Unless otherwise stated, all biochemical reagents used in this study were purchased from Sigma Aldrich, Inc. (St. Louis, MO). Antibodies used in this study include the following: β-actin (CP-01; Oncogene, San Diego, CA), histone H3, acetylated (Ac)/phosphorylated (P) histone H3 (lys9/ser10), histone H4, Ac histone H4 (Lys12), IKKβ, and phospho-RelA/p65 (ser276) (antibody catalog nos., 9715, 9711, 2592, 2591, 2370, and 3037, respectively; Cell Signaling Technology Inc., Danvers, MA), IKKα and RelA/p65 (SC-7182 and SC-372, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-acetylated histone H3 and histone H4, and IKKα (06-599, 06-598 and 05-536; Upstate, Charlottesville, VA), IKKα pS176/180 (ab17943; Abcam, Inc., Cambridge, MA). The anti-acetylated RelA/p65 antibody specific for AcLys310 or AcK310 (16
) was provided by Dr. Leonard Buckbinder at Pfizer Global R&D (Groton, CT).
Adult male C57BL/6J mice (8–10 wk, 37 ± 1.5 g; Jackson Laboratory, Bar Harbor, ME) were housed in the Inhalation Core Facility of the University of Rochester. The Animal Research Committee of the University of Rochester approved all animal experimental procedures described in this study.
Mice (six to eight per group) were used for acute (3 d) and sub-chronic (8 wk) CS exposure. The mice were placed in individual compartments of a wire cage that was placed inside an aerated plastic box connected to the smoke source. The CS was generated from 2R4F research cigarettes (total particulate matter [TPM] concentration 11.7 mg/cigarette, tar 9.7 mg/cigarette, nicotine 0.85 mg/cigarette; University of Kentucky, Lexington, KY). CS exposure was performed according to the Federal Trade Commission protocol (1 puff/min of 2-s duration and 35 ml volume) in an automatic Baumgartner-Jaeger CSM2082i CS machine (CH Technologies, Westwood, NJ). Mainstream CS was diluted with filtered air and directed into the exposure chamber. The smoke exposure (TPM per cubic meter of air, mg/m3
) was monitored in real time with a MicroDust Pro-aerosol monitor (Casella CEL, Bedford, UK) and verified daily by gravimetric sampling. The smoke concentration was set at a nominal value of approximately 300 mg/m3
TPM by adjusting the flow rate of the dilution air (13
). Sham control animals were exposed only to filtered air in the same manner for the same duration. Mice received two 1-hour exposures (1 h apart) per day for 3 days and 8 weeks (5 d/wk), and were killed 2 and 24 hours after the final exposure. Concentration of carbon monoxide in the CS-filled chamber was approximately 350 ppm. The dosimetry of carbon monoxide in CS was estimated by measuring the blood carboxyhemoglobin levels. Mice tolerated CS without the evidence of toxicity (carboxyhemoglobin, COHb levels ~17% and no body weight loss).
Tissue Harvest and Bronchoalveolar Lavage
Mice were injected with 100 mg/kg (body weight) of pentobarbiturate (Abbott laboratories, Abbott Park, IL) intraperitoneally and killed by exsanguinations. The heart and lung were removed en bloc, and the lungs were lavaged three times with 0.5 ml of 0.9% sodium chloride. The lavage fluid was centrifuged, and the cell-free supernatants were frozen at −80°C for later analysis. The bronchoalveolar lavage (BAL) fluid cell pellet was resuspended in saline, and the total cell number was determined with a hemocytometer. Differential cell count (500 cells/slide) was performed on cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA) stained with Diff-Quik (Dade Bering, Newark, DE).
The detailed procedures for hematoxylin and eosin (H&E) staining, macrophage immunohistochemistry, and analysis of pro-inflammatory mediators in BAL fluid are provided in the online supplement.
Immunohistochemical Localization of IKKα
The levels of IKKα were measured in the fixed lung sections (4 μm thick) by immunohistochemical staining using IKKα rabbit polyclonal antibody (1:100 dilution) with avidin-biotin-peroxidase complex (ABC) method followed by hematoxylin counterstaining. Appearance of dark brown color represents the presence of IKKα in the lung tissue. The numbers of IKKα-positive cells in the lung sections (five random microscopic fields per lung section in three different sections) were counted manually in a blinded manner under a magnification of ×200, and the numbers were averaged.
The human monocyte-macrophage cell line (mature monocytes-macrophages) MonoMac6, which was established from peripheral blood of a patient with monoblastic leukemia (32
), were grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/ml penicillin, 100 U/ml streptomycin, 1% nonessential amino acids, 1 mM sodium pyruvate, 1 μg/ml human holo-transferrin, and 1 mM oxaloacetic acid. These cells do not require PMA to differentiate into the macrophages, thus avoiding any stress to the cells. The cells were cultured at 37°C in a humidified atmosphere containing 7.5% CO2
Preparation of Aqueous CSE
Research-grade cigarettes (1R3F) were obtained from the Kentucky Tobacco Research and Development Center at the University of Kentucky (Lexington, KY). Tar and nicotine contents of 1R3F were 15 mg/cigarette and 1.16 mg/cigarette, respectively. CSE (10%) was prepared by bubbling smoke from one cigarette into 10 ml of culture medium supplemented with 1% FBS at a rate of one cigarette per 2 minutes as described previously (34
), with modifications (2
). The pH of the CSE was adjusted to 7.4 and was sterile filtered through a 0.45-μm filter (25-mm Acrodisc; Pall, Ann Arbor, MI). The CSE preparation was standardized by monitoring the absorbance at λ320
(optical density of 0.74 ± 0.05). The absorption spectrum observed at λ320
showed very little variation between different preparations of CSE. Freshly prepared CSE was diluted with culture medium containing 1% FBS immediately before use for each experiment. Control medium was prepared by bubbling air through 10 ml of culture medium supplemented with 1% FBS, adjusting pH to 7.4, and sterile filtered as described for CSE preparation.
MonoMac6 cells were seeded at a density of 1 × 106 cells/well (total final volume = 2 ml), grown to approximately 80 to 90% confluence in 6-well plates containing RPMI 1640 medium with 10% FBS, washed in Ca2+- and Mg2+-free PBS, and then exposed to various treatments in media containing 1% serum. All treatments were performed in duplicate. The cells were treated with CSE (0.5%, 1.0%, and 2.5%) for 1 hour at 37°C with 7.5% CO2. At the end of treatment, the cells were washed with cold, sterile Ca2+- and Mg2+-free PBS and were lysed in RIPA buffer and stored at −80°C. Culture media from these cells were collected and stored at −80°C until analyzed for IL-8 release.
The plasmids for overexpression and dominant-negative IKKα were obtained as described previously (37
). Transient transfection was performed with 1 μg of plasmids in the presence of Lipofectamine 2000 transfection reagent (product no. 11668-027; Invitrogen, Carlsbad, CA) in MonoMac6 cells. Transfection efficiency in case of both plasmids transfection was more than 80%. One day after transfection, MonoMac6 cells were treated with CSE (0.5%, 1.0%, and 2.5%). Supernatant of transfected cells was assayed for IL-8 release and cytospin slides were prepared for immunocytochemistry. Whole cell lysate was used in Western blotting analysis.
Analysis of Pro-Inflammatory Mediators in BAL Fluid, Lung Homogenates, and Cell Culture Media
Mice BAL fluid (150 μl) was analyzed for the cytokine levels using the sensitive mice Multi-Analyte Profile (version 1.6) screening by Luminex (Rules Based Medicine, co-marketed with Charles River Laboratories, Austin, TX). The assays permit simultaneous cytometric quantification of multiple chemokines/cytokines with minimal sample volume.
The levels of keratinocyte chemoattractant (KC), IL-6, monocyte chemotactic protein (MCP)-1, and granulocyte macrophage colony-stimulating factor (GM-CSF) in lung homogenates (100 μl) were measured by the Luminex 100 using the beadlyte mouse multi-cytokine beadmaster kit (Upstate) according to the manufacturer's instructions. The assays use microspheres as the solid support for immunoassays. The levels are expressed as pg/mg protein.
In MonoMac6 cells, the culture medium was collected after treatment and centrifuged at 2,500 rpm for 5 minutes to pellet the cells. The supernatant was then removed and stored at −80 °C for later analysis. IL-8 level in the supernatant was determined by enzyme-linked immunosorbent assay from the respective human IL-8 Cytoset (catalog no. CHC1303; BioSource International, Inc., Camarillo, CA) according to the manufacturer's instructions.
Nuclear Protein Isolation and Acid Extraction of Histone Proteins
One hundred milligrams of lung tissue was mechanically homogenized in 0.5 ml 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 Na orthovandate, 1% [vol/vol] NP-40, 0.4 mM phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin) on ice. The homogenate was centrifuged at 2,000 rpm in a benchtop centrifuge for 30 seconds at 4°C to remove cellular debris. The supernatant was then transferred to a 1.7-ml ice-cold microtube and further centrifuged for 30 seconds 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, 0.2 mM Na orthovandate, and 0.6 mM phenylmethylsulfonyl fluoride) and placed on the rotator in the cold room for 30 minutes. After centrifugation at 13,000 rpm in a micro Eppendorf tube for 5 minutes, the supernatant was collected as the nuclear extract and kept frozen at −80°C for Western blotting. For extraction of histone protein, pellets from the nuclear extraction were resuspended in 150 μl deionized water, 0.2 N HCl, and 0.36 N H2SO4. The histone proteins were precipitated from the supernatant, agitated overnight at 4°C, and then centrifuged at 13,000 rpm for 10 minutes, and the supernatant decanted into a fresh tube. Ice-cold acetone precipitation samples were incubated overnight at −80°C, centrifuged, and the air-dried pellets were resuspended in 50 μl deionized water.
Lung tissue homogenate samples (cytoplasmic and nuclear proteins) were separated on a 7.5%-12% SDS-PAGE. MonoMac6 cells were harvested (24 h after transfection), lysed, and the nuclear fraction was separated with 10% Igepal CA-630 lysis buffer supplemented with a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin, and PMSF). Equal amount of protein was subjected to electrophoresis on 7.5%-12% PAGE gels, electroblotted onto nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ), and then incubated overnight with primary antibodies at 4°C. The next day, membranes were washed and incubated for 1 hour at room temperature with the appropriate secondary antibody linked to horseradish peroxidase (Dako, Santa Barbara, CA); bound complexes were detected with the use of the enhanced chemiluminescence method (Jackson Immunology Research, West Grove, PA).
MonoMac6 cells (1 × 104 cells/slide) were used to prepare cytospin slides (1,500 rpm for 5 min), and fixed in 4% paraformaldehyde for 10 minutes. The cells were then permeabilized for 10 minutes in 0.3% Triton X-100 in PBS, and blocked for 1 hour using 10% normal goat serum in TBS. Samples were incubated with antibodies specific for acetylated (Ac)/phosphorylated (P) histone H3 (lys9/ser10) and IKKα using 1% BSA in TBS in a humidified chamber overnight. The primary antibodies were detected with FITC-labeled anti-mouse (MP Biomedicals, Solon, OH), or Alexa Flour 594 goat anti-rabbit secondary antibody (catalog no. A-11037; Invitrogen). Nuclei were stained with 1 μg/ml Hoechst 33342 for 1 minute. Samples without primary antibodies were used as negative controls. The coverslips were mounted onto the slides (Product no. H-1000; Vector Laboratories, Burlingame, CA) and viewed under a fluorescence microscope.
One hundred milligrams of lung tissue was homogenized in 1 mg/ml BSA with protease inhibitors (one tablet) in PBS, and cross-linked with 1% formaldehyde for 10 minutes, rinsed three times with PBS, and then 0.5 ml 2.5 M glycine was added. After a brief centrifugation, cell pellets were resuspended in SDS-lysis buffer (50 mM Tris-HCl, 1% SDS, 5 mM EDTA, 5 mM Na-butyrate, protease inhibitors). Sonication of nuclear pellet containing chromatin was performed four times for 30 seconds and one time for 15 seconds at a maximum speed using Misonix-3000 Sonicator (Misonix Inc, Farmingdale, NY). Supernatants were collected and diluted (1:10 dilution) with buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM NaButyrate, protease inhibitor) followed by preclearing the extract with 60 μl of protein A agarose/salmon sperm DNA (Cat no. 16-157; Upstate) for 3 hours at 4°C (38
). Immunoprecipitation was carried out overnight at 4°C with 1 μg of specific antibodies as mentioned above. After immunoprecipitation, 40 μl of protein A agarose/salmon sperm DNA was added and incubated for 2 hours, followed by brief centrifugation. Precipitates were washed with Paro buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), Paro buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), and Paro buffer III (0.25 M LiCl, 1% Igepal CA-630, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) for 5 minutes at 4°C. Precipitates were then washed again with Tris-buffer twice for 5 minutes each. The antigen–antibody complexes were extracted two times with 50 μl elution buffer (0.6 μg/μl proteinase K, 1% SDS, 0.1 M NaHCO3
). The eluted samples were heated at 65°C overnight to reverse formaldehyde cross-linking. The recovered DNA was purified with a QIAquick PCR purification kit (Product no. 28106; Qiagen, Valencia, CA) (38
). Samples of input DNA were also prepared in the same way as described above. PCR amplification was performed using a PTC-200 DNA engine (M. J. Research, Waltham, MA) under the following conditions: 94°C for 180 seconds; 30 to 38 cycles at 94°C for 45 seconds, 60°C for 60 seconds, and 72°C for 60 seconds; and final elongation at 72°C for 10 minutes. PCR for the input reaction was performed using 100 ng of genomic DNA. Mouse primer sequences are given in , and PCR products were analyzed on a 1.5 to 2.0% agarose gel.
PRIMER SEQUENCES USED IN CHROMATIN IMMUNOPRECIPITATION ASSAY
Protein level was measured with a BCA kit (Pierce, Rockford, IL). Protein standards were obtained by diluting a stock solution of BSA. Linear regression was used to determine the actual protein concentration of the samples.
Results are shown as means ± SEM. Statistical analysis of significance was calculated by one-way ANOVA followed by Fisher's PLSD post hoc test for multigroup comparisons (StatView 5.0; SAS Institute, Cary, NC). Statistical significance is as indicated in figure legends.