Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin–Dependent Rolling in the Multistep Leukocyte Adhesion Cascade

Abstract

Objective Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids (GSLs) expressed on human myeloid cells in regulating E-selectin mediated cell adhesion. Approach and Results A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase (UGCG) using either lentivirus delivered shRNA or CRISPR-Cas9 based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UGCG disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics based flow chamber studies demonstrated that both the UGCG knockouts and knockdowns display ~60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells (ECs), without altering cell adhesion to P-selectin. Consistent with the concept that the GSLs support slow rolling and the transition to firm arrest, inhibiting UGCG activity resulted in frequent leukocyte detachment events, skipping motion and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. Conclusions GSLs likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Migration against the direction of flow is LFA-1-dependent in human hematopoietic stem and progenitor cells

“…All primary and cell lines were cultured as described previously (Lee et al, 2014;Mondal et al, 2014Mondal et al, , 2016Stolfa et al, 2016). Briefly, KG1a cells (ATCC) were cultured as described previously in Iscove's modified Eagle's medium (IMDM) containing 20% fetal bovine serum (FBS).…”

Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion

“…When added to human leukocytes, SNAP more effectively reduced expression of the sialyl Lewis-X (sLe X , Siaα2–3Galβ14(Fucα1–3)GlcNAc) epitope on cells, compared to ONAP. SNAP also reduced cell adhesion to E-selectin, the major human selectin which binds sialofucosylated epitopes on leukocyte O-glycans, N-glycans and GSLs (Mondal et al, 2016; Stolfa et al, 2016). In mouse, SNAP treated leukocytes exhibited reduced migration to sites of inflammation and bone marrow.…”

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

“…3A). MCAM, Ninjurin-1, and CD62E, previously shown to be involved in the recruitment of specific immune cell types to the CNS (14,15,20,35,36), were also assessed and showed no significant differences between ALCAM KO and WT mouse BBB-ECs (MBECs) either in resting or stimulated conditions (Fig. S3).…”