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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.037127

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JAM-A promotes neutrophil chemotaxis by controlling integrin internalization and recycling

¹ FIRC Institute of Molecular Oncology, Milan, Italy
² Vita-Salute San Raffaele University and DIBIT San Raffaele Scientific Institute, Milan, Italy
³ Walter Brendel Center of Experimental Medicine, Ludwig-Maximilians-Universität München, Munich, Germany
⁴ Department of Biomolecular Sciences and Biotechnologies, School of Sciences, University of Milan, Milan, Italy
⁵ Mario Negri Institute of Pharmacological Sciences, Milan, Italy

View larger version (68K): [in this window] [in a new window]   Fig. 1. Impaired interstitial migration of extravasated leukocytes in vivo in JAM-A-deficient mice. (A) In vivo RLOT microscopic image of a postcapillary venule in the inflamed mouse cremaster muscle. Tissue distribution of transmigrated leukocytes was quantified in two regions of interest (ROIs), one close and one distant to the vessel (indicated by the two boxed regions). (B) Quantitative analysis of tissue distribution of transmigrated leukocytes after 60 minutes of stimulation with LTB4. In JAM-A-deficient mice, a significantly smaller proportion (41.9±4.1%) of transmigrated leukocytes is found in the ROI distant to the analyzed postcapillary venule (>25 µm distance from the venule) compared with a similar region in JAM-A+/+ mice (58.5±3.1%; n=6; mean ± s.e.m.; *P<0.05 vs JAM-A+/+).

View larger version (76K): [in this window] [in a new window]   Fig. 2. JAM-A distribution in polarized dHL60 cells. Confocal immunofluorescence analysis of polarized dHL60 (fMLP stimulation for 20 minutes). Double staining for JAM-A (red) and actin (green) is shown. The anti-JAM-A mAb was applied to living cells before paraformaldehyde fixation (see Materials and Methods). Cell nuclei were counterstained with DAPI (blue). The image represents the z-stack projection of 25 confocal sections from the basal to the apical cell side, as indicated by the arrow on the left (stack z-spacing, 2 µm). JAM-A is localized in intracellular vesicles (arrowheads), at the uropod and in ruffles at the leading edge (arrows). Scale bar: 5 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 3. JAM-A is internalized in polarized dHL60 cells. (A) Cells were seeded on fibronectin-coated coverslips, then treated or not with fMLP or PMA for 20 minutes, fixed with paraformaldehyde and stained for JAM-A (red). The nuclei of the cells were counter-stained with DAPI (blue). JAM-A is distributed in intracellular vesicles in activated cells, as shown by the confocal z-stack projections from the basal to the apical side of the cell (from the left to the right, as indicated by the arrow on the top). (B) Corresponding lateral view of JAM-A distribution in intracellular vesicles along the cell body. The images represent the maximum projection along the xz axes of confocal stacks of about 15 sections (stack z-spacing, 0.4 µm). Scale bars: 5 µm.

View larger version (26K): [in this window] [in a new window]   Fig. 4. JAM-A is internalized in neutrophils upon fMLP treatment. (A) Immunofluorescence internalization analysis of JAM-A endocytosis from the plasma membrane in fresh human neutrophils, either treated (b) or not (a) with fMLP. JAM-A internalized vesicles (red) are more abundant in activated cells. Each panel is a single projection of a z-stack of three confocal sections (stack z-spacing, 0.3 µm). Cell nuclei were counterstained with DAPI (blue). Scale bars: 5 µm. (B) Freshly purified neutrophils were labeled at 4°C with cleavable biotin and left untreated or stimulated with fMLP for 5 and 10 minutes at 37°C before surface biotin cleavage with GSH, and lysis. Biotinylated proteins were precipitated with streptavidin-agarose followed by western blot analysis with anti-JAM-A (BV22 mAb) and anti-L integrin (clone 27) antibodies. (C) Densitometric analysis of internalized JAM-A and L integrin normalized for their total amount. The figure shows one representative experiment out of five performed.

View larger version (95K): [in this window] [in a new window]   Fig. 5. JAM-A colocalizes with 5β1 and β2 integrins. Double staining of JAM-A (green) and 5β1 integrins (red) or JAM-A (red) and β2 integrin (green) in polarized dHL60 cells. The cells were seeded on fibronectin-coated coverslips, treated with fMLP for 20 minutes and incubated with anti-JAM-A and anti-β1 integrin antibodies before paraformaldehyde fixation. Each panel represents a single projection of a z-stack of about three confocal section (stack z-spacing, 0.4 µm). Merged images show JAM-A colocalization with integrins. Cell nuclei are counterstained with DAPI (blue). Scale bar: 5 µm. The figure shows one representative experiment out of five performed.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Fibronectin-induced co-clustering of JAM-A with integrins. Immunofluorescence staining of dHL60 cells incubated with either fibronectin-coated or polylysine-coated beads. After incubation with the beads, cells were fixed and double stained with antibodies against JAM-A and integrin β1. (a) JAM-A and β1 integrin co-clustering at the bead contact sites (arrows); (b) Polylysine-coated beads do not induce co-clustering (arrows). Single projections of z-stacks of two confocal sections (stack z-spacing, 0.3 µm) are shown. Scale bar: 5 µm. The figure shows one representative experiment out of four performed.

View larger version (61K): [in this window] [in a new window]   Fig. 7. Integrins induce JAM-A clustering. Immunofluorescence staining of dHL60 cells incubated with beads coated with JAM-A mAb (BV16) or a β1 integrin pAb, mouse or rabbit IgGs respectively. Cells were fixed and stained as in Fig. 6. β1-integrin-pAb-coated beads induced JAM-A clustering (arrows), whereas JAM-A-mAb-coated beads were unable to recruit integrin β1. Beads coated with either mouse or rabbit IgGs were not effective. Single projections of z-stacks of about two confocal sections (stack z-spacing, 0.3 µm) are shown. Scale bars: 5 µm. The figure shows one representative experiment out of two performed.

View larger version (77K): [in this window] [in a new window]   Fig. 8. JAM-A concentrates in intracellular vesicles in murine neutrophils. Immunofluorescence analysis of neutrophils derived from bone marrow of JAM-A+/+ and JAM-A–/– mice. Living cells were treated with an anti-murine JAM-A mAb (BV12) and then seeded on fibronectin-coated coverslips and fixed. Untreated (a,b) or WKYMVm-activated (c,d) cells are shown. The nuclei of the cells are counterstained with DAPI (blue). Murine neutrophils show JAM-A localization at intracellular vesicles, whereas, as expected, JAM-A was undetectable in JAM-A–/– cells (b,d). Scale bars: 5 µm. The figure shows one representative experiment out of four performed.

View larger version (60K): [in this window] [in a new window]   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.

View larger version (56K): [in this window] [in a new window]   Fig. 10. BAPTA-AM treatment alters the uropod length of JAM-A+/+ neutrophils. Actin immunofluorescence staining of JAM-A–/– and +/+ murine neutrophils is shown. To block integrin internalization, cells were pre-incubated with BAPTA-AM where indicated. The number of cells showing long tails was increased upon BAPTA-AM treatment of JAM-A+/+ cells, whereas uropod lengths were not altered in JAM-A–/– neutrophils. Cell nuclei are counterstained with DAPI (blue). Scale bar: 5 µm. The length of neutrophil tails was measured using Carnoy software version 2.0 for Macintosh and the frequency of cells with different tail size is reported for the two populations (JAM-A+/+, black columns; JAM-A–/–, white columns). The graph shows the means ± s.e.m. of four experiments. *P<0.01, Student's t-test.

View larger version (12K): [in this window] [in a new window]   Fig. 11. Rap-1 activation is impaired in JAM-A–/– neutrophils. A pull-down assay was performed to isolate active GTP-bound Rap-1. Freshly isolated mouse neutrophils were obtained from JAM-A+/+ and JAM-A–/– mice. 5x106 cells for each condition were left untreated or stimulated with the chemotactic peptide WKYMVm for 5 minutes at 37°C. Pull down was performed on cell lysates using the RalGDS-RBD probe (A) or the GST-negative control (C). Western blot for Rap-1 revealed impaired Rap-1 activation in both unstimulated and stimulated JAM-A–/– compared with JAM-A+/+ neutrophils. (B) Densitometric analysis of active GTP-bound Rap-1 normalized for the total amount of protein. Results in B are means ± s.e.m. of four separate experiments; A and C show a typical experiment.

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