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First published online 19 April 2005
doi: 10.1242/jcs.02340

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PECAM-1, 6 integrins and neutrophil elastase cooperate in mediating neutrophil transmigration

Cardiovascular Medicine Unit, The Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 ONN, UK

View larger version (87K): [in a new window]   Fig. 1. Transmigrated leukocytes express 6 integrins on their cell surface in IL-1ß-stimulated mouse cremaster muscles, as observed by confocal microscopy. Mouse cremaster muscles were injected i.s. with IL-1ß (30 ng) and, 4 hours later, the tissues were dissected away from the animals, fixed in 4% PF and incubated with the primary mAb against 6 integrins, GoH3. Binding of GoH3 was detected using a secondary antibody conjugated to Alexa Fluor 488. Samples were observed at RT using a Zeiss LSM 5 Pascal confocal laser-scanning microscope (using a 40x water-dipping achroplan objective with a numerical aperture of 0.75) equipped with an argon laser (excitation wavelength of 488 nm). Typically, multiple optical sections of tissue samples, running through the whole depth of the tissue, were captured and imaged using the software's automatic scanning mode. In selected experiments, z-axis stack images (collected at every 0.8-1.2 µm depth) were saved and used for three-dimensional reconstruction analysis using the LSM 5 Pascal software (version 3.2). The images show expression of 6 integrins on leukocytes in the extravascular tissue, displayed as fluorescence (left), differential-interference contrast (DIC) (middle) and merged (right) patterns. (A) This series was obtained from the mid-level section of the marked leukocyte (yellow arrow), demonstrating a pronounced ring-like expression profile, indicating cell-surface expression. (B) A three-dimensional reconstructed image of the same tissue shown in two dimensions in A, illustrating the more global-like distribution of 6 integrins on transmigrated leukocytes. Leukocytes and vessel walls are shown by yellow and red arrows, respectively. Scale bars, 10 µm.

View larger version (37K): [in a new window]   Fig. 2. The mAb GoH3 against 6 integrin and the NE inhibitor ONO-5046 suppress leukocyte transmigration through IL-1ß-stimulated cremasteric venules in vivo. (A,B) WT or NE KO mice were injected i.v. with saline, control IgG or mAb GoH3 (both at 3 mg kg–1) 15 minutes before i.s. injection of saline or IL-1ß (30 ng). 2 hours later, the jugular vein was cannulated for infusion of either saline or the NE inhibitor ONO-5046 (50 mg kg–1 bolus followed by continuous infusion of 50 mg kg–1 hour–1 using a syringe pump), the cremaster muscle was exteriorized and leukocyte responses quantified by intravital microscopy. (A,B) Leukocyte responses of firm adhesion and transmigration at 4 hours after IL-1ß administration in the different groups of animals. The data shown are the means±s.e.m. from samples of 4-12 mice per group. Statistically significant differences in responses between mice receiving i.s. saline and mice receiving i.s. IL-1ß are shown by asterisks: **P<0.01; ***P<0.001. In mice receiving i.s. IL-1ß, differences to animals injected with i.v. IgG-saline (control group) are shown by crosses:+P<0.05; +++P<0.001. (C-E) IL-1ß-stimulated cremaster muscles from mice injected with either i.v. IgG-saline (C) or GoH3/ONO-5046 (D,E) were immunostained with specific mAbs for expression of PECAM-1 (APC; green), laminin 10 (Alexa Fluor 555; red) and CD11b (Alexa Fluor 488; white). Samples were observed at RT using a Zeiss LSM 5 Pascal confocal laser-scanning microscope (using a 40x water-dipping achroplan objective with a numerical aperture of 0.75) equipped with argon (excitation wavelength of 488 nm) and helium-neon (excitation wavelengths of 543 nm and 633 nm) lasers; the figure shows merged images captured from the three channels used. (E) A high-magnification image of the specified vessel segment shown in D in order to illustrate better the localization of leukocytes within the vessel wall. This is further indicated with an accompanying schematic diagram in which the position of endothelial cells (as identified using an anti-PECAM-1 mAb) is shown by a green line, the position of the basement membrane (as identified by an anti-laminin-10 Ab) is shown by a red line and trapped leukocytes (identified using an anti-CD11b mAb) are shown in white. All images are from tissue samples stained and analysed blind with at least nine random fields per tissue being captured from three or four mice in each group. Bars, 10 µm.

View larger version (44K): [in a new window]   Fig. 3. PECAM-1 ligation enhances cell-surface expression of 6 integrins on mouse blood neutrophils in vitro as quantified by flow cytometry and observed by confocal microscopy. Blood-derived neutrophils incubated for 30 minutes in wells of 96-well plates, coated with BSA (control) or different combinations of PECAM-1, ICAM-1 and IL-8, were assayed for 6 integrins expression by flow cytometry. The binding of the primary mAb GoH3, relative to the binding of an isotype-matched control mAb, is expressed in terms of relative fluorescence intensity (RFI). (A) Cell-surface expression of 6 integrins on WT blood neutrophils after adhesion to different proteins and protein combinations. (C) Cell-surface expressions of 6 integrins on WT neutrophils incubated in wells in the presence or absence of anti-ICAM-1 or anti-ß2 mAbs, and expression on the cell surface of PECAM-1-deficient neutrophils. Results represent means±s.e.m. from three to six samples in each condition using different neutrophil preparations. Asterisks and crosses indicate significant differences from samples incubated in BSA-coated wells: *P<0.05; **P<0.01; ***P<0.001 (A,C), and significant differences from samples incubated in wells coated with PECAM-1, ICAM-1 and IL-8 (total protein being 10 µg ml–1): +P<0.05; +++P<0.001 (C). (B,D) Representative histograms from experiments shown in A,C, with the hatched histograms representing binding of control IgG. Selected samples were also analysed using a confocal laser-scanning microscope (Zeiss LSM 5 Pascal incorporating a 100x oil A-plan objective with a numerical aperture of 1.25) equipped with an argon laser (excitation wavelength of 488 nm). Expression of 6 integrins was indirectly observed using the mAb GoH3 against 6 integrins and an Alexa-Fluor-488-conjugated secondary antibody. Representative images of mid-level sections of cells from different conditions are shown. (E) WT neutrophils incubated in BSA-coated wells. (F) WT neutrophils incubated in PECAM-1/ICAM-1/IL-8 (10 µg ml–1)-coated wells. (G) PECAM KO neutrophils incubated in PECAM-1/ICAM-1/IL-8 (10 µg ml–1)-coated wells. The binding of the anti-6-integrin mAb under different experimental conditions, corrected for the binding of an isotype-matched control mAb and expressed in terms of mean fluorescence intensity (arbitrary units), is also shown (mean±s.e.m. from three to six samples in each condition using at least three different neutrophil preparations). Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 4. Cell-surface expression of NE on mouse blood neutrophils in vitro and in vivo occurs via PECAM-1-independent mechanisms. (A) Blood neutrophils from WT or PECAM KO mice were incubated for 30 minutes in 96-well plates coated with BSA or the combination of PECAM-1, ICAM-1 and IL-8 (total protein being 10 µg ml–1) before being permeabilized and immunostained for expressions of NE (red; Alexa Fluor 633) or 6 integrins (green; Alexa Fluor 488), and analysed at RT using a Zeiss LSM 5 Pascal confocal laser-scanning microscope (using a 100x oil A-plan objective with a numerical aperture of 1.25) equipped with HeNe (excitation wavelength of 633 nm) and argon (excitation wavelength of 488 nm) lasers. Images are shown of mid-level sections of representative cells from three independent experiments. Bar, 10 µm. (B,C) Cell-surface expression of NE was quantified on blood or IL-1ß-elicited peritoneal neutrophils using a fluorogenic substrate. Briefly, mice (WT or PECAM-1 KO) were injected i.p. with saline (1 ml) or IL-1ß (10 ng) and, 4 hours later, killed by CO2 asphyxiation. Purified blood neutrophils or peritoneal leukocytes were added to 96-well plates such that each well contained a total of 4x105 neutrophils. After centrifugation, the samples were fixed and assayed for NE activity. (B) A comparison between NE activity on the cell surface of WT blood and peritoneal neutrophils (significant statistical difference between samples is indicated by asterisks: ***P<0.001). (C) A comparison between NE activity on the cell surface of IL-1ß-induced transmigrated neutrophils in WT and PECAM-1 KO mice. Results represent the means±s.e.m. of samples obtained from seven to ten mice in each group.

View larger version (17K): [in a new window]   Fig. 5. Migration of murine neutrophils through laminin-coated filters is dependent on PECAM-1, 6 integrins and NE. 96-well NeuroProbe chemotaxis chambers were used to investigate neutrophil migration through laminin-coated filters. Transmigration filters (3 µm pores) were coated with 2% BSA, laminin-1 (LN-1; 15 µg ml–1) or laminin-1 coated with a combination of 20% PECAM-1 and 80% ICAM-1 (using a solution with a total protein concentration of 10 µg ml–1). Bone-marrow-derived mouse neutrophils (2x105) obtained from wild-type or PECAM-1 KO mice were placed on top of the filters with saline or IL-8 (10–7 M) in the bottom wells. Before addition to filters, cell samples were treated with saline (control), control IgG1b or IgG2a mAbs (both at 10 µg ml–1; these data were pooled because there was no significant difference between them), a mAb against mouse ß2 integrins (10 µg ml–1), the mAb GoH3 against 6 integrin (10 µg ml–1) or the NE-specific inhibitor ONO-5046 (50 µM final concentration), as indicated. After an incubation period of 3 hours at 37°C, the numbers of transmigrated cells in the bottom wells were counted by microscopy after Kimura staining. All samples were run in triplicate and the results represent means±s.e.m. from three to five independent experiments. Statistically significant responses from transmigration through BSA-treated filters are shown by asterisks (**P<0.01). Significant inhibition of transmigration through filters coated with laminin-1/PECAM-1/ICAM-1 in samples treated with GAME 46 or GoH3 compared with control mAb-treated samples are shown by hashes (#P<0.05) and significant inhibition of response by the NE inhibitor ONO-5046 compared with saline-treated cells is shown by crosses (++P<0.01).

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