Hitomi Hoshino

Preferential induction of peripheral lymph node addressin on high endothelial venule-like vessels in the active phase of ulcerative colitis.

OBJECTIVES:In the colonic mucosa with ulcerative colitis (UC), it has been suggested that L-selectin-peripheral lymph node addressin (PNAd) interaction plays a role in lymphocyte recruitment, which requires PNAd induction on high endothelial venule (HEV)-like vessels. The present study was undertaken to elucidate how these HEV-like vessels participate in the pathogenesis of UC and also to determine whether the presence of such vessels is correlated with clinical outcomes.METHODS:Biopsy specimens composed of active (N = 32) and remission (N = 12) phases of UC were subjected to immunohistochemistry for CD34, MECA-79, and HECA-452, and the immunostained sections were quantitatively analyzed. An in vitro binding assay with L-selectin•IgM chimeric protein was carried out to determine whether PNAd on HEV-like vessels formed in UC functions as an L-selectin ligand. RT-PCR was carried out to determine which enzyme is upregulated for PNAd biosynthesis on HEV-like vessels induced in the active phase of UC. Triple immunostaining for MECA-79 together with CD3 and CD20/CD79α, CD4 and CD8, or CXCR3 and ST2L was carried out to determine which lymphocyte population closely associates with these vessels.RESULTS:PNAd-expressing HEV-like vessels were preferentially induced in the active phase of UC with increased transcription of the gene encoding N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1, which directs expression of the MECA-79 epitope. Moreover, T cells, particularly CD4+ T cells, were more closely associated with these HEV-like vessels than B cells.CONCLUSIONS:T-cell recruitment via PNAd-expressing HEV-like vessels induced by expression of GlcNAc6ST-1 may play a role in UC pathogenesis.

α1,4GlcNAc-capped mucin-type O-glycan inhibits cholesterol α-glucosyltransferase from Helicobacter pylori and suppresses H. pylori growth

Helicobacter pylori infects over half of the worlds population and is thought to be a leading cause of gastric ulcer, gastric carcinoma, and gastric malignant lymphoma of mucosa-associated lymphoid tissue type. Previously, we reported that a gland mucin (MUC6) present in the lower portion of the gastric mucosa containing alpha1,4-N-acetylglucosamine (alpha1,4GlcNAc)-capped core 2-branched O-glycans suppresses H. pylori growth by inhibiting the synthesis of alpha-glucosyl cholesterol, a major constituent of the H. pylori cell wall (Kawakubo et al. 2004. Science. 305:1003-1006). Therefore, we cloned the genomic DNA encoding cholesterol alpha-glucosyltransferase (HP0421) and expressed its soluble form in Escherichia coli. Using this soluble HP0421, we show herein that HP0421 sequentially acts on uridine diphosphoglucose and cholesterol in an ordered Bi-Bi manner. We found that competitive inhibition of HP0421 by alpha1,4GlcNAc-capped core 2-branched O-glycan is much more efficient than noncompetitive inhibition by newly synthesized alpha-glucosyl cholesterol. Utilizing synthetic oligosaccharides, alpha-glucosyl cholesterol, and monosaccharides, we found that alpha1,4GlcNAc-capped core 2-branched O-glycan most efficiently inhibits H. pylori growth. These findings together indicate that alpha1,4GlcNAc-capped O-glycans suppress H. pylori growth by inhibiting HP0421, and that alpha1,4GlcNAc-capped core 2 O-glycans may be useful to treat patients infected with H. pylori.

GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis.

Background: A diffuse lymphocyte infiltrate is 1 of the characteristic features of ulcerative colitis (UC). Such lymphocyte recruitment requires lymphocyte rolling mediated by L‐selectin ligand carbohydrates (6‐sulfo sialyl Lewis X‐capped O‐glycans) and/or mucosal addressin cell adhesion molecule 1 (MAdCAM‐1) expressed on high endothelial venule (HEV)‐like vessels. The present study was undertaken to elucidate the role of MAdCAM‐1 posttranslationally modified (“decorated”) with L‐selectin ligand carbohydrates in UC pathogenesis and consequent clinical outcomes. Methods: Biopsy specimens composed of active and remission phases of UC as well as normal colonic mucosa were immunostained for CD34, MAdCAM‐1, and MECA‐79, and the immunostained sections were quantitatively analyzed. Reverse‐transcriptase polymerase chain reaction (RT‐PCR) was carried out to evaluate transcripts of MAdCAM‐1 and N‐acetylglucosamine‐6‐O‐sulfotransferases (GlcNAc6STs). CHO and Lec2 cells transfected with CD34 and MAdCAM‐1 together with enzymes involved in L‐selectin ligand carbohydrate biosynthesis were analyzed by immunofluorescence, FACS, and Western blotting to characterize the biochemical properties of GlcNAc6STs. Results: The number of MAdCAM‐1+ vessels was increased in UC, with no significant difference between active and remission phases. An increased ratio of MECA‐79+ to MAdCAM‐1+ vessels with preferential GlcNAc6ST‐1 transcripts was observed in the active phase of UC compared to the remission phase. MAdCAM‐1 protein was colocalized with L‐selectin ligand carbohydrates at the luminal surface of HEV‐like vessels in situ. GlcNAc6ST‐1 preferentially utilizes MAdCAM‐1 as a scaffold protein for GlcNAc‐6‐O‐sulfation in L‐selectin ligand carbohydrate biosynthesis. Conclusions: UC disease activity is not regulated by expression of MAdCAM‐1 protein itself, but rather by GlcNAc6ST‐1‐mediated decoration of MAdCAM‐1 protein with L‐selectin ligand carbohydrates.

Helicobacter pylori Cholesteryl α-Glucosides Contribute to Its Pathogenicity and Immune Response by Natural Killer T Cells

Approximately 10–15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18−/−) or wild-type mice, bacterial recovery significantly increased in Jα18−/− compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18−/− compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.

Prominent expression of sialyl Lewis X-capped core 2-branched O-glycans on high endothelial venule-like vessels in gastric MALT lymphoma.

High endothelial venule (HEV)‐like vessels have been observed in gastric B cell lymphoma of mucosa‐associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV‐like vessels in the latter lesion served as L‐selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)‐related glycoepitopes and found that MECA‐79−/HECA‐452+/NCC‐ST‐439+ HEV‐like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA‐79−/HECA‐452+/NCC‐ST‐439+ glycans, as well as other sLeX‐type glycans, on CD34 and evaluated L‐selectin binding to those cells, using L‐selectin–IgM chimera binding and lymphocyte adhesion assays. L‐selectin–IgM chimeras bound to CHO cells expressing 6‐sulpho‐sLeX attached to core 2‐branched O‐glycans with or without 6‐sulpho‐sLeX attached to extended core 1 O‐glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6‐sulpho‐sLeX attached to extended core 1 and/or core 2‐branched O‐glycans, as well as non‐sulphated sLeX attached to core 2‐branched O‐glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6‐sulpho‐ and non‐sulphated sLeX attached to N‐glycans and non‐sulphated sLeX attached to extended core 1 O‐glycans. These results indicate that MECA‐79−/HECA‐452+/NCC‐ST‐439+ glycans, specifically, 6‐sulpho‐ and non‐sulphated sLeXs attached to core 2‐branched O‐glycans, expressed on HEV‐like vessels in gastric MALT lymphoma function as L‐selectin ligands and likely contribute to H. pylori‐specific T cell recruitment in the progression of gastric MALT lymphoma. Copyright

Heparan Sulfate Subdomains that are Degraded by Sulf Accumulate in Cerebral Amyloid ß Plaques of Alzheimer's Disease: Evidence from Mouse Models and Patients

Alzheimers disease (AD) is characterized by extracellular cerebral accumulation of amyloid β peptide (Aβ). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aβ aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains.

KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.

A potential role for 6-sulfo sialyl Lewis X in metastasis of bladder urothelial carcinoma

OBJECTIVES It is widely accepted that sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA, also known as CA 19-9) glycans expressed on cancer cells function in E-selectin-mediated metastasis. Recently, it was reported that 6-sulfo sLeX glycans detected by the MECA-79 monoclonal antibody are expressed in roughly a quarter of gastric adenocarcinoma cases, and that these cases show a poorer prognosis than MECA-79-negative cases do. The present study was undertaken to assess expression of 6-sulfo sLeX glycans in bladder urothelial carcinoma and evaluate potential clinical implications. MATERIALS AND METHODS We analyzed 78 specimens representing bladder urothelial carcinoma, as well as 4 bladder urothelial carcinoma cell lines, by immunostaining with a battery of anticarbohydrate antibodies. We also undertook an E-selectin·IgM chimera binding assay to assess E-selectin binding to 6-sulfo sLeX expressed on bladder urothelial carcinoma cells and performed reverse transcription polymerase chain reaction and complementary DNA transfection to determine which N-acetylglucosamine-6-O-sulfotransferases function in 6-sulfo sLeX biosynthesis in those cells. Finally, we performed double-immunofluorescence staining for MECA-79 and either CD3 or CD8 to evaluate potential association between high endothelial venule (HEV)-like vessels and tumor-infiltrating T lymphocytes. RESULTS 6-Sulfo sLeX glycans were expressed in ~20% of bladder urothelial carcinoma cases, particularly in plasmacytoid and micropapillary variants. Positive cells were also bound by E-selectin·IgM chimeras in a calcium-dependent manner. Transcripts encoding N-acetylglucosamine-6-O-sulfotransferase-2 were detected preferentially in HT-1197 bladder urothelial carcinoma cells expressing 6-sulfo sLeX, and transfection of the enzyme complementary DNA into HT-1376 cells, which do not express 6-sulfo sLeX glycans, resulted in cell surface expression of 6-sulfo sLeX. Furthermore, 6-sulfo sLeX glycans were expressed in HEV-like vessels induced in and around lymphocyte aggregates formed near carcinoma cell nests. These HEV-like vessel-associated tumor-infiltrating lymphocytes were composed primarily of CD3(+) T cells, with a fraction of CD8(+) cytotoxic T cells. CONCLUSIONS Our findings indicate that 6-sulfo sLeX glycans likely play 2 roles in bladder urothelial carcinoma progression: one in lymphocyte recruitment to enhance antitumor immune responses, and the other in E-selectin-mediated tumor cell adhesion to vascular endothelial cells, which is potentially associated with metastasis.

Two distinct lymphocyte homing systems involved in the pathogenesis of chronic inflammatory gastrointestinal diseases.

Under normal and pathological conditions, lymphocyte migration into the gastrointestinal mucosa to form gut-associated lymphoid tissue is mediated by the L-selectin ligand peripheral lymph node addressin and the integrin α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venules (HEVs) and HEV-like vessels. In this review, we discuss these two distinct lymphocyte homing systems involved in the pathogenesis of chronic inflammatory gastrointestinal diseases with reference to our and others’ previously published works. We also describe a recently developed recombinant integrin α4β7 heterodimeric IgG chimera that can be used as an immunohistochemical reagent to stain functional MAdCAM-1.

An Integrin α4β7•IgG Heterodimeric Chimera Binds to Madcam-1 on High Endothelial Venules in Gut-Associated Lymphoid Tissue

Lymphocyte homing is regulated by a multistep process mediated by sequential adhesive interactions between circulating lymphocytes and high endothelial venules (HEVs). In gut-associated lymphoid tissue (GALT), the initial interactive step, “tethering and rolling,” is partly mediated by integrin α4β7 expressed on GALT-homing lymphocytes and its ligand MAdCAM-1, which is exclusively expressed on HEVs in GALT. To probe functional MAdCAM-1 in tissue sections, we developed a soluble integrin α4β7 heterodimeric IgG chimera by joining the extracellular region of mouse integrin α4 and β7 subunits to a human IgG Fc domain. Western blot analysis revealed that co-transfection of HEK 293T cells with expression vectors encoding integrin α4•IgG and β7•IgG results in the formation of α4β7•IgG heterodimeric chimeras. This complex preferentially binds to CHO cells expressing MAdCAM-1 and, to a lesser extent, to cells expressing VCAM-1, but not to cells expressing ICAM-1. Moreover, α4β7•IgG specifically binds to HEVs in GALT in situ in a divalent cation–dependent fashion and inhibits lymphocyte binding to HEVs in GALT. These findings indicate that α4β7•IgG can be used as a probe for functional MAdCAM-1 expressed on HEVs in GALT and could potentially serve as an anti-inflammatory drug inhibiting GALT-specific lymphocyte migration.