Figure 4
Figure 4. The leukocyte integrin Mac-1 promotes FcγRIIIB-mediated neutrophil recruitment to intravascular ICs and FcγRIIIB mediates IC uptake from within the vessel wall. (A-B) Mice were injected intravenously with preformed soluble ICs, and the rolling velocity and adhesion of leukocytes in the cremaster venules were evaluated. (A) Representative pictures of postcapillary venules from indicated mice with rolling (arrow) and adherent (arrowhead) neutrophils. In FcγRIIIB/γ−/− mice, IC deposition slowed the velocity of rolling neutrophils and increased their adhesion compared with γ−/− mice, and FcγRIIIB/γ−/− mice without soluble ICs (dotted line). Neutrophils of FcγRIIIB/γ−/− mice additionally deficient in Mac-1 no longer supported slow rolling and adhesion. N = 5 mice per group. (B) Wild-type mice were given soluble ICs intravenously and TNF-primed bone marrow–derived, CMFDA-labeled neutrophils from FcγRIIIB/γ−/− mice or the same lacking Mac-1 (IIIB/γ−/−/Mac1−/−) were adoptively transferred via the jugular vein. IC deposition increased adhesion of FcγRIIIB-positive neutrophils, although this was not observed with FcγRIIIB-expressing neutrophils that lacked Mac-1. N = 4 mice per group. (C) Soluble ICs generated with BSA and anti-BSA and incubated with Cy3-secondary antibody (sIC, Cy3; red) were injected intravenously together with FITC-labeled Gr-1 antibody, which labels peripheral blood neutrophils (Gr-1, FITC; green). The cremaster of anesthetized animals was exteriorized, and neutrophil-vessel wall interactions were examined by intravital confocal microscopy. Left 3 panels: Slices along the Z-axis were acquired and reconstructed into 3-D Z stacks. The merges of the 2 images are also shown. The images were subjected to surface analysis with Imaris ×64 Version 7.4.2 Software (Bitplane Scientific). Right 3 panels: Surfaces were created from Cy3 (red) and FITC channels (green). sIC (red) within the cell was visualized by creating a surface object from FITC-masked Cy3 channel in 50% transparent neutrophils. Rightmost panel: Neutrophils were cut with a clipping surface through the FITC channel to visualize Cy3 signal only within the cells. IC internalization was determined by calculating the volume ratio between the 2 surfaces objects created by FITC-masked Cy3 (red) and FITC-anti–Gr-1 (green) channels, respectively. There was an increase in the amount of ICs present within FcγRIIIB- or FcγRIIA-positive neutrophils compared with that in γ−/− neutrophils. N = 3 or 4 mice per group. **P < .01. ***P < .001, compared with IC uptake in γ−/− neutrophils. (D) Fifteen minutes after injection of BSA–anti-BSA soluble ICs, peripheral blood was collected. Gr-1–positive neutrophils were analyzed for Cy3 signal by flow cytometry. Minimal fluorescence was associated with these cells, suggesting that IC uptake did not occur in circulating neutrophils. N = 3 mice per group. n.s. indicates not significant.

The leukocyte integrin Mac-1 promotes FcγRIIIB-mediated neutrophil recruitment to intravascular ICs and FcγRIIIB mediates IC uptake from within the vessel wall. (A-B) Mice were injected intravenously with preformed soluble ICs, and the rolling velocity and adhesion of leukocytes in the cremaster venules were evaluated. (A) Representative pictures of postcapillary venules from indicated mice with rolling (arrow) and adherent (arrowhead) neutrophils. In FcγRIIIB/γ−/− mice, IC deposition slowed the velocity of rolling neutrophils and increased their adhesion compared with γ−/− mice, and FcγRIIIB/γ−/− mice without soluble ICs (dotted line). Neutrophils of FcγRIIIB/γ−/− mice additionally deficient in Mac-1 no longer supported slow rolling and adhesion. N = 5 mice per group. (B) Wild-type mice were given soluble ICs intravenously and TNF-primed bone marrow–derived, CMFDA-labeled neutrophils from FcγRIIIB/γ−/− mice or the same lacking Mac-1 (IIIB/γ−/−/Mac1−/−) were adoptively transferred via the jugular vein. IC deposition increased adhesion of FcγRIIIB-positive neutrophils, although this was not observed with FcγRIIIB-expressing neutrophils that lacked Mac-1. N = 4 mice per group. (C) Soluble ICs generated with BSA and anti-BSA and incubated with Cy3-secondary antibody (sIC, Cy3; red) were injected intravenously together with FITC-labeled Gr-1 antibody, which labels peripheral blood neutrophils (Gr-1, FITC; green). The cremaster of anesthetized animals was exteriorized, and neutrophil-vessel wall interactions were examined by intravital confocal microscopy. Left 3 panels: Slices along the Z-axis were acquired and reconstructed into 3-D Z stacks. The merges of the 2 images are also shown. The images were subjected to surface analysis with Imaris ×64 Version 7.4.2 Software (Bitplane Scientific). Right 3 panels: Surfaces were created from Cy3 (red) and FITC channels (green). sIC (red) within the cell was visualized by creating a surface object from FITC-masked Cy3 channel in 50% transparent neutrophils. Rightmost panel: Neutrophils were cut with a clipping surface through the FITC channel to visualize Cy3 signal only within the cells. IC internalization was determined by calculating the volume ratio between the 2 surfaces objects created by FITC-masked Cy3 (red) and FITC-anti–Gr-1 (green) channels, respectively. There was an increase in the amount of ICs present within FcγRIIIB- or FcγRIIA-positive neutrophils compared with that in γ−/− neutrophils. N = 3 or 4 mice per group. **P < .01. ***P < .001, compared with IC uptake in γ−/− neutrophils. (D) Fifteen minutes after injection of BSA–anti-BSA soluble ICs, peripheral blood was collected. Gr-1–positive neutrophils were analyzed for Cy3 signal by flow cytometry. Minimal fluorescence was associated with these cells, suggesting that IC uptake did not occur in circulating neutrophils. N = 3 mice per group. n.s. indicates not significant.

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