Adhesion of lymphocytes to high endothelial venules (HEV) cells is the first step in the migration of these cells from blood into lymph nodes (LN) and Peyer's patches (PP). The binding is mediated by lymphocyte homing receptors which appear to recognize organ-specific ligands on HEV cells, although very little is known about the nature of these ligands. The long term goal of the research project is to elucidate the molecular mechanisms regulating rat lymphocyte migration into lymphoid tissues. The primary objective of this proposal am to define the molecular nature and the regulation of expression of genes encoding the LN and PP HEV ligands and the corresponding receptors We have developed mAbs, designated 3C10 and 1E9, which appears to recognize the tissue-specific ligands on LN and PP HEV cells, respectively. The proposed experiments will characterize biochemically the structures of the HEV ligands on LN and PP HEV cells recognized by the mAb. Using standard molecular biology approaches, we will then isolate and characterize cDNA clones that encode the antigens in order to analyze the relationship between the molecular structure and adhesive function of HEV ligands. We will perform expression cloning by immunoselection of COS 1 cells transfected with the CDM8 expression vector containing cDNA inserts prepared from LN or PP HEV cells. Alternatively, cloning of the genes will be achieved by hybridization selection using synthetic oligonucleotides based on amino acid sequences of the antigens. The work will then be extended to test the hypothesis that cytokines regulate the expression of the 3Cl0 and 1E9 genes in LN and PP HEV cells. In parallel studies, we will extend our recent observation that additional HEV ligands may mediate the adhesion of rat L-Selectin on lymphocytes by testing directly the hypothesis that recombinant rat L-Selectin adhere to novel ligands on LN HEV cells stimulated with the cytokine IL-4. In the last specific aim, we will map the functional epitopes or domains of the PP homing receptor for the 1E9 molecules on PP HEV cells. This integrated approach should provide a comprehensive understanding of the mechanisms regulating rat lymphocyte traffic into LN and PP and may also provide insights on lymphocyte migration into chronic inflammatory lesions and lymphoproliferative malignancies.
