Molecular Regulation of IL-13 and MCP-1 Expression in Human Mast Cells by IL-1β

Mast cells play pivotal roles in IgE-mediated airway inflammation, expressing interleukin13 (IL-13) and monocyte chemoattracant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of interleukin-1 beta (IL-1β) on cytokine expression by human mast cells have not been studied well. In this report, we provide evidence that human mast cells express the type 1 receptor for IL-1 (IL-1R1). We also demonstrate that IL-1β and tumor necrosis factor alpha (TNF-α) are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1β was accompanied by the activation of IL-1 receptor associated kinase (IRAK) and translocation of the transcription factor, nuclear factor kappaB (NF-κB) into the nucleus. Accordingly, Bay-11-7082, an inhibitor of NF-κB activation inhibited IL-1β-induced IL-13 and MCP-1 expression. IL-1β also induced IL-13 promoter activity, while enhancing the stability of IL-13 mRNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1β-induced nuclear translocation of NF-κB and also the secretion of IL-13 from mast cells. Our data suggests that IL-1β can serve as a pivotal co-stimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-κB. IL-1β being a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells. This could have important implications for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma. ABBREVIATIONS IL-1RI Interleukin-1 receptor type I IL-1β Interleukin-1 beta MCP-1 Monocyte chemoattractant protein-1 IL-13 Interleukin 13 NF-κB Nuclear factor-kappa B VLA-4 Very late activating antigen-4 VCAM-1 Vascular cell adhesion molecule-1 FcεRI Fc epsilon receptor I PMA/Iono Phorbol 12-myristate 13-acetate/Ionomycin INTRODUCTION Mast cells are multifunctional, tissue-residing cells derived from bone marrow. Upon maturation, mast cells express the high affinity receptor for immunoglobulin IgE (IgE), Fc epsilon RI (FcεRI), and following cross-linkage of FcεRI by IgE and antigen, mast cell activation occurs. These activated mast cells degranulate, expressing preformed and newly synthesized mediators. These include histamine, lipid mediators, and various cytokines that regulate inflammatory responses. These have been reviewed by us recently (1). Of interest to this paper are the cytokines, interleukin-13 (IL-13) and monocyte chemoattractant protein-1 (MCP-1). Mast cells have been shown to express IL-13 (2). IL-13 has been shown to induce B lymphocyte class switching to IgE and also induce vascular cell adhesion molecule expression on endothelium cells (3,4). IL-13 has also been shown to be pivotal to airway remodeling and mucus hypersecretion in murine transgenic models (5-7). MCP-1 on the other hand is a C-C chemokine that is important in mononuclear recruitment as well as mast cell and basophil activation (8,9). Together, these cytokines have important effects on regulating allergic airway inflammation as seen in asthma and on innate immune responses (8). In this paper, we demonstrate that the monokine, IL-1β, activates the expression of both IL-13 and MCP-1 from the human mast cell line, HMC-1. We also show IL-1β induced MCP-1 expression in cord blood derived mast cells (CBDMC) and IL1β-FcεRI crosslinkage induced expression of IL-13 in CBDMC. Since IL-1β is a ubiquitous cytokine expressed in airway macrophages and in many adventitial cells such as fibroblasts, this finding has important implications for innate immunity. IL-1β mediates its effects by binding to its receptor on inflammatory cells. Two types of receptors have been cloned, the type 1 (IL-1RI) and type (IL-1RII) receptors (10). Of these two, IL-1RI is considered to be the biologically active (11-13). Before signal transduction can occur, IL-1 must bind to IL-1RI as well as a co-receptor known as IL-1 receptor accessory protein (IL1RAcP) there by creating a transmembrane heterodimeric protein complex (11,14). This interplay leads to the intracellular signaling cascade that recruits several early adaptor proteins such as MyD88 (myeloid Differentiation factor 88) and interleukin-1 receptor associated kinase (IRAK). IRAK becomes hyperphosphorylated moving into the cytoplasmic region of the cell and forms a signalsome with TNF receptor associated factor 6 (TRAF 6). This signalsome mediates signaling of many down stream events through several regulatory kinases such as the IκB kinase complex and the mitogen-activated protein kinases (13-15). This can subsequently lead to the activation NF-κB, culminating in inflammatory gene expression and subsequent molecular mast cell events (15). The presence of IL-1R on human mast cells has not been demonstrated clearly and the molecular effects of IL-1 on human mast cell signaling and gene expression have not been studied in any great detail. IL-1 has been shown to have important effects on mast cell biology. For instance, Hultner et al. recently demonstrated that IL-1 induces the secretion of Th2 cytokines, IL-3, IL-5, IL-6, and IL-9 from murine mast cells (16). Lu-Kuo and coworkers showed that IL-1β stabilized the message for IL-6 mRNA in murine mast cells (17). Hogaboam et al., demonstrated that IL-1 induced activation of rat peritoneal mast cells and expression of nitric oxide and platelet activating factor (18). However, molecular effects of IL-1 on human mast cells and the signaling pathways are unclear. Using human umbilical cord blood-derived mast cells, we recently demonstrated a profound effect of IL-1 on enhancing IgE-mediated expression of IL-5, granulocyte macrophage colony stimulating factor (GM-CSF), and IL-8 (19). In this study we have extended these observations and demonstrate the molecular effects of IL-1β on mast cells and its role in the regulation of IL-13 and MCP-1 expression from these cells. We show that IRAK and NF-κB may be involved in this induction. MATERIALS and METHODS Cell culture and stimulation HMC-1 cells were grown in RPMI 1640 (Gibco BRL, Frederick, MD) supplemented with 11.1% FBS and 1% 1M Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer solution (Gibco, Rockville, MD). Cells (1x10/ml) were stimulated for 6, 12 and/or 24 hour with various concentrations of recombinant IL-1β (1, 10, 100 ng/ml) and/or of TNF-α (1, 10, 100 U/ml; kindly provided by National Cancer Institute’s Biological Resources Branch, Rockville, MD). Activation of the cells with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) (Sigma, St. Louis, MO) and ionomycin (5μM) (Sigma, St. Louis, MO) was performed in parallel as a positive control. To investigate inhibition of NF-κB translocation, cells were treated with 10μΜ final concentration of Bay-11 7082 (Biomol, Plymouth Landing, PA) for 1.5 hrs prior to addition of IL-1β. In some experiments, cells were incubated with varying concentrations of dexamethasone (Dex, 10 and 10M) (Sigma, St. Louis, MO) for twenty-four hours prior to the addition of IL1β. Cell viability remained intact after all treatments as determined by trypan blue exclusion. Cord blood derived mast cells (CBDMC) were harvested from fresh cord blood obtained by patient consent and institutional review board (IRB) approval. Blood was diluted 1:1 PBS, layered over Lymphoprep, centrifuged, and washed with more PBS. The cells were then grown in DMEMF12 media supplemented with 20% fetal bovine serum (Atlanta Biologicals, Atlanta, GA); 5 x 10M 2-mercaptoethanol (Fisher, Pittsburgh, PA); 0.5 ml insulin-transferin-sodium selenite solution (Sigma-Aldrich, St. Louis, MO); 25 mM HEPES (Gibco, Carlsbad, CA); 300 nM PGE2 (Cayman, Ann Arbor, MI); 100 ng/ml recombinant human IL-6 (kindly provide by Amgen, Thousand Oaks, CA); and 80 ng/ml stem cell factor (kindly provided by Amgen) for about 16 weeks or until mature (19). Maturity of CBDMC was observed by anti-chymase (kindly provided by Dr. Andrew Walls, University of Southhampton, England) and anti-tryptase (kindly provided by Promega, Madison, WI) antibody staining. Crosslinking of FcεRI on CBDMC surface was done using myeloma IgE at 1 μg/mL and anti-IgE at 1.5 μg/mL. IgE was added overnight at 37C before the addition of anti-IgE. Immunocytochemistry staining For immunocytochemistry staining, cytospin preparations of resting or stimulated cells (50 ng/ml PMA and 5μM Ionomycin) were performed, and incubated with a primary rat anti-human IL-1RI Ab (Antigenix, Hunington Station, NY), followed by a 30-min incubation with a mouse anti-rat IgG FITC conjugated secondary Ab (Antigenix, Hunington Station, NY). Slides were kept in the dark until observed under fluorescence microscope. Western blotting analysis For analysis of IRAK expression, mast cells were lysed by 10 % Nonidet P-40 in hypotonic buffer, and the cytoplasmic fractions were isolated and stored at -80C. Ten mg of total protein was added to equal amounts of Laemmli’s buffer (Bio-Rad, Richmond, Va.), heated for 5 minutes at 100C, resolved on 10 % SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% bovine serum antigen (BSA) in PBS, pH 7.4 for 1 hour and incubated in 1:500 dilution of a primary mouse anti-human IRAK antibody (BD Biosciences, San Diego, CA) overnight at 4 C. After washing, membranes were incubated with peroxidase-conjugated secondary antibody (Amgen, Thousand Oaks, CA) for 1 hr and Super Signal substrate (Pierce, Rockford, IL). Immunoreactive proteins were detected by enhanced chemiluminescence. β-Actin (Santa Cruz, Santa Cruz, CA) was used as a loading control. IL-13 and MCP-1 gene and protein expression Gene expression for IL-13 or MCP-1 was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was extracted by the RNAzol technique from cultured cells according to manufacturer’s instructions (Tel-Test, Inc., Friendswood, Texas). First strand cDNA was synthesized following a reverse-transcription step at 42 C for 20 min in the presence of murine leukemia virus reverse transcriptase (2.5 U/μl), 1 mM each of the nucleotides dATP, dCTP, dGTP and dTTP, RNase inhibitor (1 U/μl), 10X PCR buffer (500 mM KCl, 100 mm TrisHCl, pH 8.3), and MgCl2 (5 mM), using oligo(dT)16 (2.5 μM) as a primer. PCR amplification was done on aliquots of the cDNA in the presence of MgCl2 (1.8 mM), each of dNTPs (0.2 mM), and AmpliTaq polymerase (1 U/50 μl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 μl. PCR consisted of 1 cycle of 95 C for 2 min, 45 cycles of 95 C for 45 s, 60 C for 45s, and 72 C for 1 min 30s, and lastly, 1 cycle of 72 C for 10 min for G3PDH and 1 cycle of 95 C for 2 min, 36 cycles of C for 45s, 60 C for 45s, and 72 C for 1 min 30s, 1 cycle of 72 C for 10 min for IL-13 (5' GGAA GCTT CTCC TCAA TCCT CTCC TGTT-3'), IL1RI (5' GAAG CTGG ACCC CTTG GTAA-3') and MCP-1 (5' AGAA CTGT GGTT CAAG AGG-3') . Twelve microliters of the amplified products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide. IL-1RI, IL-13 and MCP-1 bands were compared to expected base pair migration distances from Phi 174 Hae III DNA maker (Promega, Madison, WI). IL-13 and MCP-1 levels in cell-free culture supernatants were assayed by enzyme linked immunosorbent assay (ELISA) as previously described using commercially available kits (R&D; Systems, Minneapolis, MN) (20-22). Densitometry was done by normalizing gel band intensities to house keeping genes on Un-scan-it software (Silk Scientific, Orem, UT). mRNA stability assay for IL-1β -induced IL-13 gene expression To examine whether IL-1β regulates IL-13 gene expression posttranscriptionally, analysis of mRNA stability was performed using a transcription inhibitor, Actinomycin D, a potent inhibitor of RNA polymerase II-dependent transcription, and semi-quantitative RT-PCR analyses at various time points with the inhibitor. The cells (1x10 per condition) were treated with IL-1β (10 ng/ml) for two hrs to induce IL-13 expression, followed by extensive washes. The cells were then cultured with or without IL-1β in the presence of Actinomycin D (2 μg/ml; Sigma, St. Louis, MO). Total RNAs were isolated by the use of RNeasy kit (Qiagen, Santa Clarita, CA) at various time points after the addition of Actinomycin D. RT-PCR analysis was performed using a standard protocol and pairs of human IL-13 and G3PDH primers. DNA strands were denatured at 95 C for 45s, followed by PCR at 60 C for 45s, 72 C for 45s, for 32 cycles for IL-13 and 26 cycles for G3PDH. The intensities of PCR products on 2% ethidium bromide-containing agarose gel with optimized exposure were evaluated by OpiQuant Acquisition and Analysis (Packard Bioscience Co., Mariden, CT). The relative level of gene expression was quantified by calculating the ratio of densitometric readings (OD) of the band intensity for IL-13 and G3PDH from the same sample at each time point, and then normalized to the ratio at time 0. Induction of IL-13 promoter activity in IL-1-stimulated mast cells To investigate the minimal promoter activity of IL-13 gene in IL-1β -stimulated cells, transient transfection assays were performed using a reporter gene construct containing the minimal promoter sequence of IL-13, in which the promoter sequence (-233 to + 50, relative to the transcription initiation site) of the IL-13 gene was fused to the luciferase coding sequence. A reporter gene construct containing a minimal promoter (-217 to +51; relative to the transcription initiation site) of the IL-4 gene was included in the assays for comparison. Plasmid DNA was obtained with double-cesium chloride purification (BioServe Biotechnologies, Laurel, MD). The SuperFect reagent (Qiagen) was used for transient transfections of HMC-1 cells according to the manufacturer’s directions. Two micrograms of plasmid DNA and 8 μl SuperFect reagent were used for transfection of 1 x 10 HMC-1 cells. The transfected cells were stimulated with 10 ng/ml IL-1β. 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) as recommended by the manufacturer. Total protein content of cell lysates was determined with Bio-Rad protein assays (Bio-Rad, Hercules, CA). Nuclear factor kappa-B assays Nuclear proteins were extracted from HMC-1 by a previously described method with modification (23). HMC-1 cells were centrifuged, then washed three times in cold PBS, and collected in a 1.5 mL micro centrifuge tube. Added to this was 100μL of ice cold hypotonic buffer: 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA (ethylenediaminetetraacetic acid), 0.1 mM EGTA (ethyleneglycolttetraacetic acid), 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM b-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Cells were incubated on ice for 30 minutes and vortexed after addition of 6.25 mL of 10 % Nonidet P-40. After 2 minutes of centrifugation at 30,000 x g, the supernatants were decanted off the top and kept at -80 C. While the pellets were resuspended and vortexed every 20 minutes for 3 hr. in 60 μL 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 mM leupeptin, 50 mM NaF, 30 mM b-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Nuclear fraction protein samples were then spun down and supernatants which contain nuclear proteins were harvested and kept in -80 C until used. Total protein concentration for both samples was determined by BCA protein assay reagent. Nuclear translocation of NF-κB was analyzed by the electrophoretic mobility shift assay (EMSA). Briefly, 7 mg of nuclear protein were added to 2 μL of 1 x binding buffer (50 mg/mL of double stranded poly dI-dC, 10 mM Tris HCl pH 7.5, 50 mM NaCl, 0.5 mM EDTA , 0.5 mM DTT, 1 mM MgCl2, and 10 % glycerol), and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3') end labeled with γ-P ATP. The reaction mixture was incubated at room temperature for 20 minutes and analyzed by electrophoresis on a 5 % non denaturing polyacrylamide gel. The gel was then dried on a Gel-Drier (Bio-Rad Laboratories, Hercules CA) and exposed to Kodak X-ray film at -80 C. Statistical analysis Data shown are representative of three independent experiments. All individual experiments were done in triplicate samples. RT-PCR experiments were done three times to insure reproducibility. All values are given as the mean ± standard deviation (SD). Statistical analysis was done using the Students t-test and Statistica version 5 computer software (StatSoft, Inc Tulsa, OK). A p-value of < 0.05 was considered significant. RESULTS Mast cells express IL-1 type one receptor Several studies have shown that IL-1 receptor type I (IL-1RI) is able to transduce a signal and induce cellular activation (11-13). Figure 1 shows immunocytochemistry staining on resting HMC-1 cells (Fig 1 A and B) and human umbilical cord blood-derived mast cells (FIG 1C and D) with a rabbit antibody to IL-1RI. Figure 1A shows minimal background fluorescence from unlabelled HMC-1 cells. Figure 1B shows resting HMC-1 cells express IL-1 receptor type one (IL-1RI) constitutively. Human umbilical cord blood-derived mast cells constitutively express the IL-1RI as well (FIG C unlabelled and FIG D labeled with antibody). To confirm constitutive expression of IL-1RI in HMC-1 we performed RT-PCR on resting and PMA and ionomycin stimulated HMC-1 (Fig 1E). While constitutive gene expression of IL-1RI is seen in resting cells, activation is accompanied by induction of IL-1R1 transcripts at 12 hours. This is on keeping with the immunocytochemistry data and suggests that human mast cells may constitutively express the type 1 IL-1R. IL-1β induces IL-13 and MCP-1 gene expression in mast cells To investigate the ability of IL-1β (10 ng/ml) and TNF-α (100 U/ml) to induce IL-13 and MCP-1 gene expression in human mast cells, HMC-1 cells were activated for 6 or 12 hours with IL-1β and/or TNF-α or with PMA and ionomycin as positive control. RNA was extracted and reverse transcribed to cDNA. cDNA was amplified by PCR for 36 cycles. GAPDH was used as a housekeeping gene. To ensure equal RNA loading, RNA was quantitated by optical density readings at 260 nm, and the integrity of the 28S and 18S RNA bands determined by electrophoresis in ethidium bromide-stained 2% agarose gel. Figure 2A shows IL-13 gene expression induced by IL-1β and TNF-α at 6 and 12 hours. Resting cells expressed no expression of IL-13. PMA and ionomycin strongly and consistently induced IL-13 transcripts as would be expected. However when HMC-1 cells were activated with PMA and ionomycin, IL-13 gene expression message peaked at 6 hours and then decreased by 12 hours. On the other hand, IL-1β-activated cells intensified their expression of IL-13 mRNA from 6 to12 hours, suggesting that IL-1β may prolong IL-13 mRNA stability (2.4 fold to roughly a 6.0 fold increase respectively). In studying the effects of IL-1β and TNF-α on MCP-1 expression in HMC-1, the same treatments and procedures were used for MCP-1 as with IL-13. When HMC-1 cells were incubated with PMA and ionomycin or IL-1β MCP-1 gene expression peaked at 6 hours and diminished by 12 hour (Fig 2B). There was a 3.2 fold increase in MCP-1 gene expression in mast cells treated with IL-1β for 6 hours which decreased by 12 hours. This expression contrasted with the later expression of IL-13 transcripts, suggesting differential regulation and/or altered RNA decay with the two cytokines. Nevertheless, these data confirm that IL-1β induces IL-13 and MCP-1 transcription in human mast cells. IL-1 enhances IL-13 mRNA stability and IL-13 promoter activity In order to investigate the ability of IL-1β to stabilize IL-13 mRNA transcripts, HMC-1 cells were stimulated with IL-1β (10 ng/ml) for two hours and then Actinomycin D (Act.D) was added to stop de novo RNA synthesis, and total RNAs were harvested after additional 0, 1, 4, 8 and 12 hours in the presence or absence of IL-1β. Figure 3A shows the decay kinetics of IL-13 mRNA. In the absence of IL-1β in the culture, the relative level of IL-13 mRNA decreased to around 50% at 4-hour time point after the treatment with Act. D, and at 12-hour time point only about 10% of IL-13 mRNA remained. In contrast, the relative level of IL-13 mRNA remained stable in the entire 12-hour time period when IL-1β is present in the culture. To evaluate further the ability of IL-1β to induce IL-13 transcription at a molecular level, we transiently transfected HMC-1 cells with minimal promoter sequences of both IL-4 and IL-13 as described in the materials and methods. As shown in Fig. 3B, IL-1β was able to induce about a 2.7 fold increase in IL-13 promoter activity as compared to that seen for media control, while a slight decrease in the promoter activity of IL-4 was found in IL-1β-treated cells, but the difference did not reach statistical significance (Fig 3B). These results suggest, therefore, that the functional effect of IL1β on the expression of IL-13 is operative at both transcriptional and posttranscriptional levels. CBDMC express IL-13 and MCP-1 CBDMC were stimulated with PMA/Iono, IgE/anti-IgE, IL-1β, and IL-1β plus IgE/antiIgE for 24 hours and assayed for IL-13 and MCP-1 production by ELISA (Fig 4A and B). PMA/Ionomycin is a potent stimulator of IL-13 while FcεRI crosslinking and IL-1β alone had no effect. Untreated CBDMC produced 0.886 ± 1.53 pg/mL of IL-13 while PMA/Iono treated cells produced 580.7 ± 19.3 pg/mL (p< 0.000005 compared to untreated). IL-1β and IgE/anti-IgE crosslinkage alone both produced 0.00 pg/mL of IL-13. Crosslinking of FcεRI along with IL-1β stimulation together however greatly increased IL-13 production over untreated samples (339.7 ± 16.5 pg/mL, p< 0.00005 compared to untreated). This shows a need for both stimuli to trigger IL13 production in CBDMCs (Figure 4A). CBDMCs are also capable of producing MCP-1 but due to the growth factors needed for mast cell maturation and the highly prolific nature of these cells, MCP-1 baseline production is higher than normal. PMA/Iono treatment is a potent activator of MCP-1 (2859.6 ± 85.4 pg/mL compared to 1138.2 ± 71.2 pg/mL for untreated cells, p< 0.00002 compared to untreated). IgE/anti-IgE and IL-1β alone both significantly increased MCP-1 production over untreated levels (1715.2 ± 86.5 pg/mL and 1983.3 ± 125.1 pg/mL respectively, both p values are less than 0.001 compared to untreated). IL-1β plus IgE/anti-IgE together (2109.8 ± 117.0 pg/mL) did not have a significant enhancing effect over IL-1β alone but did however have a significant enhancing effect over IgE/anti-IgE alone (p< 0.01). IL-1 induction of IL-13 and MCP-1 secretion from mast cells HMC-1 cells were incubated with various concentration of IL-1β (1, 10, 100 ng/ml) for 24 hours and cell-free supernatants were assayed for IL-13 and MCP-1 proteins by ELISA. Constitutively, HMC-1's produced a mean value of 30.3 ± 35.9 pg/ml of IL-13 protein (Fig 5A and B). Cells stimulated with IL-1β at various concentrations showed an induction of IL-13 protein production between the 1 and 10 ng/ml concentrations (94.9 ± 17.1 pg/ml and 122.0 ± 41.0 pg/ml, respectively but the effect reached a plateau at a concentration of 100 ng/ml, 123.0 ± 46.5 pg/ml [Figure 5A]). All concentrations of IL-1β induced a significant increase in IL-13 protein production as compared to media control, (p< 0.005, Fig 5A). HMC-1 cells activated with PMA (50ng/ml) and ionomycin (5μM) produced 400.3 ± 11.1 pg/ml of IL-13 (p< 0.000001 as compared to media control, Fig 5B). Cells stimulated with TNF-α did not show significant increase in IL-13 protein production at 100 U/ml concentration (34.3 ± 35.8 pg/ml, p< 0.057 when compared to the media control, Fig 5B). The induction of IL-13 transcript in response to TNF-α in the absence of concomitant IL-13 secretion is interesting. This could be partly explained as followsexpression of cytokine transcript in the absence of secreted protein might represent complicated intracellular post-transcription/translational/secretory processes which are poorly understood. Cells stimulated with IL-1β and TNF-α together, however, showed an increase in IL-13 protein production (268.5 ± 112.6 pg/ml, p< 0.0008, Fig 5B). Similar experiments were conducted to measure MCP-1 secretion by HMC-1 cells. HMC-1 cells secreted 645.1 ± 21.8 pg/ml of MCP-1 constitutively. HMC-1 showed a dose dependent response to IL-1β: they produced 1321.0 ± 93.3, 1916 ± 691.9, and 2,118 ± 353.6 pg/ml of MCP-1 when treated with 1, 10, and 100 ng/ml (p< 0.000001, p< 0.0002, p< 0.000003, respectively, Fig 5C). IL-1β at concentrations of 100 ng/ml also increased MCP-1 significantly as compared to that induced by IL-1β at concentrations of 1 ng/ml (p< 0.05). Cells incubated with PMA and ionomycin secreted a mean value of 2005.3 ± 586.6 pg/ml of MCP-1 (p< 0.0003 as compared to media control, Fig 5D). IL-1 β alone induced a significant increase in MCP-1secretion (1916.3 ± 691.9 pg/ml, p< 0.0002, as compared to media control Fig 5D). Cells treated with TNF-α (100 U/ml) alone showed an increase in MCP-1 protein secretion, 1119 ± 165.6 pg/ml p< 0.005 as compared to media control, fig 5D. HMC-1 treated with both IL-1β and TNF-α had no further effect on MCP-1 protein production (2251 ± 489.8 pg/ml, p< 0.00002 compared to the control, Fig 5D). IL-1 regulates NF-κB nuclear translocation and IRAK induction in mast cells To further understand the molecular consequences between IL-1β and IL-1RI in mast cells, we evaluated two signaling mechanisms, IRAK and NF-κB. Immunoblot analysis demonstrated that there was only a slight expression of IRAK in untreated cells, while there was an enhanced expression in cells stimulated with PMA and ionomycin or IL-1β (10 ng/ml) at thirty minutes (Fig 6A). We next evaluated the nuclear translocation of NF-κB in these cells following activation. HMC-1 cells were treated the same as previously described and nuclear proteins separated and analyzed by EMSA. As shown in Figure 6B, nuclear translocation of NF-κB is seen following activation of mast cells by IL-1β. When cells were pre-treated with Bay-11 7082, inhibition of nuclear translocation of NF-κB was seen following IL-1-induced activation (32% decrease as seen by densitometry) (Fig 6B). These data suggest that some of the effects following the binding of IL-1 β to its receptor, IL-1R1, on mast cells, may be mediated by a pivotal transcription factor, NF-κB. To look at the functional of aspects NF-κB translocation on IL-13 expression in mast cells, we pre-incubated cells with Bay-11 7082 (10μΜ) for 1.5 hrs prior to treatment with IL-1β (10ng/ml) for 24 hours. Cell-free supernatants were harvested and protein production was determined by ELISA. Pretreatment of HMC-1 with Bay-11 7082 (10μM) significantly decreased IL-1β (10ng/ml) induced IL-13 protein production, (32.9 ± 4.3 pg/ml vs. 132.5 ± 35.5 pg/ml of IL-1β alone, p< 0.005, Fig 6C). Bay-11 7082 also decreased IL-1β induced MCP-1 protein production, (948 ± 90.2 pg/ml vs. 1440 ± 120.9 pg/ml of IL-1β alone, p = 0.017, Fig 6D) but not as profoundly as that for IL-13. The differences in sensitivities of IL-13 and MCP-1 to Bay-11 7082 could be explained by complicated underlying regulatory mechanisms that govern specific signaling for cytokines. Dexamethasone inhibits IL-13 expression in mast cells Dexamethasone is a glucocorticoid commonly used in allergic and inflammatory disease. It stabilizes the NF-κB-IκB complex by increasing IκB production. We therefore sought to determine the effects of dexamethasone on IL-1β induced cytokine synthesis and NF-κB nuclear translocation. HMC-1 cells were incubated overnight with dexamethasone at either 10 M or 10 M concentrations. The concentrations were chosen because they are in the physiological ranges when dexamethasone is administered therapeutically. At these concentrations all cells retained their viability as determined by trypan exclusion. Cells pre-treated with dexamethasone were stimulated with IL-1β (10ng/ml) and incubated for either 1 hour for nuclear fractions, or 12 and 24 hours for ELISA. Cell-free supernatants were harvested and subjected to IL-13 and MCP-1 measurements by ELISA. To investigate the mechanism of protein reduction by dexamethasone, we isolated the nuclear fractions from the treated HMC-1 at 1 hour incubation time for NF-κB analysis by EMSA. Figure 6A shows dexamethasone inhibited the nuclear translocation of NFκB. This may provide one mechanism of action of these drugs in inflammatory disease and is consistent with our earlier data demonstrating that inhibition of NF-κB results in decreased IL-13 and MCP-1 expression. Dexamethasone significantly inhibited the IL-1β induced IL-13 production in HMC-1 in a dose dependent fashion at both the 12 and 24 hour incubations (Fig. 7B). Following 12 hours of incubation, cells treated with IL-1β in the presence of dex at 10M or 10 M concentration showed a 25 % ± 6 % and 72 % ± 6 % reduction of IL-13 protein, respectively (p< 0.002 and 0.00003, respectively, as compared to IL-1 treated cells). A comparable result was also shown at the 24 hour time point. IL-1β treated cells are represented as 100%, while cells treated with IL1β in the presence of dexamethasone at 10M or 10M showed a 29 % ± 1 % and 58 % ± 4 % reduction of IL-13 protein, respectively (p< 0.002 and 0.00002, respectively, as compared to IL-1 treated cells). In similar experiments dexamethasone also showed a significant inhibition of MCP-1 protein production in a dose dependent manner at both the 12 hour and 24 hour time periods (Fig. 7C). Cells treated and incubated for 12 hours with IL-1β in the presence of dexamethasone at 10M or 10 M concentration showed a significant reduction in MCP-1 protein production, respectively (30 % ± 10 % p< 0.02 and 42 % ± 11 % p< 0.003). While Cells treated and incubation for 24 hours with IL-1β in the presence of dexamethasone at 10M or 10 M concentration showed a significant reduction in MCP-1 protein production, respectively 41.3 % ± 10 % p< 0.007 and 74 % ± 3 % p< 0.0008). DISCUSSION Our data suggests that IL-1β is not only an activator of cytokine gene transcription but also serves to stabilize cytokine mRNAs, thereby prolonging their half-life. This phenomenon has previously been described in mouse bone marrow mast cells as well as in fibroblast and Bcells but not in human mast cells (16,17). Along with enhancement of IL-13 mRNA stability we illustrate that IL-1β is capable of increasing the activity of the IL-13 promoter suggesting some specificity in the response. The results from both experiments when taken together strongly suggest that IL-1β is a potent activator of IL-13 in mast cells, which can magnify the immune response through VLA-4-VCAM-1 monocyte recruitment. This data suggest that IL-1β activates IL-13 gene expression through direct activation of the IL-13 promoter, while the IL-4 promoter activity decreased, supporting previous studies that suggest the IL-4 promoter effects may be more transient (24). IL-1β is a ubiquitous, potent proinflammatory cytokine that is capable of modulating angiogenesis, lymphokine production, monocyte recruitment, cartilage remodeling and proliferation of mesangial cells, fibroblast, and smooth muscle cells (15). IL-1β responsive activities depend on its interactions with its receptor IL-1RI and the formation of a transmembrane heterodimer complex between IL-1RI and IL-1 receptor associated protein (IL1RacP) (14). IL-1 RI is the signal transducing receptor and transduces with a high efficacy, requiring less than 10 bound ligands to produce a signal (13). The ability of IL-1β to directly activate mast cells provides a novel and pivotal pathway of the innate immune response. In infectious or inflammatory states, IL-1β can be expressed by adventitial cells such as fibroblasts, endothelium, or mononuclear cells. Our data suggests that this binding of IL-1β to IL-1RI on mast cells could lead to a cascade of events culminating in inflammatory-immune responses that may be pivotal to mucosal immunity. Mast cell activation, leading to activation of IRAK and NFκB followed by inflammatory cytokine gene expression and cellular recruitment could provide an important adjunct pathway to defend against pathogens. In the case of the airway, inflammatory cell adhesion and chemotaxis, mucus production, and IgE regulation may all culminate in chronic inflammatory responses. This is summarized in cartoon format in Figure 8. In this paper we demonstrate that the human mast cell line HMC-1 expresses IL-RI, thereby allowing IL-1β to directly activate human mast cells. Previous reports have shown that bone marrow derived murine mast cells were able to be co-activated by IL-1 and ionomycin (16). It has been previously hypothesized that induction of IL-13, which induces airway hyperreponsiveness, may be accomplished via surrounding cells (6). In this report IL-1β significantly up regulated IL-13 and MCP-1 gene expression and protein production independent of IgE, therefore implying that mast cells play a greater role in the orchestration of inflammatory diseases such as rheumatoid arthritis and atherosclerosis as well as asthma in an IgE-independent manner. We also found that IL-1β and TNF-α together increased the stimulation of IL-13 protein and MCP-1 production. This may be important as both cytokines are produced at local sites of inflammation, and could compound the severity of disease (25). We have also shown that IL-1β alone as well as IL-1β and TNF-α together signal through an NF-κB dependent pathway as do bacterial pathogens such as moraxella catarrhalis in mast cells (26). The actions of IL-1β could be attenuated by addition of Bay-11-7082 to cell culture prior to activation, by inhibiting cytokine-induced translocation of NF-κB by blocking IkapppaBalpha phosphorylation (27). Dexamethasone also inhibited NF-κB nuclear translocation, as would be expected, in response to IL-1β stimulation. Dexamethasone has been shown to inhibit MCP-1 expression in human airway smooth muscle cells (28). Dexamethasone has also been shown to inhibit IL-13 production in HMC-1 and human lung mast cells (29). In our study, dexamethasone strongly inhibited both IL-13 and MCP-1 expression from mast cells. Yet, interestingly the maximal inhibition for IL-13 was at the 24 hour time point while MCP-1 was at the 12 hour time point. This suggests differential sensitivities of signaling pathways regulating IL13 and MCP-1 to the effects of glucocorticoids. Thus, the IRAK-NF-kB pathway in mast cells could represent a dominant mechanism that regulates inflammatory gene expression. While most of these experiments were carried out in HMC-1 cells, we have shown similar enhancing effects of IL-1β on cytokine expression from human cord blood mast cells developed from umbilical cord blood-derived mononuclear cells (19). Because they use some of the same signaling molecules, like MyD88, IRAK, and TNF receptor associated factor (TRAF)-6, the Tolllike receptor-IL-1 signaling pathways may in some way converge to induce pivotal effects in human mast cells. Investigators have demonstrated that LPS acting upon toll like receptor (TLR)4 induced significant release of IL-13 in cord blood derived mast cells (14,30). Another group of investigators have also shown that LPS acting through TLR-4 can induce human mast cells to secrete TNF-α and several chemokines specific for Th2 cells and eosinophils (31). Activation of TLR-4 via LPS has also been shown to inhibit apoptosis of CBDMCs by inducing Bcl-xL (32). Studies on the regulation of the TLR pathways in human mast cells are currently underway in our laboratories. In summary, the ability of IL-1β to directly activate mast cells provides a novel and pivotal pathway of the innate immune response involving human mast cells. Binding of IL-1β expressed by adventitial cells in response to infection to IL-1RI on mast cells, leading to activation of IRAK and NF-κB followed by inflammatory cytokine gene expression and cellular recruitment could provide an important adjunct pathway to defend against pathogens. In the case of the airway, inflammatory cell adhesion and chemotaxis, mucus production and IgE regulation may all culminate in chronic inflammatory responses. These are summarized in Figure 8. ACKNOWLEDGMENTSThis work has been supported by NIH grants AI-43310 and HL-63070, RDC grant (ETSU), TheCardiovascular Research Institute (ETSU) and Amgen, Inc Thousand Oaks, CA. REFERENCES1. Krishnaswamy, G., J. Kelley, D. Johnson, G. Youngberg, W. 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Cells were then fixed and stained with a rat anti-human IL-1RI antibody and a secondary antibody of mouse anti-rat IgG with FITC conjugate oran isotype control. Pictures were obtained under fluorescence microscope at 40x magnificationat 1/4 sec exposure. (A) Unstained HMC-1 cells. (B) HMC-1 cells stained with primary andsecondary antibodies. (C.) Cord-blood derived mast cells unstained. (D.) Cord-blood derivedmast cells stained primary and secondary antibodies. (E.) For confirmation ofimmunocytochemistry results, IL-1RI gene expression was evaluated in HMC-1 cells eitherresting or activated with PMA/Iono for 12 hours prior to RNA harvest. RT-PCR analysis wasperformed using primers specific for IL-1R1 and GAPDH (housekeeping gene) and specificity ofamplification was determined by electrophoresis on 3% agarose gel.Figure 2. IL-1β and TNF-α induce IL-13 and MCP-1 gene expression Mast cells were incubated with either with PMA/Iono, IL-1β (10 ng/ml), TNF-α (100 U/ml) or IL-1β (10 ng/ml) and TNF-α (100 U/ml) for 6 and 12 hours prior to RNA harvest. Total mRNAwas extracted after 6 and 12 hrs. RT-PCR analysis was performed using either IL-13 primer orMCP-1 primer and product was determined by expected base pair migration an agarose gel.GAPDH was used as a housekeeping gene. Figure 2A shows IL-13 transcripts expressed at 6 and12 hours after activation and figure 2B shows MCP-1 transcripts at 6 and 12 hours afteractivation. IL-1-induced IL-13 mRNA expression peaks at 12 hours while IL-1-induced MCP-1expression peaks at 6 hours but persists at hour 12.Figure 3. IL-1β enhances mRNA stability and induces IL-13 promoter activity. 3A. Analysis of IL-13 mRNA stability. Cells were stimulated with IL-1β (10 ng/ml) for two hours and then Actinomycin D (Act.D) was added to stop de novo RNA synthesis, and RNAfrom cells harvested after 0, 1, 4, 8 and 12 hrs. RT-PCR analysis was performed, and the ratio ofIL-13 mRNA steady state level to that of GAPDH was calculated by densitometric analysis foreach time point, and data were expressed as the optical density (OD) ratio of the PCR productsfor IL-13 and GAPDH. Y axis demonstrates % of mRNA remaining and X axis indicates thetime points of RNA harvest following activation. Please see text for interpretation. 3B. IL-1β-induced promoter activity of IL-13. HMC-1 cells were transiently transfected with each of theluciferase reporter constructs, pGL3.IL4p bearing bp -217 to +51 (relative to the transcriptionstart site) of human IL-4 gene, and pGL3.IL13p bearing bp -233 to +50 (relative to thetranscription start site) of human IL-13 gene. Results are indicated as mean fold increase + SD induplicate. Please see text for interpretation.Figure 4. Demonstration of IL-13 and MCP-1 Production in CBDMCCBDMC were treated for 24 hours with PMA/Iono, IgE/anti-IgE, IL-1β, and IL-1β plus IgE/antiIgE. A. PMA/Iono was a good activator of IL-13 in CBDMC while IgE/anti-IgE and IL-1β had no effect. IL-1β and IgE/anti-IgE together however produced a significant increase in IL-13. (*p< 0.000005 compared to CBDMC untreated and ** p< 0.00005 compared to CBDMCuntreated) B. MCP-1 is produced in CBDMC treated with PMA/Iono (* p< 0.00002 compared to untreated). FcεRI crosslinking with IgE/anti-IgE and IL-1b also produced a significant increasein MCP-1 over control levels (** p< 0.001 compared to untreated).Figure 5. IL-1β and TNF-α induce IL-13 and MCP-1 protein production Mast cells were incubated with either with PMA/Iono, IL-1β (1, 10, 100 ng/ml), TNF-α (1, 10, 100 U/ml) or IL-1β (10 ng/ml) and TNF-α (100 U/ml) for 24 hours, cell free supernatants were harvested, and protein production was determined by ELISA. 4A and B: IL-1β induction of IL-13 protein production (*= p< 0.002, **= p< 0.002, *** = p< 0.003, # = p< 0.001, as compared tocontrol). 4A studies the dose response curve using varying concentrations of IL-1β while 4B indicates fixed concentrations of IL-1β (10 ng/ml) and TNF-α (100 U/ml). 4C and D: IL-1βinduction of MCP-1 protein production (* = p< 0.001, ** = p< 0.002, *** = p< 0.00003, # = p<0.005, ## = p< 0.001 as compared to control). 4C studies the dose response curve using varyingconcentrations of IL-1β while 4D indicates fixed concentrations of IL-1β (10 ng/ml) and TNF-α(100 U/ml).Figure 6. IL-1β signaling via IRAK and NF-κB pathways IL-1RI activation by IL-1β leads to activation of IL-1 receptor-associated kinase [IRAK]. 5A. Induction of IRAK is seen with IL-1β within 20 minutes of mast cell activation. 5B. Mast cells were stimulated with either IL-1β (10ng/ml) or pre-incubated with Bay-11 7082 (10μΜ) for 1.5 hrs prior to IL-1β stimulation. NF-κB nuclear translocation was assessed using EMSA. Bay-11 7082 inhibited nuclear translocation of NF-κB translocation as would be expected. 5C. HMC-1 mast cells were stimulated for 24 hrs with IL-1β (10ng/ml) or pre-incubated with Bay-11 (10μM) for 1.5 hrs prior to IL-1β stimulation. Cell-free supernatants analyzed for IL-13 proteinproduction by ELISA (* = p< 0.004, ** = p< 0.005). 5D. HMC-1 cells were stimulated for 24 hrs with IL-1β (10ng/ml) or pre-incubated with Bay-11 (10μΜ) for 1.5 hrs prior to IL-1βstimulation. Cell-free supernatants were harvested and assayed for MCP-1 protein production byELISA (# = p< 0.005, ## = p< 0.017).Figure 7. Dexamethasone attenuates IL-1β stimulation of IL-13 and MCP-1 production viathe NF-κB pathway. 6A. Dexamethasone inhibits nuclear translocation of NF-κB. Mast cells were stimulated witheither IL-1β (10ng/ml) or pre-incubated with dexamethasone (10M) for 24 hrs prior to IL-1β stimulation. NF-κB nuclear translocation was assessed using EMSA. Dexamethasone inhibits IL-1β induced NF-κB nuclear translocation. 6B. IL-13 production is attenuated bydexamethasone. Mast cells were treated with dexamethasone at either 10 or 10 Mconcentrations overnight then incubated with IL-1β (10 ng/ml) for 12 or 24 hr. Cell free-supernatants were harvested and protein was determined by ELISA. Significant inhibition of IL-13 production from mast cells in response to IL-1β was seen with dexamethasone [* p< 0.002, ** p< 0.002, # p< 0.00003 and ## p< 0.00002 as compared to cells treated with IL-1β alone]. Actual values of IL-13 in pg/mL for IL-1β 12 and 24 hours are 132.5 ± 35.5 and 105.9 ± 36.4respectively. Actual values of IL-13 for dex 10 and 10 are 99.38 ± 7 and 77.8 ± 7.8respectively for 12 hours and 65.3 ± 16.6 and 27.7 ± 10 respectively for 24 hours. 6C. MCP-1production is attenuated by dexamethasone. Mast cells were treated the same as for IL-13.Significant inhibition of MCP-1 production from mast cells in response to IL-1β was seen withdexamethasone [* p< 0.02, ** p< 0.007, # p< 0.003and ## p< 0.0008 as compared to cells treated with IL-1β alone]. Actual values of MCP-1 in pg/mL for IL-1β 12 and 24 hours are 1110.4 ±22.4 and 1716.3 ± 22.4 respectively. Actual values of MCP-1 for dex 10 and 10 are 792.6 ±23.5 and 68.65 ± 3.3 respectively for 12 hours and 966.27 ± 70.2 and 441.01 ± 14.2 respectivelyfor 24 hours.Figure 8. Cartoon demonstrating the effects of IL-1R1 ligation on human mast cells.Activation of IRAK is accompanied by NF-κB nuclear translocation and IL-13 and MCP-1 geneexpression. Translation and secretion of these proteins from mast cells can lead to VCAM-1 expression, IgE class witching, mucus hypersecretion and leukocyte/mononuclear cellchemotaxis. This can contribute to airway inflammation and innate immune responses.Dexamethasone as an antiinflammatory drug can inhibit activation of this pathway.