Detection of a sulfotransferase (HEC-GlcNAc6ST) in high endothelial venules of lymph nodes and in high endothelial venule-like vessels within ectopic lymphoid aggregates: relationship to the MECA-79 epitope

Abstract

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules (HEVs) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte homing. The MECA-79 monoclonal antibody reports HEV-expressed ligands for L-selectin by recognizing a critical sulfation-dependent determinant on these ligands. HEC-GlcNAc6ST, a HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope. Here, we use an antibody against murine HEC-GlcNAc6ST to study its expression in relationship to the MECA-79 epitope. In lymph nodes, the enzyme is expressed in the Golgi apparatus of high endothelial cells, in close correspondence with luminal staining by MECA-79. In lymph node HEVs of HEC-GlcNAc6ST-null mice, luminal staining by MECA-79 is almost abolished, whereas abluminal staining persists although reduced in intensity. HEV-like vessels in several examples of inflammation-associated lymphoid neogenesis, including nonobese diabetic mice, also exhibit concomitant expression of the sulfotransferase and luminal MECA-79 reactivity. The correlation extends to ectopic lymphoid aggregates within the pancreas of RIP-BLC mice, in which CXCL13 is expressed in islets. Analysis of the progeny of RIP-BLC by HEC-GlcNAc6ST-null mice establishes that the enzyme is responsible for the MECA-79 defined luminal ligands.

Figure 1

Staining of lymph nodes with an anti-HEC-GlcNAc6ST serum. An anti-HEC-GlcNAc6ST antiserum was produced by immunization of an individual rabbit with three peptides derived from the HEC-GlcNAc6ST sequence, as described in Materials and Methods. The serum was used to stain cryostat sections of mouse PN in the absence (B) or presence (D) of peptide 3 (RGKGMGQHAFHTNC). A and C show the bright-field images (hematoxylin stained) corresponding to B and D. Scale bar, 25 μm.

Figure 2

Expression of HEC-GlcNAc6ST and the MECA-79 epitope in mouse lymph node. Serial cryostat sections were prepared from PNs of wild-type (A, C, E) or −/− (B, D, F) mice. Sections were stained by immunofluorescence with MECA-79 (C, D) or the anti-HEC-GlcNAc6ST antibody (E, F). The bright-field images (A, B) establish the presence of HEVs in both wild-type and −/− lymph nodes. Scale bar, 25 μm.

Figure 3

Golgi localization of HEC-GlcNAC6ST. A cryostat section from a rat lymph node was dual stained with an antibody for a Golgi marker (B; GM-130, Cy3, red) and the anti-HEC-GlcNAc6ST antibody (C; Cy2, green). D: Overlay of the two fluorescent images; yellow indicates overlap of the two stains. A is the bright-field image of the same field showing a single HEV. Scale bar, 25 μm.

Figure 4

Contribution of HEC-GlcNAC6ST to the generation of luminal and abluminal MECA-79 epitopes. Cryostat sections of PN were prepared from wild-type (A, C) and −/− (B, D) mice that had received intravenous injections of unlabeled MECA-79. The sections were stained first with Cy3-conjugated secondary reagents to detect injected luminally bound MECA-79 and then with biotinylated MECA-79 followed by streptavidin-Cy2 to detect abluminal MECA-79 epitopes. C and D: Overlay of the two fluorescent images. A and B show the corresponding bright-field images. Scale bar, 25 μm.

Figure 5

Expression of HEC-GlcNAc6ST in MNs and PPs. Cryostat sections from MNs of wild-type mice were stained simultaneously for MECA-79 (B; Cy3, red), HEC-GlcNAc6ST (C; Cy2, green), and MAdCAM-1 (D; aminomethylcoumarin, blue). Large arrowheads in B and D indicate vessels or segments of vessels that are MAdCAM-1-negative but contain luminal MECA-79 epitopes. Arrows in B and C indicate the correspondence between expression of HEC-GlcNAc6ST and luminal MECA-79 epitopes. Small arrowheads in B, C, and D indicate a segment of a vessel that is positive for MAdCAM-1, negative for HEC-GlcNAc6ST, and in which MECA-79 is confined to the luminal aspect. In E and F, a section of PP was dual stained for MECA-79 (E; Cy3, red) and HEC-GlcNAc6ST (F; Cy2, green). Scale bar, 25 μm.

Figure 6

Expression of HEC-GlcNAc6ST in HEV-like vessels at sites of inflammation. Cryostat sections from hyperplastic AKR thymus (A–C), NOD pancreas (D–F), NOD lacrimal glands (G–I), and NOD salivary glands (J–L) were dual stained for MECA-79 (A, D, G, J; Cy3, red) and HEC-GlcNAc6ST (B, E, H, K; Cy2, green). C, F, I, and L are the respective overlays. Scale bar, 25 μm.

Figure 7

Contribution of HEC-GlcNAc6ST to luminal MECA-79 epitopes and lymphoid aggregates in the pancreas of RIP-BLC mice. Cryostat sections from infiltrated pancreas of RIP-BLC/HEC-GlcNAc6ST+/+ (A, C, and E) and RIP-BLC/HEC-GlcNAc6ST−/− (B, D, and F) mice were dual stained for MECA-79 (Cy3, red) and HEC-GlcNAc6ST (Cy2, green). Shown are the bright-field images (A, B), the MECA-79 staining (C, D), and the merged MECA-79 and anti-HEC-GlcNAc6ST staining (E, F). Scale bar, 25 μm.