Shigeharu Fukuda

Abstract The novel cytokine interferon‐γ‐inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)‐12. IGIF has been found to enhance the production of interferon‐γ (IFN‐γ) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) while inhibiting the production of IL‐10 in concanavalin A (Con A)‐stimulated PBMC. In this study, when anti‐CD3 monoclonal antibody (mAb)‐stimulated human enriched T cells were exposed to IGIF, the cytokine dose‐dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL‐2 at lower concentrations of IGIF. Neutralizing antibody against IFN‐γ had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme‐linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN‐γ; however, changes in the production of IL‐4 and IL‐10 were minimal. IGIF, but not IL‐12, significantly enhanced IL‐2 and GM‐CSF production in T cell cultures, as determined by CTLL‐2 bioassay and ELISA, respectively; however, both IGIF and IL‐12 enhanced IFN‐γ production by the T cells. When T cells were exposed to a combination of IGIF and IL‐12, a synergistic effect was observed on the production of IFN‐γ, but not on production of IL‐2 and GM‐CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL‐2‐dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL‐12 in terms of enhanced IFN‐γ production but not IL‐2 and GM‐CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin‐18.

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.

Trehalose is a disaccharide that attracts much attention as a stress protectant. In this study, we investigated the mechanism of the antioxidant function of trehalose. The spin-lattice relaxation times (T(1)) of (1)H and (13)C NMR spectra were measured to investigate the interaction between trehalose and unsaturated fatty acid (UFA). We selected several kinds of UFA that differ in the number of double bonds and in their configurations (cis or trans). Several other disaccharides (sucrose, maltose, neotrehalose, maltitol, and sorbitol) were also analyzed by NMR. The T(1) values for the (1)H and (13)C signals assigned to the olefin double bonds in UFA decrease with increasing concentration of trehalose and the changes reaches plateaus at integer ratios of trehalose to UFA. The characteristic T(1) change is observed only for the combination of trehalose and UFA with cis double bond(s). On the other hand, from the (13)C-T(1) measurements for trehalose, the T(1) values of the C-3 (C-3') and C-6' (C-6) are found to change remarkably by addition of UFA. (1)H[bond](1)H NOESY measurements provide direct evidence for complexation of trehalose with linoleic acid. These results indicate that one trehalose molecule stoichiometrically interacts with one cis-olefin double bond of UFA. Computer modeling study indicates that trehalose forms a stable complex with an olefin double bond through OH...pi and CH...O types of hydrogen bonding. Furthermore, a significant increase in the activation energy is found for hydrogen abstraction reaction from the methylene group located between the double bonds that are both interacting with the trehalose molecules. Therefore, trehalose has a significant depression effect on the oxidation of UFA through the weak interaction with the double bond(s). This is the first study to elucidate the antioxidant function of trehalose.