Hidekazu Tamauchi

The Runx1 Transcription Factor Inhibits the Differentiation of Naive CD4+ T Cells into the Th2 Lineage by Repressing GATA3 Expression

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo.

Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies were performed using cell viability analysis and showed that SIG and MTA fucoidans significantly decreased the viable number of LCC and MC cells in a dose-response fashion. Histochemical staining showed morphological changes of melanoma B16 cells after exposure to fucoidan. The observed changes were indicative of crude fucoidan induced apoptosis. Male C57BL/6JJCL mice were subjected to daily i.p. injections over 4 days with either SIG or MTA fucoidan (50mg/kg body wt.). The cytolytic activity of natural killer (NK) cells was enhanced by crude fucoidan in a dose-dependent manner as indicated by (51)Cr labeled YAC-1 target cell release. This study provides substantial indications that crude fucoidan exerts bioactive effects on lung and skin cancer model cells in vitro and induces enhanced natural killer cell activity in mice in vivo.

Suppression of Th2 Immune Responses by Mekabu Fucoidan from Undaria pinnatifida Sporophylls

Background: We demonstrated that mekabu fucoidan obtained from Undaria pinnatifida (Up) sporophylls augments the type 1 T-helper (Th1) cell response in normal BALB/c mice. In this study, we examined the effects of the fucoidan of mekabu on the type 2 T-helper (Th2) response in bronchoalveolar lavage fluid (BALF) after ovalbumin (OVA) aerosol challenge. Methods: Mekabu fucoidan (50 mg/kg) was injected intraperitoneally into BALB/c mice for 4 days, and then the mice were sensitized with 50 µg/mouse of OVA plus alum (1 mg/mouse) 1 and 8 days later. The mice were challenged with OVA delivered using a nebulizer 7, 8 and 9 days after the second challenge with OVA plus alum. After 24 h, we assessed T cell responses in BALF by measuring the amount of Th2 cytokines (IL-4, IL-5, IL-13) and γ-interferon (IFN-γ) produced by Th1 cells. Results: The production of Th2 cytokines was suppressed (p < 0.05), and the amount of IFN-γ was not increased in the mice treated with mekabu fucoidan. Anti-OVA immunoglobulin E (IgE) and IgE levels in serum determined after challenge with aerosolized OVA at the end of the experiment were lower (p < 0.05) in the treated than in the control mice. Conclusions: The pulmonary inflammation was relieved by mekabu fucoidan, which also downregulated Th2-dominated responses. These results indicate that mekabu fucoidan modulates Th2 responses and might be useful for treating allergic inflammation.

Establishment of a human hemangiosarcoma cell line (ISO-HAS)

A cell line (ISO‐HAS) has been established from tumor tissue of a human hemangiosarcoma arising on the scalp by the use of conditioned medium from a murine‐phenotypic angiosarcoma cell line (ISOS‐1). Cells have been cultured for more than 2 years with up to 100 passages. The cells retained endothelial‐cell properties, such as a characteristic cobblestone appearance at confluency, contact‐inhibited growth, active uptake of acetylated low‐density lipoprotein labeled with 1,1‐dioctadecyl 1,3,3,3,3‐tetramethyl‐indocarbocyanine perchlorate (Dil‐Ac‐LDL) and CD31 expression. However, they were weakly positive for von‐Willebrand‐factor (vWf) antigen and for binding of Ulex europaeus agglutinin‐I (UEA‐1) lectin, and lacked tube‐formation activity. These findings indicate that ISO‐HAS is a poorly differentiated endothelial cell line. ISO‐HAS cells showed accumulation of p53 protein in the nuclei, and a new‐typed p53‐gene point mutation was found in exon 7 at codon 240. When inoculated s.c. into severe‐combined‐immunodeficiency (SCID) mice, the cells showed solid‐tumor growth that caused death. These properties suggest that ISO‐HAS is a malignant endothelial cell line with high tumorigenicity. Int. J. Cancer 81: 305–308, 1999.

Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.

The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris

The role ofCD4+ and CD8+ T cells in protective immunity to Trichuris muris was studied in CD4+ or CD8+ or both CD4+ and CD8+ T cell‐depleted BALB/c mice. To assess in vivo depletion of T‐cell subsets, CD4+ and CD8+ T cells in the Peyers patches, the mesenteric lymph nodes (MLN), and the spleens of mice treated with T cell‐specific monoclonal antibodies (MoAbs) were analysed by FACS. CD4+ T cells were selectively depleted in mice injected with anti‐CD4 MoAb i.p. and injection of anti‐CD8 MoAb resulted in selective depletion ofCD8+ T cells. The expulsion ofl. muris was inhibited in CD4+ T cell‐depleted mice and numerous worms were detected in the large intestine on days 14 and 21 after infection, although no suppression of protective immunity to T. muris was observed in CD8+ T cell‐depleted mice. Moreover, there was no difference in suppression of protective immunity to T. muris between CD4+ T cell‐depleted and both CD4+ and CD8+ T cell‐depleted mice. These results indicate that CD4+ T cells play a central role in protective immunity to T. muris infection.

IL-10 controls Th2-type cytokine production and eosinophil infiltration in a mouse model of allergic airway inflammation

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P<0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.

Th2 Immune Response Plays a Critical Role in the Development of Nickel-Induced Allergic Contact Dermatitis

Background:The precise roles of T helper (Th)1-type and Th2-type cytokine responses in nickel (Ni)-induced allergic contact dermatitis have not yet been clearly defined. We investigated the involvement of Th2 cytokines in Ni-induced contact hypersensitivity reaction using GATA-3 transgenic (Tg) mice. Methods: A Ni-titanium (Ti) alloy was implanted under the skin of GATA-3 Tg mice. A Ni solution was then injected 1 month after sensitization. The ear swelling response was measured at several time points after the injection; the cytokine levels in the skin were measured at 48 h after injection, and the serum levels of IgE were measured 1 month after injection. In addition, purified CD4+ splenic cells obtained from the GATA-3 Tg mice sensitized with the Ni-Ti alloy were infused into Rag-2–/– mice, and the ear swelling response of these mice after a further challenge with Ni solution was also measured. Results:Marked ear swelling and elevated serum IgE levels and skin tissue levels of IL-4 were observed in Ni-Ti-sensitized GATA-3 Tg mice. The Rag-2–/– mice transfused with the CD4+ splenic cells from the Ni-Ti alloy sensitized GATA-3 Tg mice showed a significantly more pronounced ear swelling response than the control mice. Conclusion: We confirmed the participation of Th2-type immune reactions in Ni-induced allergy using GATA-3 Tg mice.

Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB-SCID)

We treated the patients with cutaneous hemangiosarcoma with recombinant interleukin-2 (rIL-2) immunotherapy that showed clear therapeutic effects. This immunotherapy is popular for the treatment of hemangiosarcoma in Japan. The purpose of this study is to clarify the clinical effects in an animal experiment. After establishing a SCID mouse model of human hemangiosarcoma WB-SCID, we used this model to investigate anti-tumor effects of rIL-2 and LAK cells. We demonstrated that hemangiosarcoma cells are LAK-sensitive, and LAK cells induced by rIL-2 suppress the growth of hemangiosarcoma. Our results may assure the clinical effects of rIL-2 immunotherapy on hemangiosarcoma.

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T‐Cell Line Established from the Mouse Intestinal Epithelium

Growth factor‐dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long‐term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A‐stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy‐1+ CD5–TCRαβ+ CD4+CD8 α+β– (double‐positive; DP) IEL and Thy‐1+ CD5– TCRαβ+ CD4–CD8α+β– (CD8 single‐positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T‐cell receptor (TCR)‐directed stimuli. The DP IEL cell line expressed high levels of the CD3‐TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ‐directed stimuli, which indicated that TCRαβ‐mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.