It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.
We compared E-selectin-binding cell surface ligands on bovine gamma delta T cells and human leukocytes using an E-selectin/Ig chimera. The chimera worked well in flow cytometric studies and showed that bovine gamma delta T cells were the only lymphocyte population in newborn animals that bound E-selectin chimera. Furthermore, the chimera blocked gamma delta T cell rolling on E-selectin. Chimera reacted with four potential glycoprotein ligands of 180, 200, 250, and 300 kDa in Western blot analysis of gamma delta T cell detergent lysates, and it specifically precipitated at least two of these E-selectin ligands (200 and 250 kDa) from lysates of cell surface biotinylated gamma delta T cells. Preclearing bovine gamma delta lysates of GD3.5 Ag and workshop cluster 1 did not abrogate E-selectin ligand precipitation, suggesting that these surface markers do not represent E-selectin ligands. Human neutrophils possessed three E-selectin-binding ligands of approximately 80 to 90, 130, and 230 kDa, while human lymphocytes variably possessed three ligands of 120, approximately 220 to 240, and 260 kDa. Cross-precipitation experiments confirmed the results of others that neutrophil L-selectin serves as the 80 to 90-kDa E-selectin ligand. The human lymphocyte approximately 220 to 240-kDa and 260-kDa ligands may be analogous to the bovine gamma delta T cell molecules, whereas the 120-kDa is unique to human cells. The identities of the human and bovine lymphocyte E-selectin ligands are unknown. Finally, E-selectin ligand-1 apparently may have a minimal role, if any, in lymphocyte/E-selectin interactions, since a polyclonal anti-E-selectin ligand-1 serum stained a minimal number of cutaneous lymphocyte-associated Ag-positive human lymphocytes and bovine gamma delta T cells.
In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1-negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross-immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function.
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