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

Abstract

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.

Figure 1

Schematic representation of carbohydrate structure of L-selectin ligands. 6-sulfo sLeX attached to extended core 1 and/or core 2-branched O-glycans functions as an L-selectin ligand. Epitopes for monoclonal antibodies MECA-79 (6-sulfo N-acetyllactosamine attached to extended core 1 O-glycans), HECA-452 (sLeX, regardless of GlcNAc-6-O-sulfation), and NCC-ST-439 (sLeX attached to core 2-branched O-glycans, regardless of GlcNAc-6-O-sulfation) are shown.

Figure 2

HEV-like vessels appear in chronic H. pylori gastritis (A) and gastric MALT lymphoma (B). Serial tissue sections were stained with hematoxylin and eosin (HE), and immunostained for CD34 as a marker of vascular endothelial cells, MECA-79, HECA-452, and NCC-ST-439. In chronic H. pylori gastritis, more than half of MECA-79⁺ vessels are HECA-452⁻/NCC-ST-439⁻. Conversely, in gastric MALT lymphoma, MECA-79⁻ but HECA-452⁺/NCC-ST-439⁺ vessels are frequently observed. Bar = 200 µm.

Figure 3

Frequency of MECA-79⁺, HECA-452⁺, and NCC-ST-439⁺ HEV-like vessels in chronic H. pylori gastritis (left), gastric MALT lymphoma (middle), and its accompanied reactive component (right). In chronic H. pylori gastritis, the percentage of MECA-79⁺ vessels is greater than that of HECA-452⁺ and NCC-ST-439⁺ vessels, with high statistical significance (p < 0.001). Conversely, in gastric MALT lymphoma, the percentages of MECA-79⁺, HECA-452⁺, and NCC-ST-439⁺ vessels do not differ significantly. The percentage of HECA-452⁺ and NCC-ST-439⁺ but not MECA-79⁺ vessels in gastric MALT lymphoma is greater than that seen in chronic H. pylori gastritis, with statistical significance. In addition, the percentages of MECA-79⁺, HECA-452⁺, and NCC-ST-439⁺ vessels in accompanied reactive component of gastric MALT lymphoma shows similar patterns seen in chronic H. pylori gastritis. Data are presented as means ± SEM (n = 31 in chronic H. pylori gastritis, n = 22 in gastric MALT lymphoma, n = 3 in accompanied reactive component of gastric MALT lymphoma). ***, p < 0.001; **, p < 0.01.

Figure 4

FACS analysis of CHO cell lines expressing various sugar chains. Each CHO line described in Table 1 was tested for CD34, HECA-452, MECA-79, and NCC-ST-439 reactivities. Lighter gray lines represent negative control resulting from omitting the primary antibody.

Figure 5

Expression of a particular sLeX-type glycans on CD34 obtained from CHO cell lines described in Table 1. CD34 obtained from all lines except control CHO/CD34 cells are positive for HECA-452, and such HECA-452 glycoepitopes are eliminated after treatment with N-glycanase. The MECA-79 epitope is detected in CHO cells transfected with both Core1-β3GlcNAcT and LSST (GlcNAc6ST-2). The NCC-ST-439 epitope is detected in CHO/CD34/F7/C2 with or without transfection with LSST.

Figure 6

L-selectin•IgM chimera binding assay of CHO cell lines. CHO cell lines listed in Table 1 were tested. The L-selectin•IgM chimera most robustly binds to the CHO/CD34/F7/C2/LSST and CHO/CD34/F7/C2/C1/LSST lines at almost equivalent levels. The remaining lines show only marginal L-selectin•IgM binding. Red histograms represent L-selectin•IgM chimera bindings. Blue and gray histograms indicate control experiments using cells treated with sialidase and EDTA, respectively.

Figure 7

Lymphocyte adhesion to CHO cell lines expressing various sugar chains. CHO lines listed in Table 1 were examined. Lymphocyte adhesion to CHO/CD34 (A), CHO/CD34/F7/C2 (B), and CHO/CD34/F7/C2/LSST (C) observed with Nomarski differential interference optics is shown. Arrows indicate adhered lymphocytes. Note that treatments with sialidase, Dreg-56, and EDTA completely abolish lymphocyte adhesions. Bar = 100 µm. Data are representative of three independent experiments showing similar results.

Figure 8

(A) The number of adherent lymphocytes per 100 CHO cells is shown. CHO cells expressing sLeX on a core 2-branched O-glycan backbone, regardless of GlcNAc-6-O-sulfation, and those expressing 6-sulfo sLeX attached to extended core 1 O-glycans are significantly bound by lymphocytes. Such lymphocyte adhesions are maintained after treatment with N-glycanase, but almost completely abrogated with treatments with sialidase, Dreg-56, or EDTA. ***, p < 0.001; *, p < 0.05. Data are representative of three independent experiments showing similar results. (B) The amount of sLeX expressed on each CHO cell line, as assessed by cell-ELISA. Expression of sLeX on CHO cell lines does not differ except for CHO/CD34, which serve as a control.