Hitoshi Nishimura

A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing

There are several isoforms of interleu kin (IL) ‐15 generated by alternating splicing. We reported previously that alternative IL‐15 transgenic (Tg) mice expressing an IL‐15 cDNA isoform encoding nonsecretable IL‐15 protein had an impaired ability to produce IL‐15. In this study, we found that expression of endogenous IL‐15 mRNA but not tumor necrosis factor a mRNA was severely impaired in response to lipopolysaccharide, not only in macrophages from al ternative IL‐15 Tg mice but also in RAW264.7 cells that had been transfected with alternative IL‐15 together with IL‐15 receptor α (IL‐15Rα). IL‐15 promoter activ ity was suppressed in the transfected cells. Although nuclear factor‐kB activation was not impaired, the binding activity of nuclear extracts to the interferon‐ stimulated response element of the IL‐15 promoter region was reduced in RAW264.7 cells, which had been cotransfected with alternative IL‐15 and IL‐15Rα. IL‐15 was mainly colocalized with IL‐15Rα at the cytoplasmic membrane of RAW264.7 cells, which had been cotrans fected with normal IL‐15, whereas nonsecretable IL‐15 was colocalized with IL‐15Rα in nucleus after cotrans fection with alternative IL‐15 and IL‐15Rα. These re sults suggest that nonsecretable IL‐15 generated by alternative splicing suppresses further IL‐15 gene tran scription, implying a novel autocrine regulatory mech anism for cytokine gene expression by alternative splicing.

A Novel Role of IL-15 in Early Activation of Memory CD8+ CTL after Reinfection

A rapid induction of effector functions in memory T cells provides rapid and intensified protection against reinfection. To determine potential roles of IL-15 in early expansion and activation of memory CD8+ T cells in secondary immune response, we examined the cell division and cytotoxicity of memory CD8+ T cells expressing OVA257–264/Kb-specific TCR that were transferred into IL-15-transgenic (Tg) mice, IL-15 knockout (KO) mice, or control C57BL/6 mice followed by challenge with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). In vivo CTL activities and expression of granzyme B of the transferred CD8+ T cells were significantly higher in the IL-15 Tg mice but lower in the IL-15 KO mice than those in control mice at the early stage after challenge with rLM-OVA. In contrast, there was no difference in the cell division in IL-15 Tg mice and IL-15 KO mice compared with those in control mice. In vivo administration of rIL-15 conferred robust protection against reinfection via induction of granzyme B in the memory CD8+ T cells. These results suggest that IL-15 plays an important role in early activation of memory CD8+ T cells.

NKT cells are dispensable in the induction of oral tolerance but are indispensable in the abrogation of oral tolerance by prostaglandin E

NK1.1+ αu2009β T cells (NKT cells) regulate the Th1/Th2 balance in response to dietary Ag, which may be involved in regulation of oral tolerance. OVA‐specific IgE and IgG1 Ab levels were significantly lower following an i.p. injection of OVA (in CFA) in C57BL/6 mice orally given a single, high dose (25u2004mg) of OVA than in those orally given PBS. The oral tolerance was normally induced in Jα281–/– mice which lack Vα14+ NKT cells, suggesting that NKT cells are dispensable for induction of oral tolerance. Treatment with PGE1 or PGE2 abrogated the oral tolerance in Jα281+/+ mice; this abrogation was accompanied by an OVA‐specific Th2‐dominant response. The abrogation of oral tolerance by PGE1 was not evident in Jα281–/– mice. Treatment with PGE1 induced an early increase in IL‐4 production by liver NKT cells in normal mice and neutralization of the early IL‐4 by administration of anti‐IL‐4 mAb abolished PGE1‐induced abrogation of oral tolerance. These results suggest that liver NKT cells producing IL‐4 are responsible for the down‐regulation of oral tolerance that is caused by the PGE molecules.

Soluble branched (1,4)-β-D-glucans from Acetobacter species enhance antitumor activities against MHC class I-negative and -positive malignant melanoma through augmented NK activity and cytotoxic T-cell response

We previously found that an extracellular polysaccharide, AC‐1, produced by Acetobacter polysaccharogenes composed of (1,4)‐β‐D‐glucan with branches of glucosyl residues showed a strong activity to induce production of interleukin (IL)‐12 p40 and tumor necrosis factor‐α by macrophage cell lines in vitro via Toll‐like receptor‐4 signaling. In the present study, we examined the effects of oral administration of AC‐1 on protection against 2 types of murine B16 melanoma lines, major histocompatibility complex (MHC) class I‐negative B16L and MHC class I gene‐transfected B16Kb cells. Mice were inoculated subcutaneously with B16L or B16Kb cells on day 0 and administrated intragastrically with AC‐1 or PBS once every 5 days from 1 day before tumor inoculation. The tumor growth was severely retarded in AC‐1‐treated mice after subcutaneous inoculation with B16L or B16Kb cells. The AC‐1‐treated mice showed augmented natural killer (NK) cell activity against B16L cells, and in vivo depletion of NK cells by antiasialoGM1 antibody (Ab) treatment abrogated the antitumor activity in AC‐1‐treated mice. On the other hand, AC‐1‐treated mice inoculated with B16Kb cells developed a significantly higher level of cytotoxic T‐lymphocyte response against B16Kb cells, and in vivo depletion of CD8+ T cells by anti‐CD8 mAb treatment abrogated the antitumor activity. Thus, AC‐1 augmented antitumor activity against different tumors via augmentation of different antitumor mechanisms. These results suggest a possible prophylactic application of AC‐1 for human neoplasms irrespective of expression levels of their MHC class I molecules.

A New Trisaccharide Sugar Chain Linked to a Serine Residue in the First EGF-Like Domain of Clotting Factors VII and IX and Protein Z

Recently, we determined the complete amino acid sequence of bovine factor VII (Takeya, H. et al. (1988) J. Biol. Chem. 263, 14868-14877). In the course of the studies, we found an unknown serine derivative at position 52 in the first epidermal growth factor-like domain of factor VII. A pentapeptide isolated from the S-aminoethylated factor VII contained Ser-52, which could not be identified with a gas-phase sequencer. The same results were also obtained for a pentapeptide containing Ser-53 of factor IX and protein Z. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was identified as pyridylamino-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII, IX and protein Z. Similar results were obtained for human factors VII, IX and protein Z. This is the first report of a (Xyl2)-Glc-Ser structure in glycoproteins to our knowledge. The presence of the unique trisaccharide structure in factors VII, IX and protein Z leads us to anticipate its biological role in the tissue factor pathway.