Figure 1
Figure 1. The contribution of endothelial class I PI3K activity to neutrophil trafficking and E-selectin surface distribution. Representative intravital fluorescence photomicrographs depicting neutrophil adhesion and transmigration across cremaster muscle (CM) venules in (A) WT mice and chimeric animals using (B) mechanical dissection and (C) tissue cautery 6 hours after administration of TNFα (1000 ng). Photomicrographs were acquired using an Axiotech vario microscope (Carl Zeiss MicroImaging, Thornwood, NY) fitted with a 20× water-immersion objective (LUMPlanFl, 0.5 NA; Olympus America, Center Valley, PA) coupled to a piezo driver enabling viewing in the z-axis (∼1 μm sequential sections; total 75 μm), an iXon low-light camera and IQ image acquisition software (both from Andor Technology, South Windsor, CT). To distinguish intra- versus extra-vessel cells, fluorescein isothiocyanate (FITC)–dextran (Sigma-Aldrich, St Louis, MO) was administered intravenously. Data sets were flattened along the z-axis as maximum intensity projections to enable determination of the total number of cells that had migrated into a 150 × 200 μm region on either side of the vessel wall. (D) Quantitation of the number of the migrated cells 6 hours after administration of TNF α (1000 ng,n = 3 mice per genotype). Data represent means plus or minus SEM; * indicates P less than .001. (E) Electron micrographs of immunogold-labeled E-selectin or PECAM-1 expressed on HUVECs pretreated with vehicle control or isoform-specific inhibitor (IC87114; ICOS Corp, Bothell, WA; 2 μM). Gold particles (10 nm) appear as dark dots. Electron micrographs were obtained using a CM10 transmission electron microscope (Philips Research, Briarcliff, NY). Quantification of (F) E-selectin or (G) PECAM-1 expression on endothelium in terms of the number of gold particles found in discrete groupings under the conditions described in panel E. Blockade of p110δ resulted in a 55% reduction in groupings containing 3 or more gold particles per unit area with a reduction of all groupings by 40%. Inclusion in a discrete grouping required that gold particles be no more than 30 nm apart. The percentage of gold particles that existed singly, or in groupings of 2 or 3 and more over a defined distance of 97 μm was calculated from each micrograph. Data represent means plus or minus SEM; * indicates P less than .05.

The contribution of endothelial class I PI3K activity to neutrophil trafficking and E-selectin surface distribution. Representative intravital fluorescence photomicrographs depicting neutrophil adhesion and transmigration across cremaster muscle (CM) venules in (A) WT mice and chimeric animals using (B) mechanical dissection and (C) tissue cautery 6 hours after administration of TNFα (1000 ng). Photomicrographs were acquired using an Axiotech vario microscope (Carl Zeiss MicroImaging, Thornwood, NY) fitted with a 20× water-immersion objective (LUMPlanFl, 0.5 NA; Olympus America, Center Valley, PA) coupled to a piezo driver enabling viewing in the z-axis (∼1 μm sequential sections; total 75 μm), an iXon low-light camera and IQ image acquisition software (both from Andor Technology, South Windsor, CT). To distinguish intra- versus extra-vessel cells, fluorescein isothiocyanate (FITC)–dextran (Sigma-Aldrich, St Louis, MO) was administered intravenously. Data sets were flattened along the z-axis as maximum intensity projections to enable determination of the total number of cells that had migrated into a 150 × 200 μm region on either side of the vessel wall. (D) Quantitation of the number of the migrated cells 6 hours after administration of TNF α (1000 ng,n = 3 mice per genotype). Data represent means plus or minus SEM; * indicates P less than .001. (E) Electron micrographs of immunogold-labeled E-selectin or PECAM-1 expressed on HUVECs pretreated with vehicle control or isoform-specific inhibitor (IC87114; ICOS Corp, Bothell, WA; 2 μM). Gold particles (10 nm) appear as dark dots. Electron micrographs were obtained using a CM10 transmission electron microscope (Philips Research, Briarcliff, NY). Quantification of (F) E-selectin or (G) PECAM-1 expression on endothelium in terms of the number of gold particles found in discrete groupings under the conditions described in panel E. Blockade of p110δ resulted in a 55% reduction in groupings containing 3 or more gold particles per unit area with a reduction of all groupings by 40%. Inclusion in a discrete grouping required that gold particles be no more than 30 nm apart. The percentage of gold particles that existed singly, or in groupings of 2 or 3 and more over a defined distance of 97 μm was calculated from each micrograph. Data represent means plus or minus SEM; * indicates P less than .05.

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