Junichiro Fujimoto

CD24 Induces Apoptosis in Human B Cells Via the Glycolipid-Enriched Membrane Domains/Rafts-Mediated Signaling System

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt’s lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.

Induction of Apoptosis in Normal Human Renal Tubular Epithelial Cells by Escherichia coli Shiga Toxins 1 and 2

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Kinetic Analysis of Binding between Shiga Toxin and Receptor Glycolipid Gb3Cer by Surface Plasmon Resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the “bivalent analyte” model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

The Detection of Shiga Toxins in the Kidney of a Patient with Hemolytic Uremic Syndrome

Infection of Shiga toxin (Stx)-producing Escherichia coli induces hemolytic uremic syndrome (HUS) in 10 to 15% of cases in infants and young children. Although the endothelial cell damage induced by Stx is widely believed to be a primary event of renal dysfunction in HUS, the precise mechanism remains to be elucidated. We were able to examine the kidney obtained at autopsy of a child who died after HUS associated with Stx-producing Escherichia coli O157:H7 infection, and immunohistochemistry indicated the deposition of Stx1 and Stx2 in a portion of the distal tubular epithelia. To our knowledge, this is the first report to show the presence of Stx in human tissue of a patient with HUS, and the results obtained in this study provide evidence that Stx indeed migrates into the kidney and binds to renal tubules during Stx-producing Escherichia coli infection.

Improved outcome in the treatment of pediatric multifocal Langerhans cell histiocytosis: Results from the Japan Langerhans Cell Histiocytosis Study Group-96 protocol study.

The treatment outcome of multifocal childhood Langerhans cell histiocytosis (LCH) has not been satisfactory and has resulted in poor therapeutic responses with high mortality and a high incidence of reactivation with late sequelae. To overcome these issues, the Japan LCH Study Group‐96 (JLSG‐96) protocol was conducted prospectively from 1996 to 2001 in Japan.

Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells

ABSTRACT Ewings family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor–mediated apoptosis by regulating Lyn kinase activity in human B cells

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitts lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells

Shiga toxin is a bacterial toxin consisting of A and B subunits. Generally, the essential cytotoxicity of the toxin is thought to be mediated by the A subunit, which possesses RNA cleavage activity and thus induces protein synthesis inhibition. We previously reported, however, that the binding of the Shiga toxin 1-B subunit to globotriaosyl ceramide, a functional receptor for Shiga toxin, induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Although the precise role of this signaling mechanism is not known, here we report that Shiga-toxin-mediated intracellular signals induce cytoskeleton remodeling in ACHN cells derived from renal tubular epithelial carcinoma. Using confocal laser scanning microscopy, we observed that Shiga toxin 1-B treatment induces morphological changes in ACHN cells in a time-dependent manner. In addition, the morphological changes were accompanied by the redistribution of a number of proteins, including actin, ezrin, CD44, vimentin, cytokeratin, paxillin, FAK, and α- and γ-tubulins, all of which are involved in cytoskeletal organization. The transient phosphorylation of ezrin and paxillin was also observed during the course of protein redistribution. Experiments using inhibitors for a variety of kinases suggested the involvement of lipid rafts, Src family protein kinase, PI 3-kinase, and RHO-associated kinase in Shiga toxin 1-B-induced ezrin phosphorylation. Shiga toxin 1-B-induced cytoskeletal remodeling should provide an in vitro model that can be used to increase our understanding of the pathogenesis of Shiga-toxin-mediated cell injury and the role of lipid-raft-mediated cell signaling in cytoskeletal remodeling.

A role for lipid rafts in immune cell signaling.

Cross‐linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src‐family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.

Galactosemic neuropathy in transgenic mice for human aldose reductase.

We studied the functional consequences of an enhanced polyol pathway activity, elicited with galactose feeding, on the peripheral nerve of transgenic mice expressing human aldose reductase. Nontransgenic littermate mice were used as controls. With a quantitative immunoassay, the expression level of human aldose reductase in the sciatic nerve was 791 ± 44 ng/mg protein (mean ± SE), about 25% of that in human sural nerve. When the transgenic mice were fed food containing 30% galactose, significant levels of galactitol accumulated in the sciatic nerve. Galactose feeding of nontransgenic littermate mice led to a 10-fold lower accumulation of galactitol. Galactose feeding for 16 weeks caused a significant and progressive decrease in motor nerve conduction velocity in transgenic mice to 80% of the level of galactose-fed littermate mice, which was not significantly different from that of galactose-free littermate mice. A morphometric analysis of sciatic nerve detected > 10% reduction of mean myelinated fiber size but no alterations of myelinated fiber density in galactose-fed transgenic mice compared with other groups. The functional and structural changes that develop in galactose-fed transgenic mice are similar to those previously reported in diabetic animals. The results of these studies suggest that transgenic mice expressing human aldose reductase may be a useful model not only for defining the role of the polyol pathway in diabetic neuropathy but also for identifying and characterizing effective inhibitors specific for human aldose reductase.