Figure 3.
Figure 3. ICAM-1 and the integrins Mac-1 and LFA-1 mediate neutrophil–pericyte interactions. (A) Representative 3D-reconstructed confocal image of a TNF-stimulated (3 h) cremasteric postcapillary venule immunostained for PECAM-1 (EC, blue), ICAM-1 (green), and αSMA (Pericytes, red). The region within the box is enlarged in the images below following a longitudinal 2-µm cross-section of the vessel. In the middle panel of the enlarged region, a 4% opacity filter on the PECAM-1 and αSMA channels was applied to highlight ICAM-1 expression on the endothelium (arrow) and on the pericyte layer (arrowhead). The bottom panel shows only the ICAM-1 channel. Bars, 10 µm. (B) MFI per unit volume of ICAM-1 labeling on ECs and pericytes in unstimulated control and TNF-stimulated (3 h) tissues. (C) Effect of antibody blockade of ICAM-1, Mac-1, and LFA-1 on TNF-induced abluminal neutrophil crawling (after TEM) as observed in vivo in cremasteric postcapillary venules of using 4D confocal IVM. 2 h after intrascrotal injection of TNF in αSMA-RFPcherry x Lys-EGFP-ki mice (post-TEM migration), anti–ICAM-1, Mac-1, or LFA-1 blocking or isotype-matched control antibodies were injected i.s. for 30 min before cremasteric exteriorization. Crawling paths of abluminal neutrophils are shown (example of 20 per group for clarity) as normalized for their origins (sites of TEM). (D) Effect of anti–ICAM-1, Mac-1, and LFA-1 blocking antibodies on neutrophil abluminal crawling parameters as compared with control antibody-treated animals. The three graphs show the mean speed, total distance length, and displacement length of crawling. Data were obtained after analysis of a minimum of 211 crawling cells. (E) Number of extravasated neutrophils at 4 h after TNF stimulation of cremasteric muscles in animals treated with local injection of blocking anti–ICAM-1, Mac-1, and LFA-1 mAbs or isotope match control mAb. (F) MFI of KC staining per unit volume on ECs and pericytes in unstimulated control and TNF-stimulated (3 h) cremasteric venules. Results are expressed as mean of n = 4–5 vessels per mouse with at least 5 animals per group (A and B), 5–11 vessels from 4 animals (F), and from 1 vessel per animal with at least 6 animals/groups (C–E). Significant differences between groups are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant differences between the anti–Mac-1 and anti–LFA-1 treated groups are indicated by # (P < 0.05).

ICAM-1 and the integrins Mac-1 and LFA-1 mediate neutrophil–pericyte interactions. (A) Representative 3D-reconstructed confocal image of a TNF-stimulated (3 h) cremasteric postcapillary venule immunostained for PECAM-1 (EC, blue), ICAM-1 (green), and αSMA (Pericytes, red). The region within the box is enlarged in the images below following a longitudinal 2-µm cross-section of the vessel. In the middle panel of the enlarged region, a 4% opacity filter on the PECAM-1 and αSMA channels was applied to highlight ICAM-1 expression on the endothelium (arrow) and on the pericyte layer (arrowhead). The bottom panel shows only the ICAM-1 channel. Bars, 10 µm. (B) MFI per unit volume of ICAM-1 labeling on ECs and pericytes in unstimulated control and TNF-stimulated (3 h) tissues. (C) Effect of antibody blockade of ICAM-1, Mac-1, and LFA-1 on TNF-induced abluminal neutrophil crawling (after TEM) as observed in vivo in cremasteric postcapillary venules of using 4D confocal IVM. 2 h after intrascrotal injection of TNF in αSMA-RFPcherry x Lys-EGFP-ki mice (post-TEM migration), anti–ICAM-1, Mac-1, or LFA-1 blocking or isotype-matched control antibodies were injected i.s. for 30 min before cremasteric exteriorization. Crawling paths of abluminal neutrophils are shown (example of 20 per group for clarity) as normalized for their origins (sites of TEM). (D) Effect of anti–ICAM-1, Mac-1, and LFA-1 blocking antibodies on neutrophil abluminal crawling parameters as compared with control antibody-treated animals. The three graphs show the mean speed, total distance length, and displacement length of crawling. Data were obtained after analysis of a minimum of 211 crawling cells. (E) Number of extravasated neutrophils at 4 h after TNF stimulation of cremasteric muscles in animals treated with local injection of blocking anti–ICAM-1, Mac-1, and LFA-1 mAbs or isotope match control mAb. (F) MFI of KC staining per unit volume on ECs and pericytes in unstimulated control and TNF-stimulated (3 h) cremasteric venules. Results are expressed as mean of n = 4–5 vessels per mouse with at least 5 animals per group (A and B), 5–11 vessels from 4 animals (F), and from 1 vessel per animal with at least 6 animals/groups (C–E). Significant differences between groups are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant differences between the anti–Mac-1 and anti–LFA-1 treated groups are indicated by # (P < 0.05).

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