Human mast cell line, HMC-1, was incubated with IL-1β at various concentrations for 24 hours. The cell free supernatants of the cultures were harvested and subjected to IL-6 assay. HMC-1 cultured in medium alone produced trace amounts of IL-6. The IL-6 production from HMC-1 cultures treated with IL-1β was significantly increased in a dose-dependent manner (p < 0.0001) (Fig. 1). Since there was no significant difference in the IL-6 production induced by IL-1β at concentrations of 10 and 50 ng/ml, 10 ng/ml of IL-1β has been used to induce cytokine production in HMC-1 for the rest of the experiments. Epinephrine (Epi) alone at a concentration of 10^-3 M did not induce production of IL-6 in HMC-1 (Fig. 2). When epinephrine at 10^-3 to 10^-7 M concentration was added simultaneously with IL-1β into the cultures, the production of IL-6 was enhanced significantly (p < 0.05) compared with that induced by IL-1β alone (Fig. 2). Since the physiological concentration of epinephrine in plasma is 0.11 – 0.27 × 10^-6 M 31, we decided to use epinephrine at a supramaximal concentration of 1 × 10^-5 M for the rest of the experiments. In addition to IL-6, the enhancing effect of epinephrine was also observed in the production of IL-8 and IL-13 from IL-1β-induced HMC-1 cells (Fig. 3).

To measure proatherogenic cytokine gene expression, HMC-1 were treated with IL-1β, epinephrine, and IL-1β plus epinephrine for 6 hours and harvested for transcriptional analysis via RT-PCR. IL-1β-treated HMC-1 showed increased IL-6 mRNA transcription as seen with densitometry, while epinephrine alone appeared to have no effect. When IL-1β and epinephrine were added together to HMC-1, IL-6 mRNA expression increased over IL-1β treatment alone (Fig. 4). 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. The intensity indices for IL-6 were 0.36 for the control, 0.39 for IL-1β alone, 0.33 for epinephrine alone, and 0.54 for IL-1β plus epinephrine. IL-1β activated IL-8 mRNA production but epinephrine had no effect on IL-8 transcripts. IL-1β and epinephrine treatment together further increased IL-8 mRNA production (Fig. 4). The intensity indices for IL-8 were 0.17 for the control, 0.52 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. The results with IL-13 expression showed the same pattern. IL-1β was a good inducer of IL-13 transcription while epinephrine alone only minimally induced IL-13 mRNA. The combined stimulus of IL-1β and epinephrine significantly increased IL-13 mRNA production over that seen with each stimulus alone (Fig 4). Intensity indices for IL-13 were 0.22 for the control, 0.57 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. To evaluate further the ability of epinephrine to induce IL-13 transcription at a molecular level, we transiently transfected HMC-1 cells with minimal promoter sequences as described in the materials and methods. IL-1β at 10 ng/ml significantly increased IL-13 promotor activity as detected by luciferase expression (data not shown). Epinephrine did not enhance IL-13 promoter activity suggesting that post-transcriptional mechanisms may be involved in the IL-13 induction. It is likely that epinephrine either prolongs IL-13 mRNA half life and/or enhances IL-13 secretory processes from the mast cell in response to IL-1-stimulation.

Since our previous study has shown that the effect of epinephrine on nitric oxide synthesis is mediated by β-adrenoceptors 26, β-adrenergic receptor antagonists (propranolol and atenolol) were used to block the enhancing effect of epinephrine on proatherogenic cytokine production in HMC-1. Propranolol (Pro) and atenolol (Ate) at a concentration of 1 × 10^-4 M did not affect the cell viability in the cultures (88 and 90%, respectively, while that of the medium control was 85%), nor induced production of IL-6, IL-8 or IL-13 (Fig. 3). When propranolol at 1 × 10^-4 and 1 × 10^-5 M was used in the culture, it significantly reduced the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 3A). Propranolol at 1 × 10^-4 M also significantly reduced the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 3B), and at 1 × 10^-4 and 1 × 10^-5 M significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and 0.0005, respectively, Fig. 3C). However, atenolol only significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.05, Fig. 3C), but not on IL-6 or IL-8 production (Fig. 3A and 3B).

In order to further identify whether the enhancing effect of epinephrine on proatherogenic cytokine production is through the β-adrenoceptor, HMC-1 cells were incubated with rabbit polyclonal antibodies against β₁ and β₂ adrenergic receptors followed by a FITC-labeled second antibody. By flow cytometry analysis, β₁ and β₂ adrenergic receptors were found in small amounts on HMC-1 (18.6 and 11.7% respectively) (Fig 5).

Since glucocorticoids are very effective treatment strategies for inflammatory disease, dexamethasone was used to determine its effect on atherogenic cytokine production in HMC-1. Dexamethasone (Dex, 1 × 10^-7 M) alone did not induce proatherogenic cytokine production (Fig. 6). However, Dex significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 6A). The cell viability of the cultures was not different between the medium control (70%) and Dex (64%) groups. Dex also significantly inhibited the enhancing effect of epinephrine on IL-8 and IL-13 production (p < 0.05, Fig. 6B and 6C). When Dex was included in the IL-1β-treatment, it slightly decreased the cytokine production when compared to the IL-1β alone, but the decrease was not significant (Fig. 6).

NF-κB is an important transcription factor that mediates the transcription of many proinflammatory cytokine genes. To study the role NF-κB plays in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, NF-κB activation was analyzed in HMC-1 cultures. NF-κB translocation, as seen by a shift in oligonucleotide binding in EMSA gels, was not seen in control or epinephrine treated cells (Fig. 7). A marked increase of NF-κB nuclear binding activity was observed in samples stimulated with IL-1β and IL-1β plus epinephrine for one hour but started to diminish after two hours (Fig. 7). Not only did IL-1β plus epinephrine have no further effects on NF-κB translocation over IL-1β treatment alone, it seemed to decrease after one and two hours of stimulation.

Because of its importance in cytokine signaling, phosphorylated p38 MAPK was also assayed. After 30 minutes of activation, the HMC-1 were lysed to be analyzed for p38 activation by Western blot. The presence of phosphorylated p38 was greatly increased in the epinephrine and IL-1β plus epinephrine samples (Fig. 8). IL-1β alone had small effects on p38 activation at this time point while control levels were virtually nonexistent.

To confirm the role of NF-κB and p38 MAPK in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, Bay 11, an NF-κB inhibitor 32, and SB203580, a specific inhibitor of p38 MAPK 33, were added to the cultures. By themselves, neither Bay 11 (1 × 10^-5 M) nor SB203580 (1 × 10^-5 M) affected the cell viability of the cultures (92 and 87%, respectively, while that of the medium control was 92%), nor did they induce proatherogenic cytokine production (Fig. 9). However, Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.0005 and p < 0.00005, respectively, Fig. 9A). Bay 11 decreased the IL-1β-epinephrine induced IL-8 production but not significantly, however SB203580 did significantly inhibit the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 9B). Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and p < 0.0001, respectively, Fig. 9C).

HMC-1 cell line, established from a patient with mast cell leukemia, were graciously provided by Dr. Butterfield (Mayo Clinic, Rochester, MN). These cells were maintained in RPMI 1640 media (GibcoBRL, Rockville, MD), supplemented with 5 × 10^-5 M 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM HEPES (GibcoBRL), Gentamycin 50 μg/ml, 5 μg/ml insulin transferrin, 2 mM L-glutamine, and 5% heat inactivated fetal bovine serum (Atlanta Biologicals, Atlanta, GA), at 37°C and in 5% CO₂ mixture 33. HMC-1 cells were cultured and maintained in 25 cm² flasks. To each well of a 6 well culture plate, two ml of HMC-1 mast cells at 0.75 × 10⁶ cells/ml concentration were cultured with epinephrine at 1 × 10^-5 M concentration in the presence and absence of IL-1β (10 ng/ml) for 24 hrs. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA and cell viability and numbers of the culture were analyzed.

Cytokine ELISA was performed for the following cytokines: IL-6, IL-8, and IL-13. ELISA was carried out on cell-free culture supernatants using commercially available ELISA kits, according to manufacturers instructions as earlier described (R&D Systems, Minneapolis, MN; Immunotech, Westbrook, ME; Genzyme, Cambridge, MA). Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software) 4748.

At the end of incubation, the cells were subjected to the viability count by trypan blue (TB) dye exclusion technique. Two tenths ml of cell cultures were mixed with 0.05 ml of TB, applied to hemocytometer, and counted under a microscope. The cell viability is calculated by dividing the number of live cells (unstained cells) by the total number of all cells (TB-stained and unstained cells) and expressed as a percent.

HMC-1 were treated with the appropriate reagents and allowed to incubate at 37°C before being harvested for RNA. RNA was extracted from HMC-1 (3 × 10⁶ cells) by the addition of 1 ml of RNAzol B (Tel-Test, Inc., Friendswood, Texas) 49. After shaking for 1 minute the samples were centrifuged at 12,000 × g for 15 minutes at 4°C. The aqueous layer was washed twice with 0.8 ml phenol : chloroform (1:1, v/v), centrifuged at 12,000 × g for 15 minutes at 4°C, washed once with 0.8 ml of chloroform and centrifuged at 12,000 × g for 15 minutes at 4°C again. 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 and allowed to air dry until all moisture was gone. The pellet was resuspended in DEPC water and quantitated by optical density readings at 260 nm. 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'; IL-8: 5' ATG ACT TCC AAG CTG GCC GTG GCT 3' and 5' TCT CAG CCC TCT TCA AAA ACT TCT C 3'; and IL-13: 5' GGA AGC TTC TCC TCA ATC CTC TCC TGT T 3' and 5' GCG GAT TCG TTG AAC CGT CCC TCG CGA AA 3'. Densitometry was done by normalizing target genes to house keepers using Un-Scan-It Version 5.1 software (Orem, UT). The PCR experiment was repeated twice.

HMC-1 were stimulated with PMA, IL-1β and/or epinephrine and then harvested for EMSA analysis 4950. 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 Electrophoretic Mobility Shift Assay (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. The gel was then dried on a Gel-Drier (Bio-Rad Laboratories, Hercules CA) and exposed to Kodak X-ray film at -80°C.

Cells were treated and lysed in lysis buffer (50 mM Tris HCL, 150 mM NaCl, 1 mM EDTA, 1% Triton × 100, and 0.1% SDS) to be analyzed for p38 MAPK activation by Western blot 29. Briefly, 50 μg of sample protein was diluted 1:2 with Laemmli buffer (Bio-Rad laboratories, Hercules, CA) and boiled for 10 minutes in a sand bath. The resultant sample was then run in a Bio-Rad Modular Mini Electrophoresis System (Hercules, CA) on a 10% polyacrylamide gel for 1 hour and then transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad laboratories, Hercules, CA) for 1 hour. The blot was then incubated in blocking buffer (1% BSA and 0.1% Tween in PBS) for 1 hour at room temperature with gentle agitation. Rabbit anti-human Phospho-p38 MAPK (Thr180/Tyr182) polyclonal antibody (Calbiochem, San Diego, CA) was diluted 1:1000 in blocking buffer and incubated on the blot overnight at 4°C with gentle agitation. After the primary antibody was removed the blot was washed three times for 10 minutes each with agitation in the wash buffer (0.1% Tween in PBS). The blot was then incubated in horse radish peroxidase conjugated mouse anti-rabbit Ig's antibody (human adsorbed, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:5000 in blocking buffer. The blot remained in the secondary antibody for 1 hour at room temperature. The blot was then washed again and covered with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 minutes. The blot was then exposed to acetate transparency film (Kodak, Rochester, NY) and developed. The same protocol was repeated for total p38 MAPK analysis.

Resting HMC-1 were centrifuged, washed in PBS at room temperature, and resuspended in 100 μl of PBS. The cells were incubated for 20 minutes with rabbit polyclonal anti β₁ or β₂ adrenergic receptor antibodies (Santa Cruz, Santa Cruz, CA) using normal rabbit serum as a control. The samples were washed with PBS with 0.01% sodium azide and resuspended in 100 ml PBS. FITC labeled goat anti-rabbit Ig's antibody was added to the samples and allowed to bind for 20 minutes. The samples were once again washed with PBS with 0.01% sodium azide and resuspended in 100 μl of PBS. In addition, HMC-1 were pretreated with normal rabbit serum and incubated with FITC labeled goat anti-rabbit Ig's antibody as a control for nonspecific binding 51. Cell suspensions were then gated and analyzed based on fluorescence using a Becton Dickinson FACSCalibur 4-color flow cytometer (San Diego, CA) and histograms generated on WinMDI 2.8 software (kindly provided by Joseph Trotter over the internet).

HMC-1 were treated with IL-1β (10 ng/ml), epinephrine (10^-5 M), and IL-1β plus epinephrine to investigate IL-13 promotor activity. Untreated cells were used as a control. Transient transfection assays were performed using a reporter gene construct containing the minimal promoter sequence of IL-13. The promoter sequence (-233 to + 50, relative to the transcription initiation site) of the IL-13 gene was fused to the luciferase coding sequence. Plasmid DNA was obtained with double-cesium chloride purification (BioServe Biotechnologies, Laurel, MD), while SuperFect reagent (Qiagen) was used for transient transfections of HMC-1 cells. Two micrograms of plasmid DNA and 8 μl SuperFect reagent were used for transfection of 1 × 10⁶ HMC-1 cells. Luciferase expression was monitored by chemiluminescence of cell lysates 24 hrs after transfections using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, Ann Arbor, MI).

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.