Figure 1.
Local endothelial cell calcium influx is associated with in vivo neutrophil TEM. (A) Mice expressing the calcium sensor GCaMP3 specifically in endothelial cells (VE-Cadherin Cre GCaMP3fl/fl) were lethally irradiated and their bone marrow reconstituted from CatchUpIVM with red fluorescent neutrophils. After allowing reconstitution, inflammation was induced by intrascrotal injection of IL-1β. Nonblocking fluorophore-conjugated anti-PECAM (blue) was also coinjected to visualize the vasculature. 4 h after the injection, the cremaster muscle was exteriorized and prepared for confocal intravital microscopy as detailed in the Materials and methods. The images shown are Z-projections of the 3D stacks. A full-length video is included in the supplemental material (Video 1). Arrows denote neutrophils in the process of TEM. Endothelial cell calcium influx is associated with neutrophil TEM and is localized around the transmigratory pore. Insets display a magnified view of the local PECAM pore and calcium influx. 12 mice were studied. Scale bar is 50 µm. (B) Inflammation was induced in VE-Cadherin Cre GCaMP3fl/fl mice by intrascrotal injection of IL-1β. Nonblocking fluorophore-conjugated anti-PECAM (red) and fluorescently conjugated anti-CD18 (blue) were coinjected to visualize the vasculature and circulating neutrophils, respectively. This additional approach provided validation of our adoptive bone marrow transfer model. 4 h after the injection, the cremaster muscle was exteriorized and prepared for confocal intravital microscopy as detailed in the Materials and methods. The images shown are Z-projections of the 3D stacks. A full-length video is included in the supplemental material (Video 2). Arrows denote neutrophils in the process of TEM. Endothelial cell calcium influx is associated with neutrophil TEM and is localized around the transmigratory pore. Insets display a magnified view of the local calcium influx. Scale bar is 10 µm. (C) Each colored line represents a separate transmigration event where the calcium signal was quantitated. Mean fluorescence intensity of the region of interest (dotted lines in A and B) was calculated, background corrected, and normalized to baseline as described in the Materials and methods. The beginning and completion of TEM were defined by the PECAM gap. Eight independent TEM events are shown here. (D) Intravital microscopy videos of inflammation in mice expressing the calcium sensor GCaMP3 restricted to endothelial cells were analyzed. For axial profile analysis, a 15-µm line (shown as the dotted red line) was drawn centered on the pore of a TEM event, and the pixel intensity along that line was recorded for the green channel (GCaMP3 signal). The same line was used to analyze pixel intensity for the GCaMP3 signal when the leukocyte was adherent to the endothelium before the TEM event. The individual lines were adjusted slightly to account for variations in the center of the pore. The plots were normalized to the minimum intensity value proximal to the pore. Note that this makes the relative intensities for the plots similar at the proximal 0-µm end of the plot, but retains meaningful information regarding the axial profile of the GCaMP3 signal. Five separate TEM events are shown here.

Local endothelial cell calcium influx is associated with in vivo neutrophil TEM. (A) Mice expressing the calcium sensor GCaMP3 specifically in endothelial cells (VE-Cadherin Cre GCaMP3fl/fl) were lethally irradiated and their bone marrow reconstituted from CatchUpIVM with red fluorescent neutrophils. After allowing reconstitution, inflammation was induced by intrascrotal injection of IL-1β. Nonblocking fluorophore-conjugated anti-PECAM (blue) was also coinjected to visualize the vasculature. 4 h after the injection, the cremaster muscle was exteriorized and prepared for confocal intravital microscopy as detailed in the Materials and methods. The images shown are Z-projections of the 3D stacks. A full-length video is included in the supplemental material (Video 1). Arrows denote neutrophils in the process of TEM. Endothelial cell calcium influx is associated with neutrophil TEM and is localized around the transmigratory pore. Insets display a magnified view of the local PECAM pore and calcium influx. 12 mice were studied. Scale bar is 50 µm. (B) Inflammation was induced in VE-Cadherin Cre GCaMP3fl/fl mice by intrascrotal injection of IL-1β. Nonblocking fluorophore-conjugated anti-PECAM (red) and fluorescently conjugated anti-CD18 (blue) were coinjected to visualize the vasculature and circulating neutrophils, respectively. This additional approach provided validation of our adoptive bone marrow transfer model. 4 h after the injection, the cremaster muscle was exteriorized and prepared for confocal intravital microscopy as detailed in the Materials and methods. The images shown are Z-projections of the 3D stacks. A full-length video is included in the supplemental material (Video 2). Arrows denote neutrophils in the process of TEM. Endothelial cell calcium influx is associated with neutrophil TEM and is localized around the transmigratory pore. Insets display a magnified view of the local calcium influx. Scale bar is 10 µm. (C) Each colored line represents a separate transmigration event where the calcium signal was quantitated. Mean fluorescence intensity of the region of interest (dotted lines in A and B) was calculated, background corrected, and normalized to baseline as described in the Materials and methods. The beginning and completion of TEM were defined by the PECAM gap. Eight independent TEM events are shown here. (D) Intravital microscopy videos of inflammation in mice expressing the calcium sensor GCaMP3 restricted to endothelial cells were analyzed. For axial profile analysis, a 15-µm line (shown as the dotted red line) was drawn centered on the pore of a TEM event, and the pixel intensity along that line was recorded for the green channel (GCaMP3 signal). The same line was used to analyze pixel intensity for the GCaMP3 signal when the leukocyte was adherent to the endothelium before the TEM event. The individual lines were adjusted slightly to account for variations in the center of the pore. The plots were normalized to the minimum intensity value proximal to the pore. Note that this makes the relative intensities for the plots similar at the proximal 0-µm end of the plot, but retains meaningful information regarding the axial profile of the GCaMP3 signal. Five separate TEM events are shown here.

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