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Differential protease, innate immunity, and NF-B induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice

Cooperative Research Center for Chronic Inflammatory Diseases, ¹Department of Medicine, University of Melbourne, Royal Melbourne Hospital, and ²Department of Pharmacology, University of Melbourne, Parkville; ³Marine and Atmospheric Research, Commonwealth Scientific and Industrial Research Organisation, Aspendale; and ⁴Department of Respiratory Medicine, Royal Melbourne Hospital, Parkville, Australia

Submitted 3 May 2005 ; accepted in final form 14 December 2005

	ABSTRACT

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Cigarette smoke exposure is a major determinant of adverse lung health, but the molecular processes underlying its effects on inflammation and immunity remain poorly understood. Therefore, we sought to understand whether inflammatory and host defense determinants are affected during subchronic cigarette smoke exposure. Dose-response and time course studies of lungs from Balb/c mice exposed to smoke generated from 3, 6, and 9 cigarettes/day for 4 days showed macrophage- and S100A8-positive neutrophil-rich inflammation in lung tissue and bronchoalveolar lavage (BAL) fluid, matrix metalloproteinase (MMP) and serine protease induction, sustained NF-B translocation and binding, and mucus cell induction but very small numbers of CD3+CD4+ and CD3+CD8+ lymphocytes. Cigarette smoke had no effect on phospho-Akt but caused a small upregulation of phospho-Erk1/2. Activator protein-1 and phospho-p38 MAPK could not be detected. Quantitative real-time PCR showed upregulation of chemokines (macrophage inflammatory protein-2, monocyte chemoattractant protein-1), inflammatory mediators (TNF-, IL-1), leukocyte growth and survival factors [granulocyte-macrophage colony-stimulating factor, colony-stimulating factor (CSF)-1, CSF-1 receptor], transforming growth factor-, matrix-degrading MMP-9 and MMP-12, and Toll-like receptor (TLR)2, broadly mirroring NF-B activation. No upregulation was observed for MMP-2, urokinase-type plasminogen activator, tissue-type plasminogen activator, and TLRs 3, 4, and 9. In mouse strain comparisons the rank order of susceptibility was Balb/c > C3H/HeJ > 129SvJ > C57BL6. Partition of responses into BAL macrophages vs. lavaged lung strongly implicated macrophages in the inflammatory responses. Strikingly, except for IL-10 and MMP-12, macrophage and lung gene profiles in Balb/c and C57BL/6 mice were very similar. The response pattern we observed suggests that subchronic cigarette smoke exposure may be useful to understand pathogenic mechanisms triggered by cigarette smoke in the lungs including inflammation and alteration of host defense.

chronic obstructive pulmonary disease; matrix metalloproteinases; host defense; Toll-like receptor; leukocyte

It is known from histopathology and bronchoscopy studies of humans that cigarette smoke provokes lung inflammation, even after a single exposure (40). Recently, Churg et al. (12) described an acute murine model of exposure to smoke from four cigarettes over 1 h. They observed that the resulting inflammation was associated with activation of the transcription factor NF-B and proposed that TNF-, proteolytically activated by matrix metalloproteinase (MMP)-12 (macrophage metalloelastase), was largely responsible for the ensuing responses, especially upregulation of the vascular adhesion molecule E-selectin. These studies complemented their earlier work in a single-exposure acute model implicating TNF--dependent neutrophil recruitment in the protease-dependent breakdown of connective tissue, a precursor of emphysema, and in part reconciled conflicting data on the relative importance of neutrophil- vs. macrophage-dependent disease processes by providing a rational link between these cells (10).

After chronic cigarette smoke exposure in humans over many years, histomorphometric studies have demonstrated the association between neutrophil, macrophage, and lymphocyte infiltrates of the lungs and indexes of decline in lung function or, in the case of COPD, frank destruction of lung parenchyma, hypersecretion of mucins, and thickening of small airways due to release of growth/repair factors (29). These cells are known to variously release numerous chemical mediators able to constrict airways and remodel airway walls as well as proteases able to destroy lung parenchyma, notably elastases, cathepsins, granzymes and matrix MMPs such as MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-7 (matrilysin), and MMP-12. These changes are also associated with upregulation of NF-B activity in the lung (1, 8). It is also known that long-term smoking impairs human lung host defenses, via mechanisms that are poorly characterized at the molecular level, leading to airway colonization with bacterial pathogens that contribute to exacerbations and that are positively associated with poor prognosis, disease severity, and accelerated lung function decline (53, 63). Similarly, after months of exposure of mice to smoke, a characteristic macrophage-rich lung inflammation associated with development of peripheral air space enlargement reminiscent of human emphysema occurs in a protease-dependent manner (28). Studies in gene-modified mice have strongly implicated 1) neutrophil-derived serine proteases and macrophage MMP-12, 2) _v56-integrin-dependent and Smad-3-dependent transforming growth factor (TGF)- signaling, and 3) overproduction of IFN-, IL-13, TNF-, IL-1, and Nrf-2 in the progression of this pathology (3, 10, 11, 28, 36, 47, 70, 72). Acute and long-term murine models have therefore already proven invaluable in defining pathogenic mechanisms in cigarette smoke-induced lung disease.

In view of the very acute nature of the 2-h exposure model and the impracticality of chronic 3- to 6-mo exposure models, the aim of the present study was to develop a subchronic exposure model. We have studied the dose response and kinetics of subchronic exposure to cigarette smoke over 4 days, measuring inflammation, NF-B activity, MMP induction and transcriptional regulation of genes thought to be important for inflammatory cell recruitment and survival, tissue growth/repair, proteolytic damage, and Toll-like receptors (TLRs). TLRs are innate immunity pattern recognition receptors essential for host defense against infection (50). In strain susceptibility screens comparing the Balb/c response to C57BL/6, 129SvJ, and C3H/HeJ strains, we report marked differences in susceptibility to smoke-induced inflammation but largely concordant gene profiles. By partitioning responses into lung macrophages and cell-depleted lung tissue, we find the pattern of response strongly implicates macrophages as important orchestrators of the subchronic inflammatory response. The response pattern we observed in our data suggests that subchronic exposure may be useful to understand pathogenic mechanisms triggered by cigarette smoke in the lungs, including inflammation and alteration of host defenses.

	METHODS

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Animals. Specific pathogen-free male Balb/c, C57BL/6, 129SvJ, and C3H/HeJ mice aged 7 wk and weighing 20 g were obtained from the Animal Resource Centre (Perth, Australia). The animals were housed at 20°C on a 12:12-h day-night cycle in sterile microisolators and fed a standard sterile diet of Purina mouse chow with water allowed ad libitum. The experiments described in this paper were approved by the Animal Experimentation Ethics Committee of the University of Melbourne and conducted in compliance with the guidelines of the National Health and Medical Research Council of Australia on animal experimentation.

Cigarette smoke exposure. Mice were placed in an 18-liter Perspex chamber in a class II biosafety cabinet and exposed to cigarette smoke. In dose-response experiments, mice were exposed to cigarette smoke generated from 3, 6, or 9 cigarettes/day for 4 days, delivered three times per day at 8 AM, 12 noon, and 4 PM with 1, 2, or 3 cigarettes spaced over 1 h, respectively. In pilot experiments we found that 3, 6, and 9 cigarettes/day were very well tolerated. Higher doses caused piloerection and huddling and were therefore not used, to avoid distressing the animals. Sham-exposed mice were placed in an 18-liter Perspex chamber but did not receive cigarette smoke. On the fifth day, mice were killed by an intraperitoneal overdose of anesthetic (5.6 mg ketamine, 1.12 mg xylazine; Parnell Laboratories), and the lungs were lavaged with PBS as described below. In time course experiments, mice were exposed to cigarette smoke generated from 9 cigarettes/day for 1, 2, 3, or 4 days. The nine-cigarette dose was selected because it caused maximal NF-B activation (see RESULTS). Mice received smoke from three cigarettes for 1 h, and this was done three times a day for up to 4 days. On the fifth day, mice were killed by an intraperitoneal overdose of anesthetic, and the lungs were lavaged with PBS as described below. Commercially available filter-tipped cigarettes (manufactured by Philip Morris) of the following composition were used: 16 mg of tar, 1.2 mg of nicotine, and 15 mg of CO. Smoke was generated in 50-ml tidal volumes over 10 s, by use of timed draw-back mimicking normal smoking inhalation volume and cigarette burn rate. Group sizes of 8–10 mice per treatment were used to ensure that the study was powered to detect differences in response variables at the 0.05 confidence level.

Characterization of cigarette smoke exposure: total suspended particulate mass concentration. Total suspended particulate (TSP) matter was collected on a polytetrafluoroethylene filter with a PMP support ring (47-mm Teflon, 2-µm pore size, R2PJ047, Pall) enclosed in a polypropylene holder (47-mm holder 501200, MFS Advantec), using a low-flow miniature diaphragm pump (model 222-4, SKC). The pump was calibrated to a volumetric flow rate of 50 ml/min using a National Association of Testing Authorities-certified soap bubble flowmeter (Gilibrator-2 model 800272, Sensidyne). The unexposed and then smoke-exposed filter was equilibrated at a relative humidity of 50 ± 5% and a temperature of 21 ± 1°C and weighed with a Mettler UMT5 microbalance with a resolution of 0.0001 mg. The mass was corrected for the effect of buoyancy on the weighed filter mass. The TSP mass concentration, reported in milligrams per cubic meter, was determined by dividing the difference in mass between the exposed and unexposed filter by the volume of air sampled through the filter. The mean TSP mass concentration in the chamber containing cigarette smoke generated from one cigarette, measured from 3 min 13 s to 15 min, was 419 mg/m³. The uncertainty of the mass concentration determination at 95% for a coverage of k = 2 is 2.9%. The estimated loss of TSP due to dilution of the chamber during sampling is 3.3%.

Characterization of cigarette smoke exposure: particle number concentration. Particle number concentration was measured in the chamber immediately after injection of smoke into the chamber and again at the end of the 15-min period. Number concentration for a particle size range of 0.01 to >1.0 µm was measured with a hand-held condensation particle counter (model 3007, TSI). The particle number concentration dropped from 458,192 particles/cm³ at 4 min 51 s to 373,987 particles/cm³ at 15 min. The total particle number loss was 84,205 particles/cm³, which was predominantly due to dilution during a 3-min sampling period with the condensation particle counter at 700 particles·cm^–3·min^–1.

Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed in terminally anesthetized mice as we previously described (7, 20). Briefly, lungs from each mouse were lavaged in situ with a 400-µl aliquot, followed by three 300 µl of PBS, with 1 ml of BAL fluid (BALF) recovered from each animal. Smoke exposure had no effect on the recovered volume. The total number of viable cells in the BALF was determined by using the fluorophores ethidium bromide and acridine orange (Molecular Probes, San Diego, CA) on a standard Neubauer hemocytometer with a Zeiss Axioscope fluorescence microscope. Cytospins were prepared with 200 µl of BALF at 350 rpm for 10 min on a Cytospin 3 (Shandon). Cytospin preparations were stained with DiffQuik (Dade Baxter), and cells were identified and differentiated into macrophages, mononuclear cells, epithelial cells, eosinophils, neutrophils, and lymphocytes by standard morphological criteria. Mitotic figures, an index of cell division (6), were identified by standard morphological criteria. A minimum of 2,000 cells per slide were counted. In some experiments, BAL cells were enriched for macrophages for gene profiling as described below.

Enrichment of BAL cells. In sham-exposed mice, cells in BALF consisted of >98% macrophages. However, BALF obtained from smoke-exposed mice had a mixed population of cells consisting of predominantly neutrophils (range from 25% to 75%) and macrophages (range from 30% to 75%), depending on the strain of mouse used. In some experiments, the macrophage population in BALF obtained from Balb/c and C57BL/6 mice was enriched and used for quantitative real-time PCR (QPCR). Sham- and smoke-exposed mice were lavaged with DMEM as described above, and the BALF obtained from 8 mice/treatment group was pooled and placed on an Iwaki nontreated 100-mm cell culture dish, a low-adhesion and very low-activation surface. Dishes were then incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO₂ in air. The medium was then very gently aspirated to remove nonadherent neutrophils, and the lightly adherent cells, predominantly macrophages, were detached by addition of 1 ml of 0.5% (wt/vol) trypsin (in PBS containing 1 mM EDTA) for 10 min. Four milliliters of PBS was then added to the dish, the cell suspension was removed, and the dish was washed three times with four milliliters of PBS. The four aliquots of the cell suspensions were then pooled and spun at 3,000 rpm for 5 min, and the enriched cell pellet was resuspended in 1 ml of PBS. Aliquots were then taken for total and differential cell counts as described above. The cells were then pelleted by centrifugation, the PBS was aspirated, and the cell pellet was snap frozen in liquid nitrogen and stored at –80°C until required for QPCR. The cell pellet for each treatment group consisted of 1 million cells. This enrichment process routinely harvested >80% pure macrophages (residual cells were neutrophils) within 95 min of commencing lavage.

Fluorescence-activated cell sorting. BALF from individual mice was obtained as described above and centrifuged (3,000 rpm, 5 min) to pellet cells. The cell pellets from all eight mice in each treatment group were pooled and resuspended in 0.5 ml of fluorescence-activated cell sorting (FACS) buffer (PBS + 1% FCS) and counted. Numbers of cells used in each stain were Balb/c sham, 163,000; Balb/c smoke, 673,000; C57BL/6 sham 483,000; and C57BL/6 smoke, 235,200; this equated to 60–70 µl of the 0.5-ml pooled volume. Cells were labeled with fluorophore-conjugated antibodies at preoptimized dilutions to CD3-FITC, CD4-PE, and CD8a-PE (all from Becton Dickinson) for 1 h at 4°C and then washed twice in FACS buffer and resuspended in a final volume of 0.5 ml of FACS buffer containing 1% paraformaldehyde. Data were acquired on a BD FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software. A lymphocyte gate was applied, using standard forward and side scatter profiles.

Protease expression and activity in BALF. Zymography was used to assess protease expression in response to cigarette smoke exposure as previously described (7, 20). Briefly, BALF from animals in each treatment group was pooled and concentrated by adding 250 µl of 50% trichloroacetic acid to 500 µl of pooled BALF samples and leaving at 4°C overnight. The next day samples were spun (13,000 rpm for 10 min at 4°C), and the pellet was washed twice with 300 µl of 80% diethyl ether (in 20% ethanol) and dried in air for 10 min. The pellet was then resuspended in 50 µl of 1x nonreducing buffer and heated for 10 min at 65°C, and 20 µl was loaded on SDS-PAGE minigels. SDS-PAGE minigels (10%) were prepared with the incorporation of gelatin (2 mg/ml) before casting. BALF (20 µl) was run into gels at a constant voltage of 200 V under nonreducing conditions. When the dye front reached the bottom, gels were removed and washed twice for 15 min in 2.5% Triton X-100 and incubated at 37°C overnight in zymography buffer [in mM: 50 Tris·HCl (pH 7.5), 5 CaCl₂, and 1 ZnCl₂, with 0.01% NaN₃]. The gels were then stained for 45 min with Coomassie brilliant blue R-250 and extensively destained. After destaining, zones of enzyme activity appeared clear against the Coomassie blue background. Neat BALF was also tested for net gelatinase and net serine protease activity with fluorescence-conjugated gelatin (Molecular Probes) and N-methoxysuccinyl-Ala-Ala-Pro-Val-4-nitroanilide (Sigma), respectively. The gelatin substrate (10 µg) was diluted in (mM) 50 Tris pH 7.5, 150 NaCl, and 5 CaCl₂ with 0.01% NaN₃ and incubated at room temperature for 16 h with 100 µl of neat BALF. The digested substrate has absorption/emission maxima at 495 and 515 nm. The N-methoxysuccinyl-Ala-Ala-Pro-Val-4-nitroanilide substrate (50 µg) was diluted in (mM) 50 Tris pH 7.5, 150 NaCl, and 5 CaCl₂ with 0.01% NaN₃ and incubated at room temperature for 16 h with 100 µl of neat BALF. The digested substrate has absorption maxima at 405 nm. The fluorescence intensity of the substrates was measured in a microplate reader (Victor II, Wallac) to detect quantitative differences in activity.

Preparation of nuclear lung extracts and EMSA. Nuclear extracts and EMSA were performed as previously described (7). Briefly, 10 mg of lung tissue was resuspended in 500 µl of nuclear lysis buffer 1 [in mM: 10 HEPES (pH 7.6), 15 KCl, 2 MgCl₂, 0.1 EDTA, 5 -mercaptoethanol, and 0.5 PMSF with 0.2% Nonidet P-40] for 10 min on ice. Nuclei were pelleted by centrifugation at 800 g for 30 s and lysed in 500 µl of nuclear lysis buffer 2 [in mM: 50 HEPES (pH 7.6), 400 KCl, 0.1 EDTA, 5 -mercaptoethanol, and 0.5 PMSF with 10% glycerol]. After a 30-min incubation on ice, the nuclei extract (supernatant) was retained after centrifugation for 10 min (800 g at 4°C). Protein concentrations from whole and nuclear extracts were determined with the D_C protein assay (Bio-Rad, Richmond, CA), and all extracts were stored at –80°C. For EMSA, complementary oligonucleotides used to generate double-stranded DNA for NF-B and activator protein-1 (AP-1) binding sites were annealed and radiolabeled with 5 U of Klenow fragment (Promega) in the presence of (in mM) 50 Tris·HCl (pH 7.2), 10 MgSO₄, 0.1 DTT, 0.2 dGTP, 0.2 dCTP, and 0.2 dTTP with 100 µCi [-³²P]dATP (Geneworks). Labeled probes were then separated from unincorporated isotope by size exclusion chromatography with Microspin G-25 columns (Pharmacia Biotech). Nuclear extracts (5 µg) were used in binding assays in 20-µl reactions and incubated at room temperature with labeled probe (50,000 cpm) in the presence of (in mM) 10 Tris·HCl (pH 7.5), 1 MgCl₂, 0.5 DTT, and 50 NaCl with 0.05 mg/ml poly(dI-dC)·poly(dI-dC) and 4% glycerol for 20 min. After incubation, 15 µl of binding reaction was immediately loaded on a 7.5% gel slab (37.5 acrylamide:1 bis-acrylamide) in 0.5x Tris-boric acid-EDTA and electrophoresed at 200 V for 20–30 min. Gels were then dried and exposed to autoradiography with an intensifier screen at –80°C.

RNA extraction and QPCR. Whole lungs were perfused free of blood via right ventricular perfusion with 10 ml of warmed saline, rapidly excised en bloc, blotted, and snap frozen in liquid nitrogen. Total RNA was isolated from 15 mg of whole lung tissue or the macrophage-enriched BALF cell pellet (consisting of 1 million cells) according to manufacturer instructions with an RNeasy kit (Qiagen). The purified total RNA prep was used as a template to generate first-strand cDNA with SuperScript II (Invitrogen). The reaction mix containing 1 µg of RNA, 250 ng of random hexamers (Promega), and 10 mM dNTP mix was made up to 12 µl with sterile water, heated to 65°C for 5 min, and chilled on ice for 1 min. First-strand synthesis was then performed in 20 µl of total reaction volume by adding (in mM) 50 Tris·HCl (pH 8.3), 75 KCl, 3 MgCl₂, and 10 DTT with 40 U of RNaseout and 200 U of Superscript II reverse transcriptase enzyme at 42°C for 50 min, followed by enzyme inactivation at 70°C for 15 min. cDNA was diluted 10-fold in sterile water and stored at –20°C before amplification.

In this study the QPCR technique based on the 5' exonuclease activity of the Taq polymerase was used. In addition to the sense and antisense primer, an oligonucleotide probe with a 5' fluorescent reporter dye (6FAM) and a 3' quencher dye (TAMRA) hybridizes downstream of the sense primer to the target sequence. Based on a 10-µl reaction volume performed in a 384 optical well plate, the master mixture was prepared from the TaqMan Universal Master Mix (Applied Biosystems) comprising AmpliTaq Gold DNA polymerase, Amperase UNG, dNTPs (dCTP, dGTP, dATP, and dUTP), passive reference 6-carboxy-rhodamine, MgCl₂, and buffer components in amounts undisclosed by the manufacturer. To maximize accuracy and comparability, preoptimized primers and probes were purchased from Applied Biosystems and custom configured in microfluidic card format. As an internal control, eukaryotic 18S rRNA (Applied Biosystems) was measured for use as a reference. A negative (no template) control was included in every run. The fluorescence signal was monitored online with the laser detector of the ABI Prism 7900HT sequence detection system (Applied Biosystems) under default cycling parameters for the microfluidic card format. Each assay was performed in replicates of four. The threshold cycle (C_T) value is the PCR cycle number (out of 40) at which the measured fluorescent signal exceeds a calculated background threshold identifying amplification of the target sequence value and is proportional to the number of input target copies present in the sample. C_T numbers were transformed with the C_T (threshold cycle time) and relative value method as described by Applied Biosystems and were expressed relative to 18S rRNA levels.

Preparation of whole lung cell protein extracts. Protein extracts were obtained as previously described (7). Briefly, after lavage, the lungs were perfused via the right ventricle with 5 ml of PBS to remove intravascular leukocytes, removed, snap frozen in liquid nitrogen, ground, and stored at –80°C. For preparation of whole cell extracts, 10 mg of ground lung tissue was resuspended in 500 µl of lysis buffer [in mM: 50 Tris·HCl (pH 7.5), 120 NaCl, 1 EDTA, 50 NaF, 40 -glycerophosphate, 1 benzamidine, and 0.5 PMSF with 1% (vol/vol) Nonidet P-40]. After a 15-min incubation on ice, homogenates were cleared by centrifugation for 10 min (13,000 rpm at 4°C). Protein concentrations from whole lung extracts were determined with the D_C protein assay (Bio-Rad), and all extracts were stored at –80°C.

Western blot analysis. Western blot analysis was performed as previously described (7). Briefly, whole cell extracts (30 µg) were diluted in Laemmli sample buffer, denatured at 90°C for 5 min, and subjected to SDS-PAGE on 10% gel slabs with 5% stacking gels at 200 V for 45 min. Resolved proteins were transferred onto Hybond polyvinylidene difluoride (PVDF) membrane (Amersham, Arlington Heights, IL), using a Trans-blot SD transfer cell (Bio-Rad) at 10 V for 30 min. After transfer, PVDF membranes were incubated in blocking solution [Tris-buffered saline (TBS) containing 5% (wt/vol) skim milk powder and 0.5% (vol/vol) Tween 20] for 1 h at room temperature. The activation status of Akt, Erk1/2, and p38 was then assessed with phospho-specific antibodies (Cell Signaling, Beverly, MA) that recognize activated forms of these kinases. Membranes were incubated with primary antibodies diluted in blocking solution overnight at 4°C. After primary incubation, membranes were washed three times in blocking solution and incubated for 1 h with horseradish peroxidase-conjugated anti-IgG (Bio-Rad). A final wash over 30 min with six changes in wash buffer (TBS containing 0.5% Tween 20) was performed. Immunoreactive bands were finally visualized by autoradiography with chemiluminescence (ECL, Amersham). Densitometry was performed with Kodak EDAS 1D image analysis software.

Carboxyhemoglobin measurements. To ensure that smoke exposure levels were comparable to those experienced by human smokers (69) we measured carboxyhemoglobin (CoHb). Immediately after removal from the smoke chamber, blood was collected in a heparinized blood gas syringe from the abdominal vena cava of each mouse. Eighty-five microliters was analyzed in a Radiometer ABL520 blood gas analyzer, which measures COHb across a range of 0–100%. Recovery levels were determined 16 h after the last exposure.

Histology. Histology was performed as previously described (20). Briefly, to ensure consistent morphological preservation of lungs, mice were killed by intraperitoneal anesthesia (5.6 mg ketamine, 1.12 mg xylazine) overdose and then perfusion fixed via a tracheal cannula with 4% formaldehyde at exactly 200 mmH₂O pressure. After 1 h the trachea was ligated, and the lungs were removed from the thorax and immersed in 4% formaldehyde for a minimum period of 24 h. After fixation of the lung tissue and processing in paraffin wax, sections (3–4 µm thick) were cut longitudinally through the left and right lung so as to include all lobes. Sections were stained with hematoxylin and eosin for general histopathology or Alcian blue-periodic acid Schiff (AB-PAS), with -amylase predigestion to remove glycogen, for the detection of acid and neutral mucins and identification of goblet cells. Immunostaining of paraffin-embedded lung sections was performed with specific S100A8 antibody and the avidin-biotin complex staining system (both from Santa Cruz) in accordance with the manufacturer's instructions and as previously described (5). S100A8 is a highly abundant neutrophil cytoplasmic protein (5). Briefly, rehydrated sections were subjected to antigen unmasking by heat treatment, and endogenous peroxidase activity was quenched with 1% H₂O₂ in methanol. Sections were blocked for 1 h in normal blocking serum, and primary antibody was incubated at a 1:200 dilution (1.5% blocking solution in PBS) overnight at 4°C. Control sections were incubated in the absence of primary antibody. Sections were incubated with biotinylated secondary antibody and subsequent immunoperoxidase staining using diaminobenzidine chromogen as a substrate as per manufacturer's instructions. Sections were lightly counterstained with hematoxylin.

Statistical analyses. Because data were normally distributed, they are presented as grouped data expressed as means ± SE; n represents the number of mice. Differences in total BALF cell types and differential counts were determined by one-way ANOVA followed by Dunnett post hoc test for multiple comparisons, where appropriate. In some cases, Student's unpaired t-test was used to determine whether there were significant differences between means of pairs. All statistical analyses were performed with GraphPad Prism for Windows (version 3.03). In all cases, probability levels < 0.05 (P < 0.05) were taken to indicate statistical significance.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Dose-response effect of cigarette smoke on BALF cellularity. In dose-response experiments in which Balb/c mice were exposed to cigarette smoke generated from 3–9 cigarettes/day for 4 days, there was a dose-dependent increase in the total number of cells in BALF, peaking at 9 cigarettes/day (Fig. 1A). There was a dose-dependent increase in neutrophil numbers (Fig. 1A). There was an increase in the total number of macrophages in BALF from mice exposed to 9 cigarettes/day, whereas those receiving 3 and 6 cigarettes/day had macrophage numbers similar to mice that were not exposed to cigarette smoke (Fig. 1A). In addition, smoke-exposed mice had an increased percentage of BAL macrophages in mitosis (4.8 ± 0.1%), based on nuclear mitotic figures indicating active cell division as we previously described (6), compared with sham-exposed mice (0.6 ± 0.1%; P < 0.0001, Student's unpaired t-test; see also Fig. 7B). In contrast, BALF of smoke-exposed Balb/c mice had very few and variable numbers of lymphocytes (1,121 ± 678 in 9 cigarette/day group) compared with sham-exposed mice (783 ± 528). FACS analysis of BALF cell pellets from sham-exposed Balb/c mice showed that CD3+CD4+ and CD3+CD8+ cells accounted for 0.25% and 0.27% of the total number of viable cells, respectively. Because of the large number of infiltrating neutrophils and macrophages, there was an apparent decrease in the percentage of CD3+CD4+ (0.14%) and CD3+CD8+ (0.05%) in mice exposed to cigarette smoke (9 cigarettes/day; total absolute cells/mouse = 1,202,500, averaged from pooled samples).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Cigarette smoke induces time- and dose-dependent inflammation in Balb/c mice. Bronchoalveolar lavage fluid (BALF) cellular composition was differentiated on cytospin preparations in dose-response (A) and time course (B) experiments. Data are shown as means ± SE for n = 8–9 mice per treatment group. *P < 0.05, ANOVA and Dunnett test.

View larger version (63K): [in this window] [in a new window]   Fig. 7. Effect of cigarette smoke exposure on lung cells and tissue in Balb/c mice. A and B: representative DiffQuik-stained cytospin preparations of BALF from sham-exposed mice (A) or mice exposed to smoke generated from 9 cigarettes/day for 4 days (B). Note in A that sham-exposed mouse lavage comprises >98% resting alveolar macrophages, whereas B shows smoke-exposed mouse lavage comprising activated macrophages with vacuolated cytoplasm and recruitment of neutrophils (N); inset shows a macrophage in mitosis. C and D: hematoxylin and eosin-stained paraffin sections of lungs from sham-exposed mice (C) or mice exposed to cigarette smoke (D). In D note the peribronchial infiltration of mononuclear cells and some neutrophils (thin arrow) and infiltration of alveoli with mononuclear cells (thick arrow). E and F: serial paraffin-embedded sections cut transversely along a distal bronchus (Br) from smoke-exposed mouse. E: a control section. F: S100A8-positive neutrophils found intravascularly (thin arrow) and intraluminally (thick arrow) in alveoli. V, blood vessel. G and H: Alcian blue-periodic acid Schiff-stained paraffin sections of lungs from sham mice (G) or mice exposed to cigarette smoke (H). In H note epithelial goblet (mucus) cell induction (arrow). Magnification as indicated by scale bars.

NF-B activation. EMSA studies of nuclear extracts obtained from lavaged and flushed whole lung of Balb/c mice showed a dose-dependent increase in NF-B nuclear localization peaking at 9 cigarettes/day (Fig. 2).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Cigarette smoke induces dose-dependent nuclear localization of NF-B binding in Balb/c mice. EMSA was performed on nuclear extracts from whole lung. Results are typical of 3 replicates.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Proteases are dose-dependently induced by cigarette smoke from 3, 6, and 9 cigarettes/day for 4 days in Balb/c mice. A: matrix metalloproteinase (MMP)-9 assayed by gelatin zymography under reducing conditions. The BALF from mice in each treatment group (n = 8–9) was pooled. B: net free gelatinase activity by degradation of flurogenic gelatin substrate, in neat BALF assayed from individual mice, providing a measure of the balance of protease/antiprotease. C: net serine protease activity in neat BALF from individual mice measured colorimetrically with N-methoxysuccinyl-Ala-Ala-Pro-Val-4-nitroanilide substrate. Data in B and C are means ± SE for n = 8–9 mice per treatment group. *P < 0.05, ANOVA and Dunnett test.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Kinetics of the cigarette smoke-induced proteases in Balb/c mice. Assay conditions were as described in Fig. 3. A: MMP-9 expression in pooled samples. Net free gelatinase activity measured fluorometrically (B) and net free serine protease activity measured colorimetrically (C) in BALF obtained from Balb/c mice exposed to cigarette smoke generated from 9 cigarettes/day for up to 4 days are also shown. Data in B and C are means ± SE for n = 8–9 mice per treatment group. *P < 0.05, ANOVA and Dunnett test.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effects of cigarette smoke exposure on the induction of phospho-Akt and phospho-ERK1/2 in whole lung obtained from sham- and cigarette smoke-exposed Balb/c mouse lungs. These blots are typical of 3 replicates, and densitometry is shown as means ± SE. *P < 0.05, ANOVA and Dunnett test.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Cigarette smoke dose-dependently, but differentially, induced inflammatory proteases and innate host defense genes in Balb/c mice. mRNA expression for all genes was measured simultaneously under identical conditions with microfluidic format quantitative real-time PCR (QPCR). Responses are shown as fold expression relative to 18S. Note that in some panels 2 y-axes are used to accommodate differences in fold induction. Data are means ± SE of 4 replicates. MCP-1, monocyte chemoattractant protein-1; MIP-2, macrophage inflammatory protein-2; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; GM-CSF, granulocyte-macrophage colony-stimulating factor; CSF-1, colony-stimulating factor-1; CSF-1R, CSF-1 receptor; TGF-, transforming growth factor-.

COHb levels. The levels of CoHb in the blood of Balb/c mice exposed to cigarette smoke generated from 9 cigarettes/day for 4 days were measured. COHb levels in blood from mice immediately after the last cigarette were significantly increased compared with sham-exposed mice (3.1 ± 0.6% and 0.1 ± 0.0%, respectively). This level is comparable to that observed in smokers (69). In contrast, COHb levels in blood taken from mice the day after the last cigarette were similar to COHb levels in sham-exposed mice (0.1 ± 0.0% and 0.1 ± 0.0%, respectively).

Strain-dependent effects of cigarette smoke on BALF cellularity and protease expression. We compared the effects of cigarette smoke on BALF cellularity and protease expression obtained in Balb/c mice with those obtained in C57BL/6, 129SvJ, and C3H/HeJ mice. We surveyed C57BL/6 and 129SvJ mice because they are backgrounds commonly used to generate gene knockout mice. In addition, we used C3H/HeJ mice because they have a mutation in the TLR4 receptor, thereby abolishing their ability to respond to LPS. In C57BL/6 mice exposed to cigarette smoke generated from 9 cigarettes/day for 1–4 days, there was an increase in the total number of cells in BALF from mice exposed to cigarette smoke for 2, 3, and 4 days (Fig. 8A). However, total cell numbers in BALF from mice exposed to cigarette smoke for 1 day were no different from total cell numbers in BALF from sham-exposed mice. C57BL/6 mice exposed to cigarette smoke generated from 9 cigarettes/day for 2, 3, and 4 days exhibited a significant influx of neutrophils (Fig. 8B), whereas few, if any, could be detected in the BALF of mice exposed to cigarette smoke for 1 day. Similarly, there was an increase in the total number of macrophages in BALF from mice exposed to cigarette smoke for 3 and 4 days, but there was no increase in the total number of macrophages in BALF from mice exposed to 9 cigarettes/day for 1 and 2 days (Fig. 8C). FACS analysis of BALF cell pellets for sham-exposed C57BL/6 mice (4 days) showed that CD3+CD4+ and CD3+CD8+ cells accounted for 1.42% and 0.93% of the total number of viable cells, respectively. However, as with the Balb/c mice, there was a decrease in the percentage of CD3+CD4+ (0.08%) and CD3+CD8+ (0.00%) in mice exposed to cigarette smoke (9 cigarettes/day for 4 days, total absolute cells/mouse = 420,000).

View larger version (22K): [in this window] [in a new window]   Fig. 8. Cigarette smoke induces time-dependent BALF inflammation in C57BL/6, 129SvJ, and C3H/HeJ mice. A: total cells. B: total neutrophils. C: total macrophages. D: MMP-9 expression in pooled BALF samples. Data are means ± SE for n = 8–9 mice per treatment group. *P < 0.05, ANOVA and Dunnett test. Note that Balb/c data are duplicated from Fig. 1 to enable easy comparisons across all of the mouse strains in the study.

In C3H/HeJ mice exposed to smoke generated from 9 cigarettes/day for 1–4 days, there was an increase in the total number of cells in BALF from mice exposed to cigarette smoke for 3 and 4 days. However, total cell numbers in BALF from mice exposed to cigarette smoke for 1 and 2 days were no different from total cell numbers in BALF from mice that were not exposed to cigarette smoke (Fig. 8A). C3H/HeJ mice exposed to smoke generated from 9 cigarettes/day for 3 and 4 days exhibited a significant influx of neutrophils (Fig. 8B), whereas few, if any, could be detected in the BALF of mice exposed to cigarette smoke for 1 and 2 days. Similarly, there was an increase in the total number of macrophages in BALF from mice exposed to cigarette smoke for 2, 3, and 4 days, but there was no increase in the total number of macrophages in BALF from mice exposed to 9 cigarettes/day for 1 day (Fig. 8C). In addition, we could not detect any lymphocytes in BALF of sham- or smoke-exposed mice.

In time course experiments, there was a modest increase in MMP-9 expression in BALF from C57BL/6 and C3H/HeJ mice exposed to smoke generated from 9 cigarettes/day for 2, 3, and 4 days as assessed by zymography (Fig. 8D). In contrast, BALF from mice exposed to smoke for 1 day had levels similar to those observed in sham-exposed mice. In contrast, BALF from 129SvJ mice exposed to smoke generated from 9 cigarettes/day for 2, 3, and 4 days had very little MMP-9 expression. Given that there was little or only modest increase in MMP-9 expression in C57BL/6, C3H/HeJ, and 129SvJ, whereas there was marked increase in MMP-9 expression in BALF from Balb/c mice, it was not surprising to find no or very small and variable increases in net gelatinase activity in the BALF from C57BL/6, C3H/HeJ, and 129SvJ mice exposed to smoke (data not shown).

Effect of cigarette smoke on whole lung and macrophage-enriched BALF cell pellet gene expression. In addition to investigating the expression of diverse genes in response to increasing doses of cigarette smoke in Balb/c mice, we explored the impact of cigarette smoke in whole lung and BALF cell pellets enriched for macrophages from Balb/c and C57BL/6 mice. Balb/c and C57BL/6 mice were chosen for the comparison because they represented a susceptible and resistant strain, respectively, based on BALF cellularity and protease expression profiles. Cigarette smoke exposure caused an increase of whole lung mRNAs in both Balb/c and C57BL/6 mice for proinflammatory cytokines (TNF-, IL-6), inflammatory chemokines (MIP-2), proteases (MMP-12), leukocyte survival genes (GM-CSF, CSF-1), and cyclooxygenase-2 (COX-2; Fig. 9). In contrast, there was no significant increase in mRNAs for IL-10. It was interesting to note that the strain of mouse had no impact on the levels of TNF-, CSF-1, MIP-2, IL-6, GM-CSF, and COX-2, but there was less MMP-12 in C57BL/6 mice. Enriched macrophage BALF cell pellets from cigarette-smoke exposed Balb/c and C57BL/6 mice had increased TNF-, CSF-1, MIP-2, MMP-12, and COX-2 mRNA expression. In contrast, there was no significant increase in mRNAs for IL-10, IL-6, and GM-CSF. It was interesting to note that the strain of mouse had no impact on the levels of TNF-, CSF-1, MIP-2, IL-6, GM-CSF, and COX-2, but there was less MMP-12 in C57BL/6 mice. In addition, enriched macrophage BALF cell pellets (regardless of strain) had more TNF-, CSF-1, and COX-2 mRNA but less IL-10, MIP-2, IL-6, and GM-CSF.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Gene expression profiles in whole lung and macrophage-enriched BALF cell pellets (alveolar macs) obtained from Balb/c (Balb) and C57BL/6 (B6) mice. mRNA expression for all genes was measured simultaneously under identical conditions with microfluidic format QPCR. Responses are shown as fold expression relative to 18S. Open bars, sham-exposed animals; filled bars, smoke-exposed animals. Data are means ± SE of 4 replicates. COX-2, cyclooxygenase-2.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

A trivial explanation for the responses we observed would be that they are the passive consequence of increased inflammatory cells, especially neutrophils, which in this study we identified in the lung and microcirculation with antibodies against the S100A8 protein. Neutrophils are transcriptionally less active than macrophages but not silent. However, systematic review of the data indicates that this cannot be the case. Many genes showed a high-dose (9 cigarette) turndown in level, whereas neutrophils were increasing. More importantly, there was no consistent concordance between genes known to be expressed in neutrophils (e.g., the marked discordance between TLRs 2, 3, and 4) and those known not to be expressed in neutrophils (GM-CSF, CSF-1, CSF-1R). Furthermore, the experiments that partitioned expression into lung tissue and alveolar macrophages clearly indicate that the gene changes cannot be the passive consequence of neutrophil accumulation. This interpretation is consistent with gene profiling studies of human neutrophils, which can be recovered in large numbers from COPD patient peripheral blood (52).

NF-B is induced by cigarette smoke in humans, and the intensity of NF-B activation reflects disease severity in human COPD (8, 17). We observed that NF-B is dose-dependently activated by subchronic exposure to cigarette smoke in vivo. This activation is associated with the development of a neutrophil- and macrophage-rich inflammation observed in both BALF and lung tissue. The induction of NF-B was accompanied by induction of multiple genes associated with cellular inflammation, innate immunity, and repair processes and was concordant with recent global profiles in human COPD (48), but the kinetics of these genes was, in many cases, discordant from NF-B nuclear translocation and binding. It is of some interest that NF-B activation peaked at 9 cigarettes/day. This accorded well with inflammation and protease induction, and smoke has been shown to upregulate NF-B in macrophages but not neutrophils (8) and in epithelium (17). However, gene profiling studies showed that the expression of the majority of genes peaked at 6 cigarettes/day and also showed some downturn at 9 cigarettes/day in many cases.

We therefore profiled AP-1, ERKs, MAPK kinase, and Akt activation. With the exception of ERKs, none of these signaling intermediates was activated at any dose. This finding was unexpected and contrasts sharply with the positive activation of these signaling intermediates we have reported in LPS-challenged animals (7). ERK1/2 showed a small but statistically significant upregulation that was most evident at the 3 cigarettes/day dose, consistent with the influx of neutrophils observed at this dose. In previous studies in which LPS was used as the inflammatory agonist we observed (7) sustained activation of AP-1 and ERK1 and biphasic activation of Akt but not activation of ERK2 or p38 MAPK. The absence or weaker induction of kinase activity may mean that MAPK kinase or Akt activities are either very discretely, or very transiently, induced or that they do not contribute substantially in this model. The high-dose downturn we observed in some responses also suggests the possibility of induction of counterregulatory factors that remain unidentified, but it is likely that NF-B-dependent processes drive much or all of the response pattern we observed. Discordant responses are not unexpected as NF-B regulates gene expression in concert with other transcription factors via transcriptional complexes. Recently, novel transcription factors that are likely to regulate responses to smoke in concert with NF-B and AP-1, such as Egr-1 and Nrf-2, have been described in human COPD lung tissue and rodent models (25, 48, 55). Nrf-2 seems particularly important in regulating macrophage gene programs in smoke-exposed mice (30). The results of our partitioning experiments indicate that macrophages are strongly activated in this model but also that lung tissue is activated. This is consistent with LPS inflammation models in which the anatomic nexus between macrophages and epithelium in vivo is important to achieve full NF-B activation (33). Recently it has proven possible to selectively manipulate NF-B in lung epithelium and in macrophages in LPS responses in vivo (71). A similar approach would help to further dissect the contribution of NF-B in lung responses to smoke.

Histological, bronchial biopsy, sputum, and lavage studies have shown that smokers and patients with COPD have an increased number of neutrophils (16, 56), which in COPD patients correlated with disease severity and lung function (16, 32, 54, 66). The infiltration we observed was broadly in accordance with the induction of MIP-2, a potent murine neutrophil chemotactic factor. MIP-2 levels plateaued at 6 cigarettes/day, but multiple other chemoattractants, such as KC (murine equivalent of human IL-8), could also contribute and were not measured in this study. Neutrophils secrete serine proteases, including neutrophil elastase, cathepsin G, and proteinase-3, as well as MMP-8 and MMP-9, which contribute to tissue destruction and may have other roles in host defense (2). Serine proteases are potent stimulants of mucus hypersecretion (65). The pattern of net serine protease activity we measured accorded well with S100A8-positive neutrophil number in both the dose-response and time course studies. Neutrophil survival and activation are strongly upregulated by GM-CSF (2, 27), which is predominantly produced by the lung epithelium in vivo, and it is noteworthy that GM-CSF mRNA levels mirrored neutrophil responses. In addition, we found that cigarette smoke increased mRNA levels of IL-6, which plays a critical role in mediating the transition from neutrophilic to monocytic inflammation (44).

Macrophages are markedly increased in the lung parenchyma of smokers and patients with COPD (56), where their number correlates with disease severity (16). Macrophages can be directly activated by cigarette smoke to release inflammatory mediators including TNF-, MCP-1, reactive oxygen species, and neutrophil chemotactic factors. In other models, such as LPS-induced inflammation, resident alveolar macrophages are the primary drivers of both neutrophilic and mononuclear cell infiltration of the lung via NF-B-dependent processes (33, 42). In humans there is some evidence for macrophage heterogeneity in smoke-exposed lungs. As we observed mononuclear lineage chemotactic factor induction it is likely that the increase in macrophages we observed represents both the progeny of resident alveolar macrophages and influx of blood monocytes adopting an alveolar macrophage-like morphology ("alveolar monocytes"), as has been demonstrated after LPS challenge (41). If this is the case it is likely that recruited monocytes contribute to further neutrophil accumulation (42, 43). The induction of CSF-1 and its receptor as well as GM-CSF is consistent with this interpretation as well as the observation that a substantial fraction of macrophages in smoke-exposed lungs were in mitosis. Induction of GM-CSF and CSF-1 in the lung may also trigger leukocyte release or accelerate myelopoiesis in the bone marrow, which has been known for some time to be increased by smoke (67, 68). In addition to its role in leukocyte survival and innate immunity (6), GM-CSF primes the lungs for allergic sensitization (57). The upregulation of GM-CSF we observed may help to understand why smoking enhances asthma development in atopic individuals (45). Although lymphocytes infiltrate the lungs of long-term smokers they were not prominent in this model, and FACS profiling indicates that they constitute a very small fraction of total cells in this model comprising both CD3+CD4+ and CD3+CD8+ lymphocytes. CD3+CD8+ lymphocytes have recently been directly implicated in long-term smoke-induced inflammation and lung damage, suggesting that more sustained inflammation is necessary to drive their recruitment or proliferation (51, 61). As T cell activation and proliferation in response to lung pathogens take 4–7 days this is not surprising (18, 24). These data are consistent with the reports of Guerassimov et al. (26) and D'Hulst et al. (14) showing that lymphocyte accumulation progresses, only slowing in response to smoke. It is also consistent with both a recent T cell receptor (TCR) usage study of T cells from smokers in which the high frequency of oligoclonally expanded TCR indicated local proliferation rather than nonspecific recruitment from blood and a similar recruitment pattern found for influenza and respiratory syncytial virus-specific CD8+ T cells in human lung (34, 13). This, at least in the short-term smoke model, does not appear to be a major signal for lymphocyte recruitment, in contrast to effects on neutrophils and macrophages. The recent work of Stampfli and colleagues (19, 58), however, suggests that smoke exposure predisposes lung to more rapidly and extensive accumulate lymphocytes in response to Pseudomonas aeruginosa or attenuated adenovirus challenge and impairs immunity against these pathogens.

Proteases that break down connective tissue components are found in increased amounts in people with COPD. In patients with emphysema there is an increase in BALF concentrations and macrophage expression of MMP-1 (collagenase) and MMP-9 (gelatinase) (23). There is an increase in activity of MMP-9 in the lung parenchyma of patients with emphysema (49). Alveolar macrophages from normal smokers express more MMP-9 than those from normal subjects (37), and there is an even greater increase in cells from patients with COPD (59), which have greatly enhanced elastolytic activity (60). The interest in MMPs has been heightened by the demonstration that emphysema induced by chronic cigarette smoke exposure is prevented in MMP-12^–/– mice (28). uPA and tPA have functional roles in regulating the persistence of temporary matrix, such as fibrin deposition, and are therefore of interest in inflammation; however, they were not markedly induced.

Alveolar macrophages also secrete proteolytic enzymes, including MMP-2, MMP-9, MMP-12, and cathepsin K, L, and S, which contribute to destruction of lung parenchyma (2). Macrophages are therefore the likely source of the progressively increased levels of MMP-9 and much of the net gelatinase activity we measured. Although MMP-9 is more abundant than MMP-2 in COPD, MMP-2 is more efficient in degrading matrix proteins (64). The accumulation of macrophages may also explain, in part, the very marked increase in MMP-12 message we observed; however, if this is the case the downturn in MMP-12 transcript observed at 9 cigarettes/day implies active downregulation of MMP-12 as the macrophage number was still rising at this dose. This concept was confirmed in the enriched BALF cell pellet from Balb/c mice, where similar levels of MMP-12 mRNA were found for both whole lung and the macrophage-enriched BALF cell pellet. This was also true for C57BL/6 mice. It was interesting to note that although similar numbers of macrophages in the enriched cell pellet were obtained, there was a difference between the levels of MMP-12 in Balb/c and C57BL/6 mice, suggesting that alveolar macrophages from express more MMP-12 or that there is strain difference. TGF- negatively regulates MMP-12 in the mouse lung via _v6-integrin (47), but we did not observe a reciprocal relationship between TGF- and MMP-12 signals. As TGF-1 is directly controlled by Egr-1 it is likely that this COPD-associated transcription factor was active in this model (38). In the mouse MMP-12 also has an established role in processing TNF- for secretion (11). Levels of MMP-12 protein were below detection with zymography in our study, but MMP-12 is detectable by immunocytochemistry after smoke exposure where it is localized to macrophages (R. Vlahos and S. Ivanov, unpublished observation). MMP-12 has a dominant role in other mouse models of smoke-induced lung inflammation, and it is known to be discretely localized into macrophages in human lung disease (10–12, 28, 46). Overexpression of IL-1, which was strongly induced in our study, induces emphysema and neutrophilic/monocytic inflammation in mice associated with MMP-9 and MMP-12 induction and upregulation of MIP-2 (36). The duration of exposure we used was too short to cause emphysema, but our model may have additional utility as a surrogate if the protease induction we observed is proven to predict emphysema. Churg et al. (10) observed matrix breakdown products even after a single smoke exposure, and it is probable that the proteases we observed in our study may be surrogate of longer-term peripheral air space enlargement that we, like others, have observed by extending smoke exposure over 3 mo (R. Vlahos, unpublished observation). The striking induction of proteases suggests the possibility that they may be contributing to processes other than matrix turnover, such as activation of inflammatory mediators as has been described for the proteolytic release of TNF- by TNF- converting enzyme (ADAM17) (11). Lung proteolysis is actively counteracted by antiproteases. We did not measure antiproteases in this study; however, the zymographic and fluorogenic substrate approaches we used have the advantage of defining actual net matrix-degrading activity over more sensitive but less informative methods such as immunocytochemistry and ELISA.

As well as their role in matrix turnover, MMP-12 and other MMPs are increasingly recognized for their contribution to host defense. TLR2 recognizes gram-positive bacterial endotoxin, TLR4 recognizes gram-negative endotoxin (LPS), TLR 3 recognizes viral double-strand RNA, and TLR9 recognizes bacterial and viral CpG DNA (4). It is noteworthy that MMP-12 regulates the levels of soluble CD-14, an essential component in LPS signaling in responses to gram-negative bacteria (62). However, TLR4 was not markedly upregulated. This was unexpected, as we demonstrated previously (7) that GM-CSF regulates TLR4 levels in the lung and GM-CSF was upregulated in this model. As these TLRs are variably expressed on infiltrating leukocytes in inflammation, the marked upregulation of TLR2 is unlikely to reflect a simple increase in cell number, because similar changes would have been expected for TLR3, 4, and 9. In contrast to other TLRs, TLR2 expression is regulated by NF-B (21, 22), and its marked upregulation was mirrored by NF-B translocation in our study. The increases we observed in TLR2 suggest that smoke may markedly upregulate inflammation triggered by some infectious agents via TLR2 that usually signal via NF-B. Stampfli and colleagues (19, 58) recently demonstrated such an effect with replication-deficient adenovirus and the lung-colonizing pathogen P. aeruginosa, which can induce responses via multiple TLRs. However, interaction between smoke, TLRs, inflammation, and infection is likely to be much more complex, as we have demonstrated (35) that smoke transiently represses epithelial responses to LPS. It is noteworthy that population studies clearly demonstrate that smoke exposure is a risk factor for poor lung health in general, including precipitation of asthma and increasing the susceptibility to chest infections in children (9, 15, 31). The current model may prove useful to understanding some of these mechanisms as we have observed that subchronic exposure markedly increased inflammation triggered by live influenza A (R. C. Gualano, unpublished observation).

An important determinant of susceptibility to cigarette smoke-induced lung inflammation is genetic variation (26). In this study we profiled inflammation and gene expression in a number of mouse strains. We found that C57BL/6, 129SvJ, and C3H/HeJ mice had less BALF inflammation compared with Balb/c mice. In addition, Balb/c mice had significantly more protease expression activity assessed by zymography and substrate-based assays, whereas C57BL/6 mice were markedly resistant. To further extend the comparison we compared the gene profiles of lungs obtained from susceptible Balb/c mice and resistant C57BL/6 mice. We found that cigarette smoke exposure caused a similar increase of whole lung mRNAs in both Balb/c and C57BL/6 mice for TNF-, MIP-2, IL-6, GM-CSF, CSF-1, and COX-2 but that lungs from C57BL/6 mice had slightly less MMP-12 than lungs from Balb/c mice, suggesting that there was no strain-dependent effect on the expression of the genes investigated in this study. Gene expression profiles were also explored in macrophage-enriched BALF cell pellets. Macrophage-enriched BALF cell pellets from both Balb/c and C57BL/6 mice had increased levels of TNF-, CSF-1, MIP-2, MMP-12, and COX-2 mRNA. In contrast, there was no significant increase in IL-10, IL-6, and GM-CSF mRNA. It was interesting to note that macrophage-enriched BALF cell pellets, regardless of mouse strain, had more TNF-, CSF-1, and COX-2 mRNA but less IL-10, MIP-2, IL-6, and GM-CSF than whole lung.

In summary, we have profiled a subchronic mouse model of smoke-induced lung inflammation. The pattern of responses we observed suggest that this subchronic model may prove a useful adjunct to existing acute and long-term chronic models for the identification of the molecular mechanisms by which smoke impairs lung health.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Vlahos, Dept. of Medicine, Univ. of Melbourne, Royal Melbourne Hospital, Parkville VIC 3050 Australia (e-mail: rossv{at}unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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