Our initial characterization of HEC-GlcNAc6ST−/− mice revealed a marked phenotype with decreased lymphocyte homing and reduced lymphocyte numbers in peripheral lymph nodes (26
). The observation that the 50% residual homing in these animals continues to be L-selectin dependent prompted our investigation of the L-selectin ligands that function in the absence of HEC-GlcNAc6ST.
To directly observe the effect of deletion of HEC-GlcNAc6ST on the rolling of lymphocytes in peripheral lymph node HEV, we employed intravital microscopy. We found no difference in the lymphocyte rolling fraction between HEC-GlcNAc6ST−/− and +/+ mice, but a substantial defect in lymphocyte sticking in fourth and fifth order venules of HEC-GlcNAc6ST−/− mice. Measurement of the velocity of rolling cells revealed a dramatic (fourfold) increase in rolling velocity in fourth order venules. It is plausible that this increase in rolling velocity in the null mice explains the observed defect in lymphocyte sticking in higher order venules; that is, the increase in rolling velocity may decrease the efficiency of chemokine signaling by decreasing the time of exposure of rolling cells to endothelial-associated chemokines. Analogously, when α4β7 integrin function is blocked or genetically inactivated, the velocity of lymphocyte rolling in HEV of Peyer's patches increases dramatically and sticking is greatly reduced (35
). Sulfated carbohydrates have been shown to bind chemokines, such as CCL21 (36
), whose presentation on the luminal surface of HEV is essential to induce sticking of rolling T cells (37
). Thus, it is conceivable that undersulfation of molecules on the luminal surface of HEV in HEC-GlcNAc6ST−/− mice may reduce the presentation of CCL21 in high order venules, thus contributing to the observed defect in lymphocyte sticking. Undersulfation of ligands could also affect sticking by diminishing outside-in signaling processes dependent on L-selectin ligand engagement (38
We have confirmed here that HEC-GlcNAc6ST−/− mice have dramatically reduced levels of luminal reactivity for MECA79 in lymph node HEV. Consistent with this observation, neither in vivo homing to peripheral lymph nodes nor lymphocyte rolling on HEV was affected by administration of MECA79 mAb to the null animals. Yet, rolling of lymphocytes on HEV remained L-selectin dependent. These observations provide a direct demonstration of the existence of one or more MECA79-unreactive ligand(s) for L-selectin that function(s) in lymph node HEV in the absence of HEC-GlcNAc6ST.
To investigate the L-selectin ligands present in HEC-GlcNAc6ST−/− mice, we focused on components of the PNAd complex. We purified GlyCAM-1, a secreted peripheral membrane protein, and CD34, a type I transmembrane protein, from lymph nodes. CD34 has been shown by EM immunocytochemical studies to be predominantly on the luminal surface of blood vessels (8
). GlyCAM-1 and CD34 purified from lymph nodes were significantly undersulfated in HEC-GlcNAc6ST−/− mice, incorporating 65% less sulfate on a per mass basis than the same ligands purified from wild-type mice. Consistent with the direct sulfation measurements, a similar decrease was seen in the reactivity of these ligands with the sulfation-dependent antibody MECA79. Analysis of acid hydrolysates of GlyCAM-1 from HEC-GlcNAc6ST−/− mice indicated that GlcNAc-6–SO4
was the predominant (>75% of the total) sulfated saccharide present (unpublished data). Furthermore, the ligand activity of HEC-GlcNAc6ST−/− GlyCAM-1 was blocked by MECA79, which requires GlcNAc-6–SO4
for binding (25
). Therefore, we conclude that the residual ligand activity of GlyCAM-1 is dependent on the GlcNAc-6–SO4
modification rather than other sulfation modifications.
We wanted to confirm that the defect in HEC-GlcNAc6ST−/− mice was restricted to sulfation and did not secondarily affect sialylation or fucosylation of ligands. The E-selectin–IgM binding results demonstrated that the sLex moiety was present to equivalent levels on Gly–CAM-1 from both types of mice. Equivalent binding by the fucose-specific lectin AAL was further evidence that fucosylation of GlyCAM-1 was not perturbed in HEC-GlcNAc6ST−/− mice. In contrast, GlyCAM-1 from HEC-GlcNAc6ST−/− mice was unable to support binding of L-selectin–IgM and was also defective in P-selectin–IgM binding. These results demonstrated that this GlyCAM-1 was selectively deficient in the sulfate modification critical for L-selectin and, to a lesser degree, P-selectin binding. P-selectin on activated platelets has previously been shown to recognize the posttranslational modifications of PNAd and to mediate platelet binding to HEV (39
). MECA79 treatment substantially reduced the interaction of platelets with HEV, suggesting that the recognition determinant for L- and P-selectin on PNAd are shared. Our result demonstrated that P-selectin is less stringent than L-selectin in its dependency on the GlcNAc-6–sulfate modification generated by HEC-GlcNAc6ST.
Although GlyCAM-1 from HEC-GlcNAc6ST−/− mice was unable to bind L-selectin in a stringent equilibrium binding assay, we wanted to observe its activity in the flow chamber, a more sensitive assay to determine perturbations in the formation and dissociation of bonds between L-selectin and its ligands. GlyCAM-1 from null animals was able to support lymphocyte rolling, but significantly fewer cells rolled over a range of shear stresses. Rolling velocities were significantly increased and the strength of rolling adhesions was greatly diminished. Since GlyCAM-1 from HEC-GlcNAc6ST−/− mice appears to be modified with sialic acid and fucose at levels equivalent to wild-type, the observed increase in rolling velocity and decrease in shear resistance in the flow chamber is most likely the consequence of the defect in sulfation.
The presence of GlcNAc-6–SO4
and MECA79 reactivity on GlyCAM-1 and CD34 in the absence of HEC-GlcNAc6ST indicates that a second HEV-expressed GlcNAc-6–sulfotransferase modifies components of PNAd and is capable of creating the MECA79 epitope. A strong candidate for this enzyme is GlcNAc6ST which is expressed at the mRNA level within murine lymph node HEC (40
). This enzyme can participate in the generation of both the 6-sulfo-sLex epitope (19
) and the MECA79 epitope (24
) in transfected cells.
A decrease in the GlcNAc-6–sulfation of ligands like GlyCAM-1 and CD34 could conceivably explain the increased rolling velocity that we observed by intravital microscopy in higher order venules of HEC-GlcNAc6ST−/− lymph nodes. Indeed, we confirmed that undersulfated GlyCAM-1 supports faster rolling in the flow chamber. However, our MECA79 inhibition results argue that these observations are not simply attributable to diminished sulfation of PNAd components. While MECA79 completely inhibited in vitro rolling on null mice-derived GlyCAM-1 over a range of ligand concentrations, we observed no effect of this antibody on in vivo homing, rolling, or lymphocyte arrest in lymph nodes of the null mice. It is possible GlyCAM-1 from HEC-GlcNAc6ST null mice does not accurately reflect the altered posttranslational modifications of CD34 and other integral membrane components of PNAd and therefore is not an appropriate surrogate of the PNAd-adhesive complex in these mice. Countering this possibility, we observed the same reductions in overall sulfation and the expression of the MECA79 epitope on both GlyCAM-1 and CD34 when HEC-GlcNAc6ST was absent. Therefore, we believe that the deficiency in the ligand activity of null mice-derived GlyCAM-1, as well as its susceptibility to inhibition by MECA79, applies to other members of the PNAd complex. If this generalization is valid, then the contrasting efficacies of MECA79 in vivo and in vitro in the null mice would argue for the existence of MECA79-unreactive ligands that become functionally predominant in the absence of HEC-GlcNAc6ST. We proposed such ligand activity and termed it “class 2” in our initial characterization of HEC-GlcNAc6ST null animals (26
). The present findings indicate that the class 2 ligand is based on a novel protein scaffold with distinctive posttranslational modifications. Furthermore, since MECA79 treatment potently inhibits rolling in wild-type lymph nodes and does not recapitulate the faster rolling we observed in HEC-GlcNAc6ST−/− mice, our results imply that the class 2 ligand is up-regulated in higher order venules of HEC-GlcNAc6ST−/− mice.
A MECA79-unreactive L-selectin ligand is expressed in HEV of Peyer's patches. This ligand supports fast rolling and is essential to the homing of lymphocytes to this organ (41
). Interestingly, the pattern of MECA79 staining of HEC-GlcNAc6ST−/− lymph nodes is reminiscent of that seen in Peyer's patches, a tissue in which HEC-GlcNAc6ST is not expressed (19
). MECA79 reactivity is apparent only on the abluminal aspect of the HEV of wild-type Peyer's patches, consistent with the inability of MECA79 to block homing to this organ (3
). MAdCAM-1 is proposed to be the predominant L-selectin ligand in HEV of Peyer's patches, in addition to its function as a counter receptor for α4β7 (35
). However, we have not observed an up-regulation of MAdCAM-1 expression in lymph nodes of HEC-GlcNAc6ST−/− mice (26
Additional class 2 ligand candidates are those that support rolling of lymphocytes on HEV of frozen sections of human tonsils following treatment of the sections with the mucin degrading enzyme OSGE (42
). This ligand is not blocked by MECA79, although it has reactivity with sLex-specific mAbs. Further evidence for diversity in ligands for L-selectin in human lymphoid tissues comes from studies of Michie et al. (43
). These investigators found that MECA79 inhibits the adhesion of an L-selectin+
lymphoma cell line to HEV of human peripheral nodes by only 41%. The diversity of carbohydrate determinants on PNAd components has been demonstrated using a panel of monoclonal antibodies that differentially stain human lymphoid tissues and variably cross-block MECA79 (44
). Tu et al. (45
) have described a MECA79-unreactive, sulfate and sialic acid–dependent L-selectin ligand which is expressed on human endothelial cells in culture. In contrast, cytokine-activated cardiac microvascular cells express a sulfate dependent ligand that is sialidase insensitive (46
). Additionally, a heparan-sulfate–based ligand for L-selectin has been detected on the cell surface of cultured aortic endothelial cells (47
). In vivo, a MECA79-unreactive L-selectin ligand is induced on venules in a model of dermal inflammation in the mouse (48
). Intravital microscopic studies have revealed L-selectin ligand activity in mesenteric venules of rat and rabbit after surgical exteriorization (49
) and in venules of the cremaster muscle of mouse following cytokine treatment (51
). Recent studies indicate that this cremaster ligand activity is due to leukocyte-derived PSGL-1 (52
Future efforts should be directed at identification of the class 2 ligand within lymph nodes and the nature of its posttranslational modifications. Parallel studies (53
) indicate the existence of a class 2 ligand (i.e., MECA79 unreactive) on medullary venules (order 1 and 2) within lymph nodes of wild-type mice. Whether this ligand corresponds to the herein described ligand that is up-regulated in the higher order venules of HEC-GlcNAc6ST null mice remains to be determined.