SummarySdectins represent a new family of adhesion molecules, expressed by leukocytes and endothelial cells, that are involved in the regulation of leukocyte traffic. Here we have characterized a new monoclonal antibody (mAb) (EI.-246) that recognizes both human leukocyte I~selectin (previously called LAM-1, LECAM-1, or gpgOMEI:14) and endothelial cell E-sdectin (previously called ELAM-1). Eb246 recognized a 110-kD protein expressed on cells transfected with E-sdectin c.DNA and stained many postcapillary venules in inflamed human tonsil. EL-246 also stained human peripheral blood leukocytes and showed identity with anti-bsdectin mAb in two-color flow cytometric analysis. The expression of the leukocyte EI.-246 antigen was regulated in the same manner as I..selectin and EL,246 recognized anti-L-selectin mAb affinity-purified antigen in SDS/PAGE Western blot analysis. Further, L, selectin cDNA transfectants were specifically stained by EL-246. EL-246 blocked >95% of lymphocyte adhesion to peripheral lymph node high endothelial venules and >90% of neutrophil adhesion to E-sdectin transfectants. In addition to the ED246 epitope being expressed on two different human selectins, it was detected on L-selectin from a variety of different animals. Interestingly, domain mapping studies localized the Eb246 epitope to the short consensus repeat (SCR) domains of Lsdectin. EL246 is the first mAb that recognizes two different sdectins and potentially defines a functional epitope encoded by the SCR domains. Inhibitors of selectin function targeted to this region would be expected to have the added advantage of simultaneously blocking the activity of two distinct adhesion proteins involved in inflammation.
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SummaryE-Sdectin is an inducible adhesion protein expressed by endothelial ceils and recognized by lenkocytes during their extravasation from the blood into inflamed tissues. Originally, E-sdectin was defined as a mydoid ceil-specific adhesion protein, but recent studies have shown it to be recognized by human lymphocytes as well. These lymphocytes represent a memory T cell subset and have been shown to express the HECA-452 carbohydrate epitope (CLA + lymphocytes). We extend these findings and show that ruminant 3'/8 T cells bind E-sdectin as well; and we provide preliminary evidence that this interaction is mediated by a novel glycoprotein receptor on the lymphocyte. Unlike conventional T ceils (c~/13 T cells), 3'/8 T cells from neonatal and mature animals bind E-sdectin, suggesting that prior antigen stimulation and differentiation to a memory lymphocyte are not required for this interaction. Neuraminidase treatment of the 3'/8 T cells or addition of ethylenediaminetetraacetic acid (EDTA) to the assay abrogates binding, demonstrating the importance of sialic acid and divalent cations, which is consistent with other E-sdectin-mediated adhesion events. However, previously defined E-selectin carbohydrate ligands, such as sialyl Lewis x on nentrophils and the HECA-452 epitope on human memory lymphocytes, are antigenically different than the carbohydrates on ruminant 3'/8 T cells since the mAbs CSLEX and HECA-452 do not recognize these ceils. Protease treatment of 3'/8 T ceils significantly inhibits their binding to E-sdectin; however, previously characterized adhesion glycoproteins, such as L-selectin, CD44, and CD18, are not involved in the adhesive event. An E-sdectin aflinity column purifies a single glycoprotein of 250 kD (280 kD under reducing conditions) from 3'/8 T cell detergent lysates. Nenraminidase digestion of the 250-kD product as well as EDTA abolishes binding to E-sdectin. Finally, E-sdectin expression in vivo appears to mediate 3'/8 T cell accumulation. Stimulation of bovine skin with tumor necrosis factor o~ induced an increase in E-sdectin expression that was associated with an influx of 3'/8 T cells at the same site.
Selectins constitute a three-member gene family of carbohydrate-binding adhesion proteins found on the surface of leukocytes and endothelial cells that is central to inflammation-associated leukocyte recruitment and lymphocyte recirculation. E- and P-selectin are inducible and expressed on the surface of endothelial cells under inflammatory conditions, whereas L-selectin is constitutively expressed on most circulating leukocytes. Previously, we have characterized a unique mAb (EL-246) that recognizes a common epitope on both E- and L-selectin, which is presented or determined by their short consensus repeat domains. This report defines the functional properties of EL-246 and its cognate epitope. In a novel in vitro physiologic shear system, we show that neutrophil rolling on activated HUVECs and on E-selectin cDNA transfectants is blocked 45 to 120 s after infusion of EL-246. The examination of the binding of neutrophils to E-selectin cDNA transfectants reveals that their adhesion is blocked by EL-246 treatment of either cell type. A unique Ab transfer mechanism is demonstrated in which El-246 is delivered unidirectionally from L- to E-selectin to surpass the adhesion blocked by mAbs that recognize either L- or E-selectin alone. By using flow cytometry and in vivo homing techniques, we show that pretreating bovine lymphocytes with EL-246 blocks their ability to home to mouse peripheral lymph nodes by > 65%. Cumulatively, these results suggest that EL-246 is a uniquely potent pharmacologic inhibitor of leukocyte-endothelial cell interactions that are mediated by either E- or L-selectin.
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.
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