Figure 5.

FAM3D (family with sequence similarity 3, member D) induces neutrophil adhesion and transmigration and activates Mac-1 (macrophage-1 antigen) in neutrophils. A, Representative photographs and quantification of the adhesion of DiI-labeled neutrophils to human umbilical vein endothelial cells (HUVECs) after coculture in the absence or presence of FAM3D neutralization antibody 6D7 (20 μmol/L) or an equal amount of mouse IgG for 2 hours. HUVEC monolayers were stimulated with 4 ng/mL TNF-α (tumor necrosis factor α) for 24 hours prior to coculture. n=12. One-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons, *P<0.05. B, Representative intravital microscopic photographs and quantification of leukocyte rolling and adhesion to mesentery microvessels of wild-type (WT; n=8) and FAM3D^−/− (n=8) mice 4 hours after intraperitoneal injection with or without TNF-α (500 ng for each mice). Two-way ANOVA followed by Tukey’s test for multiple comparisons, *P<0.05, ns, no significance. C, Representative photographs and quantification of DiI-stained mouse neutrophils transmigrated through a confluent layer of HUVECs in response to 4 hours of treatment with mouse FAM3D. n=12. One-way ANOVA followed by Tukey’s test for multiple comparisons, *P<0.05. D, Real-time polymerase chain reaction (PCR) quantification of mRNA levels of Mac-1 (CD11b) in mouse neutrophils stimulated by FAM3D for 4 hours. β-Actin was applied as an internal control. n=6. One-way ANOVA followed by Tukey’s test for multiple comparisons, *P<0.05. E, Representative flow cytometric analysis and quantification of activated Mac-1 (CD11b) induced by FAM3D (10 nmol/L) or phorbol-12-myristate-13-acetate (PMA; 10 nmol/L) in human neutrophils. n=6. One-way ANOVA followed by Tukey’s test for multiple comparisons, *P<0.05, ns, no significance.