A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-alpha/beta and TNF-alpha in cultured endothelial cells

Abstract

Background: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb.

Results: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells.

Conclusion: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA.

Figure 1

Double immunofluorescent detection of the MECA-79 epitope and GlcNAc6ST-2 in human lymph node and RA synovial tissues. Sections were simultaneously labelled with MECA-79 (red) and anti-GlcNAc6ST-2 (green) antibodies and photographed under appropriate filters. On the right panels, a merged image shows colocalization of both markers (yellow). (a) Lymph node. (b) RA synovium; Two MECA-79 negative and GlcNAc6ST-2 positive vessels are marked by arrows. Original magnification (a) 200×. (b) 150×. Selected areas within squared insets are shown on the lower right corner insets with higher magnification (630×).

Figure 2

Immunoperoxidase staining of RA and OA synovial tissue with anti-GlcNAc6ST-2 antibodies. RA (a-d) or OA (e, f) synovial tissue sections were immunostained with anti-human GlcNAc6ST-2 (a, b, e) or anti-mouse GlcNAc6ST-2 (c, d, f) antibody. Color was developed with DAB peroxidase substrate (brown color) and sections were counterstained with hematoxylin. Sections b and d are serial to a and c respectively, and were preincubated with human or mouse GlcNAc6ST-2 peptide to demonstrate the specificity of immunostaining. Original magnification: 400×.

Figure 3

Expression of GlcNAc6ST-2 in cultured HUVEC. (a) Real time RT-PCR quantification of GlcNAc6ST-2 mRNA in cultured HUVEC. HUVEC cultures were stimulated for 8 h with LT-αβ (LT), TNF-α (TNF), or interferon-γ (IFN) and mRNA obtained from stimulated and non-stimulated cells. Basal (non-stimulated) expression was set to 1 and the GlcNAc6ST-2/β-actin ratio in stimulated cells is shown (mean ± SD of three independent experiments, each including triplicate HUVEC cultures). (*) p < 0.05. (b) Western blot analysis of GlcNAc6ST-2 in cultured HUVEC stimulated for 24 h with LT-αβ (LT) or TNF-α (TNF), (data are representative of three independent experiments with different HUVEC lines).(c) Immunofluorescent detection of GlcNAc6ST-2 protein in cultured HUVEC endothelial cells non-stimulated (Basal) or stimulated for 24 h with LT-αβ (LT) or TNF-α (TNF). In control (CTRL) panel, anti-GlcNAc6ST-2 antibody was replaced by non-immune rabbit IgG (images are representative of three independent experiments).