Kazuhiko Haruta

Inhibitory effects of ZSTK474, a novel phosphoinositide 3-kinase inhibitor, on osteoclasts and collagen-induced arthritis in mice

IntroductionTargeting joint destruction induced by osteoclasts (OCs) is critical for management of patients with rheumatoid arthritis (RA). Since phosphoinositide 3-kinase (PI3-K) plays a critical role in osteoclastogenesis and bone resorption, we examined the effects of ZSTK474, a novel phosphoinositide 3-kinase (PI3-K)-specific inhibitor, on murine OCs in vitro and in vivo.MethodsThe inhibitory effect of ZSTK474 on OC formation was determined and compared with other PI3-K inhibitors by counting tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells after culturing murine bone marrow monocytic OC precursors, and RAW264.7 cells. Activation of Akt and expression of nuclear factor of activated T cells (NFAT) c1 in cultured RAW264.7 cells were examined. The suppressing effect of ZSTK474 on bone resorption was assessed by the pit formation assay. The in vivo effects of ZSTK474 were studied in collagen-induced arthritis (CIA) in the mouse. Oral daily administration of ZSTK474 was started either when more than half or when all mice developed arthritis. Effects of ZSTK474 were evaluated using the arthritis score and histological score of the hind paws.ResultsZSTK474 inhibited the differentiation of bone marrow OC precursors and RAW264.7 cells in a dose-dependent manner. The inhibitory effect of ZSTK474 was much stronger than that of LY294002, the most commonly used PI3-K inhibitor. In addition, ZSTK474 suppressed the bone resorbing activity of mature OCs. Moreover, oral daily administration of ZSTK474, even when begun after the development of arthritis, ameliorated CIA in mice without apparent toxicity. Histological examination of the hind paw demonstrated noticeable reduction of inflammation and of cartilage destruction in ZSTK474-treated mice. ZSTK474 also significantly decreased OC formation adjacent to the tarsal bone of the hind paw.ConclusionsThese findings suggest that inhibition of PI3-K with ZSTK474 may potentially suppress synovial inflammation and bone destruction in patients with RA.

Inhibitory effects of ZSTK474, a phosphatidylinositol 3-kinase inhibitor, on adjuvant-induced arthritis in rats.

ObjectiveWe examined the effects of ZSTK474, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on adjuvant-induced arthritis (AIA).MethodsAIA was induced in Lewis rats by subcutaneous administration of Freund’s complete adjuvant at the base of the tail on day 0. ZSTK474 was orally administered once daily from day 10. The severity of AIA was assessed by measuring the hind paw volume. The number of lymphocytes in inguinal lymph nodes (ILN) was determined by flow cytometry. The in vitro effects of ZSTK474 on the cell proliferation, and the cytokines and prostaglandin E2 (PGE2) production were evaluated by BrdU method, ELISA and cytometric beads array.ResultsZSTK474 ameliorated the progression of AIA. The temporary increases in the number of T cells in ILN, which occurred along with the appearance of arthritis, were inhibited in the ZSTK474-treated groups. In vitro studies revealed that ZSTK474 inhibited the production of IFNγ and IL-17 in concanavalin A-activated T cells. In vitro studies further revealed that ZSTK474 inhibited the proliferation and PGE2 production by fibroblast-like synovial cells (FLS).ConclusionZSTK474 demonstrated prophylactic efficacy in a rat model of rheumatoid arthritis (RA) through inhibition of T cell and FLS functions. It was suggested that the inhibitors of PI3K have therapeutic potential for RA.

Pentoxifylline induces the shedding of L-selectin on polymorphonuclear cells by stimulation via adenosine receptor as well as by the inhibition of phosphodiesterase

Abstract We investigated the effects of pentoxifylline (PTX) on the expression of L-selectin on polymorphonuclear leukocytes (PMN). PTX induced the down-regulation of L-selectin expression in dose- and time-dependent manner. The measurement of soluble L-selectin in the culture medium by ELISA indicated that the down-regulation of L-selectin expression by PTX was due to the shedding of L-selectin from PMN. The mechanism by which PTX induced the shedding of L-selectin was investigated. The concentration of intracellular cyclic AMP (cAMP) was increased after treatment of PMN with PTX. However, an elevation of cAMP induced by dibutyryl cAMP (dbcAMP) as well as other methylxanthine derivatives (caffeine, aminophylline, and theophylline) did not induce the shedding of L-selectin. Although stimulation of the adenosine receptor with 5′-N-ethylcarboxamidoadenosine (NECA) or 5′-(N-cyclopropyl)-carboxamido-adenosine (CPCA) adenosine receptor agonists did not induce the shedding of L-selectin, shedding of L-selectin was demonstrated when PMN was incubated simultaneously with rolipram, a phosphodiesterase (PDE) inhibitor, and CPCA. Moreover, shedding of L-selectin induced by PTX was attenuated by aminophylline, an adenosine receptor antagonist. These results indicated that PTX induces the shedding of L-selectin on PMN by stimulation via the adenosine receptor as well as inhibition of PDE.

Endothelial cell-binding antibodies in patients with systemic lupus erythematosus

Abstract The implications of endothelial cell-binding IgG antibodies (EC IgG) in systemic lupus erythematosus (SLE) was evaluated by determining level of EC IgG in sera from 112 SLE patients. The serum EC IgG level was determined by the cyto-ELISA method using human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and aortic endothelial cells (HAEC) as antigens. The levels of EC IgG were significantly higher among patients with SLE than among healthy control subjects (P < 0.001), and 68% (76/112) of SLE patients were shown to be EC IgG-positive. In patients with active lupus nephritis, the level of EC IgG was statistically and significantly elevated compared with those without lupus nephritis (P < 0.05). Negative correlations between EC IgG level and levels of CH50, C3, and lymphocyte count were revealed (P < 0.05, P < 0.005, and P < 0.05, respectively). When clinical course was evaluated, the levels of EC IgG correlated with disease activity. Definitive correlations in antibody levels between HUVEC and HMVEC, and between HUVEC and HAEC were revealed (both P < 0.0001). The results of this study revealed that the EC IgG in patients with SLE was involved in the onset of clinical manifestation, especially in patients with active lupus nephritis.

CD154 expression and mRNA stability of activated CD4-positive T cells in patients with systemic lupus erythematosus.

Abstract The expression of CD154 (CD40 ligand) on activated CD4+ T cells is known to be transient and tightly regulated for antigen-specific immune responses, and is increased and prolonged among patients with systemic lupus erythematosus (SLE). We investigated the regulation of CD154 expression by determining the protein and mRNA expression with PMA and ionomycin stimulation in CD4+ T cells, and confirmed their increase and prolongation in SLE T cells. Treatment with actinomycin D, a transcription inhibitor, after PMA and ionomycin stimulation was performed, and the findings revealed that the stability of CD154 mRNA increased significantly in activated SLE T cells compared with that of controls. However, alternations or abnormal sequences were not identified in the 3″ untranslated region, including AU-rich elements and CU-rich sequences, while their partial involvement in the posttranscriptional regulation of CD154 mRNA stability has been reported. With 96 h culture in vitro, the destabilization of CD154 mRNA was demonstrated, resulting in a corresponding decrease and normalization of surface expression on activated SLE T cells. We speculate that the CD154 expression on T cells from SLE patients may be increased and prolonged, with mRNA stabilization being related to a continuous stimulation in vivo.

Anterior uveitis and poststreptococcal reactive arthritis

Abstract Anterior uveitis has been reported to occur during the course of illness and/or after pharyngeal or tonsillar streptococcal infection. We recently reported the first two adult patients with poststreptococcal reactive arthritis (PSRA), both whom concomitantly developed uveitis. The clinical manifestations of our patients presented at The 5th International Symposium on Tonsils and Mucosal Barriers of Upper Airways at Wakayama in April 2003 are summarized, and the possible pathogeneses of uveitis and antibiotic prophylaxis are discussed.