Varied levels of reactivity by different E-selectin/Fc constructs with cutaneous lymphocyte-associated antigen (CLA)(+) CD4(+) T cells.
T cells utilize the vascular adhesion molecule E-selectin to enter inflamed skin. T cells identified by the mAb HECA-452 [cutaneous lymphocyte-associated antigen (CLA) T cells] are enriched in E-selectin ligand expressing cells. However, the proportion of CLA(+) T cells reactive with an E-selectin/Fc chimeric construct, as determined by flow cytometry, can vary considerably between studies. One important variable in these studies has been the E-selectin/Fc chimera used to assess ligand expression. We therefore compared the reactivity of mouse, rat, and human E-selectin/Fc from the same widely used commercial source with peripheral blood CLA(+) CD4(+) T cells, neutrophils, and the promyelocytic cell line HL-60 by flow cytometry and by shear flow assays. We observed that the binding activities of the different E-selectin/Fc chimeras were considerably different. Mouse E-selectin/Fc demonstrated the highest binding activity with human neutrophils and HL-60 cells by both assay approaches, whereas human E-selectin/Fc demonstrated the lowest binding activity. In addition, mouse E-selectin/Fc binding increased essentially in a linear manner with increasing expression of CLA by CD4(+) T cells, whereas human and rat E-selectin/Fc binding occurred with only a subset of CLA(+) CD4(+) T cells. We conclude that there is substantial variability in the reactivity of different E-selectin/Fc constructs, thus caution should be used when assessing E-selectin ligand expression with these reagents. For instance, the discordance in expression of CLA and E-selectin ligands by T cells may in part be due to the E-selectin/Fc construct being used.
Ni
Zhenya
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The Department of Veterinary and Biomedical Sciences, University of Minnesota, 295j AS/VM Bldg., 1988 Fitch Avenue, St. Paul, MN 55108, USA.
Walcheck
Bruce
B
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm / Q000032:analysis*; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D018922:HL-60 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000032:analysis*; D051379:Mice; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013314:Stress, Mechanical
D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; C063419:CTAGE1 protein, human; D019040:E-Selectin; D007141:Immunoglobulin Fc Fragments; D008562:Membrane Glycoproteins; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; D004492:Edetic Acid