Lymphocyte-HEV interactions in lymph nodes of a sulfotransferase-deficient mouse

Abstract

The interaction of L-selectin expressed on lymphocytes with sulfated sialomucin ligands such as CD34 and GlyCAM-1 on high endothelial venules (HEV) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte recruitment. HEC-GlcNAc6ST-deficient mice lack an HEV-restricted sulfotransferase with selectivity for the C-6 position of N-acetylglucosamine (GlcNAc). HEC-GlcNAc6ST-/- animals exhibit faster lymphocyte rolling and reduced lymphocyte sticking in HEV, accounting for the diminished lymphocyte homing. Isolated CD34 and GlyCAM-1 from HEC-GlcNAc6ST-/- animals incorporate approximately 70% less sulfate than ligands from wild-type animals. Furthermore, these ligands exhibit a comparable reduction of the epitope recognized by MECA79, a function-blocking antibody that reacts with L-selectin ligands in a GlcNAc-6-sulfate-dependent manner. Whereas MECA79 dramatically inhibits lymphocyte rolling and homing to lymph nodes in wild-type mice, it has no effect on HEC-GlcNAc6ST-/- mice. In contrast, in vitro rolling on purified GlyCAM-1 from HEC-GlcNAc6ST-/- mice, although greatly diminished compared with that on the wild-type ligand, is inhibited by MECA79. Our results demonstrate that HEC-GlcNAc6ST contributes predominantly, but not exclusively, to the sulfation of HEV ligands for L-selectin and that alternative, non-MECA79-reactive ligands are present in the absence of HEC-GlcNAc6ST.

Figure 1.

Intravascular staining with MECA79 and effect on in vivo homing. (A–D) MECA79 mAb was injected intravenously into HEC-GlcNAc6ST−/− or +/+ mice. 30 min later peripheral lymph nodes were dissected out and processed for immunofluorescence microscopy. Bright field images of +/+ (A) and −/− (C) lymph nodes. Intravascular staining with MECA79 of (B) +/+ and (D) −/− HEV. Magnification 200×. (E) MECA79 mAb or PBS was injected intravenously into HEC-GlcNAc6ST−/− or +/+ mice and the homing of fluorescently labeled cells was determined and normalized to produce a homing index as described in Materials and Methods. Consistent with our previous findings (26), homing in the HEC-GlcNAc6ST−/− mice was 59% relative to homing in wild-type.

Figure 2.

Lymphocyte rolling and sticking fractions in HEV of HEC-GlcNAc6ST−/− mice. (A) Rolling fractions were determined as the percentage of rolling cells in the total flux of fluorescently labeled lymphocytes. Data are based on four HEC-GlcNAc6ST−/− mice (1st-4; 2nd-5; 3rd-7; 4th-16; 5th-11 venules) and four +/+ mice (1st-2; 2nd-9; 3rd-12; 4th-16; 5th-7 venules). Bars represent mean ± SEM. (B) Sticking fractions were determined as the percentage of rolling cells that subsequently arrested (stationary ≥30 s). Data are based on the same four HEC-GlcNAc6ST−/− and four +/+ mice and venules indicated in A. Sticking was not observed in first order venules or in second order venules in +/+ mice.

Figure 3.

Lymphocyte rolling velocities in HEC-GlcNAc6ST−/− mice. (A and C) Cumulative rolling velocity curves for third order (A) and fourth order (C) venules. The percentage of cells that rolled at or below a given velocity is shown as a function of V_roll. n = cells/venules/mice. (B and D) Distribution of V_rel frequencies for third order (B) and fourth order (D) venules. V_rel was calculated as described in Materials and Methods and cells were assigned to V_rel classes ranging from >0 to <2%, 2% to <4%, and so on.

Figure 4.

Effect of MECA79 mAb on lymphocyte rolling. (A) MECA79 mAb or isotype control rat IgM was injected via a femoral artery catheter into HEC-GlcNAc6ST−/− or +/+ mice. 15 min later, fluorescently labeled lymphocytes were injected. Data are based on 2 HEC-GlcNAc6ST−/− mice injected with rat IgM (1st-2; 2nd-5; 3rd-8; 4th-5; 5th-2 venules), two +/+ mice injected with rat IgM (1st-1; 2nd-2; 3rd-3; 4th-3; 5th-3 venules), two HEC-GlcNAc6ST−/− mice injected with MECA79 (1st-3; 2nd-6; 3rd-8; 4th-8; 5th-7 venules), and 2 +/+ mice injected with MECA79 (1st-2; 2nd-5; 3rd-8; 4th-8; 5th-5 venules). Bars represent mean ± SEM. (B) Fluorescently labeled L-selectin−/− lymphocytes were injected into HEC-GlcNAc6ST−/− mice treated with MECA79. Data are based on 4 HEC-GlcNAc6ST−/− mice injected with MECA79 (1st-3; 2nd-6; 3rd-8; 4th-9; 5th-7 venules) and one HEC-GlcNAc6ST−/− mouse injected with MECA79 and L-selectin−/− lymphocytes (1st-1; 2nd-3; 3rd-5; 4th-3; 5th-2 venules).

Figure 5.

³⁵Sulfate incorporation and MECA79 reactivity of GlyCAM-1 and CD34. (A and D) Lymph nodes from HEC-GlcNAc6ST−/− and +/+ mice were incubated with Na³⁵SO₄. Immunoprecipitated GlyCAM-1 and CD34 were separated by SDS-PAGE and transferred to PVDF for Western blotting. (A) GlyCAM-1 was detected with an antipeptide Ab (CAMO5). (D) CD34 was detected with an affinity-purified polyclonal Ab. (B and E) ³⁵S-labeled immunoprecipitated ligands GlyCAM-1 (B) and CD34 (E) detected by autoradiography. (C and F) MECA79 reactivity of GlyCAM-1 (C) and CD34 (F) detected by Western blotting. (G) Relative sulfate incorporation and MECA79 reactivity of GlyCAM-1 and CD34 from HEC-GlcNAc6ST−/− mice. ³⁵S incorporation was measured by performing densitometry on autoradiographs as shown in B and E and normalized for protein based on Western blots performed in parallel with protein-specific Abs as in A and D. MECA79 reactivity was measured by performing densitometry on Western blots shown in C and F and normalized based on protein-specific Western blots performed in parallel. Data are the means ± SEMs of three independent preparations.

Figure 6.

ELISA analyses of GlyCAM-1 from HEC-GlcNAc6ST−/− mice. Equivalent amounts of HEC-GlcNAc6ST−/− and +/+ GlyCAM-1 were coated on ELISA plates. (A) Protein was quantified using an anti–GlyCAM-1 Ab. (B) Relative MECA79 reactivity. L-selectin–IgM (C), P-selectin–IgM (D), or E-selectin–IgM (E) chimeric proteins or biotinylated-AAL (F) were allowed to bind and were subsequently detected with an alkaline phosphatase-conjugated secondary Ab. EDTA (selectins) or fucose (AAL) was used to show specificity of the respective interactions. Plots are representative of three experiments performed on two independent preparations of GlyCAM-1.

Figure 7.

Jurkat cell rolling on GlyCAM-1. (A) Equivalent amounts of HEC-GlcNAc6ST−/− and +/+ GlyCAM-1 were coated on dishes. Jurkat cells were perfused through the flow chamber and the number of rolling cells was determined for each shear stress. Values represent the mean ± SD. Addition of EDTA, mAb DREG56, or sialidase treatment of −/− and +/+ GlyCAM-1 inhibited >95% of rolling. The experiment shown is representative of three independent experiments each performed in duplicate using two different fields of view. (B) The velocity of 40 rolling cells was determined for each shear stress. Values represent the mean ± SD. (C) GlyCAM-1 from HEC-GlcNAc6ST−/− and +/+ mice was coated such that nearly equivalent numbers of rolling cells were observed at wall shear stress of 1–2 dyn/cm². Dishes were treated with varying concentrations of MECA79 mAb and rolling of Jurkat T cells was determined. Data was normalized based on the number of cells that rolled in the absence of mAb. Data shown is for a shear stress of 1 dyn/cm². (D) Varying concentrations of GlyCAM-1 from HEC-GlcNAc6ST+/+ and −/− mice were coated yielding two different levels of rolling for each form of GlyCAM-1. The effect of 10 μg/ml of MECA79 on rolling was determined for each type and concentration of GlyCAM-1. Values represent the mean ± SD for shear stress of 1 dyn/cm².