Le rôle des protéines S100 dans la migration des neutrophiles au site inflammatoire

Liste des illustrations

• Illustration 2.1. : Image de microscopie électronique d’un neutrophile au repos. pg : granules primaires ; sg : granules secondaires ; N : noyau ; ce : centriole ; m : mitochondrie. (tiré de (5))
• Illustration 3.1. : Les intégrines. A. Composition des 24 hétéro-dimères. B. Structure générale des intégrines. Le « I domain » est présent sur 7 sous-unités α et joue un rôle dans la liaison des ligands. Le « I-like domain » est présent sur toutes les sous-unités β. (tiré de (87))
• Illustration 3.2. : Composition de la matrice extracellulaire. La MEC est principalement formée de polysaccharides (GAG, protéoglycanes (ex : héparane-sulfate)), de protéines fibreuses, de structure (collagène, élastine) et de protéines d’adhérence (laminine, fibronectine, vitronectine).
• Illustration 3.3. : Les étapes de la migration des neutrophiles au site inflammatoire. A. Au repos, les neutrophiles circulent librement dans le sang. B. A la suite d’une agression, l’interaction entre les sélectines et leurs ligands amène les neutrophiles à rouler le long de l’endothélium. C. Les chimioattractants libérés à la surface de l’endothélium activent les intégrines β2 à la surface des neutrophiles. D. Les intégrines β2 activées interagissent avec ICAM-1, ceci amène les neutrophiles à adhérer fermement à l’endothélium. E. Les neutrophiles migrent à travers les cellules endothéliales (via PECAM-1, CD99 et les JAMs) et traversent la membrane basale en libérant leurs granules. F. Enfin, dans le tissu, les neutrophiles se dirigent vers le site de l’agression en réponse à un gradient de facteurs chimiotactiques tout en adhérant aux composants de la MEC (via les intégrines β1 et β3).
• Illustration 4.1. : Arbre phylogénétique des protéines S100 humaines. (tiré de (216))
• Illustration 4.2. : Comparaison des regroupements de gènes des protéines S100 chez l’homme et la souris. La direction de transcription de ces gènes est indiquée par des flèches. (tiré de (216))
• Illustration 4.3. : Représentation schématique de la structure secondaire des protéines S100. Chaque boucle de liaison au Ca²⁺, L1 et L2, est entourée d’hélices α (HI et HII pour L1 et HIII et HIV pour L2). Une région charnière (H) connecte les hélices II et III. L’hélice IV est suivie d’une extension en C-terminal. (tiré de (217))
• Illustration 4.4. : Alignement multiple de séquences des protéines S100 humaines. Les résidus fixant le Ca²⁺ sont surlignés en rouge. Ceux fixant le Zn²⁺ dans S100A7 et le Cu²⁺ dans S100A12 sont surlignés en magenta. Les résidus hydrophobes qui sont essentiels à la dimérisation sont surlignés en vert. Les résidus qui sont des ligands putatifs du Zn²⁺ dans les autres protéines S100 sont surlignés en jaune (cystéine) et en bleu (histidine). (tiré de (216))
• Illustration 4.5. : Structure tri-dimensionnelle de la protéine S100A12. Les deux sous-unités représentées en orange et en rouge sont liées par des interactions hydrophobes.
• Figure 1: S100A9 increases neutrophil migration across HUVECs in response to IL-8. Increasing concentrations of S100A9 were added in the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and neutrophils were let to migrate for 2 h at 37°C. The number of transmigrated cells was determined as described in materials and methods. Data shown represent the mean + SEM of ≥ 3 experiments done on neutrophils from different donors. * p<0.05, ** p<0.01, one way ANOVA, Dunnett multiple comparison test (compared with control).
• Figure 2: Anti-S100A9 polyclonal antibodies inhibit neutrophil transmigration. S100A9 (40 μg/ml) +/- polyclonal antibodies directed against S100A9 or preimmune serum as control were added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower wells and neutrophils were let to migrate for 2 h at 37°C. The number of transmigrated cells was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors. * p<0.05, ** p<0.01, one way ANOVA, Dunnett multiple comparison test (compared with control).
• Figure 3: S100A9 prolongs neutrophil migration across endothelial cells. S100A9 (40 μg/ml) was added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and every 30 min up to 2 h the upper wells were moved to new lower wells. The number of transmigrated cells was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors.
• Figure 4: S100A9 acts more on neutrophils than on HUVECs. Neutrophils or HUVECs were preincubated separately with S100A9 (40 μg/ml) or buffer for 30 min at 37°C. After two washes, these cells were used in the transmigration assay. The number of transmigrated cells was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors.
• Figure 5: S100A9 has no effect on the adhesion molecule surface expression on HUVECs. Expression of ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and CD99 was analysed on S100A9-stimulated HUVECs by flow cytometry analysis. HUVECs were stimulated with different concentrations of S100A9 for increasing time periods, then detached with tryspin-EDTA and incubated with specific antibodies. Histograms of S100A9 stimulated cells are illustrated in gray, while unstimulated cells appear in open histograms. Nonspecific stainings are illustrated by a doted line. Data shown are from an experiment representative of 2 others done on cells from different passages.
• Figure 6: S100A9 has no effect on HUVECs permeability. The permeability was assessed using the FITC-dextran tracer added to the upper well at 1 μg/ml. A 50 μl aliquot was removed from the lower well and measured fluorescence was correlated to permeability as described in materials and methods. Data shown represent the mean + SEM of ≥ 3 experiments.
• Figure 7: S100A9 does not change neutrophil chemotactic response to IL-8. Neutrophils +/-S100A9 (40 μg/ml) were allowed to migrate in response to different concentration of IL-8 in 96-well Chemo TX chemotaxis wells. The number of migrated cells was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors.
• Figure 8: S100A9 increases and prolongs IL-8 effect on neutrophil adhesion to fibrinogen. Neutrophils were incubated with S100A9 (40 μg/ml) or IL-8 (5 ng/ml) alone or in combination and let to adhere to fibrinogen for different incubation period at 37°C. The number of adhered neutrophils was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors.
• Figure 9: S100A9 increases CD11b and CD18 expression and activation. Expression of CD11a (A), CD11b (B), CD18 (C) and its activated form (D) was analysed on S100A9 and/or IL-8-stimulated neutrophils by flow cytometry analysis. Mean fluorescence value of each histogram was determined. Data shown are from an experiment representative of 2 others done on cells from different donors.
• Figure 10: S100A9 effect on neutrophil migration is abolished by anti-CD11b and anti-CD18 antibodies. S100A9 (40 μg/ml) +/- antibodies directed against CD11a, CD11b and CD18 or their isotype control were added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and neutrophils were let to migrate for 2 h at 37°C. The number of transmigrated cells was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments done on neutrophils from different donors. * p<0.05, ** p<0.01, one way ANOVA, Dunnett multiple comparison test (compared with control).
• Figure 1: S100A9 induces neutrophil adhesion to fibronectin. Neutrophils (5 X 10⁴ cells/well) were incubated with S100A8, S100A9, S100A8/A9 or S100A12 (20 μg/mL), fMLF 10^-6M (positive control) or M199 (negative control) and let to adhere on fibronectin (A), vitronectin (B), laminin (C), and collagen (D) for 30 min at 37°C. The number of adhered neutrophils was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments performed on neutrophils from different donors. * p < 0.05, ** p < 0.01, One way ANOVA, Dunnett multiple comparison test (compared with control).
• Figure 2: S100A9 has no effect on β1 and β3 integrins, but upregulates β2 integrins surface expression. Expression of β1, β2, and β3 integrins was analyzed on S100A9-stimulated neutrophils by flow cytometry analysis. Neutrophils were stimulated with S100A9 (20 μg/mL) for 30 min at 37°C and incubated with specific Abs directed against the different subunits of β1, β2, and β3 integrins. Histograms of S100A9-stimulated neutrophils are illustrated in gray, while unstimulated neutrophils (HBSS) appear in open histograms. Nonspecific stainings are illustrated by a dotted line. Data shown are from an experiment representative of 2 others done on cells from different donors.
• Figure 3: S100A9 stimulates neutrophil adhesion to fibronectin via β2, but not β1 nor β3 integrins. Neutrophils were incubated with blocking Abs directed against A) β1 integrins, B) β3 integrins, or C) β2 integrins and their adhesion to fibronectin was stimulated with S100A9 (20 μg/mL) or M199 (negative control) for 30 min at 37°C. Data shown represent mean + SEM of ≥ 3 experiments performed on neutrophils from different donors. * p < 0.05, ** p < 0.01, One way ANOVA, Dunnett multiple comparison test (compared with isotype control).
• Illustration 1. : Le rôle de S100A9 dans la migration des neutrophiles. La protéine S100A9 libérée par les neutrophiles lors de la migration favorise la migration des cellules nouvellement arrivées sur le site inflammatoire. En effet, les protéines libérées fixées aux protéoglycanes sur l’endothélium ou dans le tissu vont activer les intégrines β2 à la surface des neutrophiles et ainsi stimuler leur adhésion aux cellules endothéliales ou à la fibronectine.