Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Tissue culture reagents were obtained from Sigma (Poole, UK). Cell culture plasticware was purchased from Falcon Labware (Becton Dickinson, Oxford, UK). Recombinant human TNFα and matched antibody pairs for IL-8, eotaxin and RANTES enzyme-linked immunosorbent assays (ELISA) were purchased from R&D Systems (DuoSet, Abingdon, UK). Antibodies were purchased from Calbiochem (heme oxygenase-1) and Biogenesis, Poole, UK (GAPDH). Protease inhibitor cocktail was obtained from Roche Diagnostic (Lewes, UK). All other chemical reagents were obtained from Sigma (Poole, UK).

Human airway smooth muscle was obtained from lobar or main bronchus from patients undergoing lung resection for carcinoma of the bronchus. The smooth muscle was dissected out under sterile conditions and placed in culture as previously described [16]. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), non-essential amino acids (1:100), penicillin (100 U/ml)/streptomycin (100 μg/ml) and amphotericin B (1.5 μg/ml) in a humidified atmosphere at 37°C in air/CO₂ (95:5 % vol/vol). At confluence, HASMC cultures exhibited a typical hill-and-valley appearance. Immunofluorescence techniques for calponin, smooth muscle α-actin and myosin heavy chain revealed that more than 95% of the cells displayed the characteristics of smooth muscle cells in culture. HASMC at passages 3–7 from 9 different donors were used in the studies described below.

Cigarette smoke extract (CSE) was prepared by combusting four full strength Marlboro cigarettes (filters removed) through a modified 60 ml syringe apparatus and passing the smoke through 100 mls of DMEM. Each cigarette yielded 5 draws of the syringe (to 60 ml mark), with each individual draw taking approximately 10 seconds to complete. This solution represents '100%' strength. Smoked medium was then passed through a 0.25 μM filter in order to sterilise the solution. Smoked medium was diluted to the required strength in DMEM and placed upon the cells immediately afterwards.

Prior to the experiments, confluent cells were growth-arrested by FCS deprivation for 24 h in DMEM supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), non-essential amino acids (1:100), penicillin (100 U/ml)/ streptomycin (100 μg/ml), amphotericin B (1,5 μg/ml), insulin (1 μM), transferrin (5 μg/ml), ascorbic acid (100 μM) and bovine serum albumin (0,1 %). Cells were then exposed to smoke (0–30%) in the presence and absence of TNFα (1 ng/ml). In additional experiments cells were pretreated with 100 μM glutathione (GSH) for 30 min before exposure to CSE.

HASMC viability was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Cells grown in 96-well plates were treated as indicated above, washed with PBS and 100 μl MTT solution (1 mg/ml) was added to each well. After 1 hour of incubation at 37°C, the MTT solution was removed and the converted dye was solubilized with 100 μl DMSO. The OD was measured using a spectrophotometer set to 550 nm. None of the conditions studied cause visual morphology markers of apoptosis over the time course studied (not shown).

Cell supernatants were harvested 24 hours after stimulation and stored at -70°C until assayed for RANTES, eotaxin and IL-8. Cytokine levels were determined by using specific sandwich enzyme-linked immunosorbent assays (ELISA) according to the manufacturers' instructions.

Total RNA was isolated from HASMC after 6 hours using the RNeasy Mini Kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. cDNA was generated by reverse transcription (RT) using random hexamers. The cDNA (42 ng/reaction) was used as a template in the subsequent polymerase chain reaction (PCR) analyses. Transcript levels were determined by real-time PCR (Rotor Gene 3000, Corbett Research, Australia) using the Sybre Green PCR Master Mix Reagent Kit (Promega, San Luis Obispo, USA). The sequence for IL-8 PCR primer were sense 5'-GCCAACACAGAAATTATTGTAAAGCTT and antisense 5'-CCTCTGCACCCA GTTTTCCTT'. Primers for GAPDH were sense 5'-ATTCCATGGCACCGT CAAGGCT and antisense 5'-TCAGGTCCACCACTGACACGT. Primers were used at a concentration of 0.5 μM for real-time PCR in each reaction. Cycling conditions for real-time PCR were as follows: step 1, 15 min at 95°C; step 2, 15 sec at 94°C; step3, IL-8: 25 sec at 60°C, GAPDH: 25 sec at 64°C; step 4, 22 sec at 72°C, with repeat from step 2 to step 4 for 40 times. Data from the reaction were collected and analysed by the complementary computer software (Corbett Research, Australia). Relative quantitations of gene expression were calculated using standard curves and normalized to GAPDH.

Confluent HASMC were exposed to CSE (0–20 %). After 24 hours of incubation, cells were rinsed with ice-cold wash buffer (PBS containing 2 mM PMSF) and scraped off the culture dish. HASMC were pelleted by centrifugation at 1000 RPM at 4°C for 5 min and lysed in radioimmunoprecipitation assay (RIPA) buffer (PBS containing 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1% Igepal and 1 tablet protease inhibitor cocktail 10 ml^-1 buffer). Samples were solubilized by sonication followed by centrifugation (10,000 × g, 4°C, 4 min). Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, USA). Lysates were boiled for 10 min and total protein extracts (40 μg/lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS-Page) on a 4–12 % acrylamide precast gel (Novex, Invitrogen, Paisley, UK). The separated proteins were transferred electrophoretically to a nitrocellulose membrane in transfer buffer (Novex) and the membrane was then blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBST) for at least 1 hour at room temperature. Blots were then incubated overnight at 4°C with an anti-HO-1 antibody in TBST containing 5% dried nonfat milk at a 1:1000 dilution. The next day, the membrane was washed 3 times with TBST and then incubated for 1 hour with a 1:2000 dilution of goat anti-mouse HRP-conjugated secondary antibody in TBST containing 5% nonfat dry milk. The membrane was then washed as before and visualized by enhanced chemiluminescent (ECL) solution (Amersham, Buckinghamshire, UK). Membranes were reprobed with a mouse anti-GAPDH monoclonal antibody (1:5000, Biogenesis, Poole, UK) in order to show the amount of protein loaded. Signals were quantified by scanning densitometry using software from Ultra-Violet Products (UVP) (Cambridge, UK). Densitometry data were normalized for GAPDH values.

Data are presented as mean ± SEM. Data were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's t test post hoc to determine statistical differences. A p value < 0.05 was considered significant. SigmaStat software (Jandel Scientific, Germany) was used for statistical analysis.

Under control culture conditions, IL-8 release from HASMC was below the detection limit of the ELISA over the 24-hour experimental period. Increasing concentrations of smoke induced a 'bell-shaped' response curve for IL-8 release by HASMC. Maximum induction of IL-8 release was seen at a concentration of 15 % CSE (baseline 0 pg/ml; 15% CSE 70.3 ± 8.6 pg/ml, p < 0.001; figure 1). However, at concentration of 20% and 30%, the release of IL-8 was lower. In order to assess whether CSE-induced upregulation of IL-8 production from HASMC at up to 15% concentration was the result of increased IL-8 gene transcription, we measured IL-8 mRNA expression by real-time PCR. Stimulation of HASMC with CSE (10%) for 6 hours led to increased IL-8 mRNA expression (Ratio IL-8/GAPDH: baseline 0.075 ± 0.03, 10% CSE 0.21 ± 0.09, figure 1). Viability of cells exposed to CSE remained unchanged up to concentrations of 15 % (104.8 ± 3.2 % of control) but declined at concentrations of 30% cigarette smoke (50.1 ± 9.7 % of control, figure 1).

The stimulatory effects of CSE were greatly inhibited by pre-treatment of cells with GSH (100 μM; 10 % CSE 270.8 ± 72.5 pg/ml, 10 % CSE + GSH 70.9 ± 10.8 pg/ml, figure 2), which quenches extracellular oxidative stress [17]. Heme oxygenase-1 is expressed in most cell types and is highly inducible by oxidative stress. In order to investigate whether CSE exposure causes an intracellular oxidative stress response in HASMC, we measured the expression of heme-oxygenase-1 levels before and after exposure to smoke by western blot analysis. HASMC expressed detectable levels of heme-oxygenase-1 when cultured under control conditions. However, heme-oxygenase-1 levels were increased when cells were treated with CSE (5–20 %; figure 2).

TNFα induced a concentration dependent release of IL-8 from HASMC at 0.1 to 10 ng/ml (not shown), with 1 ng/ml representing an approximate EC₅₀ concentration. Furthermore, TNFα (1 ng/ml) acted in synergy with CSE on the release of IL-8 from HASMC (figure 3). This synergy was observed across the concentration range of 5–15% CSE with maximum effect seen at 10% smoke (TNFα 232.1 ± 18.4 pg/ml, 10% CSE + TNFα 628.5 ± 64.2 pg/ml, p < 0.001, figure 3). This synergy was lost with CSE 20% and in fact at 30% there was inhibition of IL-8 release. In line with protein release, TNFα synergised with CSE (10%) in induction of IL-8 mRNA (figure 3). Cell viability remained unchanged in cells treated with either TNFα alone or in combination with CSE at concentrations of up to 10%. AT CSE 15%, cell viability declined mildly with further reduction seen at CSE 20–30% (Figure 3).

Similar to observations made with IL-8 release, levels of either eotaxin or RANTES were below the level of detection in medium from cells cultured under basal conditions. Similar to IL-8, incubation of cells with TNFα (1 ng/ml) for 24 hours induced increased levels of both eotaxin and RANTES released by the cells (Figure 4; RANTES 637.5 ± 84.5 pg/ml; eotaxin 177.1 ± 25.9 pg/ml). However, by contrast to IL-8, CSE at all concentrations (5%–30%) failed to induce release of either eotaxin or RANTES from HASMC (figure 4). Furthermore, CSE did not synergise with TNFα in the release of either eotaxin or RANTES. In fact, CSE inhibited the release of these chemokines when induced by TNFα (figure 4) This effect was not reversed by pre-treatment of HASMC with GSH (100 μM) before adding cigarette smoke solution (data not shown).