Biomedical subjects

Mitsuomi Hirashima

Publications and source records attributed to Mitsuomi Hirashima.

31 records · Page 2Linked to original sources

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

Oligosaccharide specificity of galectins: a search by frontal affinity chromatography.

Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.

Animals↗

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

Biological activities of ecalectin: a novel eosinophil-activating factor.

Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for beta-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the beta-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.

Antibodies↗

PubMed2002

CD35 expression on peripheral blood granulocytes of patients with atopic dermatitis.

We examined CD35 expression on granulocytes from 45 patients with atopic dermatitis (AD) (male, 21, female, 24) and 25 age and sex-matched controls (male, 15; female, 10). There was no significant difference in the peripheral blood neutrophil count between AD patients and controls, whereas the eosinophil count in AD patients was significantly higher than that of controls. The percentages of CD35 positive eosinophils and neutrophils were determined by using two-color flow cytometric analysis. As regards eosinophils, we found CD35 expression from AD patients to be lower than that of controls (P < 0.05), but there was no correlation between the CD35 expression and disease severity. In contrast, the CD35 expression on neutrophils from AD patients was much lower than that of controls (P < 0.005). Furthermore, CD35 expression on neutrophils of severe AD group was significantly higher than that of the mild AD group (P < 0.05). This suggests that the CD35 expression of neutrophils but not eosinophils reflects disease severity in AD patients and the CD35 expression on neutrophils in AD patients may associate with susceptibility to bacterial infection.

Administration, Oral↗

PubMed2002

Galectin-9 in physiological and pathological conditions.

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings.

Apoptosis↗

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

Purification and cDNA cloning of Xenopus liver galectins and their expression.

We have characterized galectin family proteins in adult tissues of Xenopus laevis and purified 14-kDa and 36-kDa proteins from the liver. The liver galectins showed comparable hemagglutination activities to those of mammalian galectins. Furthermore, we isolated five galectin cDNAs from a Xenopus liver library. These cDNAs revealed that X. laevis galectins (xgalectins) form a family consisting of at least proto and tandem repeat types based on their domain structures, like the mammalian galectin family. Two proto-type xgalectins, -Ia and -Ib, exhibited a high sequence identity (91%) with each other at the amino acid level and were most similar (49-50% identity) to human galectin-1. From their sequence similarity and ubiquitous tissue distributions, xgalectins-Ia and -Ib both seemed to be Xenopus homologues of mammalian galectin-1. Three tandem repeat-type xgalectins were newly identified. Two of them, xgalectins-IIa and -IIIa, seemed to be homologous to human galectins-4 and -9, respectively, judging from their high sequence similarities (42-50% identity). However, xgalectin-IVa seemed to be a novel type. Distributions of mRNAs of xgalectins were analyzed by northern hybridization. In addition to adult tissues, either of three tandem repeat-type xgalectins were expressed in whole embryos. Moreover, amino acid sequence analysis of liver proteins indicated that xgalectins-Ia, -IIa, and -IIIa are produced as abundant galectins in the adult liver.

Amino Acid Sequence↗

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

Quantification of galectin-7 and its localization in adult mouse tissues.

We developed a method to quantify galectin-7 extracted from adult mouse tissues by Western blot analysis. More than 0.5 ng of galectin-7 per mg of tissue was detectable by this method. The amounts of galectin-7 in tissues were determined as follows: skin, 62 +/- 3 ng/mg; esophagus, 23 +/- 8 ng/mg; stomach, 18 +/- 6 ng/mg; anus, 13 +/- 1 ng/mg; and tongue, 12 +/- 2 ng/mg. This indicates that galectin-7 production coincides with the degree of stratification of the epithelia. Interestingly, we also detected significant amounts of galectin-7, 5.9 plus minus 1.4 and 2.7 +/- 0.6 ng/mg, in the trachea and ovaries, respectively. Moreover, we found that galectin-7 is localized in the pseudostratified epithelium of the trachea and stromal epithelium of the ovaries by immunohistochemistry. Thus, galectin-7 protein might be produced primarily in stratified epithelia, but also in some wet epithelia, and plays a unique role in cell-mucus contact, or the growth of ovarian follicles.

Animals↗

PubMed2002

Association of galectin-9 with eosinophil apoptosis.

BACKGROUND: There is no information whether galectin-9 (a novel eosinophil chemoattractant) was associated with pathogenesis of eosinophilic disorders. METHODS: We assessed the expression of galectin-9 with imunostaining and in situ hybridization both in the lesion of angiolymphoid hyperplasia with eosinophilia, and peripheral blood eosinophils of eosinophilic patients (E-Eos) in comparison with those of normal volunteers (N-Eos). Regulation of expression of galectin-9 on eosinophils and the effect of galectin-9 on apoptosis of eosinophil were also evaluated. RESULTS: Many eosinophils infiltrating the site were positive for galectin-9. Surface and intracellular immunoreactive galectin-9 was more evident in E-Eos than N-Eos. When eosinophils were cultured with IL-5 in vitro, the surface galectin-9 expression of E-Eos was significantly downregulated, although that of N-Eos was not affected. Treatment of eosinophils with dexamethasone or anti-Fas antibody significantly upregulated the surface galectin-9 expression of E-Eos. In contrast, dexamethasone partially downregulated the surface galectin-9 of N-Eos, although anti-Fas antibody failed to affect on the surface galectin-9 expression. We also found that recombinant galectin-9 significantly suppressed apoptosis of E-Eos (p = 0.0431), whereas it apparently enhanced apoptosis of N-Eos (p = 0.0173). Furthermore, dexamethasone-induced apoptosis of N-Eos was significantly suppressed by galectin-9 (p = 0.0431), whereas galectin-9 failed to induce significant change in dexamethasone-induced apoptosis of E-Eos. In contrast, apoptosis induced by anti-Fas antibody in both N-Eos (p = 0.0431) and E-Eos (p = 0.0431) was enhanced by galectin-9. CONCLUSIONS: These findings suggested that galectin-9 was produced by eosinophils, and galectin-9 showed heterogeneous effects and kinetics to eosinophils, and this factor might be one of crucial factors in eosinophilic inflammation.

Angiolymphoid Hyperplasia with Eosinophilia↗

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↗

PubMedDate not supplied

Galectin-9, a novel prognostic factor with antimetastatic potential in breast cancer.

Galectin-9, a member of the beta-galactoside-binding animal lectin family, is involved in various cellular biological events, including aggregation and apoptosis, adhesion of cancer cells, and dendritic cell maturation. We recently reported the relationship between galectin-9 expression in tumor tissue and distant metastasis in breast cancer. Tumors in 42 of the 84 patients were galectin-9-positive, and tumors in 19 of the 21 patients with distant metastasis were galectin-9-negative, assessed by immunohistochemistry. The cumulative distant metastasis-free survival ratio for galectin-9-positive patients was better than for the galectin-9-negative group (p < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independent of and much more than lymph node metastasis. MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in nude mice as well as in culture. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins.

Adult↗