Masato Matsushita

BLNK mediates Syk-dependent Btk activation.

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cγ2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.

Four Tyrosine Residues in Phospholipase C-γ2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling

Activation of phospholipase C-γ2 (PLCγ2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Brutons tyrosine kinase (Btk) and Syk are required for activating PLCγ2, the exact activation mechanism of PLCγ2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCγ2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCγ2, PLCγ2-deficient DT40 cells were reconstituted with a series of mutant PLCγ2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCγ2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCγ2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCγ2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCγ2.

A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Brutons tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

Transfer of rheumatoid arthritis into severe combined immunodeficient mice. The pathogenetic implications of T cell populations oligoclonally expanding in the rheumatoid joints.

To investigate the pathogenicity of T cells infiltrating in the rheumatoid joints, mononuclear cells (MNC), predominantly T cells, isolated from either synovial fluid or synovial tissues of the patients with RA were transferred into severe combined immunodeficient (SCID) mice by intraarticular injections. According to our observations in this experimental system, patients with RA could be classified into at least two groups. In one group of patients, the infiltrating MNC induced synovial hyperplasia in the recipient SCID mice (the positive group). Whereas, in the other group no synovial hyperplasia was observed (the negative group). The induction of synovial hyperplasia observed in the positive group was prevented by an anti-human CD3 antibody (OKT3), indicating T cell mediation. Analysis of T cell receptor (TCR) V beta usage by reverse transcriptase polymerase chain reaction in the infiltrating MNC transferred into SCID mice revealed a marked skew towards the preferential use of certain V beta genes, which was not seen in the peripheral blood MNC, in only the positive group. The patterns of TCR/V beta skew were not uniform among the patients. The analysis of the PCR-amplified genes of such skewed TCR/ V beta by single strand conformational polymorphism showed distinct bands, indicating that the T cell populations expanding in rheumatoid joints of the positive group were oligoclonal. Furthermore, the enrichment of the T cell populations expressing such skewed TCR/V beta by in vitro stimulation of peripheral blood MNC of the patients with the relevant superantigen enabled the induction of synovial hyperplasia in the SCID mice. These results suggest that the pathogenic T cells could be activated locally in rheumatoid joints by certain antigens in some, but not in all patients with RA.

Dysbiosis contributes to arthritis development via activation of autoreactive T cells in the intestine.

The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis.

A case of Mikulicz’s disease with Th2-biased cytokine profile: possible feature discriminable from Sjögren’s syndrome

This article concerns a male patient with Mikulicz’s disease (MD) accompanied with marked elevation of serum immunoglobulin (Ig)G4 and IgE levels. His peripheral blood mononuclear cells (PBMC) showed markedly enhanced in vitro production of interleukin (IL)-4, IL-5, IL-13, but not interferon gamma (IFN-γ) compared with patients with Sjögren’s syndrome (SS) and healthy donors, suggesting distinct Th2 bias in this MD patient. Besides the prominent infiltration of IgG4-producing plasma cells, the enhanced expression of both CD40 and CD40 ligand (CD40L) were observed in the swollen salivary gland of the MD patient, suggesting enhanced signaling pathways for the induction of IgG4 and IgE switching. Possible differences between MD and SS in light of their underlying pathogenesis are discussed.

Hind-mid-forefoot Deformity in Hallux Valgus Deformity in Rheumatoid Arthritis: Radiographic Evaluation Grouped by Existence of Dorsal Dislocation of Second Metatarsophalangeal Joint

Hallux valgus deformity combined with dorsal dislocation of the second metatarsophalangeal joint is frequently observed in rheumatoid arthritis cases. However, hallux valgus deformity without lesser toe dislocation is also seen in rheumatoid cases. Dislocated second toe cause the loss of the lateral support on the hallux, suggesting the importance to confirm the state of lesser toe MTP joint when assessing the risk of HV recurrence after surgery, and there may be some differences in the mechanical transmission between hind-mid and forefoot based on whether dorsal dislocation of the MTP joint in the lesser toe is present, although findings are unclear. This study examined the relationship between radiographic findings from the hind, mid, and forefoot and hallux valgus angle in rheumatoid arthritis cases grouped based on the presence or absence of dorsal dislocation of the second metatarsophalangeal joint. X-rays of 160 feet and ankles with rheumatoid arthritis were evaluated for the first metatarsophalangeal Larsen grade, existence of second metatarsophalangeal dorsal dislocation, hallux valgus angle, intermetatarsal angle between the first and second intermetatarsals, shape of the first metatarsal head, position of the sesamoid, the metatarsus primus varus angle, diastasis between the base of the first and second metatarsals, angle between long axis of the talus and short axis of the navicular, internal arch angle, tibio-calcaneal angle, and calcaneal lateral offset. Based on Pearson product-moment correlation coefficient test, involvement of hindfoot deformity should always be considered when assessing hallux valgus deformity in rheumatoid arthritis patients. Although mechanism of mechanical transmission through hindfoot to Lisfranc joint seems to be different by the presence or absence of dorsal dislocation of the second metatarsophalangeal joint, Lisfranc looseness also must be considered when assessing hallux valgus including the surgery to avoid the progression or recurrence in rheumatoid arthritis cases. Dorsal dislocation of the second metatarsophalangeal joint strongly influences the exacerbation of hallux valgus in rheumatoid arthritis cases. Thus, it is may be important to achieve adequate reduction of the second metatarsophalangeal joint dislocation and make a stable metatarsophalangeal joint to avoid recurrence of hallux valgus after forefoot surgery in rheumatoid arthritis.

Early therapeutic intervention with methotrexate prevents the development of rheumatoid arthritis in patients with recent-onset undifferentiated arthritis: A prospective cohort study

Objectives. To examine whether or not earlier therapeutic intervention with methotrexate (MTX) prevents the development of rheumatoid arthritis (RA) in patients with recent-onset undifferentiated arthritis (UA) showing high anti-citrullinated peptide antibody (ACPA) titers. Methods. The patients were divided into two groups, one was treated with MTX (MTX+ group, n = 29), and the other was treated without MTX (MTX− group, n = 19), and other disease-modifying anti-rheumatic drugs were not permitted in the two groups before the primary endpoint was met. The primary endpoint is the occurrence of definite RA, and it was compared in the two groups after 1 year. Results. The percentage of patients who developed definite RA in the MTX+ group (17.2%) was significantly lower than that in the MTX− group (78.9%) (log-rank test, P < 0.001, n = 48); adjusted hazards ratio: 0.028 [95% confidence interval (CI): 0.003–0.250, P = 0.001, n = 39]. Treatment effectiveness was not decreased by major risk factors of RA onset such as smoking habits and human leukocyte antigen-DRB1 shared epitope (SE) (smoking habit, odds ratio [OR]: 0.041 [95% CI: 0.007–0.246] P < 0.001; SE, OR: 0.022 [95% CI: 0.002–0.204] P < 0.001). The safety issues were comparable between the two groups. Conclusions. This suggests that early therapeutic intervention with MTX could safely prevent the development of RA in patients with recent-onset UA showing high ACPA titers.

Elevated soluble CD14-subtype (PRESEPSIN; P-SEP) levels in rheumatoid arthritis (RA) patients with bacterial infection

Infection is a serious complication observed in the management of rheumatoid arthritis (RA) patients. The acute inflammatory marker C-reactive protein (CRP) is elevated both during infection and du...

Hemophagocytic syndrome in a patient with rheumatoid arthritis.

A 76-year-old man with rheumatoid arthritis, who had been treated with oral prednisolone and methotrexate, presented with high fever and generalized fatigability. Laboratory data demonstrated marked pancytopenia, which we first regarded as a side effect of methotrexate, and leucovorin was administered with granulocyte-colony stimulating factor and transfusions. Because no recovery was recognized, however, bone marrow aspiration was performed, by which hemophagocytic syndrome was diagnosed. After corticosteroid pulse therapy was initiated, the patient’s symptoms were rapidly attenuated and laboratory data rapidly normalized.