Ryuichi Motoda

IFN-γ-Dependent and -Independent Mechanisms in Adverse Effects Caused by Concomitant Administration of IL-18 and IL-12

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1β. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-γ level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-α was not induced. The involvement of IFN-γ in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-γ treatment of wild-type mice alleviated it. When IFN-γ-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-γ-dependent mechanisms but also IFN-γ-independent processes.

Resolution of Streptococcus mutans glucosyltransferases into two components essential to water-insoluble glucan synthesis

Streptococcus mutans has been implicated as a significant factor in the development of human dental plaque and subsequent caries formation [ 1,2]. The cariogenicity of this organism is mainly dependent on its ability to convert dietary sucrose to adhesive waterinsoluble glucans (WIG) which give a high proportion of o-(1 + 3) to o-(1 + 6) linkages [3-51. Many bacterial species produce extracellular glucosyltransferases (EC 2.4.1.5 ‘dextran-sucrase’) which catalyze the formation of water-soluble glucans (WSG), consisting of a-(1 + 6) glucosidic linkages primarily [S-9]. The dextransucrase from several strains of S. mutans have been purified and characterized [8-lo]. In contrast, attempts to purify the water-insoluble glucan-synthesizing glucosyltransferases (WIG-GTase) have consistently failed either because the enzyme remains in high Mr aggregates [9-131, or because the activity disappears during purification procedures [ 14,151. Therefore, the mechanism of WIG synthesis by S. mutans as well as the number of proteins required for its synthesis are still unknown. Here, we describe the isolation of the disaggregated WIG-GTase from the culture fluid of S. mutans strain B-l 3 (serotype d), and show an evidence that adhesive WIG are synthesized through an overall reaction by 2 protein components.

Identification of a vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human hematopoietic cell line NALM-16

An antagonistic activity against vascular endothelial growth factor (VEGF) was identified in the culture supernatants of certain human hematopoietic cell lines and the antagonistic protein was purified from NALM‐16 (B cell) culture supernatant. Amino acid sequencing of the N‐terminus and Western blot analysis confirmed that the antagonist was identical to a soluble truncated form of Flt‐1 (sFlt‐1). Seventeen of 52 leukemia and lymphoma cell lines investigated expressed sFlt‐1 mRNA, and 16 of the sFlt‐1 expressing cells also expressed VEGF and membrane‐bound Flt‐1 (mFlt‐1). This report is the first showing that sFlt‐1 can be produced by malignant hematopoietic cells, suggesting that the production of VEGF antagonist by hematopoietic cells may play some role in the regulation of VEGF activity in normal and malignant hematopoietic cell proliferation.

Independence of water-insoluble glucan synthesis and adherence of Streptococcus mutans to smooth surfaces.

Water-insoluble glucans (WIG) produced from sucrose by Streptococcus mutans glucosyltransferases (GTases) are of major importance in the adherence and colonization of the organisms on tooth surfaces and the subsequent development of dental caries [ 1,2]. In fact, mutants of S. mutans which lack the ability to synthesize WIG do not stick to glass surfaces and do not induce smoothsurface-caries formation in germ-free rats [3]. Although it has been suggested that active de novo WIG synthesis from sucrose is essential for cell adherence [4-61, the exact relationship between WIG synthesis and cell adherence, as well as these mechanisms, are still not elucidated. The adhesive WIG synthesis of S. mutans strain B-13 (serotype d) is caused by a cooperative action of 2 GTase components, GT-I and GT-S, which catalyze mainly the formation of 1,3-a-Dand 1,6-cy-D-glucosidie linkage, respectively [7,8]. at 37°C for 20h in defined medium M4 [9] with 0.5% glucose as a carbon source. The harvested cells were heated at 100°C for 20 min, washed twice with distilled water and lyophilized. GT-I and GT-S were purified from the culture fluids as in [6,7]. Homogeneity of both GTase components were confirmed by PAGE, SDS-PAGE and double immunodiffusion. In part, a 40% ethanol fraction [7] was used as a crude GTase preparation. Specific activities of the crude GTase, GT-I and GT-S, were 17.3, 25.5 and 56.4 IU/mg protein, respectively. The activity of GT-I was assayed turbidimetrically in the presence of 0.067% dextran TlO.

Transforming growth factor-β1 augments granulocyte-macrophage colony-stimulating factor–induced proliferation of umbilical cord blood CD34+ cells with an associated tyrosine phosphorylation of STAT5

OBJECTIVE Several investigators have reported that transforming growth factor (TGF)-beta(1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically support cell proliferation. However, the mechanisms involved have not been elucidated. To clarify the mechanisms of the synergistic action of TGF-beta(1) and GM-CSF, we compared the activation states of STAT5 and mitogen-activated protein kinase in CD34(+) cells and in GM-CSF-dependent hematopoietic cell lines. MATERIALS AND METHODS Human CD34(+) cells and GM-CSF-dependent cell lines (FKH-1, YNH-1, and M-07e) were stimulated with 1.25 ng/mL GM-CSF and/or 0.25 ng/mL TGF-beta(1), and 1.25 ng/mL GM-CSF and/or 0.25 ng/mL, 0.025 ng/mL TGF-beta(1), respectively, and cell proliferation was analyzed by [3H]thymidine uptake. Expression of signal transduction proteins and their phosphorylation states were determined by Western blotting. RESULTS TGF-beta(1) synergistically enhanced the GM-CSF-augmented growth of CD34(+) cells and FKH-1 cells, but inhibited the growth of YNH-1 and M-07e cells. Tyrosine phosphorylation of STAT5 induced by GM-CSF was enhanced by stimulation with the combination of TGF-beta(1) and GM-CSF (TGF-beta(1)/GM-CSF) compared with that induced by GM-CSF alone in CD34(+) cells and FKH-1 cells. However, combinations of TGF-beta(1)/GM-CSF caused inhibition of GM-CSF-induced tyrosine phosphorylation in M-07e cells. No significant difference was observed in mitogen-activated protein kinase activation between CD34(+) cells and FKH-1 cells stimulated with GM-CSF/TGF-beta(1) or GM-CSF alone. CONCLUSIONS Results suggest that TGF-beta(1) may augment GM-CSF-induced proliferation of CD34(+) cells in association with enhanced tyrosine phosphorylation of STAT5. Our data suggest a novel mechanism for the synergistic enhancement of cellular growth induced by the combination of TGF-beta(1) and GM-CSF.