Shiro Ohshima

Delayed onset and reduced severity of collagen-induced arthritis in interleukin-6-deficient mice

OBJECTIVE To investigate the roles of interleukin-6 (IL-6) in the pathogenesis of rheumatoid arthritis (RA) by studying its effect on murine collagen-induced arthritis (CIA). METHODS IL-6-deficient (IL-6-/-) mice with a genetic background of susceptibility to CIA were generated by backcrossing them with DBA/1J mice for 8 generations. Clinical and immunologic features were compared between these mice and IL-6 wild-type (IL-6+/+) littermates with CIA. RESULTS Serum IL-6 levels increased during the development of CIA in IL-6+/+ mice. Two prominent peaks were observed. The first was coincident with the onset of arthritis, and the second one was observed during exacerbation of the disease. The onset of arthritis in IL-6-/- mice was delayed for 2 weeks compared with that in IL-6+/+ mice, and the severity of arthritis, as indicated by the arthritis score, remained significantly lower in IL-6-/- mice during the entire followup period (14 weeks), although all IL-6-/- mice developed definite arthritis as did the IL-6+/+ mice. Histologic severity was also reduced in IL-6-/- mice. In addition, radiologic changes such as osteopenia and bone erosion were reduced significantly in these animals. Both humoral and cellular responses to type II collagen (CII) in IL-6-/- mice were reduced to about half those in IL-6+/+ mice. In addition, enhanced production of IL-4 and IL-10 in response to concanavalin A stimulation was observed in IL-6-/- mice. CONCLUSION IL-6 plays an important role in the development of CIA, and both suppression of specific immune responses to CII and a tendency to a shift toward a Th2 cytokine profile might contribute in part to the attenuation of CIA in IL-6-/- mice. These findings suggest that blockade of IL-6 might be beneficial in the treatment of RA.

Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils.

Background: We have previously shown that fisetin, a flavonol, inhibits IL-4 and IL-13 synthesis by allergen- or anti-IgE-antibody-stimulated basophils. This time, we investigated the inhibition of IL-4 and IL-13 production by basophils by other flavonoids and attempted to determine the fundamental structure of flavonoids related to inhibition. We additionally investigated whether flavonoids suppress leukotriene C4 synthesis by basophils and IL-4 synthesis by T cells in response to anti-CD3 antibody. Methods: Highly purified peripheral basophils were stimulated for 12 h with anti-IgE antibody alone or anti-IgE antibody plus IL-3 in the presence of various concentrations of 18 different kinds of flavones and flavonols. IL-4 and IL-13 concentrations in the supernatants were then measured. Leukotriene C4 synthesis was also measured after basophils were stimulated for 1 h in the presence of flavonoids. Regarding the inhibitory activity of flavonoids on IL-4 synthesis by T cells, peripheral blood mononuclear cells were cultured with flavonoids in anti-CD3-antibody-bound plates for 2 days. Results: Luteolin, fisetin and apigenin were found to be the strongest inhibitors of both IL-4 and IL-13 production by basophils but did not affect leukotriene C4 synthesis. At higher concentrations, these flavonoids suppressed IL-4 production by T cells. Based on a hierarchy of inhibitory activity, the basic structure for IL-4 inhibition by basophils was determined. Conclusions: Due to the inhibitory activity of flavonoids on IL-4 and IL-13 synthesis, it can be expected that the intake of flavonoids, depending on the quantity and quality, may ameliorate allergic symptoms or prevent the onset of allergic diseases.

RANKL‐Induced Expression of Tetraspanin CD9 in Lipid Raft Membrane Microdomain Is Essential for Cell Fusion During Osteoclastogenesis

We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called “lipid raft,” and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system.

Milk fat globule EGF factor 8 in the serum of human patients of systemic lupus erythematosus

Mouse milk fat globule epidermal growth factor 8 (MFG‐E8), which is secreted by a subset of activated macrophages, binds to apoptotic cells by recognizing phosphatidylserine and promotes their engulfment. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in MFG‐E8−/− mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus (hSLE). Here, we report that hMFG‐E8 bound to phosphatidylserine and an integrin αvβ3 complex. Increasing concentrations of MFG‐E8 generated a bell‐shaped response curve for the efficiency of phagocytosis. That is, in NIH3T3 and MFG‐E8−/− thioglycollate‐elicited peritoneal macrophages that do not express MFG‐E8, hMFG‐E8 enhanced engulfment at low concentrations but inhibited it at high concentrations. On the other hand, hMFG‐E8 dose‐dependently inhibited the engulfment of apoptotic cells by MFG‐E8+/+ thioglycollate‐elicited peritoneal macrophages, indicating that an excess of MFG‐E8 has an inverse effect on the engulfment of apoptotic cells. To investigate the role of MFG‐E8 in human disease, we generated two mAb against MFG‐E8 and screened human blood samples for MFG‐E8 using an ELISA. We found that some childhood‐onset and adult SLE patients carried a significant level of MFG‐E8 in their blood samples. These results suggested that the aberrant expression of MFG‐E8 is involved in the pathoetiology of some cases of hSLE.

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.

Enhanced production of osteopontin in multiple myeloma: clinical and pathogenic implications

Summary. In this study, we examined osteopontin (OPN) production in myeloma cells and plasma OPN levels in multiple myeloma (MM) patients. We assessed OPN production in bone marrow cells (BMCs) by immunocytochemistry and enzyme‐linked immunosorbent assay (ELISA). We also assessed OPN production in various B‐cell malignant cell lines, including three myeloma cell lines by reverse transcription polymerase chain reaction (RT‐PCR) and Western blotting. In addition, we measured plasma OPN concentrations by ELISA in 30 MM patients, 21 monoclonal gammopathy of undetermined significance (MGUS) patients and 30 healthy volunteers. As a result, in an immunocytochemical study, abundant OPN was detected in BMCs from overt MM patients, whereas no OPN was detected in BMCs from patients with other haematological diseases, including MGUS. Cultured BMCs from overt MM patients produced more OPN than those from patients with either smouldering MM or MGUS. Myeloma cell lines spontaneously produced OPN. Plasma OPN levels of MM patients were significantly higher than those of MGUS patients and healthy volunteers (P < 0·05). Moreover, they correlated with both progression and bone destruction of the disease (P < 0·05). These suggest that myeloma cells actively produce OPN, which possibly contributes to osteoclastic bone resorption in MM. Plasma OPN levels may be a useful biomarker for assessing bone destruction in MM and distinguishing MM from MGUS or smouldering MM.

RGS18 Acts as a Negative Regulator of Osteoclastogenesis by Modulating the Acid‐Sensing OGR1/NFAT Signaling Pathway

We showed that RGS18, a myeloid lineage‐specific RGS protein that is inhibited after activation of the RANK/RANKL system, is a negative regulator of osteoclastogenesis. RGS18 acts through an external acidosis‐sensing osteoclastogenic mechanism through the OGR1/NFAT pathway.

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.

Remission of the Renal Involvement in a Patient with Primary Sjo¨gren’s Syndrome (SS) after Pulse High-Dose Corticosteroid Infusion Therapy

Abstract: We report the case of a young female patient with primary Sjo¨gren’s syndrome (SS). In addition to sicca symptoms she also suffered from progressive renal insufficiency and renal tubular acidosis (RTA). She was treated with three sets of pulse high-dose corticosteroid infusion and subsequent low-dose corticosteroid oral administration. When the efficacy was evaluated about 6 months after the start of the therapy, dramatic improvements were seen with no adverse effects, not only in laboratory tests but also histopathologically, as indicated by the repeat kidney biopsy. This suggests that renal involvements of SS might be reversible in some cases, and that there might be a clinical benefit of pulse high-dose corticosteroid infusion therapy in SS with progressive renal involvement.

Enhanced expression and DNA binding activity of two CCAAT/enhancer‐binding protein isoforms, C/EBPβ and C/EBPδ, in rheumatoid synovium

OBJECTIVE To investigate the activation and expression of CCAAT/enhancer-binding proteins (C/EBP), especially C/EBPbeta and -delta, in rheumatoid synovium, and their pathogenic implications in rheumatoid arthritis (RA). METHODS The activation of C/EBPbeta and -delta was assessed in synovial tissues from patients with RA by electrophoretic mobility shift assay (EMSA); DNA binding activity of C/EBPs was evaluated by measuring EMSA band density. The expression and distribution of C/EBPbeta and -delta in synovial tissues were examined by immunohistochemistry analysis. As a control, synovial tissues from patients with osteoarthritis (OA) were studied. RESULTS Enhanced DNA binding activity of C/EBPbeta and -delta, 2 major members of the C/EBP family, was detected in synovial tissues from RA patients, while synovial tissues from the patients with OA showed only faint or marginal activity (mean +/- SEM arbitrary units [AU] RA 23.3 +/- 11.7 in RA versus 4.5 +/- 1.3 in OA; P < 0.05). Moreover, the binding activities of the C/EBP proteins were correlated with both serum C-reactive protein levels (r = 0.62, P < 0.05) and synovial interleukin-6 messenger RNA levels (r = 0.60, P < 0.05). In immunohistochemistry studies, C/EBPbeta and -delta were detected predominantly in the rheumatoid synovial lining cells (both CD14+ and CD14- cells). CONCLUSION C/EBPbeta and -delta may contribute to the pathology of rheumatoid synovitis.