Figure 2: Phagocytosis and adhesion of monocyte derived macrophages from individuals homozygous (77R/R) or heterozygous (77R/H) for the Mac- 1 alleles. Monocytes from buffy coats (obtained from the Scottish National Blood Transfusion Service, Ninewells Hospital) were isolated and cultured in Macrophage specific serum free media (Life technologies) with 10ng/ml GMCSF (Peprotech) for 6 days prior to experiments, resulting in differentiation of monocytes to macrophages, as analysed by flow cytometry for macrophage markers (CD14high, CD11bhigh)(not shown).
A-Cell surface expression of CD11b and CD18 was examined by flow cytometry using CD11b and CD18 specific antibodies (Biolegend) and was similar in 77R/H heterozygous cells (red) and 77RR cells (blue). Isotype control is shown in black.
B-Phagocytosis of iC3b-coated fluorescent beads.Monocyte-derived macrophages from individuals homozygous (77R/R, n=17) or heterozygous (77R/H, n=5) were challenged with fluorescent beads coated with iC3b or with IgM alone [56] for 20 min prior to washing and analysis by flow cytometry. The percentage of cells with increased fluorescence due to bead phagocytosis was measured, and that solely due to Complement -mediated phagocytosis was calculated by subtracting the percentage phagocytosis of IgM coated beads from that of iC3b coated beads.
C-Monocyte –derived macrophages were analysed in static adhesion assays [57] on plates coated with 6 μg/ml human ICAM-1 (R and D systems)as described previously [57]. Prior to adhesion cells were incubated with control IgG (blue), anti-CD11b (red) or anti-CD18 (green) to estimate the separate contributions of beta2-family integrins to cell-adhesion, and then stimulated with PdBuor magnesium to activate integrins.
D-Adhesion of 77R/H heterozygous macrophages (red) and 77R/R macrophages (blue) to ICAM-1was examined as above using monocyte-derived macrophages from individuals homozygous (77R/R, n=18) or heterozygous (77R/H, n=6) for the Mac-1 allele. No significant differences in adhesion were observed.