Biomedical subjects

Masako Seki

Publications and source records attributed to Masako Seki.

9 recordsLinked to original sources

PubMed2003

Galectin-8 modulates neutrophil function via interaction with integrin alphaM.

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.

Actins↗

PubMed2003

Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway.

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

Adjuvants, Immunologic↗

PubMed2003

Evolutional study on acetylcholine expression.

Acetylcholine (ACh) is a well-known neurotransmitter in the cholinergic nervous systems of vertebrates and insects; however, there is only indirect evidence for its presence in lower invertebrates, such as plants and fungi. We therefore investigated the expression of ACh in invertebrates (sea squirt, sea urchin, trepang, squid, abalone, nereis, sea anemone, coral and sponge), plants (arabidopsis, eggplant, bamboo shoot, cedar, hinoki, pine, podcarp, fern, horsetail and moss), fungi (yeast and mushroom) and bacteria by assaying ACh content and synthesis, focusing on the presence of two synthetic enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). Using a specific radioimmunoassay, ACh was detected in all samples tested. The levels varied considerably, however, with the upper portion of bamboo shoots having the highest content (2.9 micromol/g). ACh synthesis was also detected in all samples tested; moreover, the activity in most samples from the animal kingdom, as well as bamboo shoots and the stem of the shiitake mushroom, were sensitive to both ChAT and CarAT inhibitors. Levels of ACh synthesis were lower in samples from other plants, fungi and bacteria and were insensitive to ChAT and CarAT inhibitors. These findings demonstrate the presence of ACh and ACh-synthesizing activity in evolutionally primitive life as well as in more complex multicellular organisms. In the context of the recent discovery of non-neuronal ACh in various mammalian species, these findings suggest that ACh been expressed in organisms from the beginning of life, functioning as a local mediator as well as a neurotransmitter.

Acetylcholine↗

PubMed2003

Potential roles of galectins in myeloid differentiation into three different lineages.

Little is known about the roles of galectins, a family of beta-galactoside-binding lectins, in myeloid cell differentiation. In the present experiments, we used HL-60 cells as a model of myeloid cell differentiation. The HL-60 cells were differentiated into eosinophil-, monocyte-, and neutrophil-like cells by coculture with sodium butyrate under a mild alkaline condition, phorbol 12-myristate 13-acetate, and dimethyl sulfoxide, respectively. Thus, the expression of galectins in HL-60 cells during differentiation into three different lineages was assessed. Reverse transcriptase-polymerase chain reaction analyses revealed that undifferentiated HL-60 cells expressed galectin-1, -3, -8, -9, and -10 (identical to Charcot Leyden crystal) mRNAs, and galectin-2, -4, and -7 were negligible before and after the differentiations. We failed to detect evident changes in the mRNA levels of galectin-1 and -8 during the differentiations. However, during the eosinophilic differentiation, galectin-9 mRNA expression was gradually decreased, whereas galectin-10 mRNA expression was increased. During the course of monocytic differentiation, galectin-9 mRNA expression was down-regulated, whereas galectin-3 mRNA expression was up-regulated. Moreover, only galectin-10 mRNA expression was enhanced in the process of neutrophilic differentiation. These changes in galectin expressions were confirmed by Western blot and flow cytometry analyses. It is thus suggested that changes in the expressions of galectin-3, -9, and -10 are potentially important for myeloid cell differentiation into specific lineages.

Butyrates↗

PubMed2002

Selective eosinophil adhesion to fibroblast via IFN-gamma-induced galectin-9.

Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-gamma stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-gamma up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-gamma up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-gamma up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-gamma-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-gamma-stimulated fibroblasts as a physiological modulator at the inflammatory sites.

Adult↗

PubMed2002

Possible role of galectin-9 in cell aggregation and apoptosis of human melanoma cell lines and its clinical significance.

Galectin-9 expression was examined in 6 human melanoma cell lines. Among them, MM-BP proliferated with colony formation, but MM-RU failed. RT-PCR analysis revealed evident expression of galectin-9 mRNA in MM-BP but not in MM-RU. MM-BP expressed galectin-9 protein both on the surface and in the cytoplasm, whereas MM-RU expressed it only weakly in the cytoplasm. Exogenous galectin-9 induced in vitro both cell aggregation and apoptosis of MM-RU proliferating without colony formation. Association of galectin-9 expression in melanoma cells with prognosis of the patients bearing melanocytic tumors was further examined. Galectin-9 protein was strongly and homogeneously expressed in melanocytic nevi, but down-regulated in melanoma cells especially in metastatic lesions. High galectin-9 expression was inversely correlated with the progression of this disease, suggesting that high galectin-9 expression in primary melanoma lesions links to a better prognosis.

Annexin A5↗

PubMed2002

Regulation of galectin-9 expression and release in Jurkat T cell line cells.

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.

Animals↗

PubMed2002

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity.

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.

Base Sequence↗

PubMed2002

Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells.

Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-gamma (IFN-gamma). IFN-gamma also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-gamma-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Autoimmune Diseases↗