Recent studies from this laboratory implicate all three members of the selectin (LEC-CAM) family and the beta2-integrins in monocyte-endothelial adhesive interactions in human rheumatoid arthritis (RA). Mabs to P- selectin consistently inhibit > 90% of adhesion while Mabs to E- and L- selectin inhibit up to 50%. In addition Mabs to LFA-1, Mo1/Mac1 and their common beta2-chain inhibit 30-50%. This work constitutes the first direct evidence that selectins participate in leukocyte recruitment in RA. This section contains three inter-related projects exploring the role of selectins in monocyte-microvascular interactions.
Specific aim #1 focuses on the ligands for E- and P-selectin in monocytes and cell lines. the glycoproteins expressing two carbohydrate epitopes linked to or contained in the selectin binding sites (CSLEX-1 and HECA-452) will be isolated and characterized. Their interactions with fusion proteins containing the cell binding domains of E- and P-selectin will then be established. In addition, transfectants will be used to determine whether post- translational glycosylation introduces E- and P-selectin binding sites into L-selectin. Dr. Lowe (Section 1) will transfect full length cDNAs encoding L-selectin into several CHO-transfectants capable of generating ligands for E and P-selectins. A U937 line transfected with L-selectin will also be tested. L-selectin in the transfectants will then be examined for expression of E- and P-selectin binding sites.
Specific aim #2 defines the binding sites for monocytic L-selectin and beta2-integrins on synovial microvasculature in RA. The principle endothelial counter-receptor for L- selectin is thought to be an O-linked cluster of sialylated, fucosylated and sulfated oligosaccharides. We will use an IgG1-L-selectin chimera and immunogold labelling techniques to determine whether this or structurally related oligosaccharide are expressed on synovial venules. In addition, the effect of microvascular desialylation on the attachment of both monocytes and the IgG1-L-selectin chimera will be determined. The possible interaction between sialylated Lewisx structures on monocytic L-selectin and endogenous E- and P-selectin will be explored using the frozen section adhesion assay. Related experiments will determine whether the beta2- integrin dependent adhesion detected in preliminary studies reflects binding to ICAM-1 or to the endothelial selectins on synovial venules. The principle endothelial counter-receptor for the beta2-integrins is the ICAM- 1. However, these receptors also contain a low affinity binding site for P-selectin, the Lewisx epitope (CD15), and are potential carriers of the sialylated Lewisx structure. Thus the beta2-integrins may interact with both ICAM-1 and selectins on the surface of synovial venules.
Specific aim #3 will evaluate the specificity and potency of the selectin inhibitors synthesized in section 1 against human selectins. Their activities will be compared in selectin-specific frozen section assays, flow cytometric assays and in an assay which permits evaluation of leukocyte-endothelial adhesion at physiologic levels of shear-stress. The goal of this section is to determine the specificity of the inhibitors and identify assays which are likely to predict efficacy in vivo.
Showing the most recent 10 out of 37 publications
