Fig. 1.
Fig. 1. Effect of phagocytic stimulation on the expression of CXCR1 and CXCR2 in human neutrophils. / (A,B) PMNs were incubated alone for 15 minutes at 37°C (1) or treated with nonopsonized (2) or opsonized S cerevisiae(OSC) (3) at an OSC/PMN ratio of 2:1. (C) Dose dependence of the effect of phagocytic stimulation with OSC on the expression of CXCR1 (closed bars) and CXCR2 (open bars, mean of triplicates ± SD). (D) Time dependence of the expression of CXCR1 (▴) and CXCR2 (●) in phagocytosing neutrophils (closed symbols) and in control cells (open symbols; mean ± SD, n = 3). (E,F) Effect of phenantroline at 2 mM (line 3) on the modulation of CXCR1 (E) and CXCR2 (F) expression after OSC (2), compared with untreated PMNs (1). The expression levels in panels A, B, E, F, and H are displayed as mean fluorescence intensity (MFI) of duplicate samples; the difference between double FACScan estimates was in the range of 2% to 10%. Each PMN sample was stained with irrelevant monoclonal antibody of IgG1 isotype, and the resulting MFI was subtracted from MFI with specific mAb. To test a possible effect of the OSCs themselves on the PMN-staining procedure, OSC suspension was added to control PMNs at the end of incubation, along with sodium azide, following by a full staining procedure: no effect of OSC was detected (not shown). Results similar to those of panels A and B were obtained in 16 separate experiments; dose dependence similar to that of panel C was obtained in 3 experiments; time dependence similar to that of panel D, in 5 experiments; and the effect of phenantroline was reproduced in 4 separate experiments. (G) CXCR1 (closed bars) and CXCR2 (open bars) mRNA expression in human neutrophils after treatment of PMNs with OSC. Results are expressed as a ratio of CXCR mRNA to β2-microglobulin mRNA as external control. (H) Confocal images showing the distribution of CXCR1 and CXCR2 in PMNs incubated alone (Hi,ii; upper section), treated with IL-8 at 500 ng/mL for 15 minutes (i,ii; lower section) or in PMNs treated with OSC (iii-v); corresponding MFI data are shown below the pictures (Hiii-v) Fluorescence is shown in the lower part of the pictures, and transmission light microscopy of the same cells is shown in the upper part (pN indicates PMN phagocytosing OSC; N, nonphagocytosing PMN; OSC, nonspecific fluorescence of OSC at 543 nm).

Effect of phagocytic stimulation on the expression of CXCR1 and CXCR2 in human neutrophils.

(A,B) PMNs were incubated alone for 15 minutes at 37°C (1) or treated with nonopsonized (2) or opsonized S cerevisiae(OSC) (3) at an OSC/PMN ratio of 2:1. (C) Dose dependence of the effect of phagocytic stimulation with OSC on the expression of CXCR1 (closed bars) and CXCR2 (open bars, mean of triplicates ± SD). (D) Time dependence of the expression of CXCR1 (▴) and CXCR2 (●) in phagocytosing neutrophils (closed symbols) and in control cells (open symbols; mean ± SD, n = 3). (E,F) Effect of phenantroline at 2 mM (line 3) on the modulation of CXCR1 (E) and CXCR2 (F) expression after OSC (2), compared with untreated PMNs (1). The expression levels in panels A, B, E, F, and H are displayed as mean fluorescence intensity (MFI) of duplicate samples; the difference between double FACScan estimates was in the range of 2% to 10%. Each PMN sample was stained with irrelevant monoclonal antibody of IgG1 isotype, and the resulting MFI was subtracted from MFI with specific mAb. To test a possible effect of the OSCs themselves on the PMN-staining procedure, OSC suspension was added to control PMNs at the end of incubation, along with sodium azide, following by a full staining procedure: no effect of OSC was detected (not shown). Results similar to those of panels A and B were obtained in 16 separate experiments; dose dependence similar to that of panel C was obtained in 3 experiments; time dependence similar to that of panel D, in 5 experiments; and the effect of phenantroline was reproduced in 4 separate experiments. (G) CXCR1 (closed bars) and CXCR2 (open bars) mRNA expression in human neutrophils after treatment of PMNs with OSC. Results are expressed as a ratio of CXCR mRNA to β2-microglobulin mRNA as external control. (H) Confocal images showing the distribution of CXCR1 and CXCR2 in PMNs incubated alone (Hi,ii; upper section), treated with IL-8 at 500 ng/mL for 15 minutes (i,ii; lower section) or in PMNs treated with OSC (iii-v); corresponding MFI data are shown below the pictures (Hiii-v) Fluorescence is shown in the lower part of the pictures, and transmission light microscopy of the same cells is shown in the upper part (pN indicates PMN phagocytosing OSC; N, nonphagocytosing PMN; OSC, nonspecific fluorescence of OSC at 543 nm).

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