high-dose influenza 12 wk after the initial infection; 5 d later, we assessed the BAL inflammatory profile in these animals. That we did not observe airways inflammation in either sham- or MTS-exposed mice suggests that both groups of animals were completely protected after rechallenge (Figure 6B). This was corroborated by a complete absence of virus in the lungs of rechallenged mice (Figure 6C). Moreover, neither sham- nor MTS-exposed mice displayed any clinical symptoms after rechallenge with high-dose influenza (data not shown). The ability to mobilize and activate antigen-specific memory T cells is likely important in protecting against infection with heterologous influenza strains, where previously established immunoglobulin responses may be ineffective. To determine the effect of MTS on the establishment of influenza-specific memory T-cell responses, the percentage of influenza-specific memory T cells was assessed in the lung and spleen by tetrameric flow cytometric staining. We detected similar percentages of nucleoprotein (NP) 366–374⫹ CD62Lhi and NP366–374⫹ CD62Llo T cells in the lungs and spleens of sham- and MTS-exposed animals 12 wk after infection (Table 5), likely representing previously described central memory and effector memory T-cell populations, respectively (33). Rechallenge with high-dose influenza was associated with an equivalent expansion in the percentage of NP366–374⫹ T cells in both the lungs and spleens of sham- and MTS-exposed animals. Innate Immune Intervention

Figure 4. Type 1 IFN activity in the lung. Sham- (closed bars) and MTSexposed (open bars) mice were infected with high-dose influenza virus. At 3 d after infection, type 1 IFN bioactivity in BAL fluid was determined by plaque reduction assay. Values represent relative percent protection conferred by BAL samples at the indicated dilutions. Data show one representative experiment of two; means ⫾ SD; n ⫽ 3. Statistical analysis was performed using a t test; p ⬍ 0.05. *Statistically significant compared with sham-exposed, influenza-infected mice.

Our data show that smoking affects inflammatory processes elicited by an initial encounter with influenza virus. Therefore, recently described antiviral intervention strategies that target innate immune mechanisms may be influenced by smoking. Specifically, poly(I:C), a synthetic mimic of viral double-stranded RNA, has been shown to confer protection against subsequent viral challenge in a nonimmunized host (34). To assess whether intranasal administration of poly(I:C) elicits protection against influenza infection, sham- and MTS-exposed animals were inoculated intranasally with poly(I:C) 24 h before viral infection. Poly(I:C) administration led to a significant decrease in total cells and the number of neutrophils in both sham- and MTS-exposed animals after infection with low-dose influenza (Figure 7). Although poly(I:C) administration did not affect the total number

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Figure 5. Viral burden in the lung tissue. Sham- (closed bars) and MTS-exposed (open bars) mice were infected with either low- or high-dose influenza virus. Lung viral titers were determined at the indicated time points after infection. Data represent means ⫾ SEM; n ⫽ 5–9. Statistical analysis was performed using t test; p ⬍ 0.05. *Statistically significant compared with sham-exposed, influenza-infected mice.

of inflammatory cells in the BAL of either sham- or MTSexposed animals after high-dose influenza infection, we observed significantly fewer mononuclear cells in the airways of poly(I:C)treated, MTS-exposed animals compared with untreated, MTSexposed mice (Figure 7).

DISCUSSION The objective of this study was to investigate the impact of active smoking on the course of respiratory viral infection in mice. Animals were exposed to MTS using a smoke exposure system for small rodents that was initially developed for guinea pigs (35) and has since been adapted for mice (30). Animals were infected with one of two infectious doses of a replicating, mouseadapted influenza A virus: 2.5 ⫻ 103 pfu (low dose) or 2.5 ⫻ 105 pfu (high dose). The low infectious dose led to an airways inflammatory response, but did not induce clinical symptoms, whereas infection with the high dose induced significant clinical symptoms and was associated with a more pronounced airway inflammation. Unlike administration of low-dose influenza, the higher infectious dose resulted in significant morbidity. Animals became ruffled, hypomobile, and displayed evidence of labored breathing. Importantly, whereas all of the sham-exposed mice survived

through 1 wk of infection, MTS exposure resulted in death for three of eight animals. Our data are consistent with clinical studies in large military cohorts demonstrating a worsening of clinical symptoms and an increased incidence of influenzarelated deaths among smokers compared with nonsmokers (36–38). MTS exposure attenuated the BAL inflammatory response to low-dose influenza infection. Although the underlying mechanism behind this observation remains unclear, the cellular toxicity of cigarette smoke has previously been demonstrated (39–41). Therefore, MTS may limit the extent of viral replication, either through its effects on airway epithelial cells, the primary infectious target of the virus, or by directly impacting viral replication. Consequently, the obligation to respond to a lower viral burden may result in the recruitment of fewer inflammatory cells into the airways. Alternatively, cigarette smoke may be toxic to immune inflammatory cells, and the reduced inflammation in the BAL is a reflection of this cytotoxicity. For example, it has recently been shown that activated granulocytes are susceptible to nitric oxide, a component of cigarette smoke (42). MTS exposure led to significantly increased numbers of mononuclear cells and neutrophils in the airways of mice infected with a 2-log higher infectious dose of influenza (2.5 ⫻ 105 pfu) when compared with sham-exposed animals. The exacerbating

TABLE 4. FLOW CYTOMETRIC ANALYSIS OF THE LUNG Dose Uninfected

CD4 T cells CD3⫹/CD4⫹ CD69 CD8 T cells CD3⫹/CD8⫹ CD69⫹ GrB⫹

Low

High

Sham

MTS

Sham

MTS

Sham

MTS

10.9 ⫾ 0.9 5.7 ⫾ 2.1

10.7 ⫾ 1.2 6.0 ⫾ 1.4

8.1 ⫾ 0.8 24.4 ⫾ 1.9*

9.1 ⫾ 1.0 21.0 ⫾ 2.7*

6.1 ⫾ 0.9* 32.5 ⫾ 3.7*

5.6 ⫾ 0.2* 24.1 ⫾ 1.7*‡

10.7 ⫾ 0.6 6.1 ⫾ 1.7 6.0 ⫾ 1.0

12.2 ⫾ 0.5 3.1 ⫾ 1.8 2.7 ⫾ 0.8

11.4 ⫾ 1.6 35.8 ⫾ 2.5* 18.6 ⫾ 2.6*†

10.0 ⫾ 1.1 30.4 ⫾ 3.3* 10.5 ⫾ 1.2‡

6.5 ⫾ 0.4* 27.5 ⫾ 5.1* 6.2 ⫾ 1.5

7.5 ⫾ 0.9* 31.2 ⫾ 4.1* 4.6 ⫾ 0.3

For definition of abbreviation, see Table 1. MTS- and sham-exposed mice were infected with either low- (2.5 ⫻ 103 pfu) or high- (2.5 ⫻ 105 pfu) dose influenza A virus. At 5 and 7 d after infection, respectively, lung mononuclear cells were isolated and flow cytometric analysis was used to identify activated CD4 T cells (CD4, CD3, and CD69), CD8 T cells (CD8, CD3, and CD69), and granzyme B–expressing CD8 T cells (CD8, CD3, and GrB). Values are expressed as percentages of cells within the lymphocyte gate. Data shown represent means ⫾ SEM; n ⫽ 5–10. Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls method; p ⬍ 0.05. * Statistically significant compared to MTS-exposed, uninfected mice. † Statistically significant compared with MTS-exposed, uninfected mice. ‡ Statistically significant compared with sham-exposed, influenza infected mice.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006 Figure 6. Secondary responses to influenza A. (A ) Sham- (closed circles) and MTS-exposed (open circles) mice were infected with 2.5 ⫻ 103 pfu influenza A virus. After 6 wk, immunoglobulin levels were measured in the serum (IgG1 and IgG2a) and BAL (IgA). Data shown represent means ⫾ SEM; n ⫽ 4–6. (B ) At 12 wk after infection with low-dose virus, MTS- and sham-exposed mice were rechallenged with 2.5 ⫻ 105 pfu influenza; 5 d after rechallenge, animals were killed, BAL was performed, and the number of mononuclear cells and neutrophils was determined. Data shown represent means ⫾ SEM; n ⫽ 4–6. Statistical analysis was performed using oneway ANOVA with the StudentNewman-Keuls method; p ⬍ 0.05. *Statistically significant compared with sham- and MTS-exposed mice after secondary infection; †statistically significant compared with sham-exposed mice after primary infection. (C ) Lung viral titres from animals in B were assessed. Statistical analysis was performed using a t test; p ⬍ 0.05. *Statistically significant compared with sham- and MTS-exposed mice after secondary infection.

effect of MTS on lung inflammation was specific to BAL specimens, as we isolated similar numbers of cells and detected comparable MPO activity in the lung tissue of sham- and MTSexposed, influenza-infected mice. Therefore, in our model, it is

TABLE 5. MEMORY T CELLS IN THE LUNG AND SPLEEN NP366–374⫹

Spleen Sham MTS Sham MTS Lung Sham MTS Sham MTS

Viral Rechallenge

CD62Llo

CD62Lhi

⫺ ⫺ ⫹ ⫹

0.16 0.12 0.57 0.48

0.26 0.23 0.24 0.23

⫺ ⫺ ⫹ ⫹

0.13 0.11 3.32 2.94

0.23 0.16 0.66 0.53

Definition of abbreviations: MTS ⫽ mainstream tobacco smoke; NP ⫽ nucleoprotein. MTS- and sham-exposed mice were infected with low-dose influenza. After 12 wk, mice were rechallenged with 2.5 ⫻ 105 pfu influenza A. Lung mononuclear cells and splenocytes were isolated either before or after viral rechallenge, and flow cytometry was used to identify NP366–374⫹ CD62Lhi and NP366–374⫹ CD62Llo T cells. Shown is one representative experiment of two. Values represent percentages of cells within the lymphocyte gate.

the extent of inflammation in the BAL rather than the lung tissue that is the best indicator of clinical status. Similar viral titers in the lungs of sham- and MTS-exposed animals indicated that factors other than antigen load likely contributed to the MTS-induced increase in BAL inflammation. Specifically, MTS exposure may have stimulated the bone marrow, as indicated by the increased numbers of neutrophils observed in the peripheral blood of MTS-exposed, influenza-infected animals. It has been demonstrated, in both animal and clinical studies, that cigarette smoking stimulates the bone marrow to release neutrophils into the circulation (43, 44). Cigarette smoke may also suppress local defense mechanisms in the lung, necessitating increased recruitment of monocytes and neutrophils from the periphery to compensate for local deficiencies. This hypothesis is supported by in vitro studies showing that bronchial epithelial cells produce fewer cytokines after stimulation with viral and bacterial agents in the context of cigarette smoke and its components (14). Inflammatory cytokines are a consequence of and help to drive inflammatory processes. The elevated levels of TNF-␣, IL-6, and type 1 IFN observed in the airway, therefore, may contribute to the increased inflammatory response observed in MTS-exposed, high-dose influenza–infected animals. TNF-␣ and IL-6 are well-described proinflammatory cytokines, with the latter demonstrating neutrophil chemoattractant activity (45). Type 1 IFNs initiate inflammatory cascades through the activity of IFN stimulatory genes (46), induce neutrophil respiratory burst responses (47), and prevent neutrophil apoptosis (48, 49).

Robbins, Bauer, Vujicic, et al.: Cigarette Smoke and Antiviral Responses

1349 Figure 7. Effect of polyinosine-polycytidylic acid (poly[I:C]) on airway inflammatory responses to influenza. Sham(closed bars) and MTS-exposed (open bars) mice were administered poly(I:C) 24 h before infection with either lowdose (upper panels) or high-dose (lower panels) influenza virus. At 5 d after infection, animals were killed, BAL was performed, and the number of mononuclear cells and neutrophils was determined. Data shown represent means ⫾ SEM; n ⫽ 5–7. Statistical analysis was performed using one-way ANOVA with the StudentNewman-Keuls method; p ⬍ 0.05. *Statistically significant compared with shamexposed, uninfected mice; §statistically significant compared with MTSexposed, uninfected mice; †statistically significant compared with shamexposed, influenza-infected mice; ‡statistically significant compared with MTSexposed, influenza-infected mice.

T cells play an important role in the resolution of influenza infection in the lung. Taking the total cell number into consideration, we detected similar numbers of activated (CD69⫹) CD4 and CD8 T cells in the draining lymph nodes and lungs of shamand MTS-exposed mice after infection with low- and high-dose influenza virus (data not shown, and Table 5). We have previously reported that MTS decreased the number of activated CD4 and CD8 T cells after infection with a replication-deficient adenovirus (22), suggesting that cigarette smoke differentially impacts immune responsiveness depending on the nature of the virus. CD8 T cells kill virus-infected cells through the release of cytotoxic granules, including perforin and granzyme B. Consequently, intracellular granzyme B expression has been demonstrated to be a functional marker of antigen specificity (32). In our studies, MTS exposure resulted in a statistically lower percentage of granzyme B–expressing CD8 T cells in the lungs of animals infected with low-dose influenza compared with sham exposure. Interestingly, this was not associated with an increased viral burden in the lungs of these animals. Unexpectedly, shamand MTS-exposed mice infected with high-dose influenza exhibited decreased percentages of CD4, CD8, and granzyme B–expressing CD8 cells compared with low-dose infection. This observation is consistent with a recent report by Legge and colleagues demonstrating that high doses of influenza induce apoptosis of virus-specific CD8 T cells, a process mediated by lymph node–resident dendritic cells in an IL-12–regulated, FasLdependent manner (50). Alternatively, the observed differences in T-cell percentages between the low- and high-dose influenza– infected groups may be a reflection of the timing of the measurements, 7 versus 5 d after infection for low- and high-dose infection, respectively. Influenza virus is largely cleared from the airway lumen and from within airway epithelial cells by mucosal IgA (51, 52). Serum-derived IgG isotypes are instrumental in clearing virus from the lung parenchyma and in protecting against reinfection (53–55). We demonstrate that low-dose influenza infection induced similar levels of influenza-specific mucosal IgA and serum IgG1 and IgG2a in sham- and MTS-exposed animals. Furthermore, animals were completely protected on rechallenge (Figures 5B and 5C). Mackenzie and colleagues previously showed that short- and long-term smoke exposure either enhanced or

depressed humoral responses, respectively (56). It is difficult to directly compare this study with our own, because the smoking apparatus and protocol, as well as the infectious strain of influenza, differed markedly. The methodology for assessing humoral responses was also dissimilar between the two studies. However, our data are consistent with those from subsequent studies by Mackenzie and colleagues and others, demonstrating that MTS does not impair secondary responses to influenza in experimental animals, and may account for the efficacy of annual influenza vaccination strategies in lowering the incidence of influenza infection in both asymptomatic smokers and patients with COPD (27, 57–59). Razani-Boroujerdi and colleagues recently demonstrated that in vivo administration of nicotine, a constituent in tobacco smoke with known immunosuppressive properties, significantly inhibited the tissue inflammatory response to a virulent mouseadapted influenza virus (PR8) in rats and mice that was associated with increased viral titers in the lung (60). Although this study assessed the effect of a single tobacco constituent on viral infection, tobacco smoke contains more than 4,500 compounds in the particulate and vapor phases. Our study assessed the collective effect of all these compounds on host defenses to influenza, and suggests a differential effect for individual tobacco components and whole-smoke exposure on antiviral responses. The need for annual reformulation of influenza vaccines arises from the continuous mutation of influenza coat proteins that render previously acquired humoral immunity ineffective. It has been suggested that novel vaccination strategies will need to induce memory T-cell responses to more conserved peptides if they are to protect against variant viruses that have undergone significant antigenic drift and antigenic shift. In addition to being highly conserved, the core NP is the dominant influenza T-cell epitope (61). Using major histocompatibility complex class I–restricted, NP-specific tetramer staining, we assessed influenzaspecific memory T cells in the lungs and spleens of animals infected with low-dose influenza. We detected antigen-specific, central (CD62Lhi) and effector (CD62Llo) memory T-cell populations in both the lungs and spleens of influenza-infected mice. Furthermore, MTS exposure did not interfere with the development of influenza-specific memory T-cell responses after either initial or secondary exposure to virus. To our knowledge, this

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is the first study to incorporate tetramer technology to assess the impact of MTS on pathogen-specific T-cell responses, and suggests that smokers would benefit from novel vaccine strategies aimed at amplifying memory T-cell populations. Interaction between pathogens and the host is mediated by pattern recognition receptors, including Toll-like receptors (TLRs). Recent studies have demonstrated that TLR2 expression is altered in cigarette smoke–exposed mice, as well as in smokers and patients with COPD, suggesting that smoking impacts innate immune responsiveness (62, 63). The importance of TLR3 in the innate recognition of many viruses, including influenza, is well documented (64). We show that MTS exposure did not impact cell surface/intracellular expression of TLR3 in the lung or interfere with the ability of animals to induce an antiviral state after influenza infection (data not shown and Figure 4). Moreover, Figure 7 demonstrates that poly(I:C) administration attenuated airway inflammation after influenza infection in both sham- and MTS-exposed animals, suggesting that TLR3 signaling was not compromised by cigarette smoke exposure. An increasing number of clinical studies have demonstrated the potential benefit of using innate bacterial immunostimulants to reduce both the number and severity of acute exacerbations (65, 66). Our findings suggest that similar consideration may be given to intervention strategies aimed at inducing innate antiviral immunity. The Global Initiative for Chronic Obstructive Lung Disease states that patients with COPD mount inappropriate inflammatory responses to noxious particles or gases within cigarette smoke (67). In our study, we show that cigarette smoke exacerbates inflammatory processes elicited by viral infection. We have previously demonstrated that MTS exacerbates inflammatory responses to bacterial agents and is associated with a decline in health status (68). We postulate that this altered responsiveness to respiratory pathogens contributes to the chronic inflammation observed in smokers and the decline in health status associated with COPD exacerbation. Furthermore, the fact that MTS exposure did not lead to increases in viral burden in the lung suggests that normal pathogen clearance does not preclude the development of exaggerated and potentially harmful inflammatory responses. In summary, we show that the effect of MTS on host responses to respiratory infection with influenza A is complex and at least partly dependent on the magnitude of the infectious dose. Although MTS exposure attenuated airways inflammation in mice infected with low-dose influenza, it exacerbated inflammation, increased the production of inflammatory cytokines, and led to death in animals infected with a 2-log higher infectious dose. We believe a comprehensive understanding of the impact of cigarette smoke on host responses to respiratory infections will yield novel insight into the underlying mechanisms connecting cigarette smoking, inflammatory processes, and COPD disease progression.

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Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment : The authors thank Joanna Kasinska, Sussan Kianpour, and Suzanna Gonchorova for their expert technical support. They also thank Mary Kiriakopoulos for her secretarial assistance.

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References 1. Arcavi L, Benowitz NL. Cigarette smoking and infection. Arch Intern Med 2004;164:2206–2216. 2. Aronson MD, Weiss ST, Ben RL, Komaroff AL. Association between cigarette smoking and acute respiratory tract illness in young adults. JAMA 1982;248:181–183. 3. Regueiro CR, Hamel MB, Davis RB, Desbiens N, Connors AF Jr, Phillips RS. A comparison of generalist and pulmonologist care for patients

25.

26.

hospitalized with severe chronic obstructive pulmonary disease: resource intensity, hospital costs, and survival. SUPPORT Investigators. Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatment. Am J Med 1998;105:366–372. Gibson PG, Wlodarczyk JH, Wilson AJ, Sprogis A. Severe exacerbation of chronic obstructive airways disease: health resource use in general practice and hospital. J Qual Clin Pract 1998;18:125–133. Soler N, Torres A, Ewig S, Gonzalez J, Celis R, El-Ebiary M, Hernandez C, Rodriguez-Roisin R. Bronchial microbial patterns in severe exacerbations of chronic obstructive pulmonary disease (COPD) requiring mechanical ventilation. Am J Respir Crit Care Med 1998;157:1498–1505. Wedzicha JA. Airway infection accelerates decline of lung function in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164:1757–1758. Sherk PA, Grossman RF. The chronic obstructive pulmonary disease exacerbation. Clin Chest Med 2000;21:705–721. Toews G. Pulmonary clearance of infection. In: Pennington JE, editor. Respiratory infections: diagnosis and management, 3rd ed. New York: Raven, 1994. Reynolds HY. Modulating airway defenses against microbes. Curr Opin Pulm Med 2002;8:154–165. Tamura S, Kurata T. Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 2004;57:236–247. Li XY, Donaldson K, Rahman I, MacNee W. An investigation of the role of glutathione in increased epithelial permeability induced by cigarette smoke in vivo and in vitro. Am J Respir Crit Care Med 1994; 149:1518–1525. Witten ML, Lemen RJ, Quan SF, Sobonya RE, Roseberry H, Stevenson JL, Clayton J. Acute cigarette smoke exposure increases alveolar permeability in rabbits. Am Rev Respir Dis 1985;132:321–325. Wanner A. The role of mucus in chronic obstructive pulmonary disease. Chest 1990;97:11S–15S. Laan M, Bozinovski S, Anderson GP. Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells. J Immunol 2004;173:4164–4170. Twigg HL III, Soliman DM, Spain BA. Impaired alveolar macrophage accessory cell function and reduced incidence of lymphocytic alveolitis in HIV-infected patients who smoke. AIDS 1994;8:611–618. McCrea KA, Ensor JE, Nall K, Bleecker ER, Hasday JD. Altered cytokine regulation in the lungs of cigarette smokers. Am J Respir Crit Care Med 1994;150:696–703. Ohta T, Yamashita N, Maruyama M, Sugiyama E, Kobayashi M. Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages. Respir Med 1998;92:922–927. Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM. Suppression of human IL-1␤, IL-2, IFN-␥, and TNF-␣ production by cigarette smoke extracts. J Allergy Clin Immunol 2000; 106:280–287. Newman LS, Kreiss K, Campbell PA. Natural killer cell tumoricidal activity in cigarette smokers and in silicotics. Clin Immunol Immunopathol 1991;60:399–411. Ginns LC, Ryu JH, Rogol PR, Sprince NL, Oliver LC, Larsson CJ. Natural killer cell activity in cigarette smokers and asbestos workers. Am Rev Respir Dis 1985;131:831–834. Sopori ML, Gairola CC, DeLucia AJ, Bryant LR, Cherian S. Immune responsiveness of monkeys exposed chronically to cigarette smoke. Clin Immunol Immunopathol 1985;36:338–344. Robbins CS, Dawe DE, Goncharova SI, Pouladi MA, Drannik AG, Swirski FK, Cox G, Sta¨mpfli MR. Cigarette smoke decreases pulmonary dendritic cells and impacts antiviral immune responsiveness. Am J Respir Cell Mol Biol 2004;30:202–211. Vassallo R, Tamada K, Lau JS, Kroening PR, Chen L. Cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of Th-2 priming. J Immunol 2005;175:2684–2691. Kalra R, Singh SP, Savage SM, Finch GL, Sopori ML. Effects of cigarette smoke on immune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T cells and depletes IP3-sensitive Ca(2⫹) stores. J Pharmacol Exp Ther 2000;293:166–171. van der Strate BW, Postma DS, Brandsma CA, Melgert BN, Luinge MA, Geerlings M, Hylkema MN, van den Berg A, Timens W, Kerstjens HA. Cigarette smoke–induced emphysema: a role for the B cell? Am J Respir Crit Care Med 2006;173: 751–758. Savage SM, Donaldson LA, Cherian S, Chilukuri R, White VA, Sopori ML. Effects of cigarette smoke on the immune response: II. Chronic

Robbins, Bauer, Vujicic, et al.: Cigarette Smoke and Antiviral Responses exposure to cigarette smoke inhibits surface immunoglobulinmediated responses in B cells. Toxicol Appl Pharmacol 1991;111:523– 529. 27. Nicholson KG, Kent J, Hammersley V. Influenza A among communitydwelling elderly persons in Leicestershire during winter 1993–4; cigarette smoking as a risk factor and the efficacy of influenza vaccination. Epidemiol Infect 1999;123:103–108. 28. Goh SK, Johan A, Cheong TH, Wang YT. A prospective study of infections with atypical pneumonia organisms in acute exacerbations of chronic bronchitis. Ann Acad Med Singapore 1999;28:476–480. 29. Robbins CS, Bauer CMT, Gaschler GJ, Vujicic N, Brown EG, Sta¨mpfli MR. The effect of cigarette smoke on immune inflammatory responses to influenza a is dependent on viral dose [abstract]. Proc Am Thorac Soc 2006;3;A779. 30. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke–induced emphysema in mice. Science 1997;277:2002–2004. 31. Brown EG, Bailly JE. Genetic analysis of mouse-adapted influenza A virus identifies roles for the NA, PB1, and PB2 genes in virulence. Virus Res 1999;61:63–76. 32. Lawrence CW, Ream RM, Braciale TJ. Frequency, specificity, and sites of expansion of CD8⫹ T cells during primary pulmonary influenza virus infection. J Immunol 2005;174:5332–5340. 33. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708–712. 34. Ashkar AA, Yao XD, Gill N, Sajic D, Patrick AJ, Rosenthal KL. Tolllike receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the absence of inflammation seen with CpG DNA. J Infect Dis 2004;190:1841–1849. 35. Simani AS, Inoue S, Hogg JC. Penetration of the respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab Invest 1974;31:75–81. 36. Kark JD, Lebiush M. Smoking and epidemic influenza-like illness in female military recruits: a brief survey. Am J Public Health 1981;71: 530–532. 37. Kark JD, Lebiush M, Rannon L. Cigarette smoking as a risk factor for epidemic a(h1n1) influenza in young men. N Engl J Med 1982;307: 1042–1046. 38. Rogot E, Murray JL. Smoking and causes of death among US veterans: 16 years of observation. Public Health Rep 1980;95:213–222. 39. Phillips J, Kluss B, Richter A, Massey E. Exposure of bronchial epithelial cells to whole cigarette smoke: assessment of cellular responses. Altern Lab Anim 2005;33:239–248. 40. Nardini M, Finkelstein EI, Reddy S, Valacchi G, Traber M, Cross CE, van der Vliet A. Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells: modulation by ␣-tocopherol and ascorbic acid. Toxicology 2002;170:173–185. 41. Green GM. Mechanisms of tobacco smoke toxicity on pulmonary macrophage cells. Eur J Respir Dis Suppl 1985;139:82–85. 42. Zhang X, Moilanen E, Lahti A, Hamalainen M, Giembycz MA, Barnes PJ, Lindsay MA, Kankaanranta H. Regulation of eosinophil apoptosis by nitric oxide: role of c-Jun-N-terminal kinase and signal transducer and activator of transcription 5. J Allergy Clin Immunol 2003;112:93– 101. 43. van Eeden SF, Hogg JC. The response of human bone marrow to chronic cigarette smoking. Eur Respir J 2000;15:915–921. 44. Terashima T, Wiggs B, English D, Hogg JC, van Eeden SF. The effect of cigarette smoking on the bone marrow. Am J Respir Crit Care Med 1997;155:1021–1026. 45. Fenton RR, Molesworth-Kenyon S, Oakes JE, Lausch RN. Linkage of IL-6 with neutrophil chemoattractant expression in virus-induced ocular inflammation. Invest Ophthalmol Vis Sci 2002;43:737–743. 46. Smith PL, Lombardi G, Foster GR. Type I interferons and the innate immune response: more than just antiviral cytokines. Mol Immunol 2005;42:869–877. 47. Little R, White MR, Hartshorn KL. Interferon-␣ enhances neutrophil respiratory burst responses to stimulation with influenza A virus and FMLP. J Infect Dis 1994;170:802–810. 48. Wang K, Scheel-Toellner D, Wong SH, Craddock R, Caamano J, Akbar AN, Salmon M, Lord JM. Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-␦, and NF-␬ B signaling pathways. J Immunol 2003;171:1035– 1041. 49. Sakamoto E, Hato F, Kato T, Sakamoto C, Akahori M, Hino M, Kitagawa S. Type I and type II interferons delay human neutrophil apoptosis

1351

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2. J Leukoc Biol 2005;78:301–309. Legge KL, Braciale TJ. Lymph node dendritic cells control CD8⫹ T cell responses through regulated FasL expression. Immunity 2005;23:649– 659. Mazanec MB, Kaetzel CS, Lamm ME, Fletcher D, Nedrud JG. Intracellular neutralization of virus by immunoglobulin A antibodies. Proc Natl Acad Sci USA 1992;89:6901–6905. Mazanec MB, Coudret CL, Fletcher DR. Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol 1995;69:1339–1343. Ito R, Ozaki YA, Yoshikawa T, Hasegawa H, Sato Y, Suzuki Y, Inoue R, Morishima T, Kondo N, Sata T, et al. Roles of anti-hemagglutinin IgA and IgG antibodies in different sites of the respiratory tract of vaccinated mice in preventing lethal influenza pneumonia. Vaccine 2003;21:2362–2371. Ramphal R, Cogliano RC, Shands JW Jr, Small PA Jr. Serum antibody prevents lethal murine influenza pneumonitis but not tracheitis. Infect Immun 1979;25:992–997. Yoshikawa T, Matsuo K, Suzuki Y, Nomoto A, Tamura S, Kurata T, Sata T. Total viral genome copies and virus-Ig complexes after infection with influenza virus in the nasal secretions of immunized mice. J Gen Virol 2004;85:2339–2346. Mackenzie JS, Flower RL. The effect of long-term exposure to cigarette smoke on the height and specificity of the secondary immune response to influenza virus in a murine model system. J Hyg (Lond) 1979;83:135– 141. MacKenzie JS, MacKenzie IH, Holt PG. The effect of cigarette smoking on susceptibility to epidemic influenza and on serological responses to live attenuated and killed subunit influenza vaccines. J Hyg (Lond) 1976;77:409–417. Mancini DA, Mendonca RM, Mendonca RZ, do Prado JA, Andrade Cde M. Immune response to vaccine against influenza in smokers, non-smokers and, in individuals holding respiratory complications. Boll Chim Farm 1998;137:21–25. Wongsurakiat P, Maranetra KN, Wasi C, Kositanont U, Dejsomritrutai W, Charoenratanakul S. Acute respiratory illness in patients with COPD and the effectiveness of influenza vaccination: a randomized controlled study. Chest 2004;125:2011–2020. Razani-Boroujerdi S, Singh SP, Knall C, Hahn FF, Pena-Philippides JC, Kalra R, Langley RJ, Sopori ML. Chronic nicotine inhibits inflammation and promotes influenza infection. Cell Immunol 2004;230:1–9. Yewdell JW, Bennink JR, Smith GL, Moss B. Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natl Acad Sci USA 1985;82:1785– 1789. Vlahos R, Bozinovski S, Jones JE, Powell J, Gras J, Lilja A, Hansen MJ, Gualano RC, Irving L, Anderson GP. Differential protease, innate immunity, and NF-␬B induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol 2006;290:L931–L945. Droemann D, Goldmann T, Tiedje T, Zabel P, Dalhoff K, Schaaf B. Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients. Respir Res 2005;6:68. Guillot L, Le Goffic R, Bloch S, Escriou N, Akira S, Chignard M, Si-Tahar M. Involvement of Toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem 2005;280:5571–5580. Collet JP, Shapiro P, Ernst P, Renzi T, Ducruet T, Robinson A. Effects of an immunostimulating agent on acute exacerbations and hospitalizations in patients with chronic obstructive pulmonary disease. The PARI-IS Study Steering Committee and Research Group. Prevention of Acute Respiratory Infection by an Immunostimulant. Am J Respir Crit Care Med 1997;156:1719–1724. Li J, Zheng JP, Yuan JP, Zeng GQ, Zhong NS, Lin CY. Protective effect of a bacterial extract against acute exacerbation in patients with chronic bronchitis accompanied by chronic obstructive pulmonary disease. Chin Med J (Engl) 2004;117:828–834. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163:1256–1276. Drannik AG, Pouladi MA, Robbins CS, Goncharova SI, Kianpour S, Sta¨mpfli MR. Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection. Am J Respir Crit Care Med 2004;170:1164–1171.