Ce travail a été soutenu par le ministère de l&#39;UCSF des soins d&#39;anesthésie et de soins périopératoires.

1. Placage et l&#39;entretien des cellules endothéliales microvasculaires <ol><li> Décongeler et faire croître les cellules endothéliales microvasculaires selon les fabricants fournis instructions. Dans ce protocole, les cellules ont été cultivées dans EGM-2 médias MV (Lonza), et il est recommandé que toutes les expériences sont effectuées dans les deux semaines suivant la décongélation de minimiser toute variation due au nombre de passages. Nos expériences avec HMVEC-Lung sont effectués entre les passages 4 à 9. </li><li> Jour 1: Lorsque les cellules atteignent 80-90% de confluence, trypsiniser et remettre à 120.000 cellules par ml. Plate 36.000 cellules (0,3 ml) par puits dans 48 puits, plaque de culture tissulaire en polystyrène (s). Inclure les puits de HMVEC qui ne sera pas mis à incuber avec des neutrophiles afin d&#39;obtenir une lecture de fluorescence de fond. À moins d&#39;utiliser des plaques à 48 puits spécialement conçues pour les essais par fluorescence, des lignes ou des colonnes en alternance permettra de minimiser toute contamination lumière des puits voisins. NOTE: Les puits peuvent être pré-enduit de poly-lysiNE, collagène ou fibronectine. </li><li> Jour 2: Ajouter des milieux frais. </li><li> Jour 3: En microscopie en contraste de phase, confirment les cellules sont en bonne santé (c. apparence &quot;pavé&quot; et peu de débris cellulaires), et ils sont 100% de confluence (Figure 1). Si les cellules ne sont pas 100% de confluence, changer les médias tous les jours jusqu&#39;à ce qu&#39;ils soient prêts. </li><li> Une fois les monocouches HMVEC sont prêts pour le traitement, laver les cellules une fois avec une solution saline équilibrée de Hank (HBSS) et ajouter 0,3 ml d&#39;EGM-2 médias ou médias MV frais contenant agoniste inflammatoire dans les puits appropriés. Dans ce protocole, le HMVEC-pulmonaire ont été traités avec le TNFa [100 ng / ml] (PeproTech, New Jersey) pendant 3 heures car il s&#39;agit d&#39;un activateur bien décrit des EC 21. NOTE: Il est recommandé de temps votre expérience afin que vos cellules HMVEC activés (étape 4.1) et calcéine AM neutrophiles marquées (Étape 3.5) sont prêts à utiliser en même temps. Il nous faut environ 2,5 heures pour isoler et étiqueter neutrophILS. </li></ol> 2. Isolement des neutrophiles en utilisant Polymorphprep <ol><li> Isoler les neutrophiles, comme décrit précédemment 22, ou avec une solution de gradient densité ready-made pour isoler les granulocytes neutrophiles dans le sang total. Employant Polymorphprep suivant le protocole par Nuzzi et.al. résultats 2007 des rendements d&#39;environ 2-3 x 10 6 neutrophiles par ml de sang total 23. REMARQUE: Pour éviter la lyse des érythrocytes excessive, une sédimentation dextran pour éliminer les globules rouges peut être effectuée avant la centrifugation de gradient de densité 24. </li><li> Porter la solution à gradient de densité Polymorphprep à RT. </li><li> Recueillir 30 ml de sang total à partir d&#39;un volontaire humain sain, en présence d&#39;un anticoagulant tel que l&#39;héparine, le citrate ou l&#39;EDTA. Le sang doit être utilisé dans les 2 heures et maintenue entre 18-22 ° C. </li><li> Couche 5 ml de sang total plus de 5 ml de la solution à gradient de densité dans un tube conique de 15 ml (Figure 2A). Éviter de modifier les volumes de sang et la solution de gradient de densité, car cela pourrait modifier les conditions de centrifugation ultérieures. </li><li> Centrifuger à 450 g pendant 30 min à 18-22 ° C. Assurez-vous d&#39;accélérer lentement de haut en bas de la centrifugeuse (c.-à désactiver le frein de la centrifugeuse), et d&#39;équilibrer les tubes bien pour éviter les vibrations pour aider à réduire l&#39;activation des neutrophiles et d&#39;empêcher le mélange des couches. Temps de centrifugation ou la force centrifuge plus élevé se traduira par la couche leucocytaire inférieure, qui contient des neutrophiles, de migrer plus loin vers les globules rouges agglomérés. </li><li> Aspirer le plasma et la bande de leucocytes supérieur contenant CMSP pour empêcher la contamination de la couche contenant les neutrophiles (figure 2B). </li><li> Utiliser une pipette sérologique 1-2 ml pour enlever la couche de leucocytes polynucléaires neutrophiles inférieur contenant. Combiner les couches de neutrophiles dans 30 ml de PBS (sans Ca 2 + et Mg 2 +) dans un 50 mltube conique. </li><li> Porter le volume à 50 ml avec du PBS (sans Ca 2 + et Mg 2 +) et centrifuger à 450 g pendant 10 min à 18-22 ° C. </li></ol> Remarque: Les étapes 2.9 et 2.10 sont facultatives mais fortement recommandées. <ol start="9"><li> Aspirer le surnageant. Reprendre le neutrophiles dans 8 ml d&#39;eau stérile pendant 30 secondes pour lyser les globules rouges, ajouter immédiatement la suspension de cellules à 2 ml de 5x PBS (sans Ca 2 + et Mg 2 +) et mélanger délicatement à la main. Ne pas incuber les cellules en l&#39;eau pendant plus de 30 secondes depuis la mort de neutrophiles importante peut se produire. </li><li> Centrifuger à 250 g pendant 5 min à 18-22 ° C. </li><li> Laver les cellules une fois avec 10 ml de PBS (sans Ca 2 + et Mg 2 +) et centrifuger à 250 g pendant 5 min à 18-22 ° C. </li><li> Reprendre les neutrophiles dans 10 ml de RPMI-1640 (sans rouge de phénol) et de compter les cellules vivantes en utilisant le bleu trypanteindre méthode d&#39;exclusion. Éviter les périodes prolongées de fortes densités cellulaires supérieures à 5 x 10 6 par ml, car cela pourrait entraîner une activation des neutrophiles. </li></ol> 3. Neutrophiles étiquetage avec Calcein AM <ol><li> 6 x 10 5 cellules neutrophiles sont utilisés par puits d&#39;une plaque à 48 puits. </li><li> Après le dépouillement, transférer les cellules remises en suspension dans un tube conique de 50 ml et diluer les neutrophiles à 2-4 x 10 6 cellules par ml de rouge de phénol libre RPMI-1640. </li><li> Ajouter calcéine AM solution stock à 3 pm (ie 2,98 ul de calcéine AM stock (1 mg / ml) par ml de milieu) et bien mélanger en tapotant doucement le tube. Plus de 5 uM AM calcéine va entraîner une fuite des neutrophiles. Remarque: non fluorescent calcéine AM est clivée dans les cellules par des estérases endogènes pour produire la molécule hautement fluorescent calcéine (ie les cellules mortes ne fluorescence). </li><li> Couvrez le tube avec du papier d&#39;aluminium et laisser incuber for 30 min à 37 ° C dans l&#39;obscurité. Remarque: des temps d&#39;incubation plus longue peut entraîner une plus calcéine être libérée des neutrophiles au cours de l&#39;essai d&#39;adhésion. </li><li> Centrifugeuse à 450 g pendant 5 min à 18-22 ° C, et laver les neutrophiles 2x avec 10 ml de RPMI-1640 (sans rouge de phénol, préchauffé à 37 ° C). Après remise en suspension des cellules pour le second lavage, d&#39;abord passer les neutrophiles si un filtre stérile de 40 uM à éliminer les agrégats, puis centrifuger une fois de plus à 450 g pendant 5 min à 18-22 ° C. </li><li> Reprendre les neutrophiles dans RPMI 1640 (sans rouge de phénol, préchauffé à 37 ° C) à 2 x 10 6 neutrophiles par ml. </li></ol> 4. Essai de liaison des neutrophiles <ol><li> Laver les monocouches HMVEC 3x avec 0,5 ml de stérilisée par filtration (0,22 um) RPMI 1640 (sans rouge de phénol) contenant 3% de BSA par puits. </li><li> Aspirer les médias et ajouter 6 x 10 5 neutrophiles calcéine marqués (0,3 ml de suspension cellulaire), Soit 0,3 ml de RPMI 1640 (sans rouge de phénol) sans neutrophiles, les puits appropriés de la plaque de 48 puits. Note: Ceci est l&#39;équivalent de 8 x 10 5 neutrophiles par cm 2. </li><li> Incuber pendant 20 min à 37 ° C, 5% de CO 2 incubateur. </li><li> Nombre de neutrophiles Fluorescence (prélavage): Mesurer l&#39;intensité de fluorescence de chaque puits avec une longueur d&#39;onde d&#39;excitation de 485 nm et une longueur d&#39;onde d&#39;émission de 520 nm (jeu de filtres fluorescéine) dans un FLUOstar, ou équivalent, spectrophotomètre. Effectuez cette étape juste avant le point final d&#39;incubation Note: Dans ce protocole, calcéine excitation et la détection de la lumière émise ont été réalisés depuis le sommet des puits découverts, mais les deux peuvent également être effectuées à partir du fond des puits.. </li><li> Laver les puits contenant des monocouches HMVEC et des neutrophiles 5x avec 0,5 ml par puits de PBS (w / Ca 2 + et Mg 2 +). Retirez le papier et les lavagesen inversant la plaque et tapoter délicatement sur du papier absorbant pour enlever le liquide restant. </li><li> Rajouter 0,3 ml de RPMI 1640 (sans rouge de phénol) par puits. </li><li> Adhérent neutrophiles Fluorescence (POST lavage): Mesurer l&#39;intensité de fluorescence de chaque puits comme dans l&#39;étape 4.4. </li><li> Déterminer le respect de pour cent et adhérence relative par puits: </li></ol> Remarque: A ce stade, les images des neutrophiles calcéine-marqués peuvent être obtenus en utilisant un microscope capable fluorescence équipé de filtres fluorescéine standard. Les images de la figure 4 ont été obtenus sur un Olympus IX51 microscope inversé équipé d&#39;une caméra 2000R Retiga (Q Imaging) en utilisant Q-Capture logiciel Pro7 (Q Imaging).

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U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

Les auteurs n&#39;ont rien à révéler.

Les étapes les plus importantes pour un neutrophile succès / test d&#39;adhérence des cellules endothéliales microvasculaires sont les suivants: 1) Utilisation de faible nombre de passage (&lt;9), les cellules endothéliales saines; 2) le maintien des neutrophiles isolés à dire &lt;5 x 10 6 cellules de faible densité ( / ml) et de les utiliser dans les deux heures d&#39;isolement, et 3) le lavage fastidieuse des neutrophiles calcéine AM-étiquetés et utiliser des neutrophiles calcéine AM-é...

Depuis l&#39;endothélium vasculaire est en contact direct avec le sang circulant, il est situé de façon unique pour initier une réponse inflammatoire rapide lors de l&#39;infection ou de blessure. Les cellules endothéliales (EC) expriment motif récepteurs de reconnaissance qui reconnaissent une variété de composants bactériens conservés et des molécules de danger, et les récepteurs pour les hôtes médiateurs inflammatoires telles que le TNFa. L&#39;activation de ces récepteurs induit EC de sécréter des cytokines (par exemple IL-6, IL-8, CXCL1 et CCL2), et à réguler à la hausse des molécules d&#39;adhésion (par exemple, E / P-sélectine, VCAM-1 et ICAM-1) à leur surface cellulaire 1 , 2. Ces molécules tous facilitent la localisation des leucocytes aux sites d&#39;infection et les blessures afin d&#39;effacer l&#39;hôte des agents infectieux et de lancer la réparation des tissus 3,4. La réponse des neutrophiles à l&#39;infection implique une interaction bien coordonnée entre l&#39;endothélium vasculaire et la réponse neutrophiles. Lors de l&#39;activation CE, IL-8 est sécrété et forme un gradient intra-vasculaire sur l&#39;endothélium qui permet neutrophiles à l&#39;accueil au site de l&#39;infection ou de blessure 5,6. E / P-sélectine médiate neutrophiles capture et rouler à travers les associations relativement faibles avec glycomolecules sur la surface des cellules neutrophiles. Ces interactions, avec liaison de l&#39;IL-8 et de ses récepteurs apparentés, facilitent le solide, l&#39;attachement médiée par l&#39;intégrine et arrêt éventuel de neutrophiles à la surface des cellules endothéliales 7-10. Après l&#39;arrestation, les neutrophiles peuvent migrer hors de la vascularisation des sites spécifiques d&#39;infection à éliminer directement les agents pathogènes, de générer des pièges extracellulaires neutrophiles pour prévenir la propagation d&#39;agents pathogènes, de favoriser la guérison des plaies et libérer des facteurs supplémentaires qui recrutent d&#39;autres leucocytes tels que les monocytes, macrophages et dendritiques cellules 11-17. Décrite dans le présent rapport est une méthode in vitro pour quantifier l&#39;adhésion des neutrophiles à microVasculaire ECS après activation par l&#39;hôte médiateur inflammatoire TNF. Ce test est conçu pour évaluer l&#39;activation des EC, et non des neutrophiles. Neutrophiles humains primaires sont d&#39;abord isolés par séparation par gradient de densité, et sont ensuite marquées à la calcéine acétoxyméthylique (AM). Estérases l&#39;intérieur des cellules en direct hydrolyze calcéine AM à la molécule de calcéine hautement fluorescent avec une excitation de 492-495 nm et une émission de 513-516 nm 18. Les neutrophiles marquées par fluorescence sont ensuite incubés avec des monocouches CE et neutrophiles non adhérentes sont ensuite éliminés. La fluorescence des autres, les neutrophiles lié est ensuite mesurée à l&#39;aide d&#39;un spectrophotomètre à fluorescence, et calculé en pour cent de l&#39;apport total par fluorescence neutrophiles bien. Cette méthode présente l&#39;avantage supplémentaire que les neutrophiles liés calcéine marqués utilisés en spectrophotométrie peuvent être directement visualisées par microscopie à fluorescence pour donner une plus qualitative lire des CE acvation. Depuis cet essai est effectué dans des conditions statiques, seuls les événements très initiales qui se produisent dans la cascade de l&#39;adhésion des neutrophiles seront évalués. Ceci est confirmé dans ce rapport en utilisant des anticorps bloquants E-sélectine à montrer que l&#39;adhésion des neutrophiles au TNFa humain poumon traité CE microvasculaire (HMVEC-Lung) monocouches est considérablement réduite lorsque l&#39;interaction avec E-sélectine est perturbé. En plus de TNF, nous avons utilisé avec succès ce test pour déterminer l&#39;étendue de la veine activation humaine ombilical CE (HUVEC) par le récepteur Toll-like de 1/2 agonistes lipoprotéine associée à un peptidoglycane (PAL), murein lipoprotéine (MLP) et Pam3Cys et activation HMVEC-Lung par Pam3Cys 19,20. En outre, nous avons utilisé avec succès ce test avec les inhibiteurs de kinases et après knockdown ARNi médiée de surface et protéines cytoplasmiques dans HMVEC-Lung, ce qui suggère que cette méthode est compatible avec une variété de biochimie et screenindosages g 20. En résumé, ce test fournit une interface facile à utiliser, de façon reproductible, plus fonctionnel pour accéder à l&#39;importance de l&#39;activation CE par des médiateurs inflammatoires in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

L&#39;endothélium vasculaire joue un rôle essentiel dans la réponse inflammatoire. Pendant la phase aiguë de l&#39;inflammation, les cellules endothéliales (EC) sont activés par des médiateurs d&#39;accueil ou directement par des composants microbiens conservés ou molécules de danger dérivées de l&#39;hôte. Activés CEs cytokines express, des chimiokines et de molécules d&#39;adhésion qui mobilisent, activent et conservent leucocytes au site de l&#39;infection ou de blessure. Les neutrophiles sont les premiers leucocytes à arriver, et adhèrent à l&#39;endothélium à travers une variété de molécules d&#39;adhésion présentes sur les surfaces des deux cellules. Les principales fonctions des neutrophiles sont d&#39;éliminer directement les menaces microbiennes, de promouvoir le recrutement d&#39;autres leucocytes à travers la libération de facteurs supplémentaires, et d&#39;initier la cicatrisation des plaies. Par conséquent, leur recrutement et leur attachement à l&#39;endothélium est une étape critique dans l&#39;initiation de la réponse inflammatoire. Dans ce rapport, nous décrivons un test d&#39;adhérence in vitro en utilisant des neutrophilescalcéine AM-étiqueté neutrophiles humains primaires pour quantifier le degré d&#39;activation des cellules endothéliales microvasculaires, en conditions statiques. Cette méthode présente l&#39;avantage supplémentaire que les mêmes échantillons quantifiés par spectrophotométrie de fluorescence peuvent également être visualisés directement par microscopie de fluorescence pour une évaluation plus qualitative de la liaison des neutrophiles.

Afin d&#39;obtenir des résultats fiables et reproductibles en utilisant un test de liaison des neutrophiles, il est essentiel que la santé et la confluence des cellules endothéliales microvasculaires sont optimales le jour de l&#39;essai, comme illustré avec HMVEC-Lung à la figure 1. En outre, il est impératif que nombre passage bas microvasculaire EC sont utilisés (soit moins de 9 passages), et en conséquence, nous conseillons d&#39;effectuer toutes les expériences dans les deux semai...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

L&#39;adhésion des neutrophiles à l&#39;endothélium activé sur les sites d&#39;infection est une partie intégrante de la réponse inflammatoire de l&#39;hôte. Décrite dans le présent rapport est une analyse de liaison des neutrophiles qui permet de<em&gt; In vitro</em&gt; Quantification des neutrophiles humains primaire liaison aux cellules endothéliales activées par des médiateurs inflammatoires dans des conditions statiques.

Quantitative<em&gt; In vitro</em&gt; Test pour mesurer l&#39;adhésion des neutrophiles à Activé cellules endothéliales microvasculaires humaines primaires dans des conditions statiques

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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Research

JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

17.0K vues.  University of California, San Francisco. L'adhésion des neutrophiles à l'endothélium activé sur les sites d'infection est une partie intégrante de la réponse inflammatoire de l'hôte. Décrite dans le présent rapport est une analyse de liaison des neutrophiles qui permet de In vitro Quantification des neutrophiles humains primaire liaison aux cellules endothéliales activées par des médiateurs inflammatoires dans des conditions statiques.

Vidéo: Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Test in vitro de l&#39;adhésion bactérienne sur des mammifères cellules épithéliales

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Recherche

Immunologie et infection

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolement des cellules humaines endothéliales de veines ombilicales et leur utilisation dans l&#39;étude de la Transmigration des neutrophiles en conditions d&#39;écoulement

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Un<em&gt; In vitro</em&gt; Co-infection modèle pour étudier<em&gt; Plasmodium falciparum</em&gt;-VIH-1 dans les interactions de l&#39;homme primaires dérivées de monocytes cellules immunitaires

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Essai de coculture pour évaluer la migration transépithéliale des neutrophiles induite par l’agent pathogène

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un flux Essai d&#39;adhésion à étudier Leucocyte recrutement pour hépatiques humaines sinusoïdales endothélium dans des conditions de contrainte de cisaillement

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Human neutrophiles Chambre flux Essai d&#39;adhésion

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Statique Essai d&#39;adhésion pour l&#39;étude de l&#39;intégrine activation dans les lymphocytes T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imagerie en temps réel des jonctions endothéliales cellule-cellule Pendant neutrophiles Transmigration Sous physiologique débit

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro et in vivo modèle pour étudier l&#39;adhésion bactérienne au navire muraux dans des conditions de débit

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Dosage de Adhérence Sous contrainte de cisaillement pour l&#39;étude des lymphocytes T-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...