Minehiko Yokoyama

E prostanoid 2 (EP2)/EP4‐mediated suppression of antigen‐specific human T‐cell responses by prostaglandin E2

Prostaglandin E2 (PGE2) is a lipid mediator that displays important immunomodulatory properties, such as polarization of cytokine production by T cells. Recent investigations have revealed that the effect of PGE2 on cytokine production is greatly influenced by external stimuli; however, it is unclear whether PGE2 plays a significant role in major histocompatibility complex‐mediated antigen‐specific T‐cell responses via binding to one of four subtypes of E prostanoid (EP) receptor alone or in combination. In the present study, we sought to determine the effect of PGE2 on antigen‐specific CD4+ T‐cell responses in humans, especially in terms of receptor specificity. We used purified protein derivative (PPD) and Cry j 1 as T helper type 1 (Th1) and Th2‐inducing antigens, respectively. We generated several different Cry j 1‐ and PPD‐specific T‐cell lines (TCLs). PGE2 significantly and dose‐dependently inhibited the proliferation and subsequent production of interleukin‐4 by Cry j 1‐specific TCLs and of interferon‐γ by PPD‐specific TCLs upon antigen stimulation. Administration of EP2 receptor agonist and EP4 receptor agonist suppressed these responses in an adenylate cyclase‐dependent manner, while EP1 and EP3 receptor agonists did not. Messenger RNA for EP2, EP3 and EP4, but not EP1, receptors were detected in Cry j 1‐ and PPD‐specific TCLs, and no differences in EP receptor expression were observed between them. Furthermore, PGE2 and EP2 receptor agonist significantly inhibited interleukin‐5 and interferon‐γ production by peripheral blood mononuclear cells in response to Cry j 1 and PPD stimulation, respectively. These results suggest that PGE2 suppresses both Th1‐ and Th2‐polarized antigen‐specific human T‐cell responses via a cAMP‐dependent EP2/EP4‐mediated pathway.

Direct activation of guanine nucleotide binding proteins through a high-energy phosphate-transfer by nucleoside diphosphate-kinase

An in vitro study of phosphate-transfer, from the high-energy phosphates on the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinase to GDP on various guanine nucleotide binding proteins (G1, elongation factor alpha 1, recombinant v-rasH p21 protein, transducin, Gi and Go), revealed that the GDP acts as a phosphate-acceptor, in the presence of divalent cations (Mg2+ and Ca2+). This finding suggests that via phosphate-transfer, NDP-kinase may be responsible for the direct activation of various guanine nucleotide binding proteins through phosphate-transfer by the enzyme.

Glycoform analysis of Japanese cypress pollen allergen, Cha o 1: A comparison of the glycoforms of cedar and Cypress pollen allergens

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen (Cry j 1), the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).

PS80 interferes with the antiallergic effect of Cry-consensus peptide, a novel recombinant peptide for immunotherapy of Japanese cedar pollinosis, at very low concentration through modulation of Th1/Th2 balance

Polysorbate 80 (PS80 or Tween‐80) is often used as an additive to promote the rapid solubilization of pharmaceuticals in aqueous solutions. We investigated whether coinjection of a minimal amount of PS80 had a modulatory effect on the immunotherapeutic effects of Cry (Cryptomeria)‐consensus peptide, a novel peptide developed for the therapeutic management of Japanese cedar pollinosis, using a Cry j 1‐sensitized mouse model with experimental allergic rhinitis. Subcutaneous challenge with Cry‐consensus peptide plus 50 µg/ml of PS80 did not affect the antigen‐specific proliferation of splenocytes, but decreased the potency of Cry‐consensus peptide to inhibit antigen‐specific interleukin (IL)‐5 production by the cells significantly in comparison with challenge with Cry‐consensus peptide alone. However, there was no significant difference between the effect of Cry‐consensus peptide administration on interferon (IFN)‐γ production in the presence and absence of PS80, indicating that PS80 interfered with the T helper 1 (Th1)‐dominant T helper balance induced by Cry‐consensus peptide challenge. Moreover, the increase in the level of antigen‐specific immunoglobulin G2a (IgG2a) induced by Cry‐consensus peptide challenge was inhibited slightly but unambiguously by PS80 coinjection. These in vitro experiments indicated that PS80 induces Th2‐type differentiation of T helper cells through preferential inhibition of IFN‐γ expression relative to IL‐5 expression in splenocytes in a concentration‐dependent manner. In naïve mice, sensitization by Cry‐consensus peptide with PS80 induced antigen‐specific IL‐5 production more potently than sensitization by Cry‐consensus peptide alone, and when PS80 was added to bone marrow‐derived dendritic cells, the endocytosis of fluorescence‐labelled Cry‐consensus peptide was dramatically inhibited in a concentration‐dependent manner. Therefore, we conclude that PS80 has an immunomodulatory effect on the antigen‐specific response resulting in a shift towards Th2 predominance with respect to the antigen recognition stage. Taken together, our findings suggest that PS80 might decrease the efficacy of Cry‐consensus peptide through modulation of the efficiency of antigen endocytosis and/or of the direction of successive T helper cell differentiation.

Rapid induction of nucleoside-diphosphate kinase in HeLa S3 cells by human-type interferons

Nucleoside‐diphosphate (NDP)‐kinase can be considered to be induced by human‐type interferons (HuIFNs) rapidly, since an enzyme increase was detected within 2 h of incubation of HeLa S3 cells with HuIFNs, while incubation with heterologous mouse IFNs had no such effect. The enzyme increase induced by HuIFNs reached a plateau at 6 h after treatment. Actinomycin D (0.5 ) significantly blocked the enzyme increase induced by HuIFNs in the cells. A possible biological role of the enzyme in the IFN‐induced biochemical events is discussed.

Complete recovery of the phosphoenzyme-forming activity of nucleoside-diphosphate kinases after reconstitution of their subunits

Two distinct subunits [α‐subunit (M r 21 000, pI 7.6) and β‐subunit (M r 19000, pI 6.5)] of nucleoside‐diphosphate (NDP) kinases highly purified from HeLa S3 cells can be separated by FPLC using a Mono P column in the presence of 6 M urea and 1% pharmalyte (pH range between 5.0 and 8.0). Comparatively high [32P]‐phosphate incorporation was detected when these two subunit fractions were reconstituted in vitro. Available evidence suggests that these two enzyme subunits are necessary for the formation of phosphoenzyme, which functions as an intermediate in NDP kinase action

Trypsin-Treated β-Lactoglobulin Improves Glucose Tolerance in C57BL/6 Mice by Enhancing AMPK Activation and Glucose Uptake in Hepatocytes

It was reported that trypsin-treated β-lactoglobulin (β-LG) had a glucose-lowering effect in the oral glucose tolerance test (OGTT) in mice and a dipeptidyl peptidase-4 (DPP-4) inhibition activity in vitro. However, whether trypsin-treated β-LG improves glucose tolerance by inhibiting DPP-4 in vivo has not yet been examined, and the mechanism of the glucose-lowering effect of trypsin-treated β-LG is thus unclear. Here we investigated the detailed mechanism underlying the glucose tolerance effect of trypsin-treated β-LG. The oral administration of trypsin-treated β-LG significantly decreased the blood glucose concentrations in both the OGTT and an intraperitoneal glucose tolerance test (IPGTT). However, trypsin-treated β-LG did not increase the insulin secretion after glucose loading. Trypsin-treated β-LG potently increased the level of phosphorylated AMP-activated protein kinase (AMPK) in human hepatocellular carcinoma (HepG2) cells and in mice hepatocytes. Moreover, trypsin-treated β-LG significantly enhanced glucose uptake into the HepG2 cells. These results indicate that trypsin-treated β-LG decreases blood glucose levels after glucose loading by upregulating AMPK activation and glucose uptake in the liver, which could contribute to the reduction of postprandial hyperglycemia.