Regulation On The Surface Expression Of FcεRI And The Bioactivities Of BMMC

ObjectiveMast cells, which mainly exist in the mucous and connective tissue, are activated upon stimulation by binding IgE to the high affinity surface receptor, FcεRI, thereby leading to allergic disease. In brief, cross-linking of FcεRI with IgE/allergen complex causes immediate reaction including degranulation and the de novo synthesis of proinflammatory cytokies such as TNF-α\ IL-6 and IL-13. Therefore, mast cells play key roles in the allergic diseases by means of these bioactivators. The elucidation of regulation on the surface expression of FcεRI and the bioactivities of mast cells will be helpful to the prevention and treatment of allergic diseases.FcεRI is composed of three subunits:the IgE-bingding a-chain, and signal-transducingβ-and y-chain. In our previous studies, Elf-1 was found to recognize the nucleotide sequence around the cis-element of the FcεRI a-chain promoter in vitro. Elf-1 is a transcription factor belonging to the Ets family, which possesses a highly conserved DNA-binding region termed the Ets domain. Elf-1 cDNA was identified from human T cells as a cell-type specific transcription factor that enhanced the HIV-2 promoter. Subsequently, Elf-1 was shown to be involved in the expression of various immunorelated genes, including interleukin (IL)-2, GM-CSF, IL-5, IL-2RA, and CD4 in T cells; immunoglobulin heavy chain, blk, lyn, and CD1D1 in B cells; IL-3 in megakaryocytes; and stem cell leukemia gene in mast cells. Recently Elf-1 has been reported to inhibit the gene expression of FcεRI y-chain in human basophilic cell line KU812. However, it is remain unknown whether Elf-1, which binds to the promoter of FcεRI a-chain, can influence the the gene expression of FcεRI in mouse mast cells.In addition to transcription factors, the expression of genes is associated with the status of histone acetylation in the chromatin, which is controlled by two key regulators, histone acetyltransferase (HAT), and histone deacetylase (HDAC). Trichostatin A (TSA), one of the most potent HDAC inhibitors (HDACi), belongs to the hydroxamic acids. Increasing evidence suggests that TSA is a potential anticancer agent as it was found to inhibit the growth of and to induce differentiation and apoptosis of tumor cells. TSA and another HDACi, suberoylanilide hydroxamic acid (SAHA), have also been shown to be effective in immune disease rodent models, including SLE, graft-versus-host-disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. In addition, there is a report of therapeutic usage of TSA on allergic mouse model. However, the effects of TSA on the surface expression of FcεRI and related functions of mast cell, a key element in immune responses, have been rarely examined.In this study, we use mouse bone marrow-derived mast cells (BMMC) transfected with Elf-1 siRNA or stimulated by TSA in vitro in order to investigate the role of Elf-1 and TSA on the gene expression of FcεRI on BMMC and its related bioactivities. It would provide new therapeutic approach for mast cell-mediated allergic diseases.Methods1,Preparation of mouse BMMCMouse BMMC were obtained by culturing femoral bone marrow cells from 6-8-week-old female BALB/c mice in BMMC culture medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS,100μM 2-mercaptoethanol,10 mM sodium pyruvate,10μM MEM nonessential amino acid olution,100 U/ml penicillin, 100μg/ml streptomycin and 10% pokeweed-mitogen-stimulated spleen-conditioned medium). In brief, both femurs were separated, and bone marrow cells were collected by rinse and centrifugation at 1000rpm for 5min at 4℃. The cells were cultured with BMMC culture medium for 4 weeks.2,Transfection with Elf-1 siRNA and Elf-1 expression plasmid in vitroFive microliters of 20μM double-stranded Elf-1 siRNA, control siRNA and FITC-labeled control oligonucleotide was introduced into 1×106 of BMMC for 24h (for detection of transfection rate, gene expression of Elf-1, FcεRIα-,β-, and y-chain) and 48h (for detection of Elf-1 protein expression) with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit according to the manufacturer's instructions. One microgram of pGV-B2-aNN0.6 (reporter plasmid), or pGL3-Basic (promoter less control) was cotransfected with 25ng pRL-CMV (internal control for normalization of transfection efficiency) and siRNA into BMMC by Nucleofector II as described above. For overexpression of Elf-1, 10μg of pCR-Elf-1 or pCR3.1 control plasmid was introduced into BMMC with 5μg of reporter plasmid and 25ng of pRL-CMV by electroporation using Bio-Rad Gene PulserⅡ. The transfected cells were cultured for 24h at 37℃(for detection of the activity of the promoter of FcεRI a-chain).3,The detection of Elf-1 expressionAfter 24 h of culture after electroporation, mRNA levels of Elf-1 were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit.Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including Elf-1 (Mm00468217_ml) and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of Elf-1 were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software. In addition, After 48h of culture after electroporation,1×106 BMMC were collected, and whole cell lysates were subjected to electrophoresis on a 7.5% SDS polyacrylamide gel. Anti-Elf-1 antibody and anti-actin Ab were used as primary Abs. Alexa Fluor 680 goat antirabbit IgG and IR Dye 800 goat anti-mouse IgG (Molecular Probes) were used as secondary Abs against Elf-1 and actin, respectively. Infrared fluorescence on membranes was detected using the Odyssey infrared imaging system. The amount of protein was quantified by calculating band intensity using the Odyssey software.4,The detection of promoter activities of FcεRI a-chain in BMMCAfter 24h of cultivation, BMMC were harvested and treated with a Dual-luciferase assay kit for the measurement of luciferase activity. The luminescence was measured with Micro Lumat Plus. 5,The detection of mRNA expression of FcεRIα-,β-and y-chainAfter 24 h of culture after electroporation, mRNA levels of FcεRIα-,β-and y-chain were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including FcεRI a-chain (Mm00438867_ml), the FcεRI p-chain (Mm00442780_ml), the FcεRIγ-chain (Mm00438869_gl), and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of FcεRIα-,β-, and y-chain were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software.6,The detection of PU.1 binding to FcεRIα-chain promoterBMMC transfected with siRNA were collected and lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-PU.1 goat IgG Ab and goat IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the promoter region of the a-chain gene,-82/+1, forward primer-82/-56 (5'-GGCATAGCTGATGAGTTAACCAGATAC-3'), reverse primer+1/-22 (5'-TATGGCTTCGAAAATAGGCTTGA-3'), and TaqMan probe-51/-33 (5'-FAMCAGAAGACATTTCCTTCTC-MGB-3').7,BMMC were stimulated with TSA in vitroBMMC from BALB/c mouse was adjusted to 1×106/ml and stimulated with TSA at the final concentration of 0,2,10 and 50nM at 37℃for 24h (for detection of expression of FcεRI and functions of BMMC) and 48h (for detection of apoptosis of BMMC), respectively.8,The detection of the expression of FcεRI on BMMCBMMC transfected with siRNA or stimulated with TSA were collected and then incubated with PE-labeled anti-FcεRI a-chain Ab for 1 h at 4℃after blocking Fc receptors on the cell surface with 2.4G2. Cells were washed with PBS twice. Then the expression of FcεRI on BMMC was analyzed using a FACSCalibur flow cytometer.9,The detection of the apoptosis of BMMCBMMC stimulated with TSA were collected. For analysis of apoptotic cells, BMMC were stained with 5μg/ml propidium iodide (PI) and Annexin V-FITC for 15min at room temperature. Cells were analyzed using a FACSCalibur flow cytometer.10,β-hexosaminidase release assayAfter incubation of BMMC with the indicated concentration of TSA for 24 h at 37℃in the complete culture medium, the cells were sensitized with 1μg/ml mouse IgE for 1 h at 4℃and then resuspended in Tyrode's buffer at a concentration of 1 x 106 cells/ml, and stimulated with 1μg/ml anti-mouse IgE for 45 min at 37℃. The cell supernatants were collected, andβ-hexosaminidase activity in the supernatants and cell lysates was quantified by spectrophotometric analysis of hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucopyranoside. The percentage ofβ-hexosaminidase release was calculated using the following formula:percent release (%)=(OD of the stimulated supernatant-OD of the unstimulated supernatant)/(OD of the total cell lysate-OD of the unstimulated supernatant)×100%.11,Measurement of IL-6 production from BMMCAfter incubation with TSA for 24h at 37℃, BMMC at concentration of 1 x 106/ml were stimulated with IgE/anti-IgE cross-linking for 1h at 37℃. The total RNA was collected and reversed transcripted into cDNA. The expression of IL-6 mRNA was analyzed by Real-time PCR. TSA incubated BMMC were stimulated with IgE/anti-IgE cross-linking for 6h at 37℃and the supernatant were collected. The concentration of IL-6 in culture supernatant was determined by an ELISA kit according to the manufacturer's instructions.12,The detection of histone acetylation of IL-6 promoter in BMMCBMMC incubated with TSA were collected and then stimulated with IgE/anti-IgE cross-linking for 30min at 37℃. Cells were lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-acetyl-histone H3 rabbit IgG, anti-acetyl-histone H4 rabbit antiserum and rabbit IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the mouse IL-6 promoter (-84/-9), forward primer IL-6-84F 5'-CCCATGAGTCTCAAAATTAGAGAGTTG-3'), reverse primer IL-6-9R (5'-CAGAGCAGAATGAGCTACAGACATC-3'), and TaqMan probe IL-6-56P (5'-CTCCTAATAAATATGAGACTGGG-3').Results1,Elf-1 expression on mouse BMMC knockdown by siRNA transfection(1) The detection of transfection efficiency of Mouse Macrophage Nucleofector KitThe FITC-labeled control oligonucleotides were introduced into BMMC with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit. Transfection efficiency was confirmed to be approximately 70% by fluorescence microscopy and phase microscopy.(2) The expression of Elf-1 in siRNA transfected BMMCBMMC were transfected with Elf-1 siRNA and Control siRNA respectively.24h later, expression levels of Elf-1 mRNA were determined by real-time PCR. The amount of Elf-1 transcripts in Elf-1 siRNA-treated BMMC was less than one fifth of that in control BMMC.48h later, expression levels of Elf-1 protein were determined by Western blot. The band intensity of Elf-1 protein was markedly reduced by Elf-1 siRNA as compared with control siRNA-treated BMMC. These results indicate that Elf-1 siRNA generated in the present study successfully down regulated Elf-1 expression in BMMC.2,The effects of Elf-1 on the gene expression of FcεRI in mouse BMMC(1) The effects of Elf-1 on the promoter activity of FcεRI a-chain in mouse BMMCTo evaluate the effects of Elf-1 on FcεRI a-chain promoter activity, a reporter plasmid, pGV-B2-αNN0.6, carrying the luciferase gene under the control of the FcεRI a-chain promoter was introduced into BMMC in addition to the above-mentioned siRNA. When Elf-1 siRNA was co-introduced into BMMC with the a-chain reporter plasmid, luciferase activity derived from BMMC was significantly increased in comparison with that of control siRNA transfectants, whereas luciferase activity driven by the promoterless control plasmid, pGL3-Basic, was not markedly affected by Elf-1 siRNA. In addition, overexpression of Elf-1 by co-introduction of the Elf-1 expression plasmid decreased FcεRI a-chain-driven luciferase activity in BMMC in Elf-1 binding sequence-dependent manner. These results suggest that Elf-1 exhibits suppressive effect on the FcεRI a-chain promoter in BMMC.(2) The effects of Elf-1 on FcεRIα-,β-, and y-chain transcription in mouse BMMCAfter transfection with siRNA for 24h, the mRNA expression of FcεRIα-,β-, andγ-chain were analyzed by Real-time PCR. The amount of a-chain mRNA in Elf-1 siRNA-treated BMMC was significantly higher than that of control siRNA-treated BMMC. In contrast, significant differences were not observed inβ-andγ-chain between BMMC transfected with Elf-1 or control siRNA. Based on these results, Elf-1 leads to specific inhibition of FcεRI a-chain transcription.(3) The effects of Elf-1 on recruitment of PU.1 toward FcεRI a-chain promoter in mouse BMMCAfter transfection with siRNA for 24h, BMMC were performed to CHIP assay. Compared with that in control siRNA transfected BMMC, the amount of a-chain promoter region bound with PU.1 significantly increased in Elf-1 siRNA-treated BMMC, suggesting that Elf-1 specifically inhibits recruitment of transactivator PU.1 toward the a-chain promoter in mouse BMMC.(4) The effects of Elf-1 on the surface expression of FcεRI on mouse BMMCAfter transfection with siRNA for 48h, FACS was performed to detect the surface expression of FcεRI on mouse BMMC. Similar expression of FcεRI were shown on Elf-1 siRNA and control siRNA transfected BMMC, indicating that Elf-1 has little effect on the surface expression of FcεRI.3,The effect of TSA on the activation of mouse BMMC(1) The effects of TSA on the degranulation of mouse BMMCMouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. The release ofβ-hexosaminidase was determined. The result indicated that TSA treatment significantly suppressedβ-hexosaminidase release from BMMC activated by IgE/FcεRI cross-linking in a dose-dependent manner, whereas the degranulation ability reached approximately 50% in the absence of TSA.(2) The effects of TSA on the production of IL-6 in mouse BMMCMouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. TSA treatment significantly suppressed the expression of IL-6 mRNA in BMMC determined by Real-time PCR and secretion of IL-6 in the supernatant of BMMC determined by ELISA.(3) The effects of TSA on the surface expression of FcεRI on mouse BMMCMouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h and then performed to FACS to detect the expression of FcεRI. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the reduced expression of FcεRI, whereas,2 and 10nM TSA treatment did not.(4) The effects of TSA on the apoptosis of mouse BMMCMouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 48h and then performed to FACS to detect the apoptosis. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the increased number of apoptotic cells, whereas,2 and 1 OnM TSA treatment did not.(5) The effects of TSA on the histone acetylation of IL-6 promoter of mouse BMMCMouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab for 30min, and then performed to CHIP assay to detect the histone acetlylation of IL-6 promoter. In comparison to the TSA unstimulated BMMC,10 and 50nM TSA treatment led to the increased amount of acetylated H3 and H4 toward to mouse BMMC IL-6 promoter.Conclusions1,Mouse Macrophage Nucleofecter Kit (Amaxa) is suitable for mouse BMMC transfection.2,Elf-1 siRNA can specifically knockdown the expression of Elf-1 in BMMC.3,Elf-1 suppressed the mouse BMMC FcεRI a-chain promoter activity and transcription by inhibition of recruitment of PU.1 towards to a-chain promoter.4,Elf-1 inhibited the mRNA expression of FcεRI a-chain, but not that ofβ-and y-chain.5,Elf-1 alone had little effect on the surface expression of FcεRI on mouse BMMC.6,TSA suppressed FcεRI-mediated activation of mouse BMMC in a dose-dependent mode.7,50nM TSA treatment led to FcεRI-mediated degranulation and IL-6 production in BMMC supposedly by induction of apoptosis of BMMC and subsequently reduced expression of FcεRI on mouse BMMC.8,TSA induced the accumulation of acetylated histones on the mouse IL-6 promoter in BMMC activated by IgE/FcεRI cross-linking.