Takehiro Higashi

Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis.

Five types of dopamine receptors, termed D1 to D5, have been identified to date. The D1 and D5 receptors form the D1-like group that couples with the Galphas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Galphai class of G proteins. A D2-like-receptor (D2-like-R) antagonist L750667 induced dendritic cell (DC)-mediated Th17 differentiation. In contrast, a D1-like-R antagonist SCH23390 inhibited DC-mediated Th17 differentiation. The D1-like-Rs were expressed on both DCs and T cells, whereas D2-like-Rs were marginally expressed on CD4+CD45RA+ naïve T cells. In addition, SCH23390 had the ability to prevent experimental autoimmune encephalomyelitis (EAE) in mice. Spleen cells from EAE mice showed decreased IL-17 production, when SCH23390 was administered. Adoptive transfer of DCs treated with SCH23390 successfully prevented EAE. These findings indicate that antagonizing D1-like-Rs on DCs inhibits Th17 differentiation, thereby leading to an amelioration of EAE.

B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones

Th 17 cells represent a novel subset of CD4+ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.

Dopamine D1-Like Receptor Antagonist Attenuates Th17-Mediated Immune Response and Ovalbumin Antigen-Induced Neutrophilic Airway Inflammation

Allergic airway inflammation is generally considered a Th2-type immune response. Recent studies, however, demonstrated that Th17-type immune responses also play important roles in this process, especially in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We previously reported that dendritic cells release dopamine to naive CD4+ T cells in Ag-specific cell–cell interaction, in turn inducing Th17 differentiation through dopamine D1-like receptor (D1-like-R). D1-like-R antagonist attenuates Th17-mediated diseases such as experimental autoimmune encephalomyelitis and autoimmune diabetes. However, the effect of antagonizing D1-like-R on Th17-mediated airway inflammation has yet to be studied. In this study, we examined whether D1-like-R antagonist suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice and then elucidated the mechanism of action. DO11.10 mice were nebulized with OVA or PBS, and some mice received D1-like-R antagonist orally before OVA nebulization. D1-like-R antagonist significantly suppressed OVA-induced neutrophilic airway inflammation in DO11.10 mice. It also inhibited the production of IL-17 and infiltration of Th17 cells in the lung. Further, D1-like-R antagonist suppressed the production of IL-23 by lung CD11c+ APCs. In contrast, D1-like-R antagonist did not increase Foxp3+ regulatory T cells in the lung. D1-like-R antagonist neither suppressed nonspecific LPS-induced neutrophilic airway inflammation nor OVA-induced eosinophilic airway inflammation. These results indicate that D1-like-R antagonist could suppress Th17-mediated neutrophilic airway inflammation, raising the possibility that antagonizing D1-like-R serves as a promising new strategy for treating neutrophil-dominant severe asthma.

Intracellular galectin‐9 activates inflammatory cytokines in monocytes

Whether galectin‐9 plays a role in inflammatory responses remains elusive. The present study was designed to determine the role of intracellular galectin‐9 in activation of inflammatory cytokine genes in human monocytes. Galectin‐9 expression vector pBKCMV3‐G9 was transiently co‐transfected into THP‐1 monocytic cells along with luciferase reporters carrying gene promoters of IL‐1α (IL1A), IL‐1β (IL1B) and IFNγ. Transient transfection studies showed that galectin‐9 over‐expression activated all three gene promoters, suggesting that intracellular galectin‐9 induces inflammatory cytokine genes in monocytes. Galectin‐9 over‐expression also activated NF‐IL6 (C/EBP β) and AP‐1, but not NF‐κB. In contrast, extracellular galectin‐9 is not involved in regulation of inflammatory cytokines. Immunoprecipitation/Western blotting, using anti‐galectin‐9 Ab and anti‐NF‐IL6 Ab, showed physical association of intracellular galectin‐9 with NF‐IL6. RT‐PCR confirmed that galectin‐9 over‐expression increased IL‐1α and IL‐1β mRNA levels in THP‐1 cells. The interaction of galectin‐9 with NF‐IL6 was enhanced following LPS treatment in THP‐1 cells. Intracellular galectin‐9 synergized with LPS to activate NF‐IL6. Nuclear translocation of galectin‐9 was also observed in THP‐1 cells treated with LPS. Our results indicate that galectin‐9 is a LPS‐responsive factor, and further demonstrate that intracellular galectin‐9 transactivates inflammatory cytokine genes in monocytes through direct physical interaction with NF‐IL6.

Curdlan Induces DC-Mediated Th17 Polarization via Jagged1 Activation in Human Dendritic Cells

BACKGROUND Th17-inducing activity is carried by certain polysaccharides such as beta-glucan derived from Candia albicans. Our previous studies have shown that Th1- and Th2-inducing activities can be qualitatively evaluated by the expression patterns of Notch ligand isoforms, using human monocyte-derived dendritic cells (Mo-DCs) and some leukemic cell lines such as THP-1. The association of Th17-inducing activities with Notch ligand expression patterns has been unclear. METHODS Mo-DCs from healthy volunteers were co-cultured with HLA-DR-nonshared allogeneic CD4+ naïve T cells to induce a mixed lymphocyte reaction, in the presence of adjuvants, such as curdlan. Culture supernatants were assayed for IFNgamma, IL-5 and IL-17 by an enzyme-linked immunosorbent assay (ELISA). Notch ligand expression on Mo-DCs and THP-1 cells was evaluated by using RT-PCR. RESULTS The present study shows that curdlan, one of the beta-glucans, has the ability to induce DC-mediated Th17 differentiation. It is also interesting to note that Jagged1 mRNA in Mo-DCs and THP-1 cells is up-regulated by curdlan. Furthermore, polyclonal anti-Jagged1 antibody inhibited such DC-mediated Th17 differentiation. CONCLUSIONS This study suggests that curdlan induces human DC-mediated Th17 polarization via Jagged1 activation in DCs.

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1

High mobility group box 1 protein (HMGB1), originally described as a non‐histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte‐specific E26 transformation‐specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31‐kDa proIL‐1β protein. The −131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S‐transferase‐pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix‐turn‐helix DNA‐binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1‐binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1‐deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.

Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice.

Dopamine receptors have five isoforms, termed D1-D5. The D1 and D5 receptors form the D1-like group that couples with the Galphas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Galphai class of G proteins. In our previous studies, a D1-like-R antagonist, SCH23390, inhibited DC-mediated Th17 differentiation and exhibited preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE) in mice. We herein demonstrate in the current study that in the pancreas obtained from NOD mice, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific markers for Th17. Thereafter, NOD mice were orally administered SCH23390 from week 6 to week 26. At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively (p<0.05). A histological examination of SCH23390-treated mice exhibited a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islets from vehicle controls showed insulitis. In week 26, spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and exhibited an augmentation of IFNgamma induction and the suppression of IL-17 induction in the SCH23390-treated mice. These findings indicate that antagonizing D1-like-R suppresses IL-17 expression, thereby leading to a decreased occurrence of NOD.

Human Th17 cell clones and natural immune responses.

Immunomodulators such as lipopolysaccharides (LPS) and forskolin change the nature of dendritic cells (DCs) to induce Th1 and Th2 cells, respectively, thereby designated Th1 or Th2 adjuvants. Recent studies showed that Th17-inducing activity can be carried by certain polysaccharides such as beta-glucan derived from Candia albicans. Such activities can be scrutinized by using MLR, cAMP and possibly, differential expression of Notch ligand isoforms. In this review article, we also introduce an effective method to establish human Th17 cell clones and a transcriptome analysis using human Th subpopulations. In vivo relevance to human Th17 responses is discussed.

Evaluation of Adjuvant Activities Using Human Antigen Presenting Cells in Vitro

BACKGROUND Immunomodulators such as lipopolysaccharides (LPS) and forskolin change the nature of dendritic cells (DCs) to induce Th1 and Th2 cells, respectively, thereby designated Th1 or Th2 adjuvants. Our previous study showed that such activities can be qualitatively evaluated by expression patterns of Notch ligand isoforms, using human monocyte-derived DCs and some leukemic cell lines, such as THP-1 or KG-1. However, quantitative evaluation of the adjuvant activities was not fully established. METHODS PMA-treated human monocytic cell line THP-1 was used as a target. Cells were stimulated with various adjuvants, and the intracellular levels of cAMP were determined. RESULTS Th2 adjuvant forskolin was qualitatively evaluated by an increased expression of Delta1 in PMA-treated THP-1 cells, as reported in our previous study. In PMA-treated THP-1 cells, intracellular cAMP levels increased after stimulation with forskolin, in a dose-dependent manner. On the other hand, LPS, one of the known Th1 adjuvants, suppressed the increased cAMP level in a dose-dependent manner. CONCLUSIONS Th2- and Th1-adjuvant activities can be quantitatively evaluated by using PMA-treated THP-1 cells and PMA-treated/forskolin-stimulated THP-1 cells, respectively.

Growth suppression of human mast cells expressing constitutively active c-kit receptors by JNK inhibitor SP600125

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 showed constitutive activation of JNK/c-Jun, and the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G1 phase and apoptosis accompanied by the cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis. Following Sp600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Thus, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with mutations in the catalytic domain of c-kit.