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Gene expression profiling of dexamethasone-treated RBL-2H3 cells: induction of anti-inflammatory molecules
Immunology Letters 98 (2005) 272–279
Gene expression profiling of dexamethasone-treated RBL-2H3 cells: induction of anti-inflammatory molecules Ryosuke Nakamuraa , Haruyo Okunukia , Seiichi Ishidab , Yoshiro Saitoa , Reiko Teshimaa,∗ , Jun-ichi Sawadaa a
b
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 22 October 2004; received in revised form 26 November 2004; accepted 2 December 2004 Available online 5 January 2005
Abstract Glucocorticoids are well known for their anti-inflammatory effect through the regulation of gene expression in many types of immune cells, including mast cells. However, the genes that are involved in suppression of mast cell-mediated inflammation by glucocorticoids have not been fully identified. Therefore, we examined the dexamethasone (Dex)-responsive genes in RBL-2H3 mast cells using a high-density oligonucleotide microarray technique. Gene expression profiling revealed that the antigen-induced up-regulation of pro-inflammatory factors, including monocyte chemoattractant protein-1, was markedly inhibited by 100 nM Dex. On the other hand, Dex treatment itself caused the substantial up-regulation of many genes, including phenylethanolamine-N-methyl transferase (PNMT) and cytokine-inducible SH2-containing protein (CISH), in the mast cells. The expression of these two genes significantly increased 6 h after Dex exposure and lasted for more than 24 h. Considering that PNMT is the rate-determining enzyme in epinephrine synthesis and that CISH is a suppressor of cytokine signaling, these Dex-responsive genes may be potential anti-inflammatory factors. Thus, gene expression profiling suggested that Dex might exert its anti-inflammatory effect through two pathways in mast cells: the suppression and induction of potentially pro- and anti-inflammatory factors, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Mast cells; Dexamethasone; DNA microarray
1. Introduction Glucocorticoids are the major anti-inflammatory agents presently in clinical use. These drugs act not only on lymphocytes, but also on various types of inflammatory cells, including mast cells [1,2]. Glucocorticoids exert their effects through intracellular receptors, which act as potent transcriptional activators of genes that possess glucocorticoid responsive elements (GREs) [1]. By regulating gene expression, glucocorticoids suppress the production of pro-inflammatory proteins, such as cytokines, chemokines and some enzymatic ∗
Corresponding author. Tel.: +81 3 3700 9437; fax: +81 3 3707 6950. E-mail address: [email protected] (R. Teshima).
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.12.009
mediators [3]. A number of studies have focused on the cellular and molecular mechanisms of the anti-inflammatory effects of glucocorticoids. However, the genes that are involved in the anti-inflammatory effects on mast cells remain unidentified. Using a DNA microarray technique, we have identified several genes, including those for cytokines and signal transducers, that were up-regulated in mast cells after stimulation with antigen or an antioxidant (DTBHQ) [4]. In a previous study, we demonstrated that the mRNA expression of monocyte chemoattractant protein (MCP)-1, which is a CC chemokine that activates monocytes to infiltrate inflammatory tissues, was markedly enhanced when RBL-2H3 mast cells were stimulated with IgE and the specific antigen,
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suggesting the importance of this chemokine in mast cell-mediated inflammation. Another previous study by our group showed that antigen-induced MCP-1 up-regulation was completely suppressed by overnight treatment with 100 nM of dexamethasone (Dex) [5]. Although Dex inhibits antigen-induced mast cell activation, its inhibitory mechanism(s) has not been fully examined. In the present study, we profiled a comprehensive expression of Dex-responsive genes in RBL-2H3 mast cells using a DNA microarray technique and identified several genes that might be involved in the inhibitory mechanism(s) of Dex.
2. Materials and methods 2.1. Reagents and cells Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Co. (Tokyo, Japan), and fetal bovine serum was from Sigma (St. Louis, MO). Murine antidinitrophenyl (DNP) monoclonal IgE antibody (IgE-53-569) and dinitrophenylated BSA (DNP7 -BSA) were prepared as described previously [6]. TRIzol reagent and the SuperScript Choice system were purchased from Invitrogen Corp (Carlsbad, CA). TaqMan Gold RT-PCR kit and TaqMan Universal PCR Master Mix were purchased from Applied BioSystems (Foster City, CA). The RBL-2H3 cells were a kind gift of Dr. R.P. Siraganian (National Institute of Dental Research, National Institutes of Health, Bethesda, MD); the cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum at 37 ◦ C under 5% CO2 in a humidified atmosphere, as described previously [6]. Rabbit polyclonal IgG for bovine PNMT (CA-401 bMTrab) was purchased from Protos Biotech (New York, NY), and goat polyclonal IgG for human CIS (N19) (sc-1529) was from Santa Cruz Biotechnology (Santa Cruz, CA). These antibodies were described by the manufacture as being cross-reactive to rat PNMT and rat CISH, respectively. 2.2. Mast cell activation and assay for Ca2+ response, degranulation and MCP-1 release RBL-2H3 cells were pretreated with the indicated concentrations of Dex for 15 h and simultaneously sensitized with 1:800 diluted anti-DNP IgE; after washing twice with DMEM, they were stimulated with 10 g/ml of antigen (DNP7 -BSA) at 37 ◦ C for 30 min (for degranulation) or 3 h (for MCP-1 assay) in the presence or absence of Dex. The intracellular Ca2+ concentration ([Ca2+ ]i ) was measured using Fura-2 AM and a fluorophotometer (RF-5300PC, Shimadzu), as described previously [7]. Degranulation from the RBL-2H3 cells was measured as the activity of hexosaminidase, as described previously [7]. MCP-1 release was measured using a rat MCP-1 immunoassay kit (BioSource International, California), following the manufacturer’s protocol.
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2.3. GeneChip analysis Total RNA from the control or antigen-stimulated RBL2H3 cells (1 × 107 ) cultured in the presence or absence of 100 nM of Dex was prepared using a TRIzol reagent, according to the manufacturer’s instructions (n = 3, respectively). The RNA was processed as described previously [4] and analyzed using GeneChip Rat Genome U34A arrays, a fluidics station, and a scanner (Affymetrix). The 12 RNA samples from the four different conditions were separately hybridized to the arrays. The expression signals (Signal) were automatically calculated using Microarray Suite (MAS, Ver. 5.0; Affymetrix) with scaling to 2500 as a median, and the data were imported into GeneSpring analysis software (Ver. 6.2; Silicon Genetics, Redwood City, CA) for further analysis. All expression signals were normalized across all the genes and all the arrays using the GeneSpring standard normalization algorithm. Among the genes that were significantly up-regulated by antigen stimulation in the absence of Dex, those that were further up- or down-regulated by antigen stimulation in the presence of Dex were selected by MAS and GeneSpring as follows: (1) genes whose Detection was “P (Present)” in at least three out of six experiments (three controls and three antigen-stimulated); (2) genes whose maximum average Signal under the three conditions (control, antigen, or antigen + Dex) was more than 1000; (3) genes those passed statistical analysis (Welch’s t-test, P < 0.05); (4) genes whose average fold change in treated (antigen or antigen + Dex) to non-treated samples was more than three (for up-regulated genes) or less than one-third (for down-regulated genes). Genes that were significantly up- or down-regulated by Dex treatment only were selected using the same criteria. 2.4. Quantitative PCR RBL-2H3 (1 × 107 ) cells were treated with 100 nM of Dex for 0, 1, 3, 6, 12 and 24 h to determine the effects of the Dex treatment time. In some cases, RBL-2H3 cells were treated with the indicated concentrations of Dex and stimulated with or without antigen. The cells were then harvested and the total RNA was prepared using TRIzol reagent. The amounts of PNMT, CISH, and GAPDH mRNA were measured by TaqMan PCR using an ABI PRISMTM 7700 sequence detector (PE Applied BioSystems, Foster City, CA), according to the manufacturer’s instructions. For each experiment, 100 ng of total RNA, 0.3 M of forward primer, 0.3 M of reverse primer, and 0.3 M of fluorescent probe were used. The PCR conditions were as follows: 50 ◦ C for 2 min; 95 ◦ C for 10 min; 40 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 1 min. Primers and probes used for TaqMan analysis were as follows: PNMT forward primer, 5 -GCT TCC GGC AGG CTT TG-3 ; PNMT reverse primer, 5 -GGC CCC GAT GAG AAG GA-3 ; PNMT TaqMan probe, 5 -VIC-TCA TAT CAC GAC GCT GCT GAG GCC-Tamra-3 ; CISH forward primer, 5 -TGA GAA TGA ACC GAA GGT GCT A-3 ;
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CISH reverse primer, 5 -CCG GCA TCT TCT GTA GAT GCT-3 ; and CISH TaqMan probe, 5 -VIC-TTC CAT TAC AGC CAG TGA GGC CCG-Tamra-3 . For GAPDH, we used the TaqMan Rodent GAPDH Control Reagents VIC Probe #4308313. 2.5. Western blotting RBL-2H3 cells (1 × 107 ) were treated with 0–100 nM of Dex at 37 ◦ C for 15 h, and lysed on ice with solubilizing buffer (1% Triton X-100, 25 mM NaF, 2 mM EDTA, 10 mM Na4 P2 O7 , 20 mM Tris–HCl, 1 mM PMSF, 2 mM Na3 VO4 , 10% glycerol, 20 M leupeptin and 4 M pepstatin [pH 7.4]). The cell extracts were treated with Laemmli buffer and boiled for 5 min. The samples were then resolved by 12% polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions [8]. After 1 h of electrophoresis at 100 V, the resolved proteins were transferred to nitrocellulose membrane, and the membrane was probed with 1:1000-diluted rabbit anti-bovine PNMT polyclonal IgG or 1:750-diluted goat anti-human CIS (N-19) polyclonal IgG at room temperature for 2 h. The membranes were washed three times with 0.05% Tween 20-containing PBSs, then stained with HRPconjugated secondary antibodies and immunostain (Konica), following the manufacturer’s instructions.
3. Results 3.1. Effect of Dex on Ca2+ signaling, degranulation, and MCP-1 release When RBL-2H3 mast cells were pretreated with 100 nM of Dex for 15 h, antigen-induced Ca2+ signaling in the cells was significantly repressed, compared to that in control cells (Fig. 1a). The calculated intracellular Ca2+ concentration ([Ca2+ ]i ) 100 s after antigen stimulation was around 120 nM (net increase; 50 nM) and 70 nM (net increase; 10 nM) in the control and Dex-treated mast cells, respectively. The relative % of net increase of [Ca2+ ]i in Dex-treated cells to control cells was about 20%. The baseline level of [Ca2+ ]i in Dex-treated cells was also inhibited, indicating decrease of free cytoplasmic Ca2+ in these cells. We next measured degranulation from the cells, since degranulation is a Ca2+ dependent process [9]. We found that degranulation from the antigen-stimulated RBL-2H3 cells was dramatically inhibited by Dex in a concentration-dependent manner (Fig. 1b). The release of MCP-1 was also inhibited by Dex in a concentration-dependent manner (Fig. 1c). The total enzyme activity of -hexosaminidase was not changed by the Dex treatment, while the total production of MCP-1 was decreased by Dex treatment. The IC50 of Dex for degranulation and MCP-1 release was about 1.1 and 0.24 nM, respectively; therefore, as far as degranulation and MCP-1 release are concerned, 100 nM of Dex was sufficient to inhibit these processes.
Fig. 1. Inhibitory effects of Dex on antigen-induced mast cell activation. (a) Intracellular Ca2+ concentration changes after antigen stimulation in RBL2H3 cells. The grey and black lines represent with and without 100 nM Dex pretreatment, respectively. Representative data from independent 2 or 3 experiments are shown. Antigen-induced degranulation (b) and MCP-1 release (c) from RBL-2H3 cells pretreated with the indicated concentrations of Dex (n = 3). The solid and open symbols represent with and without antigen stimulation, respectively, and the solid and broken lines represent released and total amount, respectively. Left axis in (b), percentages of -hexosaminidase release compared to the total enzyme activity; left axis in (c), MCP-1 concentration in the supernatant (0.5 ml). Right axis in (b), total -hexosaminidase activity at an absorbance of 405 nm; (c), total MCP-1 production.
3.2. Dex-responsive genes in RBL-2H3 cells We treated RBL-2H3 cells with Dex for 18 h without antigen stimulation. The gene expression profiles were analyzed using Affymetrix Rat Genome U34A arrays, as
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Fig. 2. Dex-induced gene expression profile in mast cells. RBL-2H3 cells were treated with 100 nM of Dex for 15 h, and significantly up- or down-regulated genes were selected using GeneSpring, as described in Section 2. The genes and experimental conditions are clustered based on the similarities of their normalized expression levels using GeneSpring, and the expression levels are indicated as pseudocolors. Annotations such as the biological function and cellular localization of these genes can be obtained at the Affymetrix website (http://www.affymetrix.com/analysis/index.affx).
described previously [4]. As shown in Fig. 2, 24 genes were significantly up-regulated and four genes were downregulated in the Dex-treated mast cells. The markedly up-regulated genes included phenylethanolamine-N-methyl transferase (PNMT), cytokine-inducible SH2-containing protein (CISH). Among many other genes, relaxin hormone (preprorelaxin) and PKA (cAMP dependent-protein kinase) regulatory subunit type II were also up-regulated. PNMT is the rate-determining enzyme of epinephrine synthesis [10], and CISH, also known as CIS, is a suppressor of cytokine signaling (SOCS) family [11]. On the other hand, Dex significantly down-regulated four genes, including MCP-1 and cyclooxygenase-2 (COX-2).
Several of the genes that were down-regulated in the presence of Dex were pro-inflammatory genes, like MCP-1 and COX-2. Protein tyrosine kinases (PYK2, calcium-dependent tyrosine kinase) and certain receptors (oxidized low density lipoprotein receptor-1, epidermal membrane protein-1) were also down-regulated by Dex. On the other hand, preprorelaxin, CISH and several other known genes were included among the seven genes that were up-regulated by the antigen in both the presence and absence of Dex. Two expressed sequence tags (ESTs) were also found to be up-regulated, one of which (Probe ID: rc AI639338 at) was a rat orthologue of src-like adaptor protein (SLAP) mRNA, as revealed by RT-PCR (data not shown).
3.3. Effect of Dex on antigen-inducible genes
3.4. Dex-induced mRNA and protein expression of PNMT and CISH
RBL-2H3 cells were treated with or without 100 nM Dex for 18 h and with antigen for the last 3 h. As a result, 15 genes were significantly up-regulated after antigen stimulation. Among them, the expression of eight genes were inhibited and that of the other seven were further increased by the presence of Dex (Fig. 3).
We next performed quantitative PCR measurements and Western blot analyses for PNMT and CISH. The total RNA of RBL-2H3 cells was prepared after the same treatments as those used in the GeneChip experiments, and the mRNA expression levels of PNMT and CISH were measured by
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Fig. 3. Effect of Dex on antigen-induced gene expression profiles in mast cells. RBL-2H3 cells were stimulated with antigen for 3 h in the presence or absence of 100 nM of Dex, and significantly up- or down-regulated genes were selected as described in Section 2.
quantitative PCR (Figs. 4 and 5). As expected, the mRNA expression levels of PNMT and CISH, compared to that of the GAPDH house-keeping gene, were increased markedly according to the Dex concentration, especially at 100 nM (Figs. 4a and 5a). The mRNA expression of CISH, but not of PNMT, was also induced by antigen stimulation (Figs. 4d and 5d). Antigen stimulation caused a decrease in the Dex-induced expression of PNMT (Fig. 4d), while CISH expression was not affected (Fig. 5d). Both PNMT and CISH expression were dramatically up-regulated 6 h after treatment with 100 nM of Dex (Figs. 4b and 5b). To determine whether PNMT and CISH were expressed as proteins, we performed a Western blot analysis on these proteins and found that both PNMT (MW = 32 kDa) and CISH (MW = 36 kDa) protein expression were induced by Dex treatment in a dose-dependent manner, as shown in Figs. 4c and 5c.
4. Discussion In this study, we have performed gene expression profiling in RBL-2H3 mast cells exposed to Dex and/or a specific antigen. As a result, we revealed two aspects of the anti-inflammatory effects of Dex on mast cells. First, Dex suppresses the induction of pro-inflammatory factors like MCP-1 and COX-2, which are induced by antigen stimulation. As described previously, MCP-1 (also known as CCL2) is one of the most strongly up-regulated genes in antigenstimulated RBL-2H3 cells [4]. MCP-1 is a well-known factor involved in the infiltration of monocytes and macrophages to inflammatory tissues [12]. As shown in Fig. 1c, Dex completely inhibited MCP-1 synthesis and release in antigenstimulated RBL-2H3 cells in a dose-dependent fashion. Other pro-inflammatory cytokine genes, like interleukin (IL)-3 and IL-4, were also up-regulated by the antigen and completely
suppressed by Dex (data not shown). Moreover, Dex also suppressed the expression of a lipid mediator synthetic enzyme, COX-2. This enzyme produces prostaglandins and it has been a well-known target of steroid/non-steroid anti-inflammatory drugs [13]. These results indicate that antigen stimulation strongly induces the expression of such pro-inflammatory factors and that this induction can be suppressed by Dex treatment. Dex also suppressed the degranulation of mast cells, but not the total amount of -hexosaminidase (Fig. 1b). Our preliminary experiments revealed that the amounts of histamine and -hexosaminidase in the granules were not affected (data not shown). The reason for the Dex-induced suppression of degranulation was unclear; however, an upstream signaling molecule(s) may be involved, since the antigen stimuliinduced Ca2+ response was suppressed by Dex (Fig. 1a). In the present study using GeneChip technology, we found that the expression signal of the high-affinity IgE receptor ␣ chain was suppressed from 20,599 ± 1234 to 8088 ± 1441 when RBL-2H3 cells were treated with 100 nM Dex. A previous report has shown that the surface expression of IgE receptors on murine mast cells is affected by Dex treatment, but that mRNA expression is not [14]. This discrepancy may have been caused by the differences between mice and rats. The second aspect of the effect of Dex on mast cells that was examined in this study was the up-regulation of potentially anti-inflammatory factors. Dex treatment induced the expression of 24 genes, including relaxin, CISH, and PNMT (Fig. 3). Relaxin is an insulin-like hormone produced mainly in corpus luteum during pregnancy and is known to inhibit degranulation from mast cells [15]. As revealed by Genomatix promotor analysis software (Genomatix, Germany), the rat genome (GenBank ID: NW 047565) has potential glucocorticoid responsive elements (GREs) upstream and downstream of the relaxin sequence (−1313, −1180, −672 and +73 bp); therefore, the up-regulation of relaxin might be explained
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Fig. 4. mRNA and protein expression of PNMT. (a) mRNA expression of PNMT induced by exposure to the indicated concentrations of Dex for 15 h. (b) mRNA expression of PNMT induced by 100 nM of Dex for the indicated time periods. (c) Protein expression of PNMT induced by exposure to the indicated concentrations of Dex for 15 h. (d) mRNA expression of PNMT induced by 100 nM of Dex and/or antigen stimulation.
by the interaction of the glucocorticoid receptor (GR) and GREs. Very recently, Dschietzig et al. reported that relaxin binds directly to GR and activates its transcription, resulting in the suppression of endotoxin-induced production of inflammatory cytokines, like IL-1 and TNF-␣ [16]. Thus, the
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Fig. 5. mRNA and protein expression of CISH. (a) mRNA expression of CISH induced by exposure to the indicated concentrations of Dex for 15 h. (b) mRNA expression of CISH induced by 100 nM of Dex for the indicated time periods. (c) Protein expression of CISH induced by exposure to the indicated concentrations of Dex for 15 h. (d) mRNA expression of CISH induced by 100 nM of Dex and/or antigen stimulation.
up-regulation of relaxin may have a physiological role in the negative regulation of mast cells. We also observed the up-regulation of a chemokine receptor, CXCR4 (previously known as LCR1) (Fig. 2).
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Although CXCR4 is known to be up-regulated by Dex treatment in eosinophils [17], this is the first report to describe Dex-induced CXCR4 expression in mast cells. The physiological ligand of CXCR4, stromal cell-derived factor-1 (SDF-1), is preferentially expressed in normal tissues, rather than inflammatory tissues; therefore, the up-regulation of its receptor on eosinophils and mast cells is thought to attenuate the infiltration of these cells to inflammatory tissues. In the present study, Dex treatment caused PNMT and CISH to be up-regulated (Figs. 4 and 5). PNMT has already been reported as a glucocorticoid-inducible gene in some tissues and cell lines, such as adrenal, heart, and PC12 cells [18]; however, this is the first report of the up-regulation of PNMT in mast cells. Functional GREs have been reported upstream of rat PNMT [19], and the present results are consistent with these findings. PNMT is the rate-determining enzyme of epinephrine biosynthesis, and epinephrine analogues are known to have an inhibitory effect on IgE-mediated histamine release from human lung mast cells [20]. Furthermore, epinephrine negatively affects T and B lymphocytes [21]; therefore, PNMT is considered to be a potential negative regulator of inflammation. Although the relationship between PNMT up-regulation and the inhibitory effect of Dex on IgE-mediated mast cell activation in the present study is unclear, it seems possible that the secretion of epinephrine could affect neighboring lymphocytes. CISH is induced through cytokine-activated STAT family protein and protects STAT from successive cytokine signal transduction by interfering with the binding of JAK proteins to tyrosil-phosphorylated cytokine receptors [11]. Thus, CISH is considered to be a potential anti-inflammatory factor. We analyzed the rat genome upstream of CISH (NW 047801), but no potential GREs were found. A previous study demonstrated that CISH expression was enhanced by STAT5-mediated IL-2 signaling in murine IL-2 dependent CTLL-2 cells and that Dex synergistically enhanced the expression of CISH via IL-2 signaling but did not induce CISH by itself [22]. In the present study, however, CISH expression was dramatically enhanced 6 h after Dex treatment in RBL-2H3 mast cells, as shown in Fig. 4c. Considering that glucocorticoid receptors (GRs) bind directly to STAT5 and enhance its transcriptional activity [23], GRs may induce the transcription of CISH by interacting with STAT5. In our GeneChip results, the Detection of STAT5a1 (Probe ID: U24175 at) was “Present” in the control RBL-2H3 cells (data not shown). Therefore, this discrepancy might be due to differences in the cell types. Regardless, the present report is the first description of the up-regulation of CISH expression by Dex in mast cells. In summary, (1) we performed gene expression profiling in Dex- and/or antigen-treated RBL-2H3 mast cells and found that Dex itself up-regulated 24 genes, including PNMT and CISH, and down-regulated 4 genes, including MCP-1 and COX-2; (2) 15 genes were up-regulated by antigen; (3) among these genes, the expression of eight genes, including MCP-1 and COX-2, were suppressed, while the remaining
seven genes, including CISH and relaxin, were further upregulated by Dex; (4) Dex may exert its anti-inflammatory effect through two pathways; the suppression of potentially pro-inflammatory factors and the induction of potentially anti-inflammatory factors.
Acknowledgements This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ6) of the Pharmaceuticals and Medical Devices Agency (PMDA) and by a grant-in-aid for Scientific Research (16790074) from the Ministry of Education, Culture, Sports, Science and Technology.
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Gene expression profiling of dexamethasone-treated RBL-2H3 cells: induction of anti-inflammatory molecules Ryosuke Nakamuraa , Haruyo Okunukia , Seiichi Ishidab , Yoshiro Saitoa , Reiko Teshimaa,∗ , Jun-ichi Sawadaa a
b
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 22 October 2004; received in revised form 26 November 2004; accepted 2 December 2004 Available online 5 January 2005
Abstract Glucocorticoids are well known for their anti-inflammatory effect through the regulation of gene expression in many types of immune cells, including mast cells. However, the genes that are involved in suppression of mast cell-mediated inflammation by glucocorticoids have not been fully identified. Therefore, we examined the dexamethasone (Dex)-responsive genes in RBL-2H3 mast cells using a high-density oligonucleotide microarray technique. Gene expression profiling revealed that the antigen-induced up-regulation of pro-inflammatory factors, including monocyte chemoattractant protein-1, was markedly inhibited by 100 nM Dex. On the other hand, Dex treatment itself caused the substantial up-regulation of many genes, including phenylethanolamine-N-methyl transferase (PNMT) and cytokine-inducible SH2-containing protein (CISH), in the mast cells. The expression of these two genes significantly increased 6 h after Dex exposure and lasted for more than 24 h. Considering that PNMT is the rate-determining enzyme in epinephrine synthesis and that CISH is a suppressor of cytokine signaling, these Dex-responsive genes may be potential anti-inflammatory factors. Thus, gene expression profiling suggested that Dex might exert its anti-inflammatory effect through two pathways in mast cells: the suppression and induction of potentially pro- and anti-inflammatory factors, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Mast cells; Dexamethasone; DNA microarray
1. Introduction Glucocorticoids are the major anti-inflammatory agents presently in clinical use. These drugs act not only on lymphocytes, but also on various types of inflammatory cells, including mast cells [1,2]. Glucocorticoids exert their effects through intracellular receptors, which act as potent transcriptional activators of genes that possess glucocorticoid responsive elements (GREs) [1]. By regulating gene expression, glucocorticoids suppress the production of pro-inflammatory proteins, such as cytokines, chemokines and some enzymatic ∗
Corresponding author. Tel.: +81 3 3700 9437; fax: +81 3 3707 6950. E-mail address: [email protected] (R. Teshima).
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.12.009
mediators [3]. A number of studies have focused on the cellular and molecular mechanisms of the anti-inflammatory effects of glucocorticoids. However, the genes that are involved in the anti-inflammatory effects on mast cells remain unidentified. Using a DNA microarray technique, we have identified several genes, including those for cytokines and signal transducers, that were up-regulated in mast cells after stimulation with antigen or an antioxidant (DTBHQ) [4]. In a previous study, we demonstrated that the mRNA expression of monocyte chemoattractant protein (MCP)-1, which is a CC chemokine that activates monocytes to infiltrate inflammatory tissues, was markedly enhanced when RBL-2H3 mast cells were stimulated with IgE and the specific antigen,
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suggesting the importance of this chemokine in mast cell-mediated inflammation. Another previous study by our group showed that antigen-induced MCP-1 up-regulation was completely suppressed by overnight treatment with 100 nM of dexamethasone (Dex) [5]. Although Dex inhibits antigen-induced mast cell activation, its inhibitory mechanism(s) has not been fully examined. In the present study, we profiled a comprehensive expression of Dex-responsive genes in RBL-2H3 mast cells using a DNA microarray technique and identified several genes that might be involved in the inhibitory mechanism(s) of Dex.
2. Materials and methods 2.1. Reagents and cells Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Co. (Tokyo, Japan), and fetal bovine serum was from Sigma (St. Louis, MO). Murine antidinitrophenyl (DNP) monoclonal IgE antibody (IgE-53-569) and dinitrophenylated BSA (DNP7 -BSA) were prepared as described previously [6]. TRIzol reagent and the SuperScript Choice system were purchased from Invitrogen Corp (Carlsbad, CA). TaqMan Gold RT-PCR kit and TaqMan Universal PCR Master Mix were purchased from Applied BioSystems (Foster City, CA). The RBL-2H3 cells were a kind gift of Dr. R.P. Siraganian (National Institute of Dental Research, National Institutes of Health, Bethesda, MD); the cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum at 37 ◦ C under 5% CO2 in a humidified atmosphere, as described previously [6]. Rabbit polyclonal IgG for bovine PNMT (CA-401 bMTrab) was purchased from Protos Biotech (New York, NY), and goat polyclonal IgG for human CIS (N19) (sc-1529) was from Santa Cruz Biotechnology (Santa Cruz, CA). These antibodies were described by the manufacture as being cross-reactive to rat PNMT and rat CISH, respectively. 2.2. Mast cell activation and assay for Ca2+ response, degranulation and MCP-1 release RBL-2H3 cells were pretreated with the indicated concentrations of Dex for 15 h and simultaneously sensitized with 1:800 diluted anti-DNP IgE; after washing twice with DMEM, they were stimulated with 10 g/ml of antigen (DNP7 -BSA) at 37 ◦ C for 30 min (for degranulation) or 3 h (for MCP-1 assay) in the presence or absence of Dex. The intracellular Ca2+ concentration ([Ca2+ ]i ) was measured using Fura-2 AM and a fluorophotometer (RF-5300PC, Shimadzu), as described previously [7]. Degranulation from the RBL-2H3 cells was measured as the activity of hexosaminidase, as described previously [7]. MCP-1 release was measured using a rat MCP-1 immunoassay kit (BioSource International, California), following the manufacturer’s protocol.
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2.3. GeneChip analysis Total RNA from the control or antigen-stimulated RBL2H3 cells (1 × 107 ) cultured in the presence or absence of 100 nM of Dex was prepared using a TRIzol reagent, according to the manufacturer’s instructions (n = 3, respectively). The RNA was processed as described previously [4] and analyzed using GeneChip Rat Genome U34A arrays, a fluidics station, and a scanner (Affymetrix). The 12 RNA samples from the four different conditions were separately hybridized to the arrays. The expression signals (Signal) were automatically calculated using Microarray Suite (MAS, Ver. 5.0; Affymetrix) with scaling to 2500 as a median, and the data were imported into GeneSpring analysis software (Ver. 6.2; Silicon Genetics, Redwood City, CA) for further analysis. All expression signals were normalized across all the genes and all the arrays using the GeneSpring standard normalization algorithm. Among the genes that were significantly up-regulated by antigen stimulation in the absence of Dex, those that were further up- or down-regulated by antigen stimulation in the presence of Dex were selected by MAS and GeneSpring as follows: (1) genes whose Detection was “P (Present)” in at least three out of six experiments (three controls and three antigen-stimulated); (2) genes whose maximum average Signal under the three conditions (control, antigen, or antigen + Dex) was more than 1000; (3) genes those passed statistical analysis (Welch’s t-test, P < 0.05); (4) genes whose average fold change in treated (antigen or antigen + Dex) to non-treated samples was more than three (for up-regulated genes) or less than one-third (for down-regulated genes). Genes that were significantly up- or down-regulated by Dex treatment only were selected using the same criteria. 2.4. Quantitative PCR RBL-2H3 (1 × 107 ) cells were treated with 100 nM of Dex for 0, 1, 3, 6, 12 and 24 h to determine the effects of the Dex treatment time. In some cases, RBL-2H3 cells were treated with the indicated concentrations of Dex and stimulated with or without antigen. The cells were then harvested and the total RNA was prepared using TRIzol reagent. The amounts of PNMT, CISH, and GAPDH mRNA were measured by TaqMan PCR using an ABI PRISMTM 7700 sequence detector (PE Applied BioSystems, Foster City, CA), according to the manufacturer’s instructions. For each experiment, 100 ng of total RNA, 0.3 M of forward primer, 0.3 M of reverse primer, and 0.3 M of fluorescent probe were used. The PCR conditions were as follows: 50 ◦ C for 2 min; 95 ◦ C for 10 min; 40 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 1 min. Primers and probes used for TaqMan analysis were as follows: PNMT forward primer, 5 -GCT TCC GGC AGG CTT TG-3 ; PNMT reverse primer, 5 -GGC CCC GAT GAG AAG GA-3 ; PNMT TaqMan probe, 5 -VIC-TCA TAT CAC GAC GCT GCT GAG GCC-Tamra-3 ; CISH forward primer, 5 -TGA GAA TGA ACC GAA GGT GCT A-3 ;
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CISH reverse primer, 5 -CCG GCA TCT TCT GTA GAT GCT-3 ; and CISH TaqMan probe, 5 -VIC-TTC CAT TAC AGC CAG TGA GGC CCG-Tamra-3 . For GAPDH, we used the TaqMan Rodent GAPDH Control Reagents VIC Probe #4308313. 2.5. Western blotting RBL-2H3 cells (1 × 107 ) were treated with 0–100 nM of Dex at 37 ◦ C for 15 h, and lysed on ice with solubilizing buffer (1% Triton X-100, 25 mM NaF, 2 mM EDTA, 10 mM Na4 P2 O7 , 20 mM Tris–HCl, 1 mM PMSF, 2 mM Na3 VO4 , 10% glycerol, 20 M leupeptin and 4 M pepstatin [pH 7.4]). The cell extracts were treated with Laemmli buffer and boiled for 5 min. The samples were then resolved by 12% polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions [8]. After 1 h of electrophoresis at 100 V, the resolved proteins were transferred to nitrocellulose membrane, and the membrane was probed with 1:1000-diluted rabbit anti-bovine PNMT polyclonal IgG or 1:750-diluted goat anti-human CIS (N-19) polyclonal IgG at room temperature for 2 h. The membranes were washed three times with 0.05% Tween 20-containing PBSs, then stained with HRPconjugated secondary antibodies and immunostain (Konica), following the manufacturer’s instructions.
3. Results 3.1. Effect of Dex on Ca2+ signaling, degranulation, and MCP-1 release When RBL-2H3 mast cells were pretreated with 100 nM of Dex for 15 h, antigen-induced Ca2+ signaling in the cells was significantly repressed, compared to that in control cells (Fig. 1a). The calculated intracellular Ca2+ concentration ([Ca2+ ]i ) 100 s after antigen stimulation was around 120 nM (net increase; 50 nM) and 70 nM (net increase; 10 nM) in the control and Dex-treated mast cells, respectively. The relative % of net increase of [Ca2+ ]i in Dex-treated cells to control cells was about 20%. The baseline level of [Ca2+ ]i in Dex-treated cells was also inhibited, indicating decrease of free cytoplasmic Ca2+ in these cells. We next measured degranulation from the cells, since degranulation is a Ca2+ dependent process [9]. We found that degranulation from the antigen-stimulated RBL-2H3 cells was dramatically inhibited by Dex in a concentration-dependent manner (Fig. 1b). The release of MCP-1 was also inhibited by Dex in a concentration-dependent manner (Fig. 1c). The total enzyme activity of -hexosaminidase was not changed by the Dex treatment, while the total production of MCP-1 was decreased by Dex treatment. The IC50 of Dex for degranulation and MCP-1 release was about 1.1 and 0.24 nM, respectively; therefore, as far as degranulation and MCP-1 release are concerned, 100 nM of Dex was sufficient to inhibit these processes.
Fig. 1. Inhibitory effects of Dex on antigen-induced mast cell activation. (a) Intracellular Ca2+ concentration changes after antigen stimulation in RBL2H3 cells. The grey and black lines represent with and without 100 nM Dex pretreatment, respectively. Representative data from independent 2 or 3 experiments are shown. Antigen-induced degranulation (b) and MCP-1 release (c) from RBL-2H3 cells pretreated with the indicated concentrations of Dex (n = 3). The solid and open symbols represent with and without antigen stimulation, respectively, and the solid and broken lines represent released and total amount, respectively. Left axis in (b), percentages of -hexosaminidase release compared to the total enzyme activity; left axis in (c), MCP-1 concentration in the supernatant (0.5 ml). Right axis in (b), total -hexosaminidase activity at an absorbance of 405 nm; (c), total MCP-1 production.
3.2. Dex-responsive genes in RBL-2H3 cells We treated RBL-2H3 cells with Dex for 18 h without antigen stimulation. The gene expression profiles were analyzed using Affymetrix Rat Genome U34A arrays, as
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Fig. 2. Dex-induced gene expression profile in mast cells. RBL-2H3 cells were treated with 100 nM of Dex for 15 h, and significantly up- or down-regulated genes were selected using GeneSpring, as described in Section 2. The genes and experimental conditions are clustered based on the similarities of their normalized expression levels using GeneSpring, and the expression levels are indicated as pseudocolors. Annotations such as the biological function and cellular localization of these genes can be obtained at the Affymetrix website (http://www.affymetrix.com/analysis/index.affx).
described previously [4]. As shown in Fig. 2, 24 genes were significantly up-regulated and four genes were downregulated in the Dex-treated mast cells. The markedly up-regulated genes included phenylethanolamine-N-methyl transferase (PNMT), cytokine-inducible SH2-containing protein (CISH). Among many other genes, relaxin hormone (preprorelaxin) and PKA (cAMP dependent-protein kinase) regulatory subunit type II were also up-regulated. PNMT is the rate-determining enzyme of epinephrine synthesis [10], and CISH, also known as CIS, is a suppressor of cytokine signaling (SOCS) family [11]. On the other hand, Dex significantly down-regulated four genes, including MCP-1 and cyclooxygenase-2 (COX-2).
Several of the genes that were down-regulated in the presence of Dex were pro-inflammatory genes, like MCP-1 and COX-2. Protein tyrosine kinases (PYK2, calcium-dependent tyrosine kinase) and certain receptors (oxidized low density lipoprotein receptor-1, epidermal membrane protein-1) were also down-regulated by Dex. On the other hand, preprorelaxin, CISH and several other known genes were included among the seven genes that were up-regulated by the antigen in both the presence and absence of Dex. Two expressed sequence tags (ESTs) were also found to be up-regulated, one of which (Probe ID: rc AI639338 at) was a rat orthologue of src-like adaptor protein (SLAP) mRNA, as revealed by RT-PCR (data not shown).
3.3. Effect of Dex on antigen-inducible genes
3.4. Dex-induced mRNA and protein expression of PNMT and CISH
RBL-2H3 cells were treated with or without 100 nM Dex for 18 h and with antigen for the last 3 h. As a result, 15 genes were significantly up-regulated after antigen stimulation. Among them, the expression of eight genes were inhibited and that of the other seven were further increased by the presence of Dex (Fig. 3).
We next performed quantitative PCR measurements and Western blot analyses for PNMT and CISH. The total RNA of RBL-2H3 cells was prepared after the same treatments as those used in the GeneChip experiments, and the mRNA expression levels of PNMT and CISH were measured by
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Fig. 3. Effect of Dex on antigen-induced gene expression profiles in mast cells. RBL-2H3 cells were stimulated with antigen for 3 h in the presence or absence of 100 nM of Dex, and significantly up- or down-regulated genes were selected as described in Section 2.
quantitative PCR (Figs. 4 and 5). As expected, the mRNA expression levels of PNMT and CISH, compared to that of the GAPDH house-keeping gene, were increased markedly according to the Dex concentration, especially at 100 nM (Figs. 4a and 5a). The mRNA expression of CISH, but not of PNMT, was also induced by antigen stimulation (Figs. 4d and 5d). Antigen stimulation caused a decrease in the Dex-induced expression of PNMT (Fig. 4d), while CISH expression was not affected (Fig. 5d). Both PNMT and CISH expression were dramatically up-regulated 6 h after treatment with 100 nM of Dex (Figs. 4b and 5b). To determine whether PNMT and CISH were expressed as proteins, we performed a Western blot analysis on these proteins and found that both PNMT (MW = 32 kDa) and CISH (MW = 36 kDa) protein expression were induced by Dex treatment in a dose-dependent manner, as shown in Figs. 4c and 5c.
4. Discussion In this study, we have performed gene expression profiling in RBL-2H3 mast cells exposed to Dex and/or a specific antigen. As a result, we revealed two aspects of the anti-inflammatory effects of Dex on mast cells. First, Dex suppresses the induction of pro-inflammatory factors like MCP-1 and COX-2, which are induced by antigen stimulation. As described previously, MCP-1 (also known as CCL2) is one of the most strongly up-regulated genes in antigenstimulated RBL-2H3 cells [4]. MCP-1 is a well-known factor involved in the infiltration of monocytes and macrophages to inflammatory tissues [12]. As shown in Fig. 1c, Dex completely inhibited MCP-1 synthesis and release in antigenstimulated RBL-2H3 cells in a dose-dependent fashion. Other pro-inflammatory cytokine genes, like interleukin (IL)-3 and IL-4, were also up-regulated by the antigen and completely
suppressed by Dex (data not shown). Moreover, Dex also suppressed the expression of a lipid mediator synthetic enzyme, COX-2. This enzyme produces prostaglandins and it has been a well-known target of steroid/non-steroid anti-inflammatory drugs [13]. These results indicate that antigen stimulation strongly induces the expression of such pro-inflammatory factors and that this induction can be suppressed by Dex treatment. Dex also suppressed the degranulation of mast cells, but not the total amount of -hexosaminidase (Fig. 1b). Our preliminary experiments revealed that the amounts of histamine and -hexosaminidase in the granules were not affected (data not shown). The reason for the Dex-induced suppression of degranulation was unclear; however, an upstream signaling molecule(s) may be involved, since the antigen stimuliinduced Ca2+ response was suppressed by Dex (Fig. 1a). In the present study using GeneChip technology, we found that the expression signal of the high-affinity IgE receptor ␣ chain was suppressed from 20,599 ± 1234 to 8088 ± 1441 when RBL-2H3 cells were treated with 100 nM Dex. A previous report has shown that the surface expression of IgE receptors on murine mast cells is affected by Dex treatment, but that mRNA expression is not [14]. This discrepancy may have been caused by the differences between mice and rats. The second aspect of the effect of Dex on mast cells that was examined in this study was the up-regulation of potentially anti-inflammatory factors. Dex treatment induced the expression of 24 genes, including relaxin, CISH, and PNMT (Fig. 3). Relaxin is an insulin-like hormone produced mainly in corpus luteum during pregnancy and is known to inhibit degranulation from mast cells [15]. As revealed by Genomatix promotor analysis software (Genomatix, Germany), the rat genome (GenBank ID: NW 047565) has potential glucocorticoid responsive elements (GREs) upstream and downstream of the relaxin sequence (−1313, −1180, −672 and +73 bp); therefore, the up-regulation of relaxin might be explained
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Fig. 4. mRNA and protein expression of PNMT. (a) mRNA expression of PNMT induced by exposure to the indicated concentrations of Dex for 15 h. (b) mRNA expression of PNMT induced by 100 nM of Dex for the indicated time periods. (c) Protein expression of PNMT induced by exposure to the indicated concentrations of Dex for 15 h. (d) mRNA expression of PNMT induced by 100 nM of Dex and/or antigen stimulation.
by the interaction of the glucocorticoid receptor (GR) and GREs. Very recently, Dschietzig et al. reported that relaxin binds directly to GR and activates its transcription, resulting in the suppression of endotoxin-induced production of inflammatory cytokines, like IL-1 and TNF-␣ [16]. Thus, the
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Fig. 5. mRNA and protein expression of CISH. (a) mRNA expression of CISH induced by exposure to the indicated concentrations of Dex for 15 h. (b) mRNA expression of CISH induced by 100 nM of Dex for the indicated time periods. (c) Protein expression of CISH induced by exposure to the indicated concentrations of Dex for 15 h. (d) mRNA expression of CISH induced by 100 nM of Dex and/or antigen stimulation.
up-regulation of relaxin may have a physiological role in the negative regulation of mast cells. We also observed the up-regulation of a chemokine receptor, CXCR4 (previously known as LCR1) (Fig. 2).
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Although CXCR4 is known to be up-regulated by Dex treatment in eosinophils [17], this is the first report to describe Dex-induced CXCR4 expression in mast cells. The physiological ligand of CXCR4, stromal cell-derived factor-1 (SDF-1), is preferentially expressed in normal tissues, rather than inflammatory tissues; therefore, the up-regulation of its receptor on eosinophils and mast cells is thought to attenuate the infiltration of these cells to inflammatory tissues. In the present study, Dex treatment caused PNMT and CISH to be up-regulated (Figs. 4 and 5). PNMT has already been reported as a glucocorticoid-inducible gene in some tissues and cell lines, such as adrenal, heart, and PC12 cells [18]; however, this is the first report of the up-regulation of PNMT in mast cells. Functional GREs have been reported upstream of rat PNMT [19], and the present results are consistent with these findings. PNMT is the rate-determining enzyme of epinephrine biosynthesis, and epinephrine analogues are known to have an inhibitory effect on IgE-mediated histamine release from human lung mast cells [20]. Furthermore, epinephrine negatively affects T and B lymphocytes [21]; therefore, PNMT is considered to be a potential negative regulator of inflammation. Although the relationship between PNMT up-regulation and the inhibitory effect of Dex on IgE-mediated mast cell activation in the present study is unclear, it seems possible that the secretion of epinephrine could affect neighboring lymphocytes. CISH is induced through cytokine-activated STAT family protein and protects STAT from successive cytokine signal transduction by interfering with the binding of JAK proteins to tyrosil-phosphorylated cytokine receptors [11]. Thus, CISH is considered to be a potential anti-inflammatory factor. We analyzed the rat genome upstream of CISH (NW 047801), but no potential GREs were found. A previous study demonstrated that CISH expression was enhanced by STAT5-mediated IL-2 signaling in murine IL-2 dependent CTLL-2 cells and that Dex synergistically enhanced the expression of CISH via IL-2 signaling but did not induce CISH by itself [22]. In the present study, however, CISH expression was dramatically enhanced 6 h after Dex treatment in RBL-2H3 mast cells, as shown in Fig. 4c. Considering that glucocorticoid receptors (GRs) bind directly to STAT5 and enhance its transcriptional activity [23], GRs may induce the transcription of CISH by interacting with STAT5. In our GeneChip results, the Detection of STAT5a1 (Probe ID: U24175 at) was “Present” in the control RBL-2H3 cells (data not shown). Therefore, this discrepancy might be due to differences in the cell types. Regardless, the present report is the first description of the up-regulation of CISH expression by Dex in mast cells. In summary, (1) we performed gene expression profiling in Dex- and/or antigen-treated RBL-2H3 mast cells and found that Dex itself up-regulated 24 genes, including PNMT and CISH, and down-regulated 4 genes, including MCP-1 and COX-2; (2) 15 genes were up-regulated by antigen; (3) among these genes, the expression of eight genes, including MCP-1 and COX-2, were suppressed, while the remaining
seven genes, including CISH and relaxin, were further upregulated by Dex; (4) Dex may exert its anti-inflammatory effect through two pathways; the suppression of potentially pro-inflammatory factors and the induction of potentially anti-inflammatory factors.
Acknowledgements This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ6) of the Pharmaceuticals and Medical Devices Agency (PMDA) and by a grant-in-aid for Scientific Research (16790074) from the Ministry of Education, Culture, Sports, Science and Technology.
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