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Am J Physiol Lung Cell Mol Physiol 294: L1174-L1186, 2008. First published March 28, 2008; doi:10.1152/ajplung.00439.2007
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Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice

Hongwei Yao, Indika Edirisinghe, Saravanan Rajendrasozhan, Se-Ran Yang, Samuel Caito, David Adenuga, and Irfan Rahman

Lung Biology and Disease Program, Department of Environmental Medicine, University of Rochester Medical Center, Rochester, New York

Submitted 22 October 2007 ; accepted in final form 25 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A variety of mouse models have been used to study the pathogenesis of pulmonary emphysema/chronic obstructive pulmonary disease. The effect of cigarette smoke (CS) is believed to be strain dependent, because certain mouse strains are more susceptible or resistant to development of emphysema. However, the molecular basis of susceptibility of mouse strains to effects of CS is not known. We investigated the effect of CS on lungs of most of the commonly used mouse strains to study the molecular mechanism of susceptibility to effects of CS. C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice were exposed to CS for 3 consecutive days, and various parameters of inflammatory and oxidative responses were assessed in lungs of these mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to lung inflammatory and oxidant responses to CS exposure. The mouse strain that was more susceptible to effects of CS showed augmented lung inflammatory cell influx, activation of NF-{kappa}B and p38 MAPK, and increased levels of matrix metalloproteinase-9 and NF-{kappa}B-dependent proinflammatory cytokines compared with resistant mouse strains. Similarly, decreased levels of glutathione were associated with increased levels of lipid peroxidation products in susceptible mouse strains compared with resistant strains. Hence, we identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains could be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.

nuclear factor-{kappa}B; histone deacetylase-2; p38 MAPK; lipid peroxides; glutathione; chronic obstructive pulmonary disease

CIGARETTE SMOKE (CS) is the main etiological factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). CS triggers inflammation and oxidative stress, which play an important role in the pathogenesis of COPD. However, very little is known about the molecular mechanisms whereby CS triggers abnormal inflammation in the lung. A variety of mouse models with differential susceptibility to effects of CS have been used to study experimental emphysema/COPD. Mice of an inbred strain are genetically identical; thus mice from different strains represent an ideal model for study of the susceptibility of different phenotypes to CS-induced lung inflammation (1, 4, 23, 52). For example, it has been shown that the development of emphysema in mice is dependent on strain and CS dose (1, 4, 15, 23). However, the molecular basis of susceptibility of mouse strains to CS-induced inflammatory and oxidative responses is not known. Hence, identification of a mouse strain for acute or short-term CS exposure would be a useful model to study the molecular mechanism of inflammation and pharmacological interventions of certain agents in CS-induced lung inflammation (46). Therefore, we studied the acute effect of CS-induced inflammatory and oxidative responses in lungs of different strains and proposed that these effects would be strain and dose dependent. To investigate the susceptibility of the lung to inflammatory and oxidant responses to acute CS exposure, we exploited the effect of genetic variations present in most commonly used strains of mice, such as C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ, by exposing the animals for 3 consecutive days to CS at 80 and 300 mg/m3 total particulate matter (TPM). Furthermore, two time points (2 and 24 h after the last CS exposure) were chosen to study the lung inflammatory and oxidative responses in these strains of mice, because it has been shown that the lung inflammatory response occurs within hours after the end of the last CS exposure in C57BL/6J mice (45).

Recently, a variety of target-signaling molecules, such as mitogen-activated protein kinase (MAPK), nuclear factor-{kappa}B (NF-{kappa}B) signaling pathway, and histone deacetylases (HDACs), have been identified in response to CS, and they may be responsible for the severity of the lung inflammatory response in different strains in mice. Therefore, we investigated the molecular mechanism of susceptibility of different mouse strains to effects of CS.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult (22–25 g body wt) male C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice (Jackson Laboratory, Bar Harbor, ME) were housed in the Inhalation Core Facility at the University of Rochester for 1 wk of acclimatization before CS exposure. All animal procedures were approved by the Committee on Animal Research of the University of Rochester.

CS exposure. Eight- to 10-wk-old mice (6–8 mice per group at each time point) were used for CS exposure at two different concentrations for 3 days. Briefly, mice were housed in an individual wire cage compartment, which was placed inside an aerated plastic box, which, in turn, was connected to the smoke source. Research-grade cigarettes (2R4F, University of Kentucky, Lexington, KY; 11.7 mg TPM, 9.7 mg tar, and 0.85 mg nicotine per cigarette) were used to generate smoke, and mice were exposed according to the US Federal Trade Commission protocol (1 puff/min of 2-s duration and 35-ml volume) using an automatic cigarette-smoking machine (Baumgartner-Jaeger CSM2072i, CH Technologies, Westwood, NJ). Mainstream CS was diluted with filtered air and directed into the exposure chamber. Smoke exposure (TPM/m3 of air in chamber) was monitored in real time with an aerosol monitor (MicroDust Pro, Casella CEL, Bedford, UK) and verified daily by gravimetric sampling (14, 24, 45, 46, 51). Chamber atmosphere was also monitored for TPM by adjustment of the flow rate of the diluted medical air (14, 24, 45, 46). The 300 mg/m3 TPM protocol consisted of two 1-h CS exposures 1 h apart for 3 consecutive days, with the mice killed at 2 and 24 h after the last exposure. The 80 mg/m3 TPM protocol consisted of CS exposure for 6 h/day for 3 consecutive days, with the mice killed at 24 h after the last exposure. The levels of carbon monoxide (CO), carboxyhemoglobin, and cotinine in the 300 and 80 mg/m3 TPM protocols are shown in Table 1. No significant change of body weight between the strains of mice was observed in response to 3 days of CS exposure. Control mice were exposed to filtered air in an identical chamber according to the same protocol described for CS exposure.


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  		Table 1. Blood carboxyhemoglobin and cotinine levels and chamber concentration of CO in response to cigarette smoke exposure

 
Bronchoalveolar lavage. Mice were injected with pentobarbiturate (100 mg/kg body wt ip; Abbott Laboratories) and killed by exsanguination. The heart and lungs were removed en bloc, and the left lungs were lavaged three times with 0.5 ml of 0.9% NaCl. The lavage fluid was centrifuged, and the cell-free supernatants were frozen at –80°C for later analysis. The bronchoalveolar lavage (BAL) cell pellet was resuspended in 1 ml of 0.9% NaCl, and the total cell numbers were determined by counting on a hemocytometer. Differential cell counts (≥400 cells per slide) were performed on Cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA) stained with Diff-Quik (Dade Behring, Newark, DE).

Immunohistochemistry for macrophages and CD8+ cells. Mouse left lungs (which had not been lavaged) were inflated with 1% low-melting-point agarose at 25 cmH2O pressure and then fixed with 4% neutral buffered paraformaldehyde. Tissues were embedded in paraffin, sectioned (4 µm), and stained for immunohistochemical analysis. For macrophage immunohistochemistry, lung sections were deparaffinized and hydrated by passage through a series of xylene and graded alcohol. Endogenous peroxidase activity was quenched by exposure to 3% H2O2 in methanol for 30 min. Nonspecific binding of antibodies to the tissues was blocked by incubation of the tissue with 5% normal goat serum in 0.5% BSA in PBS for 30 min. For detection of macrophages, rat anti-mouse Mac-3 monoclonal antibody (BD Pharmingen) at a titer of 1:50 was used. A biotinylated goat anti-mouse/rabbit Ig (Dako) was used at a titer of 1:100. Tissues were incubated with primary antibody overnight at 4°C. After they were washed, the tissues were incubated with secondary antibody for 30 min. 3,3'-Diaminobenzidine (Dako) was used as peroxidase substrate. In each instance, sections from different time points were processed together, with equal time for color development. Tissues were counterstained with hematoxylin. The number of Mac-3-positive cells in lung sections (5 random microscopic fields per lung section in 3 different sections) was counted manually in a blinded manner at x200 magnification and averaged as described elsewhere (3, 13, 14, 38). Similarly, CD8+ cells were detected by immunohistochemistry in paraffin-embedded sections using a rat anti-mouse monoclonal antibody (BD Biosciences) (20). Tissues were counterstained with methyl green, and the number of CD8+T cells in the lung sections (3 random microscopic fields per lung section in 3 different sections) was also counted manually in a blinded manner.

Lipid peroxidation product assay in lung homogenate. The right lung lobe was homogenized with ice-cold 20 mM Tris·HCl (pH 7.4) and diluted to ~10% (wt/vol). The homogenates were centrifuged at 3,000 rpm for 10 min at 4°C, and the supernatants were collected. Butylated hydroxytoluene (5 mM; Sigma-Aldrich) was added to the supernatant to prevent further peroxidation, and the samples were immediately frozen in liquid nitrogen. Lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE)] were measured using a lipid peroxidation kit (Calbiochem). For measurement of MDA and 4-HNE, 200 µl of supernatant were added to 650 µl of 10.3 mM N-methyl-2-phenylindole in acetonitrile, and the tubes were vortexed; then 15.4 mM methanesulfonic acid (150 µl) was added to tubes designated for estimation of 4-HNE and 12 N HCl (150 µl) was added to tubes designated for estimation of MDA, and the tubes were incubated at 45°C for 40 min. The samples were cooled on ice, and absorbance was measured spectrophotometrically at 586 nm.

Total glutathione assay. Intracellular reduced and oxidized glutathione (GSH) levels were determined according to the method described previously (33). Briefly, the right lungs were homogenized with 0.1 M phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.1% (vol/vol) Triton X-100, and 0.6% (wt/vol) sulfosalicylic acid. The lung debris was collected by centrifugation, the supernatant was incubated with 0.2 mg/ml of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1.67 U/ml glutathione reductase in phosphate buffer-EDTA for 30 s, 0.2 mg/ml β-NADPH was added, and the rate of DTNB reduction was measured spectrophotometrically at 405 nm. Total GSH concentration in the supernatant was determined by comparison with the rate of DTNB reduction by known concentrations of GSH. Results are expressed as nanomoles of GSH per milligram of protein.

Protein extraction from lung tissue. One hundred milligrams of right lung lobe were mechanically homogenized in 0.5 ml of ice-cold buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 M EDTA, 0.2 mM NaF, 0.2 mM Na3VO4, 1% (vol/vol) NP-40, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µg/ml leupeptin]. The homogenate was centrifuged at 2,000 rpm in a bench-top centrifuge for 30 s at 4°C for removal of cellular debris. The supernatant was then transferred to a 1.7-ml ice-cold Eppendorf tube and further centrifuged for 30 s at 13,000 rpm at 4°C. The supernatant was collected as a cytoplasmic extract. The pellet was resuspended in 200 µl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 M EDTA, 1 mM DTT, 10% (vol/vol) glycerol, 0.2 mM NaF, and 0.2 mM Na3VO4] with a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin, and PMSF) (31, 50) and placed on the roller in the cold room for 30 min. After centrifugation at 13,000 rpm in an Eppendorf tube for 5 min, the supernatant was collected as the nuclear extract and stored at –80°C. Furthermore, 50 mg of lung tissue were homogenized in 0.5 ml of RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 1 mM Na3VO4, 1 mM NaF, 1 µg of leupeptin/ml, 1 µg of aprotinin/ml, and 1 mM PMSF), and the supernatants were used as whole lysate.

Assay of proinflammatory mediators in lungs. After the animals were killed, the right lung lobe was blotted dry on filter paper before homogenization to avoid the effect of water on the levels of cytokines. Fifty milligrams of lung tissue were homogenized in 0.5 ml of RIPA buffer, and the levels of proinflammatory mediators were assayed using a Bio-Plex cytokine assay kit (Bio-Rad Laboratories) according to the manufacturer's instructions. These assays permit simultaneous cytometric quantitation of multiple chemokines/cytokines with minimal sample volume. The assays utilize microspheres as the solid support for immunoassays. The level of protein in these lung homogenates was also determined, and the levels of cytokines were normalized and expressed in the samples as picograms per milligram of protein.

Protein assay. Protein level was measured with a bicinchoninic acid kit (Pierce). Protein standards were obtained by dilution of a stock solution of BSA. Linear regression was used to determine the actual protein concentration of the samples.

Western blot analysis. Lung tissue homogenate samples were separated on a 7.5–12% SDS polyacrylamide gel. Separated proteins were electroblotted onto nitrocellulose membranes (Amersham, Arlington Heights, IL) and blocked for 1 h at room temperature with 5% nonfat dry milk. The membranes were then probed with 1:1,000-diluted antibodies of anti-acetylated RelA/p65 at lysine 31D (K310) (developed and provided by Dr. Leonard Buckbinder, Pfizer), anti-RelA/p65 and anti-HDAC2 (Santa Cruz Biotechnology, Santa Cruz, CA) for determination of respective nuclear proteins, and anti-phospho-p38 MAPK and p38 MAPK (Cell Signaling Technology), and anti-matrix metallopeptidase (MMP)-9 and anti-glutamate-cysteine ligase (GCLC; Santa Cruz Biotechnology) for detection of corresponding protein. After three washing steps (10 min each), the levels of protein were detected using secondary antibody [1:5,000 dilution in 2.5% nonfat dry milk in PBS containing 0.1% (vol/vol) Tween 20 for 1 h] linked to horseradish peroxidase (Dako), and bound complexes were detected using the enhanced chemiluminescence method (Perkin Elmer). Equal loading of the sample was determined by quantitation of protein as well as by reprobing membranes for β-actin as a housekeeping protein.

EMSA. NF-{kappa}B DNA binding was determined using the EMSA detection kit (Promega, Madison, WI) in nuclear protein obtained from the lungs of different strains of mice. The sequence of NF-{kappa}B consensus oligonucleotide was 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5'. Synthetic double-stranded oligonucleotides were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase as recommended by the manufacturer. A binding mixture containing 5 µg of nuclear extract, 2.5 µl of 5x binding buffer [50 mM Tris·HCl (pH 7.5), 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 20% glycerol, and 0.25 mg/ml poly(dI-dC)·poly(dI-dC)], and 1 µl of 32P-labeled double-stranded probe in a total volume of 13.5 µl was used for the DNA-binding reaction, which was conducted at room temperature for 30 min. To ensure that the detected bands were specific for NF-{kappa}B, a 100-fold excess of unlabeled competitor (NF-{kappa}B consensus oligonucleotide) and noncompetitor (activator protein-2 consensus oligonucleotide) was added to the reaction mixture before addition of the probe. Gel tracking dye [250 mM Tris·HCl (pH 7.5), 0.2% bromphenol blue, and 40% glycerol] was loaded into lane 1, and the DNA-protein complexes were loaded into the remaining wells without tracking dye on the 6% nondenaturing polyacrylamide gel. The gel was then dried and subjected to autoradiography.

Statistical analysis. Values are means ± SE. Statistical analysis of significance was calculated using one-way ANOVA followed by Tukey's post hoc test for multigroup comparisons using StatView.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Inflammatory cell influx into lungs of different strains of mice in response to CS exposure. To determine the inflammatory response in the lungs of C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice to CS exposure at 300 mg/m3 TPM, we assessed the number of neutrophils, macrophages, and CD8+ T lymphocytes in BAL fluid (BALF) and lung tissue using Diff-Quick and immunohistochemical staining. As shown in Fig. 1A, the number of neutrophils was significantly increased in the BALF of C57BL/6J and A/J mice, whereas BAL neutrophil numbers were not changed in AKR/J, CD-1, and 129SvJ mice at 2 h after the last CS exposure. At 24 h after the last CS exposure, the number of neutrophils was increased in BALF of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice (Fig. 1B). Interestingly, the number of BALF macrophages was not altered in any of the strains of mice at 2 and 24 h after the last CS exposure (Fig. 1, D–F). However, immunohistochemical staining of macrophages and CD8+ cells showed that CS exposure at 300 mg/m3 TPM resulted in significant influx of macrophages and CD8+ cells into the lung interstitium of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice at 2 and/or 24 h after the last exposure (Figs. 2 and 3). The number of neutrophils was not significantly different in lung interstitium of all the mouse strains studied in response to CS exposure as assessed by hematoxylin-and-eosin staining (data not shown). CS exposure at 300 mg/m3 TPM recruited more inflammatory cells into the lung in C57BL/6J mice than in other strains exposed to CS and killed at 2 and 24 h after the last exposure (Figs. 1–3). These results suggest that the C57BL/6J strain is highly susceptible, the A/J, AKR/J, and CD-1 strains are moderately susceptible, and the 129SvJ strain is resistant to acute CS-mediated inflammatory cell influx into the lungs. Therefore, we further assessed the lung inflammatory response to CS exposure using a lower concentration of CS (80 mg/m3 TPM) in the most sensitive (C57BL/6J) and most resistant (129SvJ) mouse strains at 24 h after the last exposure to determine the effect of the lowest concentration of CS that is used in the literature to induce inflammatory cell influx into the lungs. The number of lavaged neutrophils was increased in C57BL/6J, but not 129SvJ, mice exposed to CS at 80 mg/m3 TPM (Fig. 1C). Moreover, fewer neutrophils were counted in BALF after CS exposure at 80 than at 300 mg/m3 TPM (Fig. 1, B and C). These data suggest that acute CS-induced neutrophil influx is concentration dependent in different mouse strains.


Figure 1
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  		Fig. 1. Neutrophil and macrophage influx into bronchoalveolar fluid (BALF) of different strains of mice in response to cigarette smoke (CS) exposure. Mice exposed to CS at 300 mg/m3 total particulate matter (TPM; A, B, D, and E) and 80 mg/m3 TPM (C and F) for 3 days were killed at 2 h (A and D) and 24 h (B, C, E, and F) after the last exposure, and left lungs were lavaged. Total cells in BALF were counted using a hemocytometer. At least 400 cells were counted in a blinded manner to determine the number of neutrophils (A–C) and macrophages (D–F) on Cytospin slides stained with Diff-Quick. Values are means ± SE (n = 4–5 per group). ***P < 0.001 vs. air. +P < 0.05; #P < 0.01; $P < 0.001 vs. CS-exposed C57BL/6J.

 

Figure 2
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  		Fig. 2. Macrophage cell influx into lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Macrophages were detected by immunohistochemical staining using the anti-mouse Mac-3 antibody in lung sections. Number of Mac-3-positive cells in lung sections (5 random microscopic fields per lung section in 3 different sections) was counted manually in a blinded manner. Images represent results of ≥3 separate experiments. Arrows indicate macrophages. Insets: Mac-3-positive macrophages (arrows) in mouse lungs. Original magnification x200. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed groups. #P < 0.01, $P < 0.001 vs. CS-exposed C57BL/6J.

 

Figure 3
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  		Fig. 3. CD8+ lymphocyte infiltration into lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 24 h after the last exposure. CD8+ lymphocytes were detected by immunohistochemical staining using rat anti-mouse monoclonal antibody in the lung. Number of CD8+ cells in lung sections (5 random microscopic fields per lung section in 3 different sections) was counted manually in a blinded manner. Images represent results of ≥3 separate experiments. Arrows indicate CD8+ lymphocyte. Insets: CD8+-positive lymphocytes (arrows) in mouse lungs. Original magnification x200. *P < 0.05; ***P < 0.001 vs. respective air-exposed groups.+P < 0.05; $P < 0.001 vs. CS-exposed C57BL/6J.

 
Inflammatory mediators in lungs of different strains of mice in response to CS exposure. CS is a strong inflammatory stimulus that induces the release of proinflammatory mediators such as IL-6, the chemokine keratinocyte chemoattractant (KC), and monocyte chemoattractant protein-1 (MCP-1) in the lung tissue (18, 24, 46, 51). Therefore, we investigated whether these different strains of mice exhibited a differential pattern of cytokines/chemokines in lungs in response to CS exposure at 300 mg/m3 TPM. Similar to the pattern of inflammatory cell influx in different mouse strains, the levels of KC, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-2 (MIP-2), and IL-6 were significantly increased in C57BL/6J, A/J, AKR/J, and/or CD-1, but not 129SvJ, mice exposed to CS at 2 h after the last exposure (Fig. 4A). However, there was no change in the levels of MCP-1, TNF-{alpha}, and IL-10 in lungs of all the strains of mice studied in response to CS at 2 h after the last exposure (Fig. 4A). Although a significant increase in the level of IL-13 was observed in C57BL/6J mice, a similar exposure to CS did not alter the level of IL-13 in the lungs of other strains of mice at 2 h after the last exposure (Fig. 4A). At 24 h after the last exposure, CS increased the lung levels of KC, MCP-1, TNF-{alpha}, MIP-2, IL-6, and IL-13 in C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice (Fig. 4B). The levels of GM-CSF and IL-10 were significantly increased in C57BL/6J mice, but not in the other strains, at 24 h after the last exposure (Fig. 4B). Remarkably, the level of IP-10 was increased in all strains of mice exposed to CS at 24 h after the last exposure (Fig. 4B). These results are consistent with the inflammatory cell profiles in lungs of different strains of mice exposed to CS and further suggest that the C57BL/6J strain is supersusceptible, the A/J, AKR/J, and CD-1 strains are mildly susceptible, and the 129SvJ strain is resistant to acute CS-mediated release of proinflammatory mediators. We also found that CS exposure at 80 mg/m3 TPM led to increased release of KC, MCP-1, IP-10, TNF-{alpha}, MIP-2, IL-6, and IL-13 in C57BL/6J, but not 129SvJ, mice and that the level was less than in mice exposed to 300 mg/m3 TPM (Fig. 4, B and C), confirming that the CS-mediated acute lung inflammatory response is concentration dependent.


Figure 4
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  		Fig. 4. Levels of proinflammatory mediators in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM (A and B) and 80 mg/m3 TPM (C) for 3 days and killed at 2 h (A) and 24 h (B and C) after the last exposure, and lungs were homogenized. Proinflammatory mediators in lung homogenates were measured by a Luminex assay. KC, keratinocyte chemoattractant; MCP-1, monocyte chemoattractant protein-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; IP-10, interferon-inducible protein-10; MIP-2, macrophage inflammatory protein-2. Values are means ± SE (n = 3–4 per group). *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed group. +P < 0.05; #P < 0.01; $P < 0.001 vs. respective C57BL/6J group.

 
Activation of NF-{kappa}B and levels of HDAC2 in lungs of different strains of mice in response to CS exposure. NF-{kappa}B activation has been shown to play a key role in CS-mediated lung inflammation (26, 45, 50, 51). However, the activation of NF-{kappa}B in susceptible and resistant mouse strains is not known. It has been recently shown that NF-{kappa}B acetylation leads to changes in its DNA-binding and transcriptional activities, resulting in sustained proinflammatory gene transcription (6, 51). We studied the nuclear levels of total NF-{kappa}B and its acetylated form in lungs of different mouse strains in response to CS exposure to determine the underlying mechanism of CS-mediated susceptibility in mouse strains. CS exposure led to an increase in the levels of total and acetylated RelA/p65 in C57BL/6J, A/J, AKR/J, and/or CD-1 mice, whereas the levels of total and acetylated RelA/p65 were not altered in 129SvJ mice, at 2 and 24 h after the last exposure (Fig. 5, A and B). This finding was confirmed by the EMSA data showing increased NF-{kappa}B DNA-binding activity in lungs of C57BL/6J compared with A/J, AKR/J, CD-1, and 129SvJ mice at 2 h after the last CS exposure (Fig. 5C). It has been shown that the level/activity of HDAC2 was decreased in lungs of patients with COPD (16) and that decreased HDAC2 was associated with acetylation of RelA/p65 in macrophages and alveolar epithelial cells in response to CS exposure (26, 50). Therefore, we assessed the level of HDAC2 in these different strains of mice exposed to CS at 300 mg/m3 TPM. We found that the level of HDAC2 was decreased in lungs of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice in response to CS at 2 and 24 h after the last exposure (Fig. 5, A and B). These results suggest that CS exposure decreased the level of HDAC2 that was associated with acetylation of RelA/p65 and subsequent release of proinflammatory mediators in the lung.


Figure 5
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  		Fig. 5. Levels of total and acetylated RelA/p65 and histone deacetylase-2 (HDAC2) and activation of NF-{kappa}B in lungs of different mouse strains in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Levels of total and acetylated (Ac K310) RelA/p65 and HDAC2 in nuclear fractions were analyzed by Western blotting (A and B), and β-actin was used as an indicator for equal protein loading. Lamin B and {alpha}-tubulin were used as nuclear and cytoplasmic markers, respectively. No bands of {alpha}-tubulin were observed in the nuclear fraction. NF-{kappa}B DNA binding was determined in the nuclear protein by EMSA (C). Migration of free probe is not shown. NSC, nonspecific competitor; SC, specific competitor. Each lane represents results of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed group.

 
Phosphorylated and total levels of p38 MAPK in lungs of different strains of mice exposed to CS. It has been shown that the level of p38 MAPK was increased in lungs of patients with COPD and rats exposed to CS (24, 40). The differential activation of p38 MAPK may be responsible for differential susceptibility of various mouse strains to CS. Therefore, we determined the phosphorylated and total levels of p38 MAPK by Western blotting in lungs of different strains of mice exposed to CS at 300 mg/m3 TPM. The phosphorylated and total levels of p38 MAPK were increased in lungs of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice in response to CS at 2 and 24 h after the last exposure (Fig. 6). These data suggest that activation of p38 MAPK is involved in susceptibility or resistance to the CS-induced lung inflammatory response in these different strains of mice.


Figure 6
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  		Fig. 6. Phosphorylated and total levels of p38 MAPK (p-p38 and p38, respectively) in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Phosphorylated and total levels of p38 MAPK in lungs were analyzed by Western blotting, and β-actin was used as an indicator for equal protein loading. Western blots are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed group.

 
Level of MMP-9 in lungs of different strains of mice in response to CS exposure. Various studies have shown that MMPs, particularly increased levels of MMP-9 and MMP-12, are involved in CS-induced alveolar wall destruction (8, 10, 44, 49). Therefore, we determined the level of MMP-9 in lungs of different strains of mice exposed to CS at 300 mg/m3 TPM. We found that the protein level of MMP-9 was increased in C57BL/6J, A/J, AKR/J, and/or CD-1 mice exposed to CS at 2 and 24 h after the last exposure (Fig. 7). However, CS did not alter the protein level of MMP-9 in 129SvJ mice at 2 and 24 h after the last exposure (Fig. 7), suggesting that MMP-9 is differentially regulated in different mouse strains in response to CS exposure.


Figure 7
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  		Fig. 7. Levels of matrix metalloproteinase-9 (MMP-9) in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Level of MMP-9 in whole lysate of lung tissue was analyzed by Western blotting, and β-actin was used as an indicator for equal protein loading (housekeeping control). Western blots are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed group.

 
Levels of lipid peroxidation products (4-HNE + MDA) and GSH in lungs of different mouse strains in response to CS exposure. Lung macrophages and neutrophils of smokers have been shown to release an increased amount of reactive oxygen species (29). Therefore, we assessed the lung levels of lipid peroxidation products (4-HNE and MDA) in different strains of mice exposed to CS at 300 mg/m3 TPM. CS resulted in an increase in levels of 4-HNE and MDA in C57BL/6J, A/J, AKR/J, and/or CD-1, but not 129SvJ, mice at 2 and 24 h after the last exposure (Fig. 8). The level of GSH was significantly decreased in lungs of C57BL/6J and A/J mice but was not altered in lungs of AKR/J, CD-1, and 129SvJ mice at 2 h after the last CS exposure (Fig. 9). On the contrary, CS increased the lung levels of GSH in C57BL/6J and CD-1, but not A/J, AKR/J, and 129SvJ, mice at 24 h after the last exposure (Fig. 9). These results suggest that CS-induced lipid peroxidation and GSH depletion are strain dependent, which might contribute to susceptibility of these mice to CS-induced oxidative stress (at least in response to acute CS exposure).


Figure 8
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  		Fig. 8. Levels of lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE)] in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Levels of 4-HNE and MDA were measured spectrophotometrically in lung homogenates. Values are means ± SE (n = 3–4 per group). **P < 0.01; ***P < 0.001 vs. respective air-exposed mice.

 

Figure 9
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  		Fig. 9. Level of GSH in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h (A) and 24 h (B) after the last exposure. Level of GSH was measured spectrophotometrically in lung homogenate. Values are means ± SE (n = 3–4 per group). *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed mice.

 
Levels of GCLC in lungs of different mouse strains in response to CS exposure. It is known that the biosynthesis of GSH is a two-step process involving GCLC and GSH synthetase and that GCLC catalyzes the first and rate-limiting step of GSH biosynthesis. We determined the levels of GCLC in lungs of these strains of mice in response to CS exposure (300 mg/m3 TPM). The lung level of GCLC increased in all five strains of mice at 2 h after the last CS exposure (Fig. 10), suggesting that GCLC expression responded to CS at 2 h after the last exposure in an attempt to increase GSH biosynthesis.


Figure 10
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  		Fig. 10. Level of glutamate-cysteine ligase (GCLC) in lungs of different strains of mice in response to CS exposure. Mice were exposed to CS at 300 mg/m3 TPM for 3 days and killed at 2 h after the last exposure. Levels of GCLC in the cytosolic fraction were analyzed by Western blotting, and β-actin was used as a housekeeping control. Western blots are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective air-exposed group.

 

   DISCUSSION
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 MATERIALS AND METHODS
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Recent studies have shown that genetic variability is an important determinant of CS-induced susceptibility to emphysema (1, 4, 15). However, the knowledge of the effect of CS exposure on inflammatory and oxidative responses in lungs of different strains of mice is still lacking. Therefore, we chose the five most commonly used strains of mice, C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ, exposed them to two different concentrations of CS for 3 days, and killed them at two different times after the last CS exposure. The aim of the present study was to determine the susceptibility of these mouse strains to acute CS exposure and study the molecular changes. Therefore, we assessed various parameters of inflammation, oxidative stress, and signaling molecules that regulate inflammation and oxidative stress in lungs of these mice. We found significantly different susceptibilities to CS-induced lung inflammatory response and oxidative stress in all five strains of mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to the lung inflammatory response after acute CS exposure, which was consistent with a previous finding that 3 days of CS exposure led to more neutrophils in BALF of C57BL/6J than 129SvJ mice (48). However, our data contradict the previous observation of relative susceptibility to emphysema, i.e., the greater increase in mean linear intercept in A/J and AKR than C57BL/6J mice exposed to CS 5 days/wk for 6 mo (15). The reason underlying this discrepancy is not clear, but it may be related to different exposure time and dose of CS in these different strains of mice. As mentioned above, more neutrophils were observed in BALF of C57BL/6J than 129SvJ mice exposed to CS for 3 days, whereas the number of BALF neutrophils was very similar between C57BL/6J and 129SvJ mice after 4 days of CS exposure (48). Therefore, the dose and time of CS exposure play an important role in development of CS-induced lung inflammatory response in these different strains of mice.

CS exposure leads to lung inflammation, with an increase in inflammatory cell influx, such as macrophages, neutrophils, CD8+ T lymphocytes, and dendritic cells, and increased release of proinflammatory mediators in C57BL/6J (TH1 response) or Balb/C (TH2 response) mice (2, 25, 45). Our data show that the influx of macrophages, neutrophils, and CD8+ T lymphocytes into the lungs was increased in C57BL/6J, A/J, AKR/J, and/or CD-1 mice in response to CS exposure at 300 mg/m3 TPM. Furthermore, CS recruited more inflammatory cells in lungs of C57BL/6J mice than the other susceptible strains of mice and increased proinflammatory mediator release in lungs of C57BL/6J mice exposed to CS for 3 days at 2 and 24 h after the last exposure. However, the number of inflammatory cells in lungs of 129SvJ mice was not altered in response to CS exposure, which further confirms that the 129SvJ strain is resistant to the acute CS-induced lung inflammatory response. Similar results were obtained in terms of susceptibility to lung inflammatory cells influx by LPS aerosolization in C57BL/6J and 129 SvJ mouse strains (Yao Hw and Rahman I, unpublished observations). It has been suggested that macrophage activation, rather than the increase in macrophage numbers, is crucial for initiation of the acute lung inflammatory response to CS (9). This may be a reason for our finding that the number of macrophages in BALF was not increased, whereas the number of Mac-3-positive macrophages in lung interstitium was increased, in mice exposed to CS.

In the present study, the number of neutrophils was much higher in BALF of C57BL/6J, A/J, AKR/J, and CD-1 mice killed at 24 h than at 2 h after the last CS exposure, i.e., 5 times higher in C57BL/6J and 25 times higher in CD-1 mice. This finding was corroborated by a previous study showing that acute CS exposure (3 days) led to an increase (~6-fold) in the number of lavaged neutrophils in C57BL/6J mice at 24 h compared with 4 h after the last exposure (45). These data suggest that acute effects of CS exposure (within hours after the end of CS exposures) are related to suppression of the number of neutrophils recovered in the lavage, possibly due to capillary trapping, or increased adhesion, which would reduce the numbers recovered from the air spaces. The other reason would be that the higher release of chemokines at 24 h than at 2 h after the last CS exposure would attract more neutrophils into the lung interstitium in response to CS.

We also compared the lung inflammatory response to CS at two different concentrations, i.e., 80 and 300 mg/m3 TPM, using the most sensitive (C57BL/6J) and resistant (129SvJ) strains. Similar to CS exposure at 300 mg/m3 TPM, we were unable to observe any significant increase in the inflammatory response (neutrophil influx and cytokine release) of 129SvJ mice to CS exposure at 80 mg/m3 TPM, confirming that the 129SvJ strain is resistant to the CS-induced acute lung inflammatory response. Although neutrophil influx and cytokine release were increased in C57BL/6J mice exposed to CS at 80 mg/m3 TPM, the inflammatory response (neutrophil influx and cytokine release) was less than at 300 mg/m3 TPM. These data are corroborated by previous studies showing that CS-induced lung inflammation and emphysema are concentration and time dependent (21, 22). Another possibility would be that the effect of CS exposure on the CS-mediated lung inflammatory response is dose and rate dependent, because there was less inflammatory response in lungs (neutrophil influx and cytokine release) in C57BL/6J mice exposed to CS at 80 than at 300 mg/m3 TPM, although the total exposure time was similar: 1,800 and 1,440 mg·m–3·h–1 at 300 and 80 mg/m3 TPM, respectively.

We further studied the molecular changes in the lungs in these different mouse strains to compare the results of inflammatory response by CS exposure. MMP-9, an NF-{kappa}B-dependent protease, is derived from activated macrophages (48). MMP-9 expression is greater in alveolar macrophages from cigarette smokers than from normal subjects (19) and even greater in cells from patients with COPD (41), which have greatly enhanced elastolytic activity (42). In the present study, CS exposure increased the protein level of MMP-9, which paralleled significant macrophage influx in these sensitive strains (C57BL/6J, A/J, AKR/J, and CD-1), but not in 129SvJ mice, in response to acute CS exposure. Because of the proinflammatory mediator release and MMP-9 level in these strains of mice, we proposed that these mice must exhibit different activation of NF-{kappa}B in response to CS exposure. As expected, the total level of RelA/p65 in the nucleus was increased in lungs of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice in response to CS exposure. Recent studies have shown that degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B are not sufficient to promote maximal NF-{kappa}B transcriptional activity. Rather, the NF-{kappa}B complex must undergo additional posttranslational modifications involving site-specific phosphorylation and acetylation (5–7). For example, acetylation of the lysine 310 residue of RelA/p65 is required for full transactivation of the NF-{kappa}B complex (6). Interestingly, CS exposure induced the acetylation of RelA/p65 (the main subunit of NF-{kappa}B) on the lysine 310 residue, which was consistent with the increased level of total RelA/p65 and NF-{kappa}B DNA-binding activity in lungs of C57BL/6J, A/J, AKR/J, and CD-1 mice in response to CS exposure. Therefore, the mechanism underlying the susceptibility or resistance of these strains of mice to CS-mediated acute lung inflammation may be due to the varying degree of NF-{kappa}B activation, particularly via acetylation of RelA/p65. Our previous data demonstrated that RelA/p65, but not acetylated RelA/p65, interacts with HDAC2 and that RelA/p65 becomes available or retained in the nucleus for proinflammatory gene transcription when HDAC2 is decreased in response to CS (50). We showed that the level of HDAC2 was decreased in lungs of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice in response to CS exposure. Thus the activation of RelA/p65 and the differential response of HDAC2 to CS might contribute to the sensitivity or resistance of these five strains of mice to the CS-mediated acute lung inflammatory response. It has been shown that NF-{kappa}B is activated by p38 MAPK (17, 43). We found increased p38 MAPK activation in lungs of C57BL/6J mice compared with A/J, AKR/J, CD-1, and 129SvJ mice exposed to CS. Therefore, it is possible that differential activation of p38 MAPK and the NF-{kappa}B pathway is involved in the susceptibility of these mice to the CS-mediated inflammatory response.

An imbalance between oxidants and antioxidants has been shown to occur in smokers (32, 35) in association with the lung inflammatory response. We previously showed that leukocytes isolated from patients with COPD generate elevated levels of superoxide anions, which were associated with increased systemic levels of inflammatory mediators (27, 28, 30, 36). 4-HNE, a highly reactive and diffusible end product of lipid peroxidation, is a known marker of oxidative stress and can attack target cells far from the site of the original free radical event (11, 12). In the present study, we attempted to investigate whether any variation in the extent of lipid peroxidation in response to CS exposure is a factor in susceptibility to the CS-induced response in all five strains of mice. Indeed, our data show that CS exposure results in an increase in 4-HNE and MDA contents in lung of C57BL/6J, A/J, AKR/J, and CD-1, but not 129SvJ, mice at 2 and 24 h after the last exposure. Furthermore, the decreased level of the antioxidant GSH in lungs of C57BL/6J and A/J mice exposed to CS (killing and) may be due to direct and rapid conjugation of highly electrophilic β-carbonyl chemical compounds in CS with GSH (34, 39, 47). Therefore, it is likely that the imbalance of oxidants and antioxidants renders C57BL/6J, A/J, AKR/J, and CD-1 mice susceptible to acute CS-mediated lung inflammation. It is interesting to note that the level of GSH was increased in C57BL/6J and CD-1 mice, but not in other strains, at 24 h after the last CS exposure, which may implicate a transient antioxidant adaptive response to CS-induced free radical/oxidant stress in C57BL/6J and CD-1 mice. A possible explanation for increased GSH contents in C57BL/6J and CD-1 mice could be the rebound effect as a transient compensatory mechanism whereby GCLC (a rate-limiting enzyme in GSH biosynthesis) is upregulated (18, 37). Indeed, the level of GCLC in lungs was increased in C57BL/6J and CD-1 mice in response to CS exposure. However, the reasons for an increase in the level of GCLC at 2 h but no increase in the level of GSH at 24 h after the last CS exposure in A/J, AKR/J, and 129SvJ mice remain to be determined. It is possible that the highest level of GSH appears between 2 and 24 h after the last CS exposure in A/J, AKR/J, and 129SvJ strains and decreases to the basal level at 24 h after the last exposure. Taken together, our data suggest that the lung GSH levels may not be the sole determinant of susceptibility to CS in different mouse strains, because CS produces overwhelming oxidative and inflammatory responses in the lung.

Overall, on the basis of our data on the inflammatory cell influx and proinflammatory mediator release in lungs, we categorized the C57BL/6J strain as highly sensitive, the A/J, AKR/J, and CD-1 strains as moderately sensitive, and the 129SvJ strain as resistant to lung inflammatory and oxidative responses after acute CS exposure: C57BL/6J > A/J > AKR/J > CD-1 > 129SvJ. The mechanism underlying this finding is linked to differential activation of NF-{kappa}B and p38 MAPK and/or a decreased level of HDAC2. The other possibility is that the imbalance between oxidants and antioxidants in lungs is the reason for the susceptibility or resistance of these mice to the acute CS-mediated lung inflammatory response. Thus we have identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains would be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-085613, National Institute of Environmental Health Sciences Grant ES-01247, Institute for Science and Health, and Toxicology Training Grant ES-07026.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Rahman, Dept. of Environmental Medicine, Lung Biology and Disease Program, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 850, Rochester, NY 14642 (e-mail: irfan_rahman{at}urmc.rochester.edu)

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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 GRANTS
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