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. 2018 Aug 10;293(38):14689–14706. doi: 10.1074/jbc.RA118.004589

Figure 6.

Figure 6.

P-selectin and hyaluronic acid-binding assay in CD11b+ and CD3ϵ+ peripheral blood subsets in male Apoe−/− and Neu1hypoApoe−/− mice. Hypomorphic sialidase Apoe−/− peripheral blood displayed no difference in P-selectin binding in the CD11b+ (A and B) subsets (p = 0.70), but a significant reduction of P-selectin binding in the CD3ϵ+ (C and D) subsets was observed (p = 0.04). Peripheral blood was incubated with 4 μg of P-selectin-human IgG chimera for 1 h and then incubated with anti-human IgG-AF488, anti-CD11b-APC, and anti-CD3ϵ-Pacific Blue. Samples were analyzed on an LSR II, n = 8 Apoe−/− and n = 9 Neu1hypoApoe−/−; the data were analyzed using a two-tailed Student's t test. E–I, HA binding on Apoe−/− and Neu1hypoApoe−/− peripheral blood. E and F, the CD11b+ monocytes and neutrophils exhibited significantly reduced hyaluronic acid binding (p = 0.027), whereas the CD3ϵ+ cells (G–I) displayed two distinct populations that bound hyaluronic acid at different amounts, HAlo (H) and HAhi (I), and showed significantly reduced hyaluronic acid binding in Neu1hypoApoe−/− cells (p = 0.042 for the HAhi group and p = 0.011 for the HAlo group, two-tailed Student's t test). J and K, leukocyte recruitment in the hepatic central vein of WT and Neu1hypo animals was analyzed by intravital microscopy following sham treatment with helper-dependent adenovirus expressing bacterial β-Gal (HD-Adβ-gal), or rescue with helper-dependent adenovirus expressing NEU1 sialdiase (HD-AdSial), and TNFα treatment to activate leukocyte extravasation. J, the flux of leukocytes rolling along the endothelium was quantified as the number of cells per min per field of view. Hypomorphic sialidase mice showed a significant increase in the flux of rolling leukocytes, which was reduced following rescue with HD-AdSial. K, the number of leukocytes adherent to the endothelium was quantified per field of view. The number of firm cell adhesion events was significantly impeded with hypomorphic NEU1 sialidase expression, and significantly increased after HD-AdSial-mediated overexpression. *, p < 0.05; **, p < 0.01; ***, p < 0.001.