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.
Bibliographical noteFunding Information:
This study was supported by the Grants HL61613 and AR049333 (BW) from the National Institutes of Health.
- Adhesion molecules
- T cells