Characterization of the bovine peripheral lymph node homing receptor: a lectin cell adhesion molecule (LECAM)

Abstract

The phenomenon of tissue-specific homing of lymphocyte populations has been most clearly shown in larger domestic animals, such as the sheep and cow, yet the molecular interactions which control these processes in these animals have not been defined. Here we tested the cross-reactivity of four anti-human peripheral lymph node homing receptor (LECAM-1) (also known as LAM-1, LEC-CAM-1, Leu-8, TQ-1, or human equivalent of gp90 MEL-14) antibodies on bovine lymphocytes. These antibodies stained all bovine neutrophils and monocytes, and variable numbers of peripheral blood lymphocytes, as determined by flow cytometry. In young calves (less than 1 month old) virtually all circulating lymphocytes expressed LECAM-1, whereas the percentage of positive lymphocytes in older animals (greater than 1 year) varied from 17%-67%. Bovine LECAM-1 was rapidly lost from the cell surface of PMA-activated and chymotrypsin-treated cells. Anti-LECAM-1 monoclonal antibody blocked greater than 80% of bovine lymphocyte binding to peripheral lymph node high endothelial venules (HEV). Since the lectin domain of LECAM-1 is thought to mediate lymphocyte-HEV adhesion, we sought to establish further the similarity of the bovine, mouse, and human molecules by comparing nucleotide sequences in this region of the molecule. The polymerase chain reaction (PCR) was used to specifically clone the bovine lectin domain from single-strand cDNA. Subsequent sequencing showed an identity of greater than 80% at the nucleotide level with the human and mouse molecules. The predicted amino acid sequences were also highly conserved. Though striking similarities were seen between the bovine, mouse and human molecule, indicating evolutionary conservation of this family of proteins, notable differences were detected. The nucleotide sequence of the bovine lectin domain predicts one additional N-linked glycosylation site compared to mouse and human. Preliminary analysis suggested a more tissue-restricted expression of LECAM-1 in the compared to the human and mouse, which correlates with a better separation of lymphocyte homing phenotypes seen in these larger animals. Virtually all peripheral lymph node lymphocytes in 6-month-old calves expressed LECAM-1, whereas, ileal Peyer's patch lymphocytes were predominantly negative. Finally, by testing anti-human LECAM-1 antibodies in a different species we have established the co-expression of antigenic epitopes on leucocyte LECAM-1 and a molecule(s) expressed by endothelial cells.