Ken Mei Sakata

Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis

The effects of galectin-9 on a mouse collagen-induced arthritis (CIA) model were assessed to clarify whether galectin-9 suppresses CIA by regulating T cell immune responses. Galectin-9 suppressed CIA in a dose-dependent manner, and such suppression was observed even when treatment was started on 7 days after the booster, indicating its preventive and therapeutic effects. Galectin-9 induced the decreased levels of pro-inflammatory cytokines, IL-17, IL-12, and IFNgamma in the joint. Galectin-9 induced the decreased number of CD4(+) TIM-3(+) T cells in peripheral blood. Galectin-9-deficient mice became susceptible to CIA may be by increased number of CD4(+) TIM-3(+) T cells and decreased number of Treg cells. We further found that galectin-9 induces differentiation of naive T cells to Treg cells, and it suppresses differentiation to Th17 cells in vitro. The present results suggested that galectin-9 ameliorates CIA by suppressing the generation of Th17, promoting the induction of regulatory T cells.

Galectin-9 ameliorates immune complex-induced arthritis by regulating FcγR expression on macrophages

Galectin-9 up-regulated Fc gamma RIIb expression of mouse peritoneal macrophages in vitro but down-regulated Fc gamma RIII expression. Galectin-9-treated macrophages stimulated with immune complexes (IC) produced less TNFalpha and IL-1 beta but more IL-10 than PBS-treated macrophages. Macrophage enhancing effects on IC-induced C5a and neutrophil chemotactic activity were also diminished for galectin-9-treated macrophages. In galectin-9-treated mice, the severity of IC-induced arthritis was reduced, as were pro-inflammatory cytokine levels in inflamed joints and serum C5a. Fc gamma RIIb expression of macrophages from galectin-9-treated mice was up-regulated, whereas Fc gamma RIII expression was down-regulated. Macrophages from galectin-9-treated mice produced less TNFalpha and IL-1 beta but more IL-10 than PBS-treated mice. Disease severity of galectin-9-transgenic mice was milder than wild-type mice, whereas that of galectin-9-deficient mice was exaggerated. Furthermore, macrophage Fc gamma RIIb expression in galectin-9-deficient mice was down-regulated, while Fc gamma RIII expression was up-regulated. These results suggest that galectin-9 suppresses IC-induced inflammation partly by regulating Fc gamma R expression on macrophages.

FAS (CD95)-TRANSDUCED SIGNAL PREFERENTIALLY STIMULATES LUPUS PERIPHERAL T LYMPHOCYTES

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas‐transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti‐Fas monoclonal antibodies (mAb) (imCH‐11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis (u2009p < 0.003 and p < 0.005, respectively). The soluble form of CH‐11 and other immobilized anti‐Fas mAb (UB‐2, ZB‐4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH‐11 induced IL‐2 and IL‐6 mRNA expression. However, imCH‐11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas‐mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH‐11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas‐transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas‐mediated peripheral immune homeostasis.

Galectin-9 accelerates transforming growth factor β3-induced differentiation of human mesenchymal stem cells to chondrocytes

Galectin-9 (Gal-9), a beta-galactoside binding lectin, plays a crucial role in innate and adaptive immunity. In the rat collagen-induced arthritis model, administration of Gal-9 induced repair of existing cartilage injury even when joints were already swollen with cartilage destruction. We thus attempted to explore the role of Gal-9 in chondrocyte differentiation utilizing human mesenchymal stem cell (MSC) pellet cultures. During chondrogenesis induced by transforming growth factor beta3 (TGFbeta3), MSCs strongly expressed endogenous Gal-9. Expression of Gal-9 peaked on day 14 and the neutralization of endogenous Gal-9 resulted in the reduced chondrogenesis, indicating possible involvement of Gal-9 in TGFbeta-mediated chondrogenesis. In pellets, addition of Gal-9 significantly enhanced TGFbeta3-induced chondrogenesis, as evidenced by increasing proteoglycan content, but not cell proliferation. In the absence of Gal-9, collagen expression by MSCs switched from type I to type II on 28 days after stimulation with TGFbeta3. When MSCs were co-stimulated with Gal-9, the class switch occurred on day 21. In addition, Gal-9 synergistically enhanced TGFbeta3-induced phosphorylation of Smad2, though Gal-9 did not itself induce detectable Smad2 phosphorylation. These results suggest that Gal-9 has a beneficial effect on cartilage repair in injured joints by induction of differentiation of MSCs into chondrocytes.

Spontaneous production of eosinophil chemotactic factors by T lymphocytes from patients with subcutaneous angioblastic lymphoid hyperplasia with eosinophilia

Subcutaneous angioblastic lymphoid hyperplasia with eosinophilia (SALH) was reviewed with respect to eosinophil chemotaxis. Lymphoid cells separated from the granuloma spontaneously released at least two different eosinophil chemotactic factors (ECF): low-molecular-weight and high-molecular-weight ECF according to the profile on gel filtration (LMW-ECF, about 500; HMW-ECF, 45,000 to 70,000). The cells, however, failed to produce chemotactic activity for macrophages and neutrophils. By analysis with monoclonal antibodies against lymphocyte subpopulations, the granuloma T cells, probably OKT4-positive cells, were shown to be responsible for spontaneous production of these two ECF. Furthermore, the blood mononuclear leukocytes were separated from the patients with SALH. An ECF closely resembling HMW-ECF was also spontaneously produced by the blood OKT4-positive T lymphocytes, whereas no LMW-ECF was released. Mononuclear leukocytes from healthy donors, however, could produce an ECF resembling HMW-ECF and chemotactic activities for macrophages and neutrophils by stimulation with concanavalin A (Con A). Protein synthesis appeared to be essential for spontaneous ECF and for Con A-induced ECF production. These results suggest that the granuloma OKT4-positive T lymphocytes of the patients with SALH are in activated condition to release LMW- and HMW-ECF, whereas the blood OKT4-positive T lymphocytes are in activated condition to release only HMW-ECF. Such spontaneous and prolonged production of HMW-ECF by the cells can be one of the diagnostic means of SALH.

MONOCYTE RESCUE OF HUMAN T CELLS FROM APOPTOSIS IS CD40/CD154 DEPENDENT

The induction of T‐cell apoptosis is regulated in part by monocytes (CD14+ cells). Human peripheral blood monocytes inhibited the spontaneous cell death of activated Tu2003cells in vitro. The inhibition of T‐cell apoptosis did not require autologous monocytes. Inhibition required direct contact with monocytes and was not due to a soluble factor. Furthermore, treatment of monocytes with actinomycinu2003D, cycloheximide and paraformaldehyde abrogated the anti‐apoptotic activity of these cells. Blocking antibody to CD40 and CD154 (CD40 ligand) decreased the ability of monocytes to aid in T‐cell survival, whereas, blocking LFA‐1/I‐CAM‐1, Fas ligand and the CD4/major histocompatibility complex class II interaction did not affect the influence of monocytes on T‐cell survival. This shows that monocytes rescue of activated Tu2003cells from apoptosis is dependent upon CD40/CD154 interaction.

Combined anti-tumor necrosis factor-α therapy and DMARD therapy in rheumatoid arthritis patients reduces inflammatory gene expression in whole blood compared to DMARD therapy alone

Periodic assessment of gene expression for diagnosis and monitoring in rheumatoid arthritis (RA) may provide a readily available and useful method to detect subclinical disease progression and follow responses to therapy with disease modifying anti-rheumatic agents (DMARDs) or anti-TNF-α therapy. We used quantitative real-time PCR to compare peripheral blood gene expression profiles in active (“unstable”) RA patients on DMARDs, stable RA patients on DMARDs, and stable RA patients treated with a combination of a disease-modifying anti-rheumatoid drug (DMARD) and an anti-TNF-α agent (infliximab or etanercept) to healthy human controls. The expression of 48 inflammatory genes were compared between healthy controls (N = 122), unstable DMARD patients (N = 18), stable DMARD patients (N = 26), and stable patients on combination therapy (N = 20). Expression of 13 genes was very low or undetectable in all study groups. Compared to healthy controls, patients with unstable RA on DMARDs exhibited increased expression of 25 genes, stable DMARD patients exhibited increased expression of 14 genes and decreased expression of five genes, and combined therapy patients exhibited increased expression of six genes and decreased expression of 10 genes. These findings demonstrate that active RA is associated with increased expression of circulating inflammatory markers whereas increases in inflammatory gene expression are diminished in patients with stable disease on either DMARD or anti-TNF-α therapy. Furthermore, combination DMARD and anti-TNF-α therapy is associated with greater reductions in circulating inflammatory gene expression compared to DMARD therapy alone. These results suggest that assessment of peripheral blood gene expression may prove useful to monitor disease progression and response to therapy.

TGF-β1 Null Mutation Leads to CD154 Upregulated Expression in Affected Tissues

The TGF-β1(–/–) mouse is a murine model for systemic autoimmune disease. The aim of this study is to elucidate the immunological mechanism that leads to multifocal tissue inflammation and autoantibody production in TGF-β1(–/–) mice. Heart, lung, liver, and salivary gland from TGF-β1(–/–) were assessed for CD154 expression by RT-PCR and immunohistochemistry. Compared to wild-type littermates, CD154 expression was elevated in all tissues studied. Furthermore, IL-12 mRNA was expressed in the salivary gland and heart of TGF-β1(–/–) mice and not in wild-type littermates. This suggests that the CD154 pathway is activated in these tissues. This shows that TGF-β1 regulates CD154 expression leading to spontaneous IL-12 production and autoimmunity.