Figure 8.
Figure 8. Plerixafor demarginates neutrophils from the lung through increase in net neutrophil release. (a) A schematic representation of the simultaneous blood sampling method. Arrows indicate the direction of blood flow (blue = venous blood entering the lung; red = arterial blood exiting the lung). X indicates the carotid artery and inferior vena cava sites of blood collection. Mice received PBS, G-CSF, or plerixafor s.c., followed by the collection of blood samples from the cannulated carotid artery and vena cava 2 h after treatment (n = 5–7 per group per time point). Net neutrophil numbers released from lung were calculated using the formula indicated. (b, Left) Kinetics of circulating neutrophil numbers in WT mice in response to i.p. injection of 1 mg/kg epinephrine treatment (n = 4 mice per group per time point). All data are shown as mean ± SEM. (b, Right) Net neutrophil release from lungs 2 h after saline or epinephrine treatment. Each symbol corresponds to one mouse (n = 7–9 per group) and the bars represent mean neutrophil numbers in each treatment group. A Student’s two-tailed unpaired t test was performed. **, P < 0.01. (c) Kinetics of net neutrophil release from lungs of mice treated s.c. plerixafor (left) or G-CSF (right; n = 6–8 per group per time point). Control group consists of mice treated s.c. with PBS. All data are shown as mean ± SEM. A Student’s two-tailed unpaired t test was performed. **, P < 0.01; n.s. = not significant. (d, Left) A representative flow cytometry plot showing the endothelial marker, CD31, and GFP expression in lung cells of WT (top) and CXCL12-GFP (bottom) mice (n = 3 per group). (d, Right) Confocal images of lung sections from CXCL12-GFP demonstrating GFP expression in subpleural area (apical; left) and secondary bronchiole (middle, right). Images are representative of three independent experiments. Bars, 25 µm. C: capillary; Al: Alveoli; Br: Bronchiole; Bv: blood vessel. (e, Top) The experimental procedure for intravital imaging of the lung. Mice were pretreated for 90 min with PBS, G-CSF, or plerixafor s.c., followed by imaging using spinning-disk confocal microscopy. Five different fields from each lung were imaged for 5 min each. (e, Bottom) Quantification of neutrophils adhering to the pulmonary capillaries over the 5-min observation period in PBS-, G-CSF–, and plerixafor-treated mice (n = 6 per group). Each symbol corresponds to data obtained from an individual mouse and the bars represent the mean number of adherent neutrophils in each treatment group. See also Video 8. (f, Top) Experimental procedure for intravital multiphoton imaging of skeletal muscle involved preparation of the LysM-GFP mouse, followed by 1 h of imaging after s.c. injection of PBS, G-CSF, or plerixafor. (f, Bottom) The duration of neutrophil adherence measured in 100-µm lengths of muscle capillaries (3–6 capillaries, 4–7 µm in diameter) of LysM-GFP mice treated s.c. with PBS, G-CSF, or plerixafor (n = 3 per group). All data are shown as mean ± SEM. One-way ANOVA analysis was performed in e and f. *, P < 0.05; **, P < 0.01; n.s. = not significant. See also Video 9.

Plerixafor demarginates neutrophils from the lung through increase in net neutrophil release. (a) A schematic representation of the simultaneous blood sampling method. Arrows indicate the direction of blood flow (blue = venous blood entering the lung; red = arterial blood exiting the lung). X indicates the carotid artery and inferior vena cava sites of blood collection. Mice received PBS, G-CSF, or plerixafor s.c., followed by the collection of blood samples from the cannulated carotid artery and vena cava 2 h after treatment (n = 5–7 per group per time point). Net neutrophil numbers released from lung were calculated using the formula indicated. (b, Left) Kinetics of circulating neutrophil numbers in WT mice in response to i.p. injection of 1 mg/kg epinephrine treatment (n = 4 mice per group per time point). All data are shown as mean ± SEM. (b, Right) Net neutrophil release from lungs 2 h after saline or epinephrine treatment. Each symbol corresponds to one mouse (n = 7–9 per group) and the bars represent mean neutrophil numbers in each treatment group. A Student’s two-tailed unpaired t test was performed. **, P < 0.01. (c) Kinetics of net neutrophil release from lungs of mice treated s.c. plerixafor (left) or G-CSF (right; n = 6–8 per group per time point). Control group consists of mice treated s.c. with PBS. All data are shown as mean ± SEM. A Student’s two-tailed unpaired t test was performed. **, P < 0.01; n.s. = not significant. (d, Left) A representative flow cytometry plot showing the endothelial marker, CD31, and GFP expression in lung cells of WT (top) and CXCL12-GFP (bottom) mice (n = 3 per group). (d, Right) Confocal images of lung sections from CXCL12-GFP demonstrating GFP expression in subpleural area (apical; left) and secondary bronchiole (middle, right). Images are representative of three independent experiments. Bars, 25 µm. C: capillary; Al: Alveoli; Br: Bronchiole; Bv: blood vessel. (e, Top) The experimental procedure for intravital imaging of the lung. Mice were pretreated for 90 min with PBS, G-CSF, or plerixafor s.c., followed by imaging using spinning-disk confocal microscopy. Five different fields from each lung were imaged for 5 min each. (e, Bottom) Quantification of neutrophils adhering to the pulmonary capillaries over the 5-min observation period in PBS-, G-CSF–, and plerixafor-treated mice (n = 6 per group). Each symbol corresponds to data obtained from an individual mouse and the bars represent the mean number of adherent neutrophils in each treatment group. See also Video 8. (f, Top) Experimental procedure for intravital multiphoton imaging of skeletal muscle involved preparation of the LysM-GFP mouse, followed by 1 h of imaging after s.c. injection of PBS, G-CSF, or plerixafor. (f, Bottom) The duration of neutrophil adherence measured in 100-µm lengths of muscle capillaries (3–6 capillaries, 4–7 µm in diameter) of LysM-GFP mice treated s.c. with PBS, G-CSF, or plerixafor (n = 3 per group). All data are shown as mean ± SEM. One-way ANOVA analysis was performed in e and f. *, P < 0.05; **, P < 0.01; n.s. = not significant. See also Video 9.

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