Enhanced effects of cigarette smoke extract on inflammatory cytokine expression in IL-1β-activated human mast cells were inhibited by Baicalein via regulation of the NF-κB pathway

The baicalein was purchased from Sigma (St. Louis, MO). The HMC-1 cell line, established from a patient with mast cell leukemia, was graciously provided by Dr. Joseph H. Butterfield (Mayo Clinic, Rochester, MN). IL-1β and ELISA kits of IL-6, and IL-8 were purchased from R&D (Minneapolis, MN). RPMI 1640 media and HEPES were obtained from GibcoBRL (Rockville, MD). 2-mercaptoethanol was purchased from Sigma (St. Louis, MO). Fetal bovine serum was obtained from Atlanta Biologicals (Atlanta, GA). RNA-BEE was purchased from Tel-Test, Inc. (Friendswood, Texas). Gene Amp RNA PCR Core Kit was purchased from Applied Biosystems (Branchburg, NJ).

An ignited cigarette was placed in a 3.1 L bell-shaped glass vessel, the Sidestream smoke (Ss) within the vessel was pumped out of the bell and collected onto quartz fiber filters. Mainstream smoke (Ms) was collected directly from the cigarette onto quartz fiber filters using a puff volume of 35 ml in 2 seconds at a rate of 8 puffs per minute. The filters were weighed before and after smoke collection and the increase in weight was recorded as cigarette smoke weight. The cigarette smoke was extracted from the filters with RPMI 1640 medium to a concentration of 5 mg/ml.

HMC-1 cells were cultured and maintained in RPMI 1640 media with 5 × 10^-5 2-mercaptoethanol, 10 mM HEPES, gentamycin 50 μg/ml, 5 μg/ml insulin, transferrin and sodium selenite, 2 mM L-glutamine, and 5% heat inactivated fetal bovine serum in a 37°C incubator with 5% CO₂. The cell cultures were maintained in 75 cm² flasks (Corning) [22].

Two ml of HMC-1 mast cells at 1 × 10⁶ cells/ml concentration were cultured with or without different concentrations of both Ms and Ss cigarette smoke extract in the presence or absence of IL-1β (10 ng/ml) for 24 hrs [13]. A group of HMC-1 cells stimulated with both IL-1β (10 ng/ml) and CSE was also treated with BAI. The cultures were carried out in triplicate. At the end of incubation, supernatants were harvested for measuring IL-6 and IL-8 by ELISA and cell viability and numbers of the culture were analyzed. The cell viability was determined by trypan blue dye exclusion. Trypan blue dye (0.4%) was added to cell samples in a ratio of 1:2.5 and preparations were viewed with a standard light microscope [13]. The ratio of live to dead cells (cell viability) was determined. The cell viabilities of the drug groups and the medium control cultures in this study were ranging from 90 to 98%. BAI, IL-1β, and CSE at the concentrations used in this study appeared to have no toxic effect to the HMC-1 cultures.

Cytokine ELISA was performed for IL-6 and IL-8. ELISA was carried out on cell-free culture supernatants using commercially available ELISA kits, according to manufacturer's instructions as earlier described. Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software) [13].

HMC-1 were treated with the appropriate reagents and allowed to incubate at 37°C with 5% CO₂ for 6 hours before being harvested for RNA. RNA was extracted from HMC-1 (3 × 10⁶ cells) by the addition of 1 ml of RNA-BEE. After the addition of chloroform and shaking for 1 minute the samples were centrifuged at 12,000 × g for 15 minutes at 4°C to achieve phase separation. Isopropanol was added to the aqueous phase, and the preparation was frozen at -20°C overnight. The following day, the samples were centrifuged at 12,000 × g for 30 minutes at 4°C. The RNA pellet was washed with 1 ml 75% ethanol containing DEPC and allowed to air dry. The pellet was resuspended in DEPC water and quantitated by optical density readings at 260 nm. Reverse Transcriptase Polymer Chain Reaction (RT-PCR) was performed with a Gene Amp RNA PCR Core Kit according to manufacturer's instructions. cDNA was synthesized with murine leukemia virus reverse transcriptase (2.5 U/μl), 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3), 1 mM each of the nucleotides dATP, dCTP, dGTP and dTTP; RNase inhibitor (1 U/μl), MgCl₂ (5 mM), and oligo(dT)₁₆ (2.5 μM) as a primer. The samples were incubated at 42°C for 20 minutes, 99°C for 20 minutes, and 5°C for 5 minutes in a DNA thermocycler (Perkin-Elmer Corp., Norwalk, CT) for reverse transcription. PCR of cDNA was done with MgCl₂ (1.8 mM), each of the dNTPs (0.2 mM), AmpliTaq polymerase (1 U/50 μl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 μl. Cycles consisted of 1 cycle of 95°C for 2 min, 35 cycles of 95°C for 45 sec, 60°C for 45 sec, and 72°C for 1 min 30 sec, and lastly, 1 cycle of 72°C for 10 min. Ten microliters of the sample were electrophoresed on a 2% agarose gel and stained with ethidium bromide for viewing. Primer sequences used are as follows: HPRT: 5' CGA GAT GTG ATG AAG GAG ATG G 3' and 5' GGA TTA TAC TGC CTG ACC AAG G 3'; IL-6: 5' ATG AAC TCC TTC TCC ACA AGC GC 3' and 5' GAA GAG CCC TCA GGC TGG ACT G 3'; and IL-8: 5' ATG ACT TCC AAG CTG GCC GTG GCT 3' and 5' TCT CAG CCC TCT TCA AAA ACT TCT C 3'. Densitometry was done by normalizing target genes to house keepers using Un-Scan-It Version 5.1 software (Orem, UT) [21].

HMC-1 were stimulated with IL-1β, CSE, and/or BAI for 30 minutes, and then harvested for isolation of nuclear and cytoplasmic proteins according our previously reported method [21]. Nuclear translocation of NF-κB was analyzed by the electrophoretic mobility shift assay (EMSA) using the nuclear proteins [23‐26]. Cells were washed with PBS and mixed with one hundred microliters of hypotonic buffer which contains: 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na₃VO₄, and 20 mM p-nitrophenyl phosphate. Cells were incubated over ice for 30 minutes and then vortexed after the addition of 6.25 μl of 10% of Nonidet P-40. After 2 minutes of centrifugation at 30,000 × g, supernatants were kept at -80°C while the pellets were collected and vortexed every 20 minutes for 3 hours in 60 ml of a hypertonic salt solution: 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 12 mM DTT, 1 mM PMSF, 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na₃VO₄, and 20 mM p-nitrophenyl phosphate. Nuclear translocation of NF-κB was analyzed by the EMSA using the nuclear fraction. Seven micrograms of nuclear protein were added to 2 ml of binding buffer (Promega, Madison, WI), and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3') (Promega, Madison, WI) end labeled with γ-P32 ATP (Amersham Biosciences, Piscataway, NJ). The samples were incubated at room temperature for 20 minutes and run on a 5% nondenaturing polyacrylamide gel for 2 hours. A supershift assay using antibodies to P65 and P50 was performed to confirm NF-κB binding specificity as previously described [23‐26].

Cytoplasmic proteins (40 μg) were mixed with 2x SDS sample buffer, heated at 95°C for 5 min, and separated by SDS-polyacrylamide (12.5%) gel electrophoresis [24, 27]. The separated proteins were transferred onto Hybond enhanced chemiluminescence membranes (Amersham) and then incubated with an appropriate rabbit primary antibody [IκBα antibody (Santa Cruz Biotechnology) or phosphorylated IκBα antibody (New England Biolabs)] in Tris-buffered saline - 0.05% Tween 20 containing 5% nonfat dry milk for 1 - 2 hours at room temperature. After they were washed three times in Tris-buffered saline - 0.05% Tween 20, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical) for 1 hour at room temperature. After three washes in PBS, the conjugated peroxidase was visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham). The protein signals of IκBα were quantified by scanning densitometry (Genomic Solutions).

All experiments were done in triplicate. The data were analyzed by Student's two-tailed t-test using Statistica software (StatSoft, Inc., Tulsa, OK). All data were reported as means ± SE. A p-value of less than 0.05 was considered significant.

HMC-1 cells were cultured with IL-1β (10 μg/mL) and various concentration of CSE for 24 hours. The medium alone did not induce IL-6 and IL-8 production in HMC-1 cells (Figures 1, 2, 3 and 4). As the previous report, IL-1β at 10 ng/mL concentration markedly induced IL-6 and IL-8 production from HMC-1, while the mainstream (Ms) and sidestream (Ss) CSE either did not or only produced trace of cytokines (Figures 1, 2, 3 and 4). The effect of Ms CSE on production of IL-6 was shown in Figure 1. The Ms CSE at 31.25 to 250 μg/mL concentrations significantly increased IL-6 production in IL-1β-activated HMC-1 cells. The Ss CSE also increased production of IL-6 in IL-1β-activated HMC-1 cells (Figure 2). Similarly, both Ms and Ss CSE increased production of IL-8 in a dose-dependent fashion in IL-1β-activated HMC-1 cells (Figures 3 and 4).

The effect of BAI on production of IL-6 and IL-8 from IL-1β-activated HMC-1 cells was studied. BAI at concentrations of 1.8, 3.6, 7.5, 15, and 30 μM have been proved to be non-toxic to HMC-1 [28]. Two mL of HMC-1 at 1 × 10⁶ cells/mL were cultured with the above mentioned concentrations of BAI in the presence or absence of IL-1β (10 ng/mL) for 24 hrs. The culture supernatants were collected and assayed for cytokines by ELISA. BAI alone did not induce cytokine production from HMC-1. However, BAI at 15 and 30 μM concentrations significantly decreased the IL-6 production to 192.7 ± 18.7 and 74.6 ± 14.6 pg/mL, respectively (p < 0.0005 and p < 0.00005, respectively) (Figure 5A). BAI at all tested concentrations (1.8 to 30 μM) significantly decreased the IL-1β-induced IL-8 production, in a dose-dependent manner, to 316.4 ± 1.3, 177.4 ± 13.2, 147.6 ± 5.4, 54.9 ± 3.3, and 46.9 ± 4.4 pg/mL, respectively (p < 0.05 for 1.8 μM, p < 0.0005 for 3.6 μM, and p < 0.00005 for all the rest) (Figure 5B). Since BAI at 30 μM was the most effective concentration in inhibition of cytokine production in IL-1β-activated HMC-1, we decided to only use this concentration in experiments with CSE-treated HMC-1.

In order to investigate the effect of BAI on the enhancing effects CSE has on IL-6 and IL-8 production in IL-1β-activated mast cells, we treated the HMC-1 cells with or without IL-1β and CSE in the presence or absence of BAI (30 μM). BAI significantly inhibited the enhancing effect of both Ms and Ss CSE on production of IL-6 in IL-1β-activated HMC-1 (Figures 6 and 7). Similarly, BAI also significantly inhibited the enhancing effect of both Ms and Ss CSE on production of IL-8 in IL-1β-activated HMC-1 (Figures 8 and 9).

To study effects of BAI on inflammatory cytokine gene expression, the experiments were performed using IL-1β-activated HMC-1. HMC-1 were treated with IL-1β (10 μg/mL) and CSE in the presence or absence of BAI (30 μM) for 6 hours and harvested for transcriptional analysis via RT-PCR. IL-1β-treated HMC-1 increased IL-6 and IL-8 mRNA transcription (Figure 10). The intensities of the cytokine and house keeping gene (HPRT) bands were measured by densitometry, and the ratio of the cytokine to the house keeping gene was calculated and assigned as the intensity index. In the presence of Ms (0.125 mg/mL) or Ss (0.125 mg/mL) CSE, the expression of IL-6 and IL-8 was increased. However, in the present of BAI, the expression of IL-6 and IL-8 was remarkably decreased (Figure 10).

NF-κB is an important transcription factor that mediates the transcription of many proinflammmatory cytokine genes [29, 30]. In order to study the role that NF-κB plays in the inhibitory effect of BAI on inflammatory cytokine production, NF-κB activation was analyzed in HMC-1 cultured with CSE and IL-1β (10 μg/mL) in the presence or absence of BAI (30 μM). NF-κB translocation, as seen by a shift in oligonucleotide binding in EMSA gels, was increased in the IL-1β-activated HMC-1 (Figure 11). With the addition of Ms or Ss CSE (0.125 mg/mL) treatment, NF-κB translocation was even higher than that of the only IL-1β-treated group (Figure 11). In the presence of BAI, NF-κB translocation was decreased when compared to that of the IL-1β and CSE-activated HMC-1 (Figure 11).

The activation of NF-κB requires phosphorylation and proteolytic degradation of the inhibitory protein IκBα [31]. To determine whether the inhibitory activity of BAI is due to its effect on IκBα phosphorylation and degradation, we used Western blot analysis to examine the cytoplasmic levels of IκBα in HMC-1 after treatment with Ms or Ss CSE (both 0.125 mg/mL) and IL-1β (10 μg/mL) in the presence or absence of BAI (30 μM). The data showed that in the presence of IL-1β, the IκBα protein levels were decreased in HMC-1 (Figure 12). In cell treated with IL-1β and Ms or Ss CSE, the IκBα protein levels were further decreased when compared to that of the only IL-1β-activated group (Figure 12). With the addition of BAI, the IκBα protein levels were markedly increased when compared to that of the IL-1β- and CSE-activated HMC-1 (Figure 12).