Fig. 4.

HRG's spherical-shape-inducing and adhesion-increasing effects on purified human neutrophils. (A, H, and I) Purified human neutrophils were labeled with calcein-AM (green) and Hoechst33342 (blue) for 20 min. The neutrophils were incubated with reagents (1 μM) on polystyrene surface (A) or a monolayer of EA.hy926 (H and I) for 60 min and the neutrophil shape was observed under a fluorescent microscope (A and I) or a differential interference contrast microscope (H). (B and J). The rounding of neutrophils was analyzed by the In Cell Analyzer. Form factor (max diameter/ min diameter) was determined. One unit represents an ideal spherical shape. ^⁎P < 0.05, ^⁎⁎⁎P < 0.001 vs. HBSS. ^†P < 0.05, ^†††P < 0.001 vs. BSA. ^‡‡P < 0.01, ^‡‡‡P < 0.001 vs. HSA. ^§§§P < 0.001 vs. fMLP. (C and K) After gentle washing of the microtiter plate with HBSS twice, the residual cell numbers were counted and expressed as percentages of the initial cell numbers. ^⁎⁎P < 0.01, ^††P < 0.01, ‡‡P < 0.01, and §§P < 0.01 vs. HBSS, BSA, HSA, and fMLP, respectively. (D) The concentration-effect curve was drawn by the plot of a spherical shape at different concentrations of HRG. The maximal roundness at 1 μM of HRG was expressed as 100%. (E) Effects of polyclonal antibody against HRG on HRG-induced induction of spherical shape in neutrophils. Anti-HRG Ab (300 μg/ml) or control IgG was added simultaneously with HRG and the incubation continued for 1 h. (F) Quantitative analysis of the effects of specific antibodies against HRG on neutrophil shape. ^⁎⁎⁎P < 0.001 and ^†††P < 0.001 vs. HBSS and HRG + IgG groups, respectively. (G) Effects of specific antibody against HRG on adhesion of neutrophils. ^⁎⁎⁎P < 0.001 and ^†P < 0.05 vs. HBSS and HRG + IgG, respectively. The results shown are the means ± SEM of three experiments (B, C, D, F, G, J, and K). Scale bars, 20 μm. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.