Biomedical subjects

B Walcheck

Publications and source records attributed to B Walcheck.

21 records · Page 2Linked to original sources

PubMed1992

Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin.

Selectins 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) (EL-246) that recognizes both human leukocyte L-selectin (previously called LAM-1, LECAM-1, or gp90MEL-14) and endothelial cell E-selectin (previously called ELAM-1). EL-246 recognized a 110-kD protein expressed on cells transfected with E-selectin cDNA and stained many postcapillary venules in inflamed human tonsil. EL-246 also stained human peripheral blood leukocytes and showed identity with anti-L-selectin mAb in two-color flow cytometric analysis. The expression of the leukocyte EL-246 antigen was regulated in the same manner as L-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 greater than 95% of lymphocyte adhesion to peripheral lymph node high endothelial venules and greater than 90% of neutrophil adhesion to E-selectin transfectants. In addition to the EL-246 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 EL-246 epitope to the short consensus repeat (SCR) domains of L-selectin. EL-246 is the first mAb that recognizes two different selectins 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.

Animals↗

PubMed1992

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

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.

Amino Acid Sequence↗

PubMed1992

Rapid activation-independent shedding of leukocyte L-selectin induced by cross-linking of the surface antigen.

L-selectin (also termed LAM-1, Leu-8,TQ-1, gp90MEL-14, peripheral lymph node homing receptor and LECAM-1) is an adhesion protein thought to be important in leukocyte entry into lymphoid tissues and sites of inflammation. We, as well as others, have shown that leukocyte activation by chemotactic factors results in rapid shedding (release) of L-selectin from the cell surface. Here we have used flow cytometry, enzyme-linked immunosorbent assay, and SDS-PAGE/Western blot analysis to determine whether cross-linking of L-selectin in the absence of activation causes shedding. We found that rapid loss of leukocyte L-selectin expression (down-regulation) could be induced by treating cells with a chemical cross-linker [bis (sulfosuccinimidyl) suberate]. L-selectin down-regulation via cross-linking could occur at 4 degrees C and in the absence of detectable cell activation (increased expression of CD11b/18). The loss of L-selectin expression was due to shedding of the molecule from the leukocyte cell surface. Cross-linking of L-selectin with specific monoclonal antibodies also caused loss of surface expression of L-selectin at 37 degrees C. Finally, shed L-selectin was detected in the plasma of healthy adults whose peripheral blood leukocytes demonstrated no obvious signs of activation. Our results suggest that activation-independent shedding of leukocyte L-selectin may occur in vivo and a possible mechanism could involve cross-linking of leukocyte L-selectin. This provides a novel mechanism for rapid regulation of expression of a leukocyte-endothelial cell adhesion receptor.

Animals↗