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Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb(3)), globotriaosylsphingosine (lyso-Gb(3)), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb(3) isoforms (various fatty acids) and lyso-Gb(3) analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb(3) and lyso-Gb(3) levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb(3) and particular lyso-Gb(3) analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb(3) and lyso-Gb(3) isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb(3) massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb(3) accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb(3) or lyso-Gb(3) may play major roles in the pathogenesis of Fabry disease, and that Gb(3) and lyso-Gb(3) are not responsible for the pathology in all tissues or cell types.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

OBJECTIVE B cells play an important role in the pathogenesis of autoimmune diseases. The role of Bruton's tyrosine kinase (Btk) in cytokine-induced human B cell differentiation and class-switch recombination remains incompletely defined. This study analysed the effect of Btk on human activated B cells. METHODS Purified B cells from healthy subjects were stimulated with B cell receptor (BCR) and other stimuli with or without a Btk inhibitor and gene expression was measured. The B cell line BJAB was used to assess Btk-associated signalling cascades. Phosphorylated Btk (p-Btk) in peripheral blood B cells obtained from 10 healthy subjects and 41 patients with RA was measured by flow cytometry and compared with patient backgrounds. RESULTS IL-21 signalling, in concert with BCR, CD40 and BAFF signals, led to robust expression of differentiation- and class-switch DNA recombination-related genes and IgG production in human B cells, all of which were significantly suppressed by the Btk inhibitor. Although phosphorylation of STAT1 and STAT3 was induced by co-stimulation with IL-21, BCR and CD40, STAT1 phosphorylation in the nucleus, but not in the cytoplasm, was exclusively impaired by Btk blockade. High levels of p-Btk were noted in B cells of RA patients compared with controls and they correlated significantly with titres of RF among RF-positive patients. CONCLUSION The findings elucidate a model in which Btk not only plays a fundamental role in the regulation of BCR signalling, but may also mediate crosstalk with cytokine signalling pathways through regulation of IL-21-induced phosphorylation of STAT1 in the nuclei of human B cells. Btk appears to have pathological relevance in RA.

Additional file 2: Fig. S1. Representative images of hematoxylin–eosin–stained sections of the sensory epithelium from chicken utricles. (A) Epithelium from a control (saline-injected) chicken. (B) Epithelium from a gentamicin sulfate-injected chicken on day 14 postinjection. Extruded hair cells (arrowheads) and phagocytosis (arrows) are indicated. Fig. S2. Representative images of BrdU incorporation in the utricle as analyzed by immunohistochemistry in control chickens (A) and aminoglycoside-treated chickens on days 2 (B), 7 (C), and 14 (D) after the last gentamicin injection. Fig. S3 Relative expression of the ATOH1 gene. Increased ATOH1 mRNA levels were detected in supporting cells 3 h after aminoglycoside injection and continued for the next 14 days (*p

Knockout mouse model of Fabry disease (Ohshima, 1997) is widely used, and has been proven an invaluable tool to investigate disease pathogenesis and therapies. However, the importance of genetic background of this mouse line has been underestimated, and in many publications WT control mice were chosen incorrectly. Our aim was to examine the effects of genetic background on biochemical and functional abnormalities of Fabry mice. We characterized Fabry mice in B6/129 mixed background (~75% B6) and Fabry mice on a purely C57BL/6 strain (10 generations backcrossing), compared with WT mice on an identical genetic background. Both Fabry-B6/129 and Fabry-B6 developed progressive cardiac and renal hypertrophy and impaired thermosensation. However, Fabry-B6/129 exhibited earlier onset and more severe phenotypes than Fabry-B6. Cardiac and/or renal hypertrophy appeared at age of 6 months in Fabry-B6/129, but at 12 months in Fabry-B6. At 12 months, kidney-to-body weight ratio increased by 50% in Fabry-B6/129 (vs. WT), but only 15% in Fabry-B6. The onset of the thermosensation abnormality was at age 2 months in Fabry-B6/129 and 7 months in Fabry-B6. Cardiac expression of hypertrophy marker, atrial natriuretic peptide (ANP), was upregulated in Fabry-B6/129, but not in Fabry-B6. Despite these differences, globotriaosylceramide (Gb3) and lyso-Gb3 levels in the heart, kidney and dorsal root ganglia were similar between Fabry-B6/129 and Fabry-B6. However, Gb3 level in liver (organ unaffected in Fabry disease) was twofold higher in Fabry-B6/129 compared to Fabry-B6. Additionally, several parameters including body weight, organ weight, kidney Gb3 and cardiac ANP level were different between WT-B6/129 and WT-B6. In conclusion: 1) genetic background has a significant impact on the severity and onset of phenotypes in Fabry mice 2) the lack of correlation between phenotype and Gb3/lyso-Gb3 levels in disease-relevant organs suggests that the effect of genetic background on the phenotype is determined by other genetic and epigenetic factors.

Activation of the transforming growth factor (TGF) α/epidermal growth factor receptor (EGFR)-mediated signaling pathway is a common mechanism for dysregulated growth of head and neck squamous cell carcinoma (HNSCC). c-Cbl-interacting protein of 85 kDa (CIN85) is an adaptor protein that facilitates EGFR internalization. Little is known, however, about a role of CIN85 in EGFR signaling as well as its relevance to tumor development and progression of HNSCC. Here, we demonstrate that CIN85 is highly expressed in HNSCC tumor samples compared with adjacent normal tissues, and this overexpression is significantly correlated with advanced clinical stage. The experiments using CIN85-overexpressing and knockdown HNSCC cell lines showed that CIN85 promotes HNSCC growth and facilitates EGFR internalization without apparently affecting phosphorylation of EGFR. Moreover, CIN85 promoted TGF-α-induced activation of Ras and phosphorylation of downstream molecules such as c-Raf, MEK, and extracellular signal-regulated kinase, leading to expression of c-Myc that is critical for sustained proliferation of HNSCC. Taken together, these findings suggest that CIN85 not only controls EGFR internalization but also promotes the EGFR-mediated tumor development and progression, and thus, CIN85 may serve as a potential therapeutic target in a subset of HNSCC.

Clinical characterization of patients with RA included in the study. (DOCX 18 kb)

Analysis of osteoclast differentiation using RAW-Venus1. (A) Differentiation into Venus positive pre-osteoclasts and osteoclasts were induced by RANKL in a dose-dependent manner. TNF-α exerted synergistic effects on RANKL-induced osteoclast differentiation (magnification × 20). (B) Both anti-RANKL Ab and OPG strongly reduced the number of Venus positive cells, indicated the inhibition of osteoclast differentiation (magnification × 20). (C) Venus positive cells were tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (magnification × 40). Cells were stained using a commercial TRAP assay (Sigma-Aldrich, St Louis, MO, USA). (A-C) Cells were cultured at 4.5 × 104 cells/ml for 3 days in a 96-well plate. No stim No stimulation. (PDF 304 kb)

Phenotypic analysis of RANKL+ and RANKL− effector memory B cells. Purified switched-memory B cells from HC were stimulated with BCR/CD40 and IFN-γ for 48 hours. RANKL+ and RANKL− cells were analyzed for expression of CD20, CD21, CD95, CD11c, CCR1 and CCR5 using respective Abs (all from BioLegend). Representative data are shown (n = 3–4). (PDF 177 kb)

Objective Activation of B cells is a hallmark of systemic lupus erythematosus (SLE). Syk and TRAF6 are key signaling molecules in B-cell activation through BCR and CD40/TLR, respectively. Nevertheless, whether expression of Syk and TRAF6 is altered in SLE B cells remains unknown. Methods Phosphorylation and/or expression of Syk and TRAF6 were analyzed by flow cytometry in peripheral blood mononuclear cells isolated from SLE patients. Results Pronounced phosphorylation and expression of Syk were noted in B cells from SLE patients compared with healthy donors. Levels of Syk phosphorylation correlated with the disease activity score. TRAF6 was significantly over-expressed in B cells of SLE patients as compared with healthy donors, and significant correlation of levels of TRAF6 expression and Syk phosphorylation was observed in SLE patients. Levels of TRAF6 expression were more pronounced in CD27+ memory B cells than in CD27-naïve B cells. In vitro treatment of SLE B cells with a Syk inhibitor (BAY61-3606) reduced Syk phosphorylation as well as TRAF6 expression. Conclusion Our results suggest that the activated Syk-mediated TRAF6 pathway leads to aberrant activation of B cells in SLE, and also highlight Syk as a potential target for B-cell-mediated processes in SLE.

By the current study, we tried to find out the interactive mechanisms enrolled by Hsp70 and Hsp90 following the 6-hydroxydopamine (6-OHDA)-induced oxidative stress. Of heat shock protein (Hsp) family, we have previously evaluated the effects of Hsp90 gene silencing on in vitro model of Parkinson's disease and its influence on controlling the mechanisms of cell survival. Here, we extended our study to Hsp70 silencing short interfering RNA (siRNA) oligonucleotides, transfected into Pheochromocytoma (PC12) cells with/without exposure to 6-OHDA stress. In order to determine the probable effects of Hsp70 silencing on apoptotic factors, we assessed Bcl2/Bax ratio, nuclear level of PARP, and cleavage of caspase-3 under 6-OHDA stress condition. The results showed deteriorated effect of Hsp70 siRNA on apoptosis in cells exposed to only 6-OHDA. This is, at least in part, in consequence of upregulation of Hsp90, both at messenger RNA (mRNA) and protein levels. These data highlight the critical role of Hsp70 for cell survival under 6-OHDA stress condition. It could be a suggestive issue for supervision of caspase cascades by survival roles of Hsps as Hsp70 silencing resulted in apoptosis phenomenon. Convergence of Hsp70 anti-apoptotic and 6-OHDA pro-apoptotic pathways may explain intensified apoptosis following Hsp70 silencing. In addition, nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor, has been previously studied in detoxification of oxidative stress. For this issue, we tried to elucidate Hsp70 silencing impact on Nrf2, which has been shown to regulate the transcription of Hsp70, unspecifically. Besides, our investigations revealed that Hsp70 siRNA did not affect the level of Nrf2 during 6-OHDA exposure. But, it is still a dealing question and other investigations are needed to have a comprehensive perception of Hsp family signaling functions.

The aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

Myeloid cell leukemia-1 (MCL-1) is an essential survival factor for hematopoiesis. In humans, hematopoietic stem cells (HSCs) express MCL-1 at the highest level in response to FMS-like tyrosine kinase-3 (FLT3) signaling. We here show that this FLT3-dependent stem cell maintenance system also plays a critical role in survival of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). The CD34+CD38− LSC fraction expresses high levels of FLT3 as well as MCL-1, even compared with normal HSCs. Treatment with FLT3 ligand induced further MCL-1 up-regulation in LSCs in all AML cases tested. Interestingly, the group of samples expressing the highest levels of MCL-1 constituted AML with FLT3–internal tandem duplications (ITD). In FLT3-ITD AML cell lines, cells expressed a high level of MCL-1, and an inhibition of MCL-1 induced their apoptotic cell death. A tyrosine kinase inhibitor suppressed MCL-1 expression, and induced apoptosis that was reversed by the enforced MCL-1 expression. Finally, transduction of FLT3-ITD into HSCs strongly activated MCL-1 expression through its signal transducer and activator of transcription 5 (STAT5)–docking domains. This effect was completely abrogated when STAT5 activation was blocked. Thus, the acquisition of FLT3-ITD ensures LSC survival by up-regulating MCL-1 via constitutive STAT5 activation that is independent of wild-type FLT3 signaling.

BCR signaling plays a critical role in purging the self-reactive repertoire, or in rendering it anergic to establish self-tolerance in the periphery. Differences in self-reactivity between human naive and IgM+ memory B cells may reflect distinct mechanisms by which BCR signaling dictates their survival and death. Here we demonstrate that BCR stimulation protected naive B cells from apoptosis with induction of prosurvival Bcl-2 family proteins, Bcl-xL and Mcl-1, whereas it rather accelerated apoptosis of IgM+ memory B cells by inducing proapoptotic BH3-only protein Bim. We found that BCR-mediated PI3K activation induced the expression of Mcl-1, whereas it inhibited Bim expression in B cells. Phosphorylation of Akt, a downstream molecule of PI3K, was more sustained in naive than IgM+ memory B cells. Abundant expression of T cell leukemia/lymphoma 1 (Tcl1), an Akt coactivator, was found in naive B cells, and enforced expression of Tcl1 induced a high level of Mcl-1 expression, resulting in prolonged B cell survival. In contrast, Galectin-1 (Gal-1) was abundantly expressed in IgM+ memory B cells, and inhibited Akt phosphorylation, leading to Bim up-regulation. Enforced expression of Gal-1 induced accelerated apoptosis in B cells. These results suggest that a unique set of molecules, Tcl1 and Gal-1, defines distinct BCR signaling cascades, dictating survival and death of human naive and IgM+ memory B cells.

Hirofumi Tsuzuki, Yojiro Arinobu, Kohta Miyawaki, Ayako Takaki, Shun-ichiro Ota, Naoko Ueki, Yuri Ota, Siamak Jabbarzadeh Tabrizi, Mitsuteru Akahoshi, Hiroaki Niiro, Hiroshi Tsukamoto, Takahiko Horiuchi, Shoichiro Ohta, Kenji Izuhara, MD, PhD, Hiroyuki Fukui, Koichi Akashi; Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka City, Japan, Clinical Education Center, Kyushu University Hospital, Fukuoka City, Japan, Department of Rheumatology, Internal medicine and connective tissue disorders, Shimonoseki City Hospital, Shimonoseki City, Japan, Division of Nephrology and Rheumatology, Fukuoka University Hospital, Fukuoka City, Japan, Department of Internal Medicine, Kyushu University Beppu Hospital, Beppu City, Japan, Division of Medical Biochemistry, Department of Biomolecular Sciences, Saga Medical School, Saga City, Japan, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima City, Japan. RATIONALE: A dried root of Sophora flavescens (S. flavescens) is a traditional Chinese herbal medicine used for the treatment of allergic diseases. Previous studies have demonstrated that S. flavescens possesses antiinflammatory activities and suppresses allergic inflammation such as allergic rhinitis. However, the mechanism of anti-asthmatic effect of S. flavescens is not yet clarified. METHODS: To clarify the effect of S. flavescens on asthma, we analyzed OVA-induced asthma mice treated with S. flavescens extract. We also conducted flow cytometry, cell count assay and gene expression analysis to evaluate the effect of S. flavescens on eosinophil hematopoiesis. To reveal the effect of S. flavescens on eosinophil chemotaxis, we performed transwell migration assay. RESULTS: Lung eosinophilic inflammation inOVA-induced asthmamice was suppressed by the treatment of S. flavescens. In addition, S. flavescens inhibited allergen-induced increment of eosinophil progenitors (EoPs) in the bonemarrow. In vitro, S. flavescens suppressed IL-5-induced expansion of EoPs and eosinophil lineage specification directly. Moreover, IL-5 expression was suppressed in CD4 T cells isolated from asthmatic mice treatedwith Sophora flavescens. In terms of eosinophil migration, S. flavescens suppressed eosinophil chemotaxis for eotaxin-1, one of themajor chemokines for eosinophil migration. CONCLUSIONS: S. flavescens suppressed eosinophil accumulation in the lung of asthmatic mice by inhibiting both eosinophil hematopoiesis and migration. S. flavescens might be valuable for the treatment of eosinophil-related diseases such as asthma.

Background B cells play a pivotal role in pathological processes of autoimmune diseases. Btk, which mediates BCR signaling, is considered as a suitable target for the treatment of autoimmune diseases. However, the underlying mechanisms of B cell activation mediated by Btk remain elusive. Objectives The aim of this study is to investigate the mechanism underlying human B cell activation and to assess the role of Btk in this process. Methods Purified B cells from healthy donors were stimulated with BCR, sCD40L/BAFF, and sIL-21 with/without a Btk inhibitor. B cell line, BJAB was utilized to assess the molecular mechanism. Results The expression of AICDA, encoding a RNA-editing deaminase and involved in class-switch recombination of Ig, was slightly induced by IL-21 stimulation alone in human primary B cells. Likewise, under BCR and/or CD40/BAFF stimulation, very little AICDA expression was induced. However, combinatorial stimulation with BCR, CD40/BAFF and IL-21 induced robust expression of AICDA, BCL6 and XBP1 and IgG production. A Btk inhibitor (ONO-A) significantly abrogated the expression to the extent of IL-21 alone in a dose-dependent manner. To assess the mechanism by which Btk regulates the genes induced by IL-21 in human B cells, we set up the Btk knock-down (KD) cell line by using BJAB. The transient exposure of IL-21 induced both p-STAT1 and p-STAT3 in BJAB. However, in Btk KD BJAB, p-STAT1 was not induced in the nucleus, while it was induced in the cytoplasm. Furthermore, IL-21-induced expression of T-bet significantly decreased in Btk-KD cells, compared to mock transferred BJAB, indicating that phosphorylation of STAT-1 by IL-21 affected its target gene T-bet , resulting in robust activation of B cells through Btk. Conclusions Our results suggest that Btk activation induced by BCR and CD40/BAFF amplify IL-21 signaling to induce robust expression of differentiation- and class-switch DNA recombination (CSR)-related genes and IgG production in human B cells. Btk-mediated signal directly regulate p-STAT1 translocation from the cytoplasm to the nucleus, thereby allowing efficient propagation of IL-21-signal critical for human B cell differentiation and CSR. These results underscore the potential role of Btk in B cell-mediated pathological processes in autoimmune diseases. Disclosure of Interest S.-P. Wang: None Declared, S. Iwata: None Declared, K. Yamaoka: None Declared, H. Niiro: None Declared, S. Nakayamada: None Declared, S. Tabrizi: None Declared, S. Kubo: None Declared, M. Kondo: None Declared, K. Akashi: None Declared, Y. Tanaka Grant/research support from: Bristol-Myers Squibb, MSD K.K., Chugai Pharma Co., Ltd., Mitsubishi-Tanabe Pharma Co., Ltd., Astellas Pharma Inc., Abbott Japan Co., Ltd., Eisai Co., Ltd. and Janssen Pharmaceutical K.K., Speakers bureau: Mitsubishi-Tanabe Pharma Co., Ltd., Abbott Japan Co., Ltd., Eisai Co., Ltd., Chugai Pharma Co., Ltd., Janssen Pharma K.K., Santen Pharma Co., Ltd., Pfizer Japan Inc., Astellas Pharma Inc., Daiichi-Sankyo Co., Ltd., GlaxoSmithKline K.K., Astra-Zeneca, Otsuka Pharma Co., Ltd., Actelion Pharma Japan Ltd., Eli Lilly Japan K.K., Nippon Kayaku Co., Ltd., UCB Japan Co., Ltd., Quintiles Transnational Japan Co. Ltd., Ono Pharma Co., Ltd.

Upon B-cell receptor (BCR) ligation by autoantigens, B cells normally undergo deletion (apoptosis), inactivation (anergy), or receptor editing. These processes are finely tuned by positive and negative signals downstream of B cell receptor (BCR). Aberrant regulation of BCR signals may thus lead to activation and expansion of autoreactive B cells. B cell adaptors function as molecular conduits to effectively link upstream signals to specific downstream branches. Cbl is an E3 ubiquitin ligase that is highly expressed in self-tolerant lymphocytes. Notably, B cell-specific ablation of Cbl in mice manifests SLE-like autoimmune disease, suggesting a pivotal role of Cbl in BCR-mediated tolerance induction. A novel Cbl-interacting adaptor termed CIN85 (c-Cbl interacting protein of 85 kDa) was recently identified, however little is known about a role of CIN85 in B cell function. In this study we demonstrate that upon BCR stimulation, CIN85 strongly associated with Cbl as well as BLNK. This association required the SH3 domain of CIN85. The experiments using CIN85-overexpressing and CIN85 knockdown B cells showed that CIN85 is critical for Cbl phosphorylation, which in turn regulates BCR-induced calcium flux and activation of Syk and PLCγ2. CIN85 knockdown in human primary B cells resulted in their enhanced survival and proliferation by upregulating expression of bcl-2 family and cyclin genes following BCR stimulation. Together, these findings suggest a critical role of CIN85 in Cbl-mediated regulation of B cell function after BCR stimulation.

Background The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T–B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL+ effector B cells and their impacts on osteoclast differentiation. Methods Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. Results RANKL expression was accentuated in CD80+CD86+ B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3+RANKL+ effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL+ effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. Conclusions Our current findings have shed light on the generation mechanism of pathogenic RANKL+ effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0957-6) contains supplementary material, which is available to authorized users.