The cDNA encoding of a phospholipase A(2) inhibitor (PLIalpha) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIalpha. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIalpha. The PLIalpha cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIalpha expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIalpha fusion protein underwent folding to form a trimeric structure like the intact PLIalpha, and showed inhibitory activity against the group II acidic PLA(2) from A. blomhoffii siniticus venom; although its binding constant (1/K(i)) value was 30-fold lower than that of the natural PLIalpha. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIalpha against the acidic PLA(2). Furthermore, the carbohydrate chains of the natural PLIalpha were found to play an important role in the inhibitory activity against the basic PLA(2).

Infection of inbred mouse strains with Leishmania major is a well characterized model for analysis of T helper (Th)1 and Th2 cell development in vivo. In this study, to address the role of costimulatory molecules CD27, CD30, 4-1BB, and OX40, which belong to the tumor necrosis factor receptor superfamily, in the development of Th1 and Th2 cells in vivo, we administered monoclonal antibody (mAb) against their ligands, CD70, CD30 ligand (L), 4-1BBL, and OX40L, to mice infected with L. major. Whereas anti-CD70, anti-CD30L, and anti-4-1BBL mAb exhibited no effect in either susceptible BALB/c or resistant C57BL/6 mice, the administration of anti-OX40L mAb abrogated progressive disease in BALB/c mice. Flow cytometric analysis indicated that OX40 was expressed on CD4(+) T cells and OX40L was expressed on CD11c(+) dendritic cells in the popliteal lymph nodes of L. major-infected BALB/c mice. In vitro stimulation of these CD4(+) T cells showed that anti-OX40L mAb treatment resulted in substantially reduced production of Th2 cytokines. Moreover, this change in cytokine levels was associated with reduced levels of anti-L. major immunoglobulin (Ig)G1 and serum IgE. These results indicate that anti-OX40L mAb abrogated progressive leishmaniasis in BALB/c mice by suppressing the development of Th2 responses, substantiating a critical role of OX40-OX40L interaction in Th2 development in vivo.

TWEAK, a new member of the tumor necrosis factor (TNF) family, induces cell death in some tumor cell lines, but its physiological functions are largely unknown. In this study, we investigated the expression and function of TWEAK in human peripheral blood mononuclear cells (PBMCs) by using newly generated anti-human TWEAK mAbs. Although freshly isolated PBMCs expressed no detectable level of TWEAK on their surfaces, a remarkable TWEAK expression was rapidly observed on monocytes upon stimulation with interferon (IFN)-gamma but not with IFN-alpha or lipopolysaccharide. Cytotoxic activity of IFN-gamma-stimulated monocytes against human squamous carcinoma cell line HSC3 was inhibited partially by anti-TWEAK mAb alone and almost completely by combination with anti-TRAIL (TNF-related apoptosis-inducing ligand) mAb. These results revealed a novel pathway of monocyte cytotoxicity against tumor cells that is mediated by TWEAK and potentiated by IFN-gamma.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a proapoptotic member of the TNF family of type II membrane proteins, which constitutes one component of T cell cytotoxicity. In this study, we investigated the expression and function of TRAIL in human peripheral blood T (PBT) cells. Although freshly isolated PBT cells did not express a detectable level of TRAIL on their surface, a remarkable TRAIL expression was rapidly induced on the surface of both CD4(+) and CD8(+) PBT cells upon stimulation with anti-CD3 monoclonal antibody and type I interferons (IFNs). This enhancement of TRAIL expression was a unique feature of type I IFNs (IFN-alpha and IFN-beta), and neither type II IFN (IFN-gamma) nor various other cytokines enhanced TRAIL expression on anti-CD3-stimulated PBT cells. Type I IFNs have been used for clinical treatment of renal cell carcinomas (RCCs), and we found that most RCC cell lines were susceptible to TRAIL-induced apoptosis. Type I IFNs substantially augmented cytotoxic activity of anti-CD3-stimulated PBT cells against RCC cell lines in a TRAIL-dependent manner. These results indicate a unique feature of type I IFNs to regulate TRAIL-mediated T cell cytotoxicity, which may be involved in the antitumor effects of type I IFNs against various tumors.

A new member of the mammalian period gene family, mPer3, was isolated and its expression pattern characterized in the mouse brain. Like mPer1, mPer2 and Drosophila period, mPer3 has a dimerization PAS domain and a cytoplasmic localization domain. mPer3 transcripts showed a clear circadian rhythm in the suprachiasmatic nucleus (SCN). Expression of mPer3 was not induced by exposure to light at any phase of the clock, distinguishing this gene from mPer1 and mPer2. Cycling expression of mPer3 was also found outside the SCN in the organum vasculosum lamina terminalis (OVLT), a potentially key region regulating rhythmic gonadotropin production and pyrogen-induced febrile phenomena. Thus, mPer3 may contribute to pacemaker functions both inside and outside the SCN.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.

TGF-beta signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-beta receptor induces phosphorylation of two such proteins, Smad2 and Smad3, which form hetero-oligomeric complex(es) with Smad4/DPC4 that translocate to the nucleus, where they then regulate transcriptional responses. However, the mechanisms by which the intracellular signals of TGF-beta are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signalling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses.

CD40 activates nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) subfamily, including extracellular signal-regulated kinase (ERK). The CD40 cytoplasmic tail interacts with tumor necrosis factor receptor-associated factor (TRAF)2, TRAF3, TRAF5, and TRAF6. These TRAF proteins, with the exception of TRAF3, are required for NF kappa B activation. Here we report that transient expression of TRAF6 stimulated both ERK and NF kappa B activity in the 293 cell line. Coexpression of the dominant-negative H-Ras did not affect TRAF6-mediated ERK activity, suggesting that TRAF6 may activate ERK along a Ras-independent pathway. The deletion mutant of TRAF6 lacking the NH2-terminal domain acted as a dominant-negative mutant to suppress ERK activation by full-length CD40 and suppress prominently ERK activation by a deletion mutant of CD40 only containing the binding site for TRAF6 in the cytoplasmic tail (CD40 delta 246). Transient expression of the dominant-negative H-Ras significantly suppressed ERK activation by full-length CD40, but marginally suppressed ERK activation by CD40 delta 246, compatible with the possibility that TRAF6 is a major transducer of ERK activation by CD40 delta 246, whose activity is mediated by a Ras-independent pathway. These results suggest that CD40 activates ERK by both a Ras-dependent pathway and a Ras-independent pathway in which TRAF6 could be involved.

Members of the transforming growth factor-beta (TGF-beta) family transmit signals from membrane to nucleus via intracellular proteins known as Smads. A subclass of Smad proteins has recently been identified that antagonize, rather than transduce, TGF-beta family signals. Smad7, for example, binds to and inhibits signaling downstream of TGF-beta receptors. Here we report that the C-terminal MAD homology domain of murine Smad7 (mSmad7) is sufficient for both of these activities. In addition, we show that mSmad7 interacts with activated bone morphogenetic protein (BMP) type I receptors (BMPR-Is), inhibits BMPR-I-mediated Smad phosphorylation, and phenocopies the effect of known BMP antagonists when overexpressed in ventral cells of Xenopus embryos. Xenopus Smad7 (XSmad7, previously termed Smad8) and mSmad7 are nearly identical within their bioactive C-domain, but have quite distinct N-domains. We found that XSmad7, similar to mSmad7, interacted with BMP and TGF-beta type I receptors and inhibited receptor-mediated phosphorylation of downstream signal-transducing Smads. However, XSmad7 is a less efficient inhibitor of TbetaR-I-mediated responses in mammalian cells than is mSmad7. Furthermore, overexpression of XSmad7 in Xenopus embryos produces patterning defects that are not observed following overexpression of mSmad7, suggesting that mSmad7 and XSmad7 may preferentially target distinct signaling pathways. Our results are consistent with the possibility that the C-domain of antagonistic Smads is an effector domain whereas the N-domain may confer specificity for distinct signaling pathways.

Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1) is known to function as a negative feedback regulator of cytokine signaling, but it is unclear whether it is involved in other biological events. Here, we show that SSI-1 participates and plays an important role in the insulin signal transduction pathway. SSI-1-deficient mice showed a significantly low level of blood sugar. While the forced expression of SSI-1 reduced the phosphorylation level of insulin receptor substrate 1 (IRS-1), SSI-1 deficiency resulted in sustained phosphorylation of IRS-1 in response to insulin.Furthermore, SSI-1 achieves this inhibition both by binding directly to IRS-1 and by suppressing Janus kinases. These findings suggest that SSI-1 acts as a negative feedback factor also in the insulin signal transduction pathway through the suppression of IRS-1 phosphorylation.

Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.

No abstract available

The contribution of antibody and/or immune-complex to the pathogenesis of immunologically-mediated glomerulonephritis is not fully understood, although it has been recently clarified that Fc receptors (FcRs) play critical roles in the inflammatory cascade. We therefore re-evaluated the classical model of glomerulonephritis, anti-glomerular basement membrane antibody-induced glomerulonephritis (Anti-GBM GN), from the standpoint of FcRs and also investigated the residual FcR-independent mechanisms.

Transforming growth factor (TGF)-beta has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-beta contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-beta/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-beta in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-beta/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-beta was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-beta/Smad signaling in mature T cells as a regulatory component of allergic asthma.

Leukotriene (LT) B4 is a well-known inflammatory mediator and is implied to play some roles in glomerulonephritis. Although LTA4 hydrolase, a final-step key enzyme to produce LTB4, is located in glomerular mesangial cells, as well as in leukocytes, platelets, and endothelial cells, its precise distribution in the kidney other than in mesangial cells remains unknown. Therefore, we have investigated the localization of mRNA, protein, and enzyme activity of LTA4 hydrolase in the rat kidney.

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor-beta (TGF-beta) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it was phosphorylated by TbetaR-II kinase. TGF-beta1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-beta1 stimulation. Similar results were obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interacted with Smad4 after TbetaR activation in transfected COS cells. In addition, we observed TbetaR-activation-dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction.