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RECEPTORS AND SIGNAL TRANSDUCTION
Department of Biomolecular Science and Orthopedics, Fukushima Medical University School of Medicine, Fukushima, Japan
Submitted 22 July 2007 ; accepted in final form 15 October 2007
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ABSTRACT |
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. EphrinB1/Fc also activated synovial cells established from patients with RA to produce IL-6. Tyrosine phosphorylation of EphB1 was induced in these cells by ephrinB1/Fc. The CpG islands in the 5' upstream regulatory region of the ephrinB1 gene were hypomethylated in RA patients compared with those of normal donors. These results suggest that ephrinB1 and EphB1 receptors play an important role in the inflammatory states of RA, especially by affecting the population and function of T cells. Inhibition of the ephrinB/EphB system might be a novel target for the treatment of RA. synovial cells; T cells; cytokine; DNA methylation
Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors, consisting of A and B types, are the largest family of transmembrane receptor tyrosine kinases (19, 23). Eph receptor-interacting proteins (ephrins) are the ligands to Eph receptors. The ephrin ligands also have two types: the glycosylphosphatidylinositol (GPI)-anchored A type and transmembrane B type. The representative functions of the ephrin/Eph signaling pathway are the development of immune networks including T cell development and migration (26). Recent reports have suggested that ephrinB1 signaling is essential in T cell-T cell interactions during T cell activation, whereas ephrinA1 provokes T cell migration (1, 27). In addition, it has been reported that the ephrin/Eph receptor system plays important signaling roles in inflammation processes and the pathogenesis of RA (16, 25). Based on these findings, we attempted to examine ephrinB molecules in T cells and synovial cells derived from RA in this study. Here, we demonstrate that the expression level of ephrinB1 is significantly higher in synovial fibroblasts and exudate lymphocytes in patients with RA compared with those in osteoarthritis (OA). mRNA and protein levels of ephrinB1 are also higher in peripheral blood lymphocytes (PBLs) prepared from patients with RA than those from normal controls. Furthermore, a recombinant ephrinB1/Fc fusion protein stimulates normal PBLs to exhibit enhanced migration and production of TNF- and also RA synovial cells to produce IL-6. These results suggest that high expression levels of ephrinB1 might be closely associated with the process of RA pathogenesis.
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MATERIALS AND METHODS |
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Histological analysis. Synovial tissues were fixed with 4% paraformaldehyde in PBS for 24 h at 4°C. After being blocked with 3% normal goat serum, 6-µm-thick sections were incubated with rabbit anti-ephrinB1 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight in a moist chamber at 4°C. After being rinsed with PBS, sections were incubated with biotin-labeled goat anti-rabbit IgG followed by avidin-biotin peroxidase complex. Positive signals were developed using a 3,3'-diaminobenzidine substrate kit (Vecstain Elite ABC kit, Vector Laboratories, Burlingame, CA). For immunofluorohistochemistry, sections were incubated with rabbit anti-ephrinB1 and mouse anti-CD3 (Santa Cruz Biotechnology) antibodies overnight at 4°C. After being washed thoroughly, sections were incubated with Alexa fluor 488-labeled anti-rabbit IgG and Alexa fluor 555-labeled anti-mouse IgG antibodies (Invitrogen, Carlsbad, CA) for 1 h.
Analysis of mRNA levels. The level of ephrinB1 mRNA was assessed by RT-PCR. Total RNA was extracted from PBL samples derived from RA patients and normal donors using an ISOGEN kit (Nippon Gene, Tokyo, Japan), and cDNA was prepared from total RNA using the SuperScript III First Strand System (Invitrogen) according to the manufacturer's instructions. Aliquots (0.5 µl) of the resultant mixtures were subjected to PCR amplification with 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 3 min. This condition was chosen so that none of the RNA analyzed reached a plateau at the end of the amplification protocol. PCR products were electrophoresed using 1.5% agarose gels and stained with ethidium bromide, and intensities of the bands were quantified using NIH Image software. The ratio between ephrinB1 and β-actin was calculated to normalize for initial variations in the sample concentration. The primers used for PCR amplifications were as follows: human ephrinB1 sense, 5'-CGTGTTGGTCACCTGCAATAG-3'; human ephrinB1 antisense, 5'-GCTTCCATTGGATGTTGAGGTAA-3'; human β-actin sense, 5'-CATGTACGTTGCTATCCAGGC-3'; and human β-actin antisense, 5'-CTCCTTAATGTCACGCACGAT-3'.
Western blot analysis. Protein fractions were prepared from the PBL samples using an ISOGEN kit (Nippon Gene) according to the manufacturer's instructions. An aliquot (50 µg protein) was subjected to SDS-PAGE and analyzed by Western blot analysis using anti-ephrinB1 antibody (Santa Cruz Biotechnology) and anti-GAPDH antibody (Chemicon, Temecula, CA). A horseradish peroxidase-conjugated antibody was used as the secondary antibody (Bio-Rad Laboratories, Hercules, CA), and positive bands were visualized by an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The intensity of each band was quantified using NIH Image software. For immunoprecipitation, cells were solubilized in a lysis buffer consisting of 20 mM Tris·HCl (pH 7.5), 1% Nonidet P-40, 1 mM EDTA, 50 mM NaF, 50 mM β-glycelophosphate, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. Cell lysates were incubated for 2 h at 4°C with anti-phosphotyrosine antibody 4G10 (Upstate, Lake Placid, NY). Immune complexes were collected with protein G-Sepharose (Zymed, San Francisco, CA). After being washed five times, proteins were solubilized by heating in 30 µl SDS sample buffer and subjected to SDS-PAGE.
Arthritis model. All animal experiments were carried out with the approval of the Fukushima Medical University Review Board and in concordance with Institutional Animal Care and Use Committee regulations. Balb/c female mice were obtained from Charles River Laboratories Japan and housed in a controlled environment with free access to food and water. Collagen antibody-induced arthritis (CAIA) was induced using arthrogen-collagen-induced arthritis antibody kits (Chondrex, Redmond, WA) according to the manufacturer's instructions. In brief, a cocktail of anti-collagen type II monoclonal antibody mix (0.5 mg each) was injected intraperitoneally into 6-wk-old mice on day 0, followed by an intraperitoneal injection of 25 µg LPS on day 3. Mice were killed for analyses by exsanguinations under anesthesia with pentobarbital at day 10.
Bioassays for ephrinB1.
For the preparation of the ephrinB1/Fc fusion protein, the extracellular domain of ephrinB1 was subcloned into a human immunoglobulin Fc fusion protein expression vector (kindly provided by Drs. Takashi Saito and Arata Takeuchi) and transfected into COS7 cells. The protein was purified from the culture supernatant using protein A-agarose (Upstate). PBL migration was determined in 24-well plates with 5-µm-pore size cell culture inserts (Corning, Corning, NY). PBLs from normal donors suspended in RPMI-1640 at 2.5 x 105 cells/ml were treated with various amounts of ephrinB1/Fc or control Fc fragment for 1 h, and 100-µl aliquots of cell suspension was added to the upper inserts; 600-µl aliquots of medium containing stromal cell-derived factor (SDF)-1, a typical chemotactic factor for lymphocytes, were added to the lower chambers. After an incubation for 3 h, the numbers of cells that had passed through the membrane into the lower chamber were quantified by counting five random fields. The production of TNF- or IL-4 from normal PBLs and IL-6 from fibroblast-like synovial cells were evaluated using human ELISA kits (Pierce, Rockford, IL). PBLs from normal donors suspended in RPMI-1640 at 5 x 105 cells/ml or synovial cells from RA patients (18) seeded at 5 x 104 cells/well in 24-well plate were starved for 24 h and then treated with various amounts of ephrinB1/Fc or control Fc fragment for 48 h. Culture supernatants were used for the evaluation.
Bisulfite sequencing analysis. Bisulfite modification of genomic DNA was performed as previously described (24). Briefly, 5 µg genomic DNA suspended in 50 µl water was incubated for 10 min at 95°C and then for 30 min at 37°C with 1.5 µl of 10 M NaOH. The solution was further incubated overnight at 55°C after the addition of 310 µl of 5 M sodium bisulfite, 2.5 µl of 0.1 M hydroquinone, and 136 µl water in a final volume of 500 µl. After modification, DNA was purified using a Wizard clean-up kit (Promega, Madison, WI) and desulfonated with 0.3 M NaOH for 15 min at 37°C. Bisulfite-treated DNA was amplified using the following primers: human ephrinB1 sense (–205 to –181), 5'-GGATTGAGAGGGATTTAATTTTAAT-3'; human ephrinB1 antisense (–25 to –48), 5'-CAAAACCAACCCTACTCTCTTAAC-3'; mouse ephrinB1 sense (–332 to –308) 5'-GTTTGTTTGTTTTAGGTTAGTGGGT-3'; and mouse ephrinB1 antisense (–48 to –69), 5'-ACACAAAACCAACCCTACTCCT-3'. PCR amplification was performed for 35 cycles of 30 s at 95°C, 1 min at 50°C, and 3 min at 72°C, with a final extension at 72°C for 10 min. PCR products were cloned into the pGEM-T-easy vector (Promega), and at least eight clones for each independent sample were sequenced.
Luciferase assay. The luciferase reporter plasmid containing the human ephrinB1 promoter sequence from –1000 to +1 relative to the start site of transcription was generated using pGL3-Basic (Promega). COS7 cells (5 x 104) plated on 24-well plates were transfected with 0.4 µg of reporter construct and 5 pg of pRL-TK vector (Promega) as an internal control using Polyfect Transfection Reagent (QIAGEN, Hilden, Germany). After an incubation for 48 h, relative luciferase activities were determined by a dual-luciferase reporter assay system (Promega). For the preparation of methylated and mock methylated vector, reporter vector (1.2 µg) was incubated with or without 4 units of SssI methylase (New England Biolabs, Ipswich, MA) and 160 µM S-adenocylmethionine for 2 h at 37°C, followed by an incubation for 20 min at 65°C to inactivate the methylase.
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RESULTS |
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added to the lower chamber, whereas control Fc had no effect on the migration of PBLs (Fig. 3A). EphrinB1 itself possessed no chemotactic activity against PBLs, because PBLs in the upper chamber did not respond to ephrinB1/Fc added to the lower chamber. This activity of ephrinB1 was attenuated by genistein, a selective inhibitor of protein tyrosine kinases. It has been reported that EphB1 is a receptor of ephrinB1, so that tyrosine phosphorylation of EphB1 was analyzed using normal PBLs. As shown in Fig. 3C, tyrosine phosphorylation was enhanced by ephrinB1/Fc, and genistein inhibited this phosphorylation. In addition, we also detected a 60-kDa band of EphB1, which was produced upon stimulation with ephrinB1. These results are consisitent with recent findings (6) suggesting that the ephrinB1/EphB1 signaling system might be involved in the stimulation of the lymphocytes.
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in a dose-dependent manner. Production levels in ephrinB1/Fc-stimulated PBLs were significantly higher than those in Fc-treated PBLs. The stimulation of the TNF- production was suppressed by genistein. A moderate stimulation was observed in PBLs treated with Fc at high concentrations, although the underlying mechanism was unclear. On the other hand, no stimulation in the production of IL-4 was observed by ephrinB1/Fc at any dose examined (data not shown). It is well known that the activation of T helper cell type 1 (Th1), rather than T helper cell type 2 (Th2), is predominant in patients with RA. Since TNF- is typical of Th1-type T cells, and IL-4 of Th2-type T cells, the ephrinB1/EphB1 system should play an important role in the activation of Th1-type T cells. We also investigated the effect of ephrinB1 on the production of cytokines in synovial cells. As shown in Fig. 4A, ephrinB1/Fc, but not Fc, stimulated the production of IL-6 in synovial cells derived from RA patients in a dose-dependent manner. The production of IL-6 was significantly suppressed by genistein, suggesting an important role of receptor tyrosine kinase EphB1 for the production of IL-6. Indeed, EphB1 of synovial cells was phosphorylated at tyrosine residues by ephrinB1/Fc, and genistein inhibited the phosphorylation of EphB1 (Fig. 4B). These results suggest that ephrinB1 expressed on the T cell surface might be involved in the activation of synovial cells via EphB1 stimulation, which per se induces an expansion of inflammation in RA.
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DISCUSSION |
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-dependent migration of PBLs in a dose-dependent manner (Fig. 3A). Furthermore, the production of TNF- in PBLs and that of IL-6 in RA synovial cells were also stimulated by ephrinB1/Fc (Fig. 4, A and B). These biological effects, namely, enhanced migration and cytokine production, were suppressed by genistein. EphB1 receptor on PBLs and RA synovial cells was actually phosphorylated by ephrinB1/Fc, and this phosphorylation was inhibited by genistein (Figs. 3B and 4B). Since EphB1 is highly expressed on some T cells and synovial cells in RA synovial tissues (data not shown), it is conceivable that ephrinB1 expressed on T cells derived from patients with RA interacts with EphB1 on neighboring T cells or other types of cells, such as synovial cells. Tight adhesion and arrest are important steps in the targeting and transmigration of leukocytes, and surface densities of ephrinB1 correlated closely with EphB1-coupled cell attachment (15, 22). T cell-T cell or T cell-synovial cell interactions might be early and critical events in acute and chronic inflammation.
RA is the most common form of inflammatory arthritis and is characterized by a disordered synovial microenvironment in which there is hyperplasia of resident stromal cells and a heavy infiltration of hematopoietic cells (5). The inflammatory process is usually tightly regulated, involving both mediators that initiate and maintain inflammation and mediators that shut the process down. In the states of chronic inflammation, an imbalance between the two mediators leaves inflammation unchecked, resulting in cellular damage and synovial tissue destruction. Thus, ephrinB1/EphB signaling might be involved in the imbalance of immune regulation and the aberrant accumulation of inflammatory lymphocytes in synovial tissues of patients with RA (25). In this context, it is quite interesting that the ephrinB1/Fc fusion protein stimulated the production of TNF-, but not IL-4, in PBLs derived from normal donors (Fig. 4A). The TNF- production seemed to be totally dependent on ephrinB1. TNF- is a cytokine that plays a central role in the pathogenesis of RA, and anti-TNF- antibodies strongly suppress inflammatory reactions in RA joints. Taken together, it is possible that ephrinB1-EphB interactions occur in RA synovial tissues, resulting in situ in activating Th1-type T cells to exhibit enhanced production of inflammatory cytokines such as TNF-.
It is noteworthy that DNA hypomethylation is involved in the high expression of ephrinB1 in PBLs derived from patients with RA. Both human (Fig. 5A) and mouse (Suppl. Fig. A) ephrinB1 genes possess a CpG island 200
400 bp upstream of the transcription start site.1 The results clearly demonstrate that the methylation status of the CpG island of ephrinB1 promoters was lower in RA patients (Fig. 5, B and C) and CAIA mice (Suppl. Figs. B and C) than in normal controls. DNA methylation of this promoter region affects transcription activity (Fig. 5D). Indeed, high expression levels of ephrinB1 were observed in PBL samples derived from RA patients (Fig. 2, A and B), and ephrinB1-positive CD3 cells were abundant in the synovial tissues of RA patients (Fig. 1B). These changes were detected in all PBL and tissue samples from patients with RA examined and were significant compared with samples derived from normal controls and patients with OA. In addition, similar results were also obtained using an experimental RA model, CAIA mice. These findings suggest that methylation states are closely associated with expression levels of Th1-type T cells.
There are several binding sites in this CpG region for methyl-CpG-sensitive transcription factors such as activator protein-2, STAT, c-Myb, and Ets. Methylation of CpG in binding sites for these factors prevents transcription activity (4, 8). A cluster of Sp1-binding sites is also located in this region. Sp1 family members act as positive regulators of TATA-less promoters (3). The Sp1 protein can bind methylated CpG sites but not activate transcription when the sites are covered with methyl-CpG-binding proteins (20). Thus, methylation of the promoter region of the ephrinB1 gene affects the expression level of ephrinB1, which might be associated with controlling the mechanism of inflammation and, per se, an imbalance in T cell function. The mechanism underlying the hypomethylation of the ephrinB1 gene in RA or CAIA lymphocytes is not clearly understood. Our results reveal that the stimulation of PBLs prepared from normal donors with ephrinB1/Fc did not affect the methylation status (unpublished data). On the other hand, it has been reported that some ephrins in rats are induced in circulating lymphocytes by the administration of LPS (17). Thus, it is quite natural to hypothesize that some environmental factors, such as LPS, induce an ephrinB1-positive subset of T cells, which frequently interact with EphB receptor-positive stroma or Th1-type T cells. This interaction activates EphB receptor-expressing cells to produce excess amounts of inflammatory cytokines. Further careful studies are required to identify ephrinB1-positive T cells and to gain an understanding of their function in the pathogenesis of RA.
In conclusion, ephrinB1-positive T cells are abundant in synovial tissues of patients with RA, and high expression levels of ephrinB1 were observed in blood lymphocytes derived from RA patients, probably due to hypomethylation in the promoter region of the ephrinB1 gene. EphrinB1 is involved in enhancing the migration and production of TNF- of lymphocytes and production of IL-6 of synovial cells. These results suggest that ephrinB1-positive T cells play an important role in the pathogenesis of RA. Further studies on the function and regulation of ephrinB1 may lead to an understanding of the etiology of RA and to the development of effective therapies for RA.
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ACKNOWLEDGMENTS |
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Present address of A. Kamataki: Dept. of Pathology, Iwate Medical Univ. School of Medicine, Iwate, Japan.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental material for this article is available online at the American Journal of Physiology-Cell Physiology website. 
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, Methylated C; 
, nonmethylated C. C: summary of the methylation status at each CpG site in normal and RA subjects. Statistical significance of the differences between normal and RA groups was determined by 
2-test (**P < 0.01). D: effect of methylation of the ephrinB1 promoter on promoter activity. The luciferase construct was incubated in the presence or absence of SssI methylase and transfected into COS7 cells. After an incubation for 48 h, relative luciferase activities were determined in triplicate by a dual-luciferase reporter assay system (means and SD). Statistical significance of the differences between methylated and control groups was calculated by Student's t-test (**P < 0.01).