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 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 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 UDP-Glucose Ceramide Glucosyltransferase 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 UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase 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: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Keywords: cell adhesion molecule; endothelial cell; flow shear stress; inflammation; leukocyte.

Figure 1. Rolling of human and mouse neutrophils on E- and P-selectin

2×10⁶/mL WT mouse neutrophils (panel A, D), human neutrophils (B, E) or HL-60 cells (C, F) were perfused over substrates bearing either E- (top panels), or P-selectin (bottom panels) at 1 dyne/cm². Substrates were composed of either E-selectin expressing IL-1β stimulated HUVECs (A-C) or physisorbed recombinant P-selectin (D-F). In some cases, the cells were treated with 1mg/mL pronase for 1h at 37°C prior to perfusion. Individual bars in each panel present the rolling (dark) and adherent (light) cell density 2 min after the start of perfusion. 2PH1 and KPL-1 are blocking mAbs against murine and human PSGL-1, respectively. * and † represent statistically significant differences for rolling and adherent cells, respectively (P<0.05 with respect to all other treatments except *s and †s are not different from each other).

Figure 2. Silencing UGCG in HL-60s

A. pLKO.1 lentiviral vector with DsRed reporter and UGCG shRNA was used to create stable UGCG^–HL-60 cells. B. TLC of UGCG enzyme assay shows activity reduction in the UGCG^–HL-60 cell lysate (lane 2) compared to WT HL-60 (lane 1). C6-NBD-GlcCer product standards serve as positive control (lanes 3, 4). Negative controls either lack cell lysate (lane 5) or contain GDP-Fuc in place of UDP-Glc (lane 6). C. Flow cytometry histograms showing cell-surface expression of CLA/HECA-452 (left), sialyl Lewis-X/CD15s/CSLEX-1 (middle) and Lewis-X/CD15 (right). Black-empty and shaded histograms correspond to WT and UGCG^–HL-60s. Dashed-empty histograms correspond to isotype controls. Silencing UGCG reduced cell-surface CLA and sLe^X expression. D-E. Rolling and adherent cell density (D) and cumulative rolling velocity distribution (E) data for WT and UGCG^–HL-60s perfused over IL-1β stimulated HUVECs at 1 dyn/cm². Statistical symbols are same as Fig. 1. Cells with reduced UGCG activity displayed ~60% reduced rolling and adherent cell density and ~2 fold higher rolling velocity. Pronase further reduced the number of adherent UGCG^–HL-60s. Numbers in brackets present ‘(number of experiments/total number of cells analyzed)’.

Figure 3. UGCG knockout HL-60s

A. Schematic of CRISPR-Cas9 editing vector used to create knockout HL-60 cells lacking UGCG. Top row presents WT HL-60 UGCG sequence while bottom row shows 16bp chromosomal deletion in the UGCG-KO cells. Bold-underlined text highlights the guide RNA sequence and the bold-italicized text denotes the protospacer adjacent motif (PAM/NGG). Only one UGCG allele was sequenced since UGCG is present on chromosome-9, and spectral karyotyping of HL-60 demonstrates the loss of the second copy due to the chromosome-9 translocation. B. Flow cytometry histograms present cell surface expression of CLA/HECA-452, sLe^X/CSLEX-1, Le^X/CD15 and VIM-2/CD65s epitopes on WT (black-empty histogram) and UGCG-KO HL-60s (gray-filled). Dashed-empty histogram is isotype control. Mean ± SEM data for WT and KO cells is presented in inset. C. L-PHA-FITC (left), Mal-II-biotin (middle) and PNA-FITC (left) lectin binding to WT (black-empty) and UGCG-KO (gray-filled) HL-60s measured using flow cytometry. Prior to PNA-FITC staining, the cells were desialylated using α2,3/6/8/9 neuraminidase from A. ureafaciens. In all panels, dashed (- -) histograms correspond to cells alone without lectins. Cells incubated with Mal-II-biotin were subsequently detected using α-biotin-FITC. Hatched peaks indicate negative controls: WT cells treated with neuraminidase (middle) and without neuraminidase (right). UGCG knockouts display reduction in CLA and sLe^X epitopes (*P<0.05 with respect to WT), but no major change in lectin binding. D. Partial MALDI-TOF MS spectra of permethylated N-glycans of WT (upper panel) and UGCG-KO (lower panel) HL-60 cells. Spectra are from the 50% acetonitrile fraction. Red peaks correspond to decreased sialylated structures, while green peaks correspond to increased bisected structures, in the UGCG-KO HL-60s (lower panel). The increase in bisected N-glycans in the UGCG-KO is supported by comparing the relative intensity of ions at m/z 3748 to 3503 (blue peaks). In WT, the ratio of these peaks is 0.356. In the UGCG-KO it is 1.183, thus suggesting a ~232% increase in bisected structures in the UGCG-KO HL-60s. Full spectra are shown in Supplemental Fig. IV. E. MALDI-TOF MS spectra of permethylated O-glycans of WT (top) and UGCG-KO (bottom) HL-60 cells. Spectra are from the 35% acetonitrile fraction. Annotated structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M+Na]⁺. Putative structures are based on composition, tandem MS/MS (data not shown), and biosynthetic knowledge. Cartoons that include sugar symbols outside a bracket have not been unequivocally defined. Letters “m” and “M” in bold characters indicate minor and major abundances respectively.

Figure 4. E-selectin mediated rolling of UGCG-KO HL-60s

A-B. Rolling and adherent cell density (panel A) and rolling velocity distribution (B) data for UGCG-KO HL-60s measured under conditions in Fig. 2D and 2E, respectively. Statistical symbols are same as Fig. 1. In addition, ** denotes statistically significant difference (P<0.05) in cell rolling with respect to all other treatments. UGCG-KO HL-60s display ~60% reduced rolling and adherent cell density, with ~3 fold higher median rolling velocity compared to WT HL60s. Pronase further increased rolling velocity. C. Individual columns present total number of cell tethers formed in a 4 min interval. Number of tethers did not vary significantly with cell type or upon pronase treatment. D. Tethered cells (normalized to 100%) were classified into: i. adherent, ii. rolling or iii. detached cells as defined in Methods.Cell detachment was prominent in UGCG-KO HL-60 cells (For detached cells: ‡P<0.05 with respect to all treatments except ‡s are not different from each other, and #P<0.05 with respect to all other treatments). Number of adherent cells is also highest for WT HL60s (†P<0.05 with respect to all treatments except †s are not different from each other). E. Instantaneous rolling velocity and F. cumulative distance travelled with time for five representative cells for each treatment. WT HL-60s rolled stably at 3-15 μm/s, whereas all the other cell types show abrupt increases in rolling velocities as seen in the intermittent peaks up to 250μm/s. Detachment events are depicted by up arrows in panel f.

Figure 5. Studies with differentiated HL-60 cells

HL-60s were differentiated towards granulocytes by culturing cells in growth medium containing 1.3% DMSO for 5 days. A. Cell surface glycan and glycoprotein expression over the course of differentiation. B. Rolling and adherent cell density data for differentiated cells binding to stimulated HUVEC monolayers at 1dyn/cm² with or without pronase treatment. Experimental method and statistical symbols are identical to Fig. 1. C.-D. % Transmigration, under shear (C) and static (no flow, D) conditions. Plot quantifies the % of total interacting cells in a FOV that transmigrated during a 20 min interval. ‡ and # denote P<0.05 with respect to all treatments, except these symbols are not different from each other. Increased cell detachment of UGCG-KO and UGCG^–HL-60s reduced transmigration under shear.

Figure 6. Neutrophils derived from human hematopoietic stem cells (hHSCs)

hHSCs were differentiated to mature neutrophils over 12 days. ‘UGCG^–-Neu’ were generated using lentivirus carrying a DsRed reporter and UGCG shRNA. Control cells generated using a vector lacking the shRNA was called ‘DsRed-Neu’. A. Transduction efficiency quantified based on % cells that were DsRed positive (bottom-right quadrant). B. Rolling and adherent cell density data for DsRed⁺ neutrophils derived from hHSCs on IL-1β stimulated HUVECs. * and † denote statistically lower rolling and adherent cell density on E-selectin upon silencing UGCG (P<0.05 with respect to WT). Data are presented for hHSCs derived from 4 different human donors.