Lymphocyte recognition of lymph node high endothelium. V. Isolation of adhesion molecules from lysates of rat lymphocytes.
Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). Investigation of lymphocyte surface molecules mediating this interaction has been carried out using an in vitro assay in which lymphocytes adhere selectively to HEV when overlaid onto sections of peripheral LN. Using this assay we have previously isolated a thoracic duct lymph component termed High Endothelial Binding Factor (HEBF), which is detected by its ability to block HEV sites where lymphocytes attach. In the current study, we present evidence that anti-HEBF antibody recognizes surface molecules of lymphocytes involved in adhesion to high endothelium. Rabbit antibody was produced against HEBF isolated from lymph by sequential ion exchange [diethylaminoethyl (DEAE)-cellulose and carboxymethyl (CM)-Sepharose] and Sephacryl S-200 gel filtration. Anti-HEBF F(ab')2 bound to 60 to 70% thoracic duct lymphocytes (TDL), spleen, and LN cells but to only 2 to 9% thymus and bone marrow cells (indirect immunofluorescence). In addition, biologically active HEBF was isolated from lymph and detergent lysates of lymphocytes by antibody affinity chromatography. Comparable amounts of the factor were isolated from lysates of TDL, LN, and spleen cells whereas little or no HEBF was detected in lysates of thymus or bone marrow cells or in serum. HEBF obtained from TDL appeared to be a glycoprotein because it was trypsin sensitive, bound to lentil lectin, and was eluted with alpha-methyl-D-mannoside. The finding that HEBF was not recovered from lysates of trypsinized TDL indicates that the activity was mediated by components expressed on cell surface membranes. Immunoprecipitation studies revealed that anti-HEBF antibody recognized radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells that resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography into three major bands with m.v. of 230,000, 210,000, and 92,000 (nonreduced) and one major band with m.w. of 70,000 and two minor bands with m.w. of 92,000 and 45,000 (reduced). These observations indicate that HEBF is a surface membrane component that is not class-specific. It is suggested that lymphocyte surface HEBF is composed of high endothelial adhesion molecules that mediate cell-cell interactions involved in entry of lymphocytes from blood into peripheral lymph nodes.