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Fig. 9. Integrin internalization is altered in JAM-A^–/– neutrophils. (A) Confocal immunofluorescence analysis of integrin β1 in mouse JAM-A^+/+ and JAM-A^–/– neutrophils. Cells were seeded on fibronectin-coated coverslips, stimulated or not with the chemotactic peptide WKYMVm, fixed and stained. The images are single projections of z-stacks of about four confocal sections (stack z-spacing, 0.3 µm). Note the higher expression of β1 integrins at the plasma membrane of JAM-A^–/– than JAM-A^+/+ neutrophils. Cell nuclei are counterstained with DAPI (blue). Scale bars: 5 µm. (B) Immunofluorescence internalization assay of integrin in JAM-A^+/+ and JAM-A^–/– neutrophils. Cells were seeded on fibronectin-coated coverslips, treated with WKYMVm and then surface-labeled with the anti-integrin-β1 antibody. Cells were then incubated at 37°C to allow integrin internalization. The integrin-antibody complexes at the plasma membrane were removed by acid washing, whereas the internalized complexes were detected by confocal microscopy under permeabilizing conditions. Single projections of z-stacks of about five confocal sections (stack z-spacing, 0.3 µm) are shown. The amount of internalized integrin is reduced in JAM-A^–/– compared with JAM-A^+/+ neutrophils. Cell nuclei are counterstained with DAPI (blue). Scale bar: 5 µm. (C) FACS recycling assay was used to quantify integrin recycling to the cell surface after internalization. The amount of integrin on the cell surface before acid wash was higher in JAM-A^–/– neutrophils (white bars) compared with JAM-A^+/+ neutrophils (black bars). Acid washing abrogated surface staining in both cell types. The amount of integrin recycled to the cell surface after acid wash was higher in the presence than in the absence of JAM-A. MFI, mean fluorescence intensity. Data are means ± s.e.m. of triplicates from one representative experiment out of three performed. ^*P<0.05, by Student's t-test. (D) Distribution of β1 integrins in neutrophils was analyzed in the mouse cremaster muscle after 4 hours of stimulation with LTB4 using immunostaining and confocal deconvolution microscopy. Representative confocal microscopic images of β₁ integrin (green) localization in transmigrated neutrophils in the cremaster muscle of JAM-A^+/+ (a,b) and JAM-A^–/– mice (c,d). A projection of z-planes covering 2.5 µm is shown (projection of ten z-planes; z-spacing, 250 nm). Comparative membrane staining was done using a CD45 antibody (red). A significantly smaller amount of internalized integrins was observed in neutrophils of JAM-A^–/– mice. Scale bar: 5 µm.

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