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Cigarette Smoke Exposure Attenuates Cytokine Production by Mouse Alveolar Macrophages

¹ Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, and ² Department of Medicine, McMaster University, Hamilton, Ontario, Canada; and ³ AstraZeneca, Lund, Sweden

Correspondence and requests for reprints should be addressed to Martin Stämpfli, McMaster University, Department of Pathology and Molecular Medicine, 1200 Main Street West, Hamilton, ON, L8N 3Z5 Canada. E-mail: stampfli{at}mcmaster.ca

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Alveolar macrophages (aMs) play a central role in respiratory host defense by sensing microbial antigens and initiating immune-inflammatory responses early in the course of an infection. The purpose of this study was to investigate the effect of cigarette smoke exposure on aMs after stimulation of innate pattern recognition receptors (PRRs) in a murine model. To accomplish this, C57BL/6 mice were exposed for 8 weeks using two models of cigarette smoke exposure, nose-only or whole-body exposure, and aMs isolated from the bronchoalveolar lavage. After stimulation of aMs with pI:C, a mimic of viral replication, and bacterial cell-wall constituent LPS, aMs from cigarette smoke–exposed mice produced significantly attenuated levels of the inflammatory cytokines TNF- and IL-6, and the chemokine RANTES. This attenuation was specific to the aM compartment, and not related to changes in aM viability or expression of Toll-like receptor (TLR)3 or TLR4 between groups. Furthermore, aMs from smoke-exposed mice had decreased cytokine RNA as compared with aMs from sham-exposed mice. Mechanistically, this was associated with decreased nuclear translocation of the proinflammatory transcription factor NF-B, and increased activator protein-1 nuclear translocation, in aMs from smoke-exposed mice. Attenuated cytokine production was reversible after smoking cessation. Cigarette smoke exposure also attenuated TNF- production after stimulation with nucleotide-oligomerization domain–like receptor agonists, showing that the effect applies more broadly to other PRR pathways. Our data demonstrate that cigarette smoke exposure attenuates aM responses after innate stimulation, including pathways typically associated with bacterial and viral infections.

Key Words: alveolar macrophage • Toll-like receptor • chronic obstructive pulmonary disease • cigarette smoke • inflammation

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   Given the central role of alveolar macrophages early in the course of an infection, attenuated Toll-like receptor function may predispose smokers to respiratory infections and bacterial colonization.

 
Chronic obstructive pulmonary disease (COPD) is reaching epidemic levels (1). Etiologically, COPD is largely associated with cigarette smoking, and epidemiological studies as early as 1982 have shown an increased risk of respiratory infection even in young, asymptomatic smokers (2). Further along in the disease progression of individuals who develop COPD, acute, often self-limiting, bacterial and viral infections are a significant cause of symptom exacerbation, often leading to hospitalization (3, 4). Taken together, this is indicative of cigarette smoke having deleterious effects on innate immunity.

Central to protecting the host early during an infection is the recognition of patterns common to large classes of pathogens by means of pattern-recognition receptors (PRRs). PRRs include members of the prominent Toll-like receptor (TLR) family (5), as well as the nucleotide-oligomerization domain (NOD) family, otherwise called the NOD-like receptor (NLR) family, intracellular receptors recognizing structures from bacterial peptidoglycan (6). TLRs are found on the cell surface and in endosomes of many different cell types. To date there have been approximately 11 TLRs identified in mice and humans with corresponding synthetic or naturally occurring ligands. Included in this are TLR4, which recognizes lipopolysaccharides (LPS) from gram-negative bacteria (7); TLR3, which recognizes double-stranded RNA found during viral infections, or synthetic polyinosinic-polycytidylic acid (pI:C) (8); and TLR9, which recognizes CpG DNA motifs (9). Upon stimulation of TLRs, intracellular signaling pathways are activated, resulting in the nuclear translocation of transcription factors including NF-B, IRF3, and activator protein (AP)-1 (10). In turn, this leads to the up-regulation and production of cytokines and chemokines important for initiating antibacterial or antiviral immune responses.

Due to their strategic positioning in the lumen of the airways, alveolar macrophages (aMs) play a central role in innate respiratory host defense (11). Although there is evidence that cigarette smoking may increase the number of aMs, further clinical and experimental evidence indicates that these cells may be functionally impaired (12–18). Of particular interest, recently Berenson and coworkers have shown decreased production of inflammatory cytokines upon stimulation of aMs from patients with COPD with antigens from Haemophilus influenzae—the most commonly isolated bacteria during exacerbations in COPD (19). However, the mechanisms leading to the functional impairment of aMs, such as pathways of recognition or PRR expression and function, as well as transcription and translation of cytokines, is less well understood.

The purpose of this study was to investigate the effect of cigarette smoke exposure on PRR-mediated responses by aMs in a murine model. To accomplish this, we used two different models of smoke exposure: nose-only and whole-body exposure. We demonstrate that cigarette smoke exposure decreased inflammatory cytokine production by aMs upon stimulation with bacterial LPS or muramyl di- and tri- peptides, as wells as pI:C, a mimic of viral replication. To our knowledge, the finding that cigarette smoke exposure attenuates cytokine production after pI:C stimulation is novel and shows that the immunologic effects of cigarette smoke on aMs are not limited to bacterial antigens. Mechanistically, we demonstrate that this observation is lung specific, reversible, and is associated with dysregulated activation of transcription factors following TLR stimulation. Similar to pI:C and LPS stimulation, we observed decreased cytokine production after stimulation with muramyl di- and tri- peptides, ligands for members of the NLR family. Collectively, these data indicate that cigarette smoke has a general impact on aM activation through PRRs, including pathways typically associated with bacterial or viral infections. Some of the results in this study were previously reported in abstract form (20).

	MATERIALS AND METHODS

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Animals
Female C57BL/6 mice (6–8 wk old) were purchased from Charles River Laboratories (Montreal, PQ, Canada) and kept in a 12-hour light-dark cycle with unlimited access to food and water. Cages, food, and bedding were autoclaved, and all animal manipulations were performed in a laminar flow hood by personnel who were gloved, gowned, and masked. The McMaster University Animal Research Ethics Board approved all experiments described in this study.

Cigarette Smoke Exposure
Nose-only exposure. Mice were exposed to two 1R3 reference cigarettes (Tobacco and Health Research Institute, University of Kentucky, Lexington, KY) daily for 5 days per week using a smoke exposure system that is described in detail elsewhere (21). In an initial lead-up period, animals were accustomed to 1 cigarette in the first and to two cigarettes in the second week. To control for handling, groups of mice were placed into restrainers only and exposed to room air (sham-exposure).

Whole-body exposure. Mice were exposed to the smoke from twelve 2R4F reference cigarettes with the filters removed, twice daily, 5 days per week using an SIU48 (PROMECH LAB AB, Vintrie, Sweden). No lead-up period is required in the whole-body exposure system. In an initial acclimatization period, mice were accustomed to the restrainers over a 3-day period. Specifically, on Day 1 mice were placed into the restrainers for 20 minutes, on Day 2 for 30 minutes, and on Day 3 for 50 minutes. Control animals were exposed to room air only.

Carboxyhemoglobin Measurement
Immediately, or 24 hours, after sham or smoke exposure, blood was drawn in clinitubes (Radiometer, Copenhagen, Denmark) for Carboxyhemoglobin (COHb) measurement by the McMaster University Medical Centre core lab.

Macrophage Isolation and Culture
Mice were anesthetized 18 to 24 hours after their last smoke exposure and killed by exsanguination before excision of the lungs. Tracheas were cannulated and bronchoalveolar lavage (BAL) was collected from sham- and smoke- exposed mice by instilling 0.5 ml of PBS into the lungs through the trachea three times. After each instilment, fluid was collected and pooled. Peritoneal lavage was collected by instilling 5 ml of PBS into the peritoneum via syringe and needle, and fluid was collected with a disposable plastic pipet. Cells were counted by trypan blue dye exclusion. Cytospins for differential cell counts were prepared and stained with Hema 3 (Biochemical Sciences Inc., Swedesboro, NJ). Standard hemocytologic criteria were used to classify mononuclear cells, neutrophils, and eosinophils. At least 500 cells were counted per cytospin. Based on trypan blue dye exclusion and differential cell counts, equal numbers of aMs were allowed to adhere for 2 hours at 37°C and 5% CO₂ and washed three times with warm PBS to remove nonadherent cells. Details of aM numbers adhered in the different experiments are provided in the specific method sections below. Adherent cells were cultured in 100 µl of RPMI supplemented with 10% FBS (Sigma-Aldrich, Oakville, ON, Canada), 1% L-glutamine, 1% penicillin/streptomycin (Invitrogen, Grand Island, NY), and 0.1% β-mercaptoethanol (Invitrogen) (cRPMI) and stimulated with either 1 µg/ml LPS (Invivogen, San Diego, CA) or 10 µg/ml of pI:C or 1–50 µg/ml of CpG (Sigma-Aldrich) for 24 hours. Cells were similarly stimulated with 10 µg/ml muramyl di- or muramyl tri- peptides, generously provided by Anthony Coyle and Jose Lora (Millennium Pharmaceutical, Inc., Cambridge, MA).

Measurement of Cytokines and Nitric Oxide
A quantity of 5 x 10⁴ aMs was adhered to a flat-bottom 96-well plate and cultured in 100 µl of cRPMI. After 24 hours of culture, cell supernatants were collected and levels of TNF-, IL-6, RANTES (regulated on activation, normal T cells expressed and secreted) (R&D, Minneapolis, MN), and IFN-β (PBL Biomedical Laboratories, Piscataway, NJ) were measured by enzyme-linked immunosorbent assay (ELISA) as per the manufacturers' instructions and measured on an ELISA plate reader. The limit of detection for the assays were 5.1, 1.6, 2, and 15.6 pg/ml, respectively. Nitric oxide was measured by examining the breakdown products of nitric oxide by Griess reactions as described in detail elsewhere (22).

Macrophage Viabiltiy and Metabolic Activity
For survival cell counts, 1 x 10⁵ aMs were adhered to glass slides in flat-bottom 12-well plates. Cells were then counted by trypan dye exclusion counts after 24 hours of culture. For metabolic activity quantification, adherent cells were cultured in 96-well flat-bottom plates in media or stimulated with pI:C and LPS for 24 hours. After this time, MTT activity was assessed by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) kit according to the manufacturers' instructions (Chemicon, Billerica, MA). Briefly, adherent cells were incubated in 100 µl of cRPMI and 10 µl of MTT reagent for 4 hours at 37°C and 5% CO₂. Subsequently isopropanol development solution was added and MTT activity measured at OD570 on an ELISA plate reader.

TaqMan Real-Time RT-PCR
A quantity of 1 x 10⁵ aMs was adhered to a flat-bottom 96-well plate. Cells were stimulated with LPS, and RNA was isolated at the indicated time points specified in RESULTS and in the figure legends with the RNeasy mini Kit and the optional DNase step (Qiagen, Mississauga, ON, Canada). RNA was quantified and purity checked using the Agilent 2100 Bio-Analyzer machine operated by the 2100 expert software (Agilent, Palo Alto, CA). Reverse transcription was completed on similar amounts of RNA per group using a RETROscript kit (Ambion, Austin, TX). Mitochondrial ribosomal protein L32 (L32), TLR3, TLR4, TNF-, IL-6, and RANTES primers and FAM-labeled probes were purchased from Applied Biosystems (Foster City, CA), and PCR was performed in duplicate or triplicate with Universal PCR Master Mix in the ABI PRISM 7900HT Sequence Detection System operated by Sequence Detector Software version 2.2 (Applied Biosystems). PCR was performed in single plex and analysis was completed by first normalizing gene expression to levels of the housekeeping gene L32 in the same sample (CT) and then compared with the control group (CT). With this method, RNA extracted from unstimulated aMs, isolated from sham-exposed mice, have a relative fold induction (R) defined as 1. Experimental groups are expressed as fold change over this control condition.

Immunofluoresence
For immunofluorescence assays, 2.5 x 10⁴ aMs were allowed to adhere per well on glass slides using chamber slide systems (Nalge Nunc Int., Rochester, NY) for 2 hours. Cells were then washed twice with warm PBS to remove nonadherent cells. Slides were placed in pre-chilled acetone at –20°C for 20 minutes and stored at –70°C. To minimize nonspecific binding, cells were incubated with 20% normal goat and normal donkey serum for 1 hour at room temperature. Slides were stained with TLR3 (Imgenex Corp., San Diego, CA) and TLR4 (eBiosciences, San Diego, CA) primary antibodies for 1 hour. Slides were washed with PBST (0.05% Tween20) and stained with Alexa-flour 633– and Alexa-fluor 488–conjugated secondary antibodies (Molecular Probes, Invitrogen) for TLR3 and TLR4, respectively. Control slides were incubated with either primary or secondary antibody alone. Slides were washed and nuclei were stained with SYTO3 in the mounting media (Vector, Burlingame, CA). Fluorescent pictures were taken by confocal microscopy (Leica, Richmond Hill, ON, Canada) with the LSM 510 software. Quantification was performed by analyzing the sum of the TLR stain divided by the sum of the nuclear stain for individual pictures with Northern Eclipse software (Empix Imaging, Mississauga, ON, Canada).

Nuclear Isolation and NF-B and AP-1 ELISAs
A quantity of 2 x 10⁶ aMs was adhered to flat-bottom 12-well plates. After 2 hours of adherence and stimulation with LPS for the indicated times, nuclear isolation was preformed with a nuclear isolation kit (Active Motif, Carlsbad, CA) as per the manufacturer's instructions. Extracts were re-suspended in a final volume of 20 µl. Nuclear extracts were used in a TransAM NF-B ELISA and TransAM c-JUN ELISA (Active Motif) and run according to the manufacturer's instructions. Analysis shown is the sample OD540 divided by the positive control OD540, multiplied by 100, thereby giving percent of positive control signal. The positive control, stimulated Jurkat cell extracts, was provided with the kit, and 2 µg of extract was loaded per well.

Data Analysis
Data are expressed as mean ± SD or SEM as indicated in the figure legends. Statistical analysis was performed using Student's t test unless otherwise stated. Differences were considered statistically significant when P < 0.05.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Nose-Only Cigarette Smoke Exposure
C57BL/6 mice were exposed to the smoke from two cigarettes a day, 5 days per week for 8 weeks. We observe carboxyhemoglobin (COHb) levels immediately following smoke exposure of 13.58% ± 2.47%, compared with 3.77% ± 0.93% in sham-exposed mice (Table 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Bronchoalveolar lavage (BAL) cellular profile and cytokine production by alveolar and peritoneal macrophages. C57BL/6 mice were sham- and smoke-exposed for 8 to 10 weeks. A and B represent the BAL total cell number and cellular differential. (C–F) aMs and pMs were isolated and cultured in media alone or stimulated with 10 µg/ml pI:C, 1 µg/ml LPS, or 10 µg/ml CpG for 24 hours and levels of cytokines measured in cell supernatants by ELISA. (C and D) TNF- production by aMs and pMs, respectively. E and F represent IL-6 and RANTES production from aMs. In all panels, aMs from smoke- or sham-exposed mice are shown in solid or open bars, respectively. (A and B) Data represent mean ± SEM of six independent experiments, with five mice pooled per group in each experiment. (C–F) AMs and pMs were isolated in three to five separate experiments, with five animals per experimental group. Data shown is mean ± SEM. Statistical analysis was performed with Student's t test *P < 0.05.

TNF-, IFN-β, Nitric Oxide, IL-6, and RANTES Production by aMs after pI:C, LPS, or CpG Stimulation

To further characterize the specificity of the effect of cigarette smoke on aM cytokine production after TLR stimulation, we measured the levels of the cytokine IL-6 and the chemokine RANTES in supernatants from stimulated aM isolated from sham- or smoke-exposed mice. As shown in Figures 1E and 1F, the levels of IL-6 and RANTES produced by aMs from smoke-exposed mice is decreased compared with aMs from sham-exposed mice. Thus, the decreased response from aM isolated from smoke-exposed mice is not limited to TNF-, but includes IL-6 and RANTES.

We did not observe production of TNF-, IL-6, IFN-β, NO, RANTES, or up-regulation of iNOs from any group after stimulation with the TLR9 ligand CpG (Figures 1C, 1E, 1F, and data not shown).

TNF- Production by Peritoneal Macrophages after pI:C, LPS, or CpG Stimulation
To investigate whether the effect of cigarette smoke exposure on cytokine production after TLR stimulation was specific to aMs, peritoneal macrophages (pMs) were isolated in parallel to aMs. Similar to aMs, pMs were cultured in media alone or stimulated with pI:C, LPS, or CpG, supernatants collected after 24 hours of culture, and levels of TNF- measured by ELISA. As demonstrated in Figure 1D, in contrast to the significant decrease observed in aMs from smoke-exposed mice, smoke exposure did not affect the production of TNF- from pMs. In contrast to aMs, pMs produce TNF- after CpG stimulation.

aM Survival and Viability in Culture
One explanation for the observed decrease in cytokine production by smoke-exposed aMs may be decreased aM viability as a result of smoke exposure, and consequently a result of culturing fewer live, viable cells. To address this, aMs were isolated and cultured as previously for cell viability and metabolic activity measurements. As shown in Table 2, aMs from sham- and smoke-exposed mice have similar cell numbers after 24 hours of culture, as determined by trypan blue dye exclusion counts. Similarly, on stimulation with pI:C or LPS, aMs from sham- and smoke-exposed mice have similar MTT activity after 24 hours of culture. Together, these observations indicate that decreased cytokine production from aMs isolated from smoke-exposed mice after stimulation with TLR agonists likely was not a result of increased death or decreased viability in culture.

aM Expression of TLR3 and TLR4

View larger version (16K): [in this window] [in a new window]   Figure 2. TLR3 and TLR4 expression in alveolar macrophages. C57BL/6 mice were sham- and smoke-exposed for 8 weeks. (A) RNA was isolated from aMs and levels of TLR3 and TLR4 RNA expression was measured by real-time quantitative PCR (TaqMan). Expression of TLR3 and TLR4 in sham-exposed mice were defined as 1 and levels in smoke-exposed mice were expressed relative to this. (B) BAL cells were adhered to glass slides, and TLR3, TLR4, and nuclei were stained. The top and bottom panels show aMs from sham- and smoke-exposed mice, respectively. Nuclear stains are shown in the left panels, TLR3 in the middle panels, and TLR4 in the right panels. (C) Immunofluoresencent picures were quantified using Northern Eclipse. In all panels, aMs from smoke- or sham-exposed mice are shown in solid or open bars, respectively.

TNF-, IL-6, or RANTES RNA Expression in aMs after LPS Stimulation
To investigate if decreased cytokine production by aMs from smoke-exposed mice was upstream of protein translation, aMs from sham- and smoke-exposed mice were isolated as previously and cultured with media alone or stimulated with LPS. After 2, 6, or 24 hours of culture, RNA was extracted from adherent cells. Levels of TNF-, IL-6, and RANTES were measured by real-time quantitative PCR. As shown in Figure 3, TNF-, IL-6, and RANTES mRNA from smoke-exposed mice were reduced compared with the levels observed in sham-exposed mice. This is indicative of aMs from smoke-exposed mice having decreased ability to up-regulate transcriptionally expression of TNF-, IL-6, and RANTES RNA after stimulation.

View larger version (11K): [in this window] [in a new window]   Figure 3. Ex vivo alveolar macrophage TNF-, IL-,6 and RANTES RNA expression. C57BL/6 mice were sham- and smoke-exposed for 8 weeks. AMs were isolated and cultured in media alone or stimulated with 1 µg/ml LPS for 2, 6, or 24 hours. RNA was isolated and levels of (A) TNF- RNA, (B) IL-6 RNA, and (C) RANTES expression was measured using real-time quantitative PCR (TaqMan). Data represent mean ± SEM, n = 10 (A), n = 5 (B and C). Statistical analysis was performed with Student's t test, *P < 0.05. For all panels, aMs from sham-exposed mice are open squares and aMs from smoke-exposed mice are solid circles.

NF-B and AP-1 Nuclear Translocation in aMs after LPS Stimulation

View larger version (11K): [in this window] [in a new window]   Figure 4. NF-B and AP-1 nuclear translocation in alveolar macrophages. C57BL/6 mice were sham- and smoke-exposed for 8 weeks. aMs were cultured in media alone or stimulated with 1 µg/ml LPS for 30, 60, or 90 minutes and nuclear extracts were isolated. Levels of (A) p65 and (B) c-Jun nuclear translocation were measured by ELISA. Shown is one representative of two experiments. Data represent mean ± SD (technical error), with 10 mice pooled per experimental group. For all panels, aMs from sham-exposed mice are open squares and aMs from smoke-exposed mice are solid circles.

TNF- Production by aMs after Muramyl-Dipeptide and Muramyl-Tripeptide Stimulation

View larger version (7K): [in this window] [in a new window]   Figure 5. Ex-vivo TNF- production by alveolar macrophages. C57BL/6 mice were sham- and smoke-exposed for 8 to 10 weeks. aMs were isolated and cultured in media alone or stimulated with 10 µg/ml M-DAP or 10 µg/ml T-DAP for 24 hours and levels of TNF- measured in cell supernatants by ELISA. In all panels, aMs from smoke- or sham-exposed mice are shown in solid or open bars, respectively. Data represent mean ± SEM, n = 2; in each experiment, five animals were pooled per group. Statistical analysis was performed with Student's t test, *P < 0.05.

In BAL from whole-body smoke-exposed mice, there is a significant increase in the total cell number, as compared with sham-exposed mice (Figure 6A). Isolated cells from the BAL of whole-body smoke- or sham-exposed mice are greater than 95% mononuclear cells (sham: 99.88 ± 0.29; smoke: 96.96 ± 4.82; n = 5 per group). Similar to nose-only exposure, the balance of the remaining cells was neutrophils, and no difference in cellular composition in the BAL was observed between groups (Figure 6B).

View larger version (14K): [in this window] [in a new window]   Figure 6. BAL cellular profile and cytokine production by alveolar and peritoneal macrophages. C57BL/6 mice were sham- and smoke-exposed for 8 to 10 weeks. A and B represent the BAL total cell number and cellular differential. (C and D) aMs and pMs were isolated and cultured in media alone or stimulated with 10 µg/ml pI:C, 1 µg/ml LPS, or 10 µg/ml CpG for 24 hours and levels of cytokines measured in cell supernatants by ELISA. C and D represent TNF- production by aMs and pMs, respectively. In all panels, aMs from smoke- or sham-exposed mice are shown in solid or open bars, respectively. Data represent mean ± SEM, n = 5 per experiment. Statistical analysis was performed with Student's t test *P < 0.05.

TNF- Production by aMs and pMs after pI:C, LPS, and CpG Stimulation

Similar to aMs, pMs were cultured in media alone or stimulated with pI:C, LPS, or CpG, supernatants collected after 24 hours of culture, and levels of TNF- measured by ELISA. As demonstrated in Figure 6D, in contrast to the significant decrease observed in aMs from smoke-exposed mice, whole-body smoke exposure did not affect the production of TNF- from pMs.

Time Course and Reversibility of the Cigarette Smoke Exposure on aM Function
Next we investigated the minimal duration of cigarette smoke exposure necessary to observe attenuated cytokine production by aMs. Mice were sham- or smoke-exposed for 1, 2, 4, or 8 weeks. At each time point, aMs were isolated from the BAL and stimulated with LPS for 24 hours. Levels of TNF- were measured in cell supernatants. As shown in Figure 7A, we observed no difference in TNF- production between aMs isolated from sham- and smoke-exposed mice after 1 or 2 weeks of smoke exposure (P = 0.91 and P = 0.22, respectively). After 4 and 8 weeks, we observed significantly decreased TNF- in the cell supernatants of aMs isolated from smoke-exposed mice (P = 0.004 and P = 0.018, respectively).

View larger version (23K): [in this window] [in a new window]   Figure 7. Cytokine production by alveolar and peritoneal macrophages. C57BL/6 mice were sham- and smoke-exposed for 1, 2, 4, 8, 10, and 12 weeks. aMs were isolated and cultured in media alone or stimulated with 1 µg/ml LPS for 24 hours and levels of TNF- measured in cell supernatants by ELISA. (A) TNF- production by aMs after 1, 2, 4, and 8 weeks of smoke-exposure. (B) Mice were sham or smoke exposed for 8 weeks and aMs were isolated and cultured for 1 week before stimulation with 1 µg/ml LPS for 24 hours. Levels of TNF- were measured in cell supernatants by ELISA. In A and B, aMs from smoke- or sham-exposed mice are shown in solid or open bars, respectively. (C) Mice were sham- and smoke-exposed for 8 weeks, and after this time mice continued their exposure regime (open bars, sham exposure; solid bars, smoke exposure) or ceased smoke exposure (shaded bars, cessation). After 2 or 4 weeks aMs were isolated and cultured in media alone or stimulated with 1 µg/ml LPS, and levels of TNF- were measured in cell supernatants by ELISA (C). Data represent mean ± SEM, n = 3–5 per experiment. Statistical analysis was performed with Student's t test *P < 0.05 (A, C) or one-way ANOVA (B.) *P < 0.05 compared with sham, P < 0.05 compared with smoke.

To investigate whether the effects of cigarette smoke exposure on aMs are reversible in vivo, mice were sham- or smoke-exposed for 8 weeks. Subsequently, smoke-exposed mice were divided into two groups: the first continued their smoke-exposure regime (smoke), while the second were exposed to room air (cessation). After 2 or 4 weeks of smoking cessation, aMs were isolated and cultured in medium alone or stimulated with LPS. After 24 hours of culture, cell supernatants were collected and levels of TNF- measured by ELISA. After 2 weeks of smoking cessation, we observe significantly decreased TNF- in both the smoke-exposed and the cessation group, as compared with the sham-exposed group (Figure 7B). No difference was observed between the smoke and cessation groups. After 4 weeks of smoking cessation aMs regained normal function; we observed similar levels of TNF- in supernatants of aMs from the cessation group as compared with the sham group. Together, these findings indicate that smoking cessation may reverse attenuated aM function, but only after a sufficient period of time.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
aMs are fundamental to respiratory host defense by sensing microbial agents early in the course of an infection and initiating immune inflammatory responses. In this study, we investigated the effect of cigarette smoke exposure on the production of inflammatory cytokines after ex vivo stimulation of aMs. To this end, mice were exposed to mainstream cigarette smoke using two distinct smoke-exposure systems. The first, a nose-only exposure system, is widely used to study smoke-induced emphysema in small rodents. With this system Hautamaki and coworkers demonstrated the requirement of macrophage elastase for cigarette smoke–induced emphysema (21). The second system, a whole-body exposure system, has been developed more recently.

Similar to human smokers (23), mice exposed to smoke in either of these model systems have an increase in the number of cells isolated from the BAL, with aMs representing greater than 95% of cells. Importantly, based on carboxyhemoglobin blood measures, levels of smoke exposure are similar to that reported clinically (24). Although we observed increased number of aMs in the BAL of smoke-exposed mice, ex vivo cytokine production after stimulation was attenuated. More specifically, we observed significantly decreased production of the inflammatory cytokines TNF- and IL-6, and the chemokine RANTES, after stimulation with the TLR ligands pI:C or LPS, as well as the NLR ligands muramyl di- or tripeptides. Our findings are in line with previous clinical and experimental observations, including a recent report in which aMs from patients with COPD were stimulated with bacterial antigens from H. influenzae (19). That cigarette smoke exposure also attenuates cytokine production after NLR stimulation, as well as pI:C stimulation, is novel and shows the effect of cigarette smoke is not limited to bacterial antigens. Therefore, cigarette smoke likely has a general impact on immune activation through PRRs, including pathways typically associated with viral infections. Along these lines, the fact that RANTES is decreased is of particular interest, as it indicates that cigarette smoke impairs not only the MyD88-dependent pathway of TLR4 signaling, but likely also the MyD88 independent pathway—a pathway associated with the production of type 1 interferons (25).

Despite the production of RANTES, we failed to measure any appreciable level of IFN-β production by aMs. As demonstrated in peritoneal macrophages, downstream events from IRF3 activation (via TLR3 or TLR4 stimulation) include the production of the type 1 interferon IFN-β. IFN-β then has paracrine and autocrine function through STAT1 to up-regulate the expression of inducible nitric oxide (iNOs) and ultimately the production of NO (26, 27). Consistent with the lack of IFN-β production by aM, we observed neither induction of iNOs or the production of NO, after LPS or pI:C stimulation (data not shown). This observation is in agreement with a previous report by Punturieri and colleagues, in which the authors demonstrated that aMs do not produce IFN or NO in response to TLR3 or TLR4 ligands alone (28). Notably, this unresponsiveness is maintained in aMs isolated from smoke-exposed animals.

While we observed robust cytokine production by aMs after pI:C or LPS stimulation, we were unable to demonstrate any response to CpG. This observation is consistent with the lack of TLR9 expression by aMs in either sham- or smoke-exposed mice (data not shown). In agreement with this, a previous study has demonstrated that aMs, unlike peritoneal macrophages, do not respond to CpG stimulation (29), likely indicating the specialized function of aMs in respiratory host defense.

The impact of cigarette smoke was lung specific, as no differences were observed between peritoneal macrophages isolated from sham- and smoke-exposed mice in their production of TNF-. Similarly, a recent clinical study indicates that cytokine production from aMs stimulated with H. influenzae antigens from individuals with COPD are impaired, but not peripheral blood monocytes (19).

On further characterization of the observed attenuated effect, we sought to control for cell viability in culture, as smoke exposure may be detrimental to aM survival, leading to stimulation of less cells. Our observations indicate that decreased cytokine production by aMs from smoke-exposed mice is not a result of increased cell death due to cellular toxicity. In agreement with this, numerous clinical studies have shown that aMs from smokers may have an increased life span, despite exposure to toxic components contained within cigarette smoke (30–33). Therefore, mechanisms relating to decreased aM production of cytokines are likely unrelated to direct toxic effects on cell survival by cigarette smoke.

Of the potential mechanisms leading to decreased cytokine production in response to pI:C and LPS, decreased surface expression of TLR4 and endosome expression of TLR3 as a result of smoke exposure may be hypothesized. However, with these experimental approaches we did not observe any changes in TLR3 or TLR4 expression between sham- and smoke-exposed mice. Specifically, no difference was seen on the RNA level as assessed by TaqMan or the protein level as assessed by immunofluorescent microscopy. Together these data indicate that the decrease in cytokine production by aMs from smoke-exposed mice is likely downstream of TLR3 and TLR4 expression. Indeed, while numerous studies have shown impaired function of aMs from smokers or patients with COPD, a previous report demonstrated no difference in TLR4 expression on aMs (34).

pI:C may stimulate aMs via alternate pathways than TLR3, including cytoplasmic RNA helicases, such as retinoic acid–inducible gene I (RIG-I) (35). Therefore, while attenuated cytokine production after LPS, T-DAP, or M-DAP stimulation can be attributed to effects on TLR4, NOD1, and NOD2 pathways, respectively, attenuated cytokine production after pI:C stimulation may involve multiple pathways.

TLR stimulation leads to the activation of intracellular signaling pathways, resulting in the nuclear translocation of the transcription factors NF-B, IRF3, and AP-1 (10). The functional unit of NF-B, usually a heterodimer of the p65 and p50 subunits, is held in its inactive form in the cytoplasm by the inhibitory molecule, IkB (36). Upon activation, the IkB molecule is degraded, allowing free NF-B to enter the nucleus and initiate pro-inflammatory gene transcription (37). In our hands, we observe decreased levels of p65 after stimulation with LPS in nuclear extracts isolated from aMs from smoke-exposed mice. Furthermore, this is associated with decreased TNF-, IL-6, and RANTES RNA after stimulation at a later time point, indicating effects of smoke exposure at the transcriptional level. However, we acknowledge that TNF- expression is regulated at the level of transcription as well as translation. Our data does not exclude the possibility that attenuated TNF- production may be, in part, due to the effect of cigarette smoke on TNF- protein translation. Together these data intimate that decreased production of cytokines by aMs may be due to attenuated activation of NF-B. Clinically this is of particular relevance, as recently it has been demonstrated that rhinovirues, an important cause of exacerbations in COPD, activate aMs in an NF-B–dependent manner (38). While there are likely critical differences between antigens and replicating pathogens, this indicates the importance of understanding the effect of cigarette smoke on attenuated signaling pathways in aMs for bacterial and viral infections.

AP-1 proteins play a large role in the expression of many of the genes involved in proliferation and cell cycle progression, and include a mixture of heterodimeric complexes of proteins from the Fos and Jun families. In contrast to decreased p65 nuclear translocation, we observed increased nuclear associated c-Jun in aMs isolated form smoke-exposed mice. This observation is at variance to a recent report by Laan and colleagues (39). The authors showed that impaired production of cytokines in an epithelial cell line after culture in cigarette smoke–conditioned media was associated with decreased nuclear translocation of the AP-1 (39), while NF-B nuclear translocation was not affected. These findings demonstrate that cigarette smoke may differentially impact aMs and epithelial cells. However, this may also be accounted for by differences in the experimental approach, primary cells versus a cell line or human versus murine cells. Expression and production of RANTES in macrophages is generally believed to be dependent on IRF3 activation (26). That we observed decreased RANTES expression in aMs isolated from smoke-exposed animals suggest that cigarette smoke also attenuates IRF 3 activation.

Overall, we show that aMs from smoke-exposed mice have a basal restraint on cytokine production after TLR stimulation, associated with dysregulated activation of transcription factors. Mechanistically there are several possible explanations. Cigarette smoke has been to shown to contain oxygen free radicals that may directly damage signaling pathways. Alternatively, cigarette smoke has been shown to contain significant levels of biologically active LPS (40), and repeated LPS stimulation is associated with the induction of negative regulators of TLR signaling and LPS tolerance (41). Therefore, smoking may be associated with the expression of negative regulators of TLR activation. On the other hand, several components of cigarette smoke have been shown to exert direct immunosuppressive activity on aMs, including acrolein and NKK (42, 43).

Importantly, attenuated aM function was reversible, as aMs regained normal function 4 weeks after smoking cessation. This may either be a consequence of individual aMs regaining function over time, or that the attenuated aMs were replaced by new macrophages via natural turnover. Ex vivo, aMs did not regain normal function when rested in culture medium for 1 week, indicating the attenuated phenotype persisted. Attempts to rest the cells longer (2 or 4 wk) resulted in markedly decreased TNF- production in either group (data not shown); hence, we cannot rule out either hypothesis.

Despite evidence for attenuated aM cytokine production, we have previously observed increased inflammation and cytokine expression in smoke-exposed animals after in vivo infection with either replication-competent influenza virus (44) or live replicating Pseudomonas aeruginosa (45). Increased in vivo inflammation and cytokine expression appears to be at variance with the ex vivo observations reported in the current manuscript. A complex and multilayered defense system protects the host against microbial agents, through a combination of physical barriers, and innate and adaptive immune mechanisms. Based our own findings and studies from other labs (reviewed in Ref. 46), we postulate that cigarette smoke suppresses resident respiratory host defense mechanisms, including aMs. As a consequence, respiratory pathogens are dealt with inefficiently, necessitating the recruitment of immune-inflammatory cells from the circulation to compensate for this local deficiency. This may explain the increased inflammation observed in our in vivo studies.

In summary, we show that cigarette smoke exposure compromises the ability of aMs to produce inflammatory cytokines in response to TLR and NLR stimulation. Attenuated production of inflammatory cytokines is associated with dysregulated activation of the transcription factors NF-B and AP-1. Together, our findings indicate that aMs from smoke-exposed mice have decreased ability to transcriptionally up-regulate inflammatory mediators and initiate innate immune inflammatory responses.

	Acknowledgments

 
The authors gratefully acknowledge the expert technical support of Joanna Kasinska and Sussan Kianpour, and the secretarial assistance of Mary Kiriakopoulos.

	Footnotes

 
The research described in this article was supported in part by the Canadian Institutes of Health Research and AstraZeneca, Sweden. M.R.S. is a holder of a CIHR New Investigator award.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0053OC on September 13, 2007

Conflict of Interest Statement: M.L. has been employed by AstraZeneca R&D Lund since 2001. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 16, 2007

Accepted in final form August 23, 2007

	References

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 

1. Lopez AD, Murray CC. The global burden of disease, 1990–2020. Nat Med 1998;4:1241–1243.[CrossRef][Medline]
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.[Abstract/Free Full Text]
3. Papi A, Bellettato CM, Braccioni F, Romagnoli M, Casolari P, Caramori G, Fabbri LM, Johnston SL. Infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations. Am J Respir Crit Care Med 2006;173:1114–1121.[Abstract/Free Full Text]
4. Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: A state-of-the-art review. Clin Microbiol Rev 2001;14:336–363.[Abstract/Free Full Text]
5. Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol 2006;117:979–987. (quiz 988).[CrossRef][Medline]
6. Strober W, Murray PJ, Kitani A, Watanabe T. Signalling pathways and molecular interactions of nod1 and nod2. Nat Rev Immunol 2006;6:9–20.[CrossRef][Medline]
7. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, et al. Defective lps signaling in c3h/hej and c57bl/10sccr mice: Mutations in tlr4 gene. Science 1998;282:2085–2088.[Abstract/Free Full Text]
8. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3. Nature 2001;413:732–738.[CrossRef][Medline]
9. Wagner H. Bacterial cpg DNA activates immune cells to signal infectious danger. Adv Immunol 1999;73:329–368.[Medline]
10. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511.[CrossRef][Medline]
11. Lambrecht BN. Alveolar macrophage in the driver's seat. Immunity 2006;24:366–368.[CrossRef][Medline]
12. Holt PG. Immune and inflammatory function in cigarette smokers. Thorax 1987;42:241–249.[Free Full Text]
13. 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.[Medline]
14. 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.[Abstract]
15. 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.[CrossRef][Medline]
16. Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM. Suppression of human il-1beta, il-2, ifn-gamma, and tnf-alpha production by cigarette smoke extracts. J Allergy Clin Immunol 2000;106:280–287.[CrossRef][Medline]
17. Hagiwara E, Takahashi KI, Okubo T, Ohno S, Ueda A, Aoki A, Odagiri S, Ishigatsubo Y. Cigarette smoking depletes cells spontaneously secreting th(1) cytokines in the human airway. Cytokine 2001;14:121–126.[CrossRef][Medline]
18. Tetley TD. Macrophages and the pathogenesis of copd. Chest 2002;121(5, Suppl)156S–159S.[CrossRef][Medline]
19. Berenson CS, Wrona CT, Grove LJ, Maloney J, Garlipp MA, Wallace PK, Stewart CC, Sethi S. Impaired alveolar macrophage response to haemophilus antigens in chronic obstructive lung disease. Am J Respir Crit Care Med 2006;174:31–40.[Abstract/Free Full Text]
20. Gaschler GJ, Zavitz CCJ, Stampfli MR. Chronic cigarette smoke exposure impairs toll like receptor 3 and 4 function in mouse alveolar macrophages [abstract]. 2006;A545.
21. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997;277:2002–2004.[Abstract/Free Full Text]
22. Xing Z, Zganiacz A, Santosuosso M. Role of il-12 in macrophage activation during intracellular infection: Il-12 and mycobacteria synergistically release tnf-alpha and nitric oxide from macrophages via ifn-gamma induction. J Leukoc Biol 2000;68:897–902.[Abstract/Free Full Text]
23. Jeffery PK. Structural and inflammatory changes in copd: a comparison with asthma. Thorax 1998;53:129–136.[Medline]
24. Stewart RD, Baretta ED, Platte LR, Stewart EB, Kalbfleisch JH, Van Yserloo B, Rimm AA. Carboxyhemoglobin levels in american blood donors. JAMA 1974;229:1187–1195.[Abstract/Free Full Text]
25. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, Akira S. Lipopolysaccharide stimulates the myd88-independent pathway and results in activation of ifn-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001;167:5887–5894.[Abstract/Free Full Text]
26. Gao JJ, Filla MB, Fultz MJ, Vogel SN, Russell SW, Murphy WJ. Autocrine/paracrine ifn-alphabeta mediates the lipopolysaccharide-induced activation of transcription factor stat1alpha in mouse macrophages: Pivotal role of stat1alpha in induction of the inducible nitric oxide synthase gene. J Immunol 1998;161:4803–4810.[Abstract/Free Full Text]
27. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, et al. Role of adaptor trif in the myd88-independent toll-like receptor signaling pathway. Science 2003;301:640–643.[Abstract/Free Full Text]
28. Punturieri A, Alviani RS, Polak T, Copper P, Sonstein J, Curtis JL. Specific engagement of tlr4 or tlr3 does not lead to ifn-beta-mediated innate signal amplification and stat1 phosphorylation in resident murine alveolar macrophages. J Immunol 2004;173:1033–1042.[Abstract/Free Full Text]
29. Suzuki K, Suda T, Naito T, Ide K, Chida K, Nakamura H. Impaired toll-like receptor 9 expression in alveolar macrophages with no sensitivity to cpg DNA. Am J Respir Crit Care Med 2005;171:707–713.[Abstract/Free Full Text]
30. Skold CM, Hed J, Eklund A. Smoking cessation rapidly reduces cell recovery in bronchoalveolar lavage fluid, while alveolar macrophage fluorescence remains high. Chest 1992;101:989–995.[CrossRef][Medline]
31. Agius RM, Rutman A, Knight RK, Cole PJ. Human pulmonary alveolar macrophages with smokers' inclusions: their relation to the cessation of cigarette smoking. Br J Exp Pathol 1986;67:407–413.[Medline]
32. Reiter C. Fluorescence test to identify deep smokers. Forensic Sci Int 1986;31:21–26.[CrossRef][Medline]
33. Marques LJ, Teschler H, Guzman J, Costabel U. Smoker's lung transplanted to a nonsmoker. Long-term detection of smoker's macrophages. Am J Respir Crit Care Med 1997;156:1700–1702.[Abstract/Free Full Text]
34. 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.[CrossRef][Medline]
35. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T. The rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses. Nat Immunol 2004;5:730–737.[CrossRef][Medline]
36. Gilmore TD, Morin PJ. The i kappa b proteins: members of a multifunctional family. Trends Genet 1993;9:427–433.[CrossRef][Medline]
37. Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, Maniatis T. Signal-induced site-specific phosphorylation targets i kappa b alpha to the ubiquitin-proteasome pathway. Genes Dev 1995;9:1586–1597.[Abstract/Free Full Text]
38. Laza-Stanca V, Stanciu LA, Message SD, Edwards MR, Gern JE, Johnston SL. Rhinovirus replication in human macrophages induces nf-kappab-dependent tumor necrosis factor alpha production. J Virol 2006;80:8248–8258.[Abstract/Free Full Text]
39. 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.[Abstract/Free Full Text]
40. Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W. Bacterial endotoxin is an active component of cigarette smoke. Chest 1999;115:829–835.[CrossRef][Medline]
41. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 2005;5:446–458.[CrossRef][Medline]
42. Li L, Holian A. Acrolein: a respiratory toxin that suppresses pulmonary host defense. Rev Environ Health 1998;13:99–108.[Medline]
43. Therriault MJ, Proulx LI, Castonguay A, Bissonnette EY. Immunomodulatory effects of the tobacco-specific carcinogen, nnk, on alveolar macrophages. Clin Exp Immunol 2003;132:232–238.[CrossRef][Medline]
44. Robbins CS, Bauer CM, Vujicic N, Gaschler GJ, Lichty BD, Brown EG, Stampfli MR. Cigarette smoke impacts immune inflammatory responses to influenza in mice. Am J Respir Crit Care Med 2006;174:1342–1351.[Abstract/Free Full Text]
45. Drannik AG, Pouladi MA, Robbins CS, Goncharova SI, Kianpour S, Stampfli MR. Impact of cigarette smoke on clearance and inflammation after pseudomonas aeruginosa infection. Am J Respir Crit Care Med 2004;170:1164–1171.[Abstract/Free Full Text]
46. Sopori ML, Kozak W. Immunomodulatory effects of cigarette smoke. J Neuroimmunol 1998;83:148–156.[CrossRef][Medline]

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