HCELL Is the Major E- and L-Selectin Ligand Expressed on Human Hematopoietic Progenitor Cells and Colon Carcinoma Cells.

Selectins are a class of cell surface proteins specialized to mediate adhesive interactions under hydrodynamic shear flow conditions, and are critical in the trafficking of hematopoietic progenitor cells (HPC) into the bone marrow and dissemination of cancer cells in hematogenous metastasis, among other biological events. More specifically, E-selectin expressed by endothelial cells has been shown to mediate shear-resistant tethering/rolling of cells recruited from the fluid stream, while L-selectin expressed by distinct leukocyte subsets has been variously shown to mediate lymph node homing, as well as the formation of heterotypic cell aggregates in bulk flow. We have previously reported that the primitive CD34+ human hematopoietic progenitor cell (HPC) line KG1a exhibits stronger E-/L-selectin ligand activities than HL60, a more differentiated CD34− human hematopoietic cell line. These findings were attributed to the HPC/KG1a restricted expression of the sialofucosylated CD44 glycoform termed HCELL (Hematopoietic Cell E-/L-selectin Ligand), since both cell lines express approximately equivalent levels of CD44 and the well-characterized selectin ligand P-selectin glycoprotein ligand-1 (PSGL-1). To directly assess whether HCELL functions as the high affinity E-/L-selectin ligand on KG1a cells, HCELL expression was silenced using lentiviral siRNA targeting CD44, and the E-/L-selectin ligand activities were subsequently compared between untreated and transduced KG1a cells. CD44 siRNA transduction of KG1a cells decreased CD44 (i.e. HCELL) expression levels >90% mean fluorescence intensity (MFI) relative to untreated and vector alone-transduced cells as measured by flow cytometry. CD44 targeting was specific, as expression of PSGL-1 was not reduced upon CD44 siRNA transduction, nor were CD29, CD49d, or CD49e levels affected. Functionally, at venous shear stress levels (≤4 dyn/cm²), CD44 siRNA-transduced KG1a cells rolled markedly faster than untreated and vector alone-transduced cells on a monolayer of E-selectin transfected Chinese hamster ovary (CHO-E) cells. At arterial and pathological shear (>4 dyn/cm²), control cells maintained rolling interactions on CHO-E cells, whereas CD44 siRNA-transduced cells readily detached from the monolayer. Comparable results were obtained for L-selectin-dependent peripheral blood mononuclear cell rolling on CD44 siRNA-transduced versus untreated and vector alone-transduced KG1a cells. Furthermore, the HCELL-negative HL60 cells supported similarly weak binding of E- and L-selectin as CD44 siRNA-transduced KG1a cells. Collectively, the data directly show that HCELL is the predominant ligand that stabilizes shear-resistant HPC binding to E- and L-selectin. We have also previously reported that the human colon carcinoma cell line LS174T expresses HCELL. To examine whether HCELL on LS174T cells functions as a selectin ligand, we again suppressed expression through targeted gene silencing by lentiviral CD44 siRNA transduction. Similar to KG1a cells, specific CD44/HCELL silencing on LS174T cells was achieved (>90% reduction in MFI relative to untreated and vector controls), resulting in significant inhibition of E- and L-selectin binding capabilities at physiologically relevant shear stress levels. In summary, these findings demonstrate that HCELL is the most potent E-/L-selectin ligand mediating shear-resistant adhesive interactions on human cells.