isolation-human-umbilical-vein-endothelial-cells-their-use-study

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, ...

University of Calgary 

Fps/Fes and Fer comprise a distinct subfamily of cytoplasmic protein-tyrosine kinases, and have both been implicated in the regulation of innate immunity. Previous studies showed that Fps/Fes-knockout mice were hypersensitive to systemic lipopolysaccharide (LPS) challenge, and Fer-deficient mice displayed enhanced recruitment of leucocytes in response to localized LPS challenge. We show here for the first time, a role for Fps in the regulation of leucocyte recruitment to areas of inflammation. Using the cremaster muscle intravital microscopy model, we observed increased leucocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-knockout mice relative to wild-type animals subsequent to localized LPS challenge. There was also a decreased vessel wall shear rate in the post-capillary venules of LPS-challenged Fps/Fes-knockout mice, and an increase in neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry to quantify the expression of surface molecules, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex vivo with LPS. These observations provide important insights into the observed in vivo behaviour of leucocytes in LPS-challenged Fps/Fes-knockout mice and provide evidence that the Fps/Fes kinase plays an important role in the innate immune response.

The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation.

&#x4F9D;&#x8D56;&#x7684; Mac-1 &#x722C;&#x7F51;&#x662F;&#x767D;&#x7EC6;&#x80DE;&#x62DB;&#x8058;&#x68AF;&#x7EA7;&#x9075;&#x5FAA; LFA 1 &#x4F9D;&#x8D56;&#x7C98;&#x9644;&#xFF0C;&#x4E4B;&#x524D;&#x79FB;&#x6C11;&#x4E2D;&#x7684;&#x65B0;&#x6B65;&#x9AA4;&#x3002;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x7C98;&#x9644;&#x901A;&#x8FC7; LFA 1 &#x8868;&#x660E;&#xFF0C;&#x4FC3;&#x4F7F;&#x7EC6;&#x80DE;&#x9AA8;&#x67B6;&#x7684;&#x91CD;&#x7EC4;&#xFF0C;&#x901A;&#x8FC7;&#x4F9D;&#x8D56;&#x4E8E; Vav1 &#x7684;&#x4FE1;&#x53F7;&#xFF0C;&#x548C;&#x76EE;&#x524D;&#x7684;&#x7814;&#x7A76;&#x8C03;&#x67E5;&#x7684; Vav1 &#x4E8B;&#x4EF6;&#x7279;&#x522B;&#x6CE8;&#x610F;&#x4F53;&#x5185;&#x7684;&#x767D;&#x7EC6;&#x80DE;&#x62DB;&#x8058;&#x8FC7;&#x7A0B;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x7ACB;&#x5373;&#x4E0B;&#x6E38;&#x7684; LFA 1 &#x4F9D;&#x8D56;&#x7C98;&#x9644;&#x3002;&#x6D3B;&#x4F53;&#x548C;&#x65CB;&#x8F6C;&#x78C1;&#x76D8;&#x5171;&#x805A;&#x7126;&#x663E;&#x5FAE;&#x955C;&#x7528;&#x6765;&#x8C03;&#x67E5;&#x4E0E;&#x76AE;&#x8DEF;&#x53E3;&#x91CE;&#x751F;&#x578B;&#x548C; Vav1(-/-) &#x5C0F;&#x9F20;&#x4F53;&#x5185;&#x8840;&#x7BA1;&#x5185;&#x722C;&#x884C;&#x3002;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x91CE;&#x751F;&#x578B;&#x8D34;&#x58C1;&#x51E0;&#x4E4E;&#x7ACB;&#x5373;&#x5F00;&#x59CB;&#x722C;&#x5782;&#x76F4;&#x4E8E;&#x8840;&#x6D41;&#x91CF;&#x901A;&#x8FC7; Mac 1 &#x76F4;&#x5230;&#x4ED6;&#x4EEC;&#x5230;&#x8FBE;&#x90A3;&#x91CC;&#xFF0C;&#x4ED6;&#x4EEC;&#x5E38;&#x5E38;&#x6539;&#x53D8;&#x65B9;&#x5411;&#x76AE;&#x4EA4;&#x754C;&#x5904;&#x3002;&#x5E94;&#x7528;&#x526A;&#x5207;&#x65F6;&#xFF0C;&#x8FD9;&#x4E00;&#x6A21;&#x5F0F;&#x7684;&#x722C;&#x7F51;&#x7684;&#x5782;&#x76F4;&#xFF0C;mechanotactic &#x88AB;&#x6982;&#x62EC;&#x4F53;&#x5916;&#x3002;&#x5728;&#x9C9C;&#x660E;&#x7684;&#x5BF9;&#x6BD4;&#xFF0C;Vav1(-/-) &#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x7684;&#x8FD0;&#x52A8;&#x4E2D;&#x7684;&#x6D41;&#x52A8;&#x65B9;&#x5411;&#x59CB;&#x7EC8;&#x662F;&#x548C;&#x4F3C;&#x4E4E;&#x66F4;&#x88AB;&#x52A8;&#xFF0C;&#x5982;&#x679C;&#x5355;&#x5143;&#x683C;&#x88AB;&#x62D6;&#x5728;&#x4F53;&#x5185;&#x548C;&#x4F53;&#x5916;&#x7684;&#x6D41;&#x52A8;&#x65B9;&#x5411;&#x3002;Vav1(-/-) &#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x7684; 80%&#x4EE5;&#x4E0A;&#x79FB;&#x52A8;&#x72EC;&#x7ACB; Mac-1&#xFF0C;&#x53EF;&#x4EE5;&#x5206;&#x79BB;&#x4E0E; LFA 1 Abs&#x3002;&#x65E0;&#x6CD5;&#x91CA;&#x653E; uropod &#x5E38;&#x88AB;&#x63D0;&#x5230;&#x7684; Vav1(-/-) &#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#xFF0C;&#x5BFC;&#x81F4;&#x5927;&#x5927;&#x7EC6;&#x957F;&#x7684;&#x5C3E;&#x5DF4;&#x3002;Vav1(-/-) &#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x672A;&#x80FD;&#x505C;&#x6B62;&#x6216;&#x6309;&#x7167;&#x8DEF;&#x53E3;&#xFF0C;&#x6700;&#x7EC8;&#x5206;&#x79BB;&#xFF0C;&#x5BFC;&#x81F4;&#x8F83;&#x5C11;&#x7684;&#x56DE;&#x6D41;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x3002;Vav1(-/-) &#x578B;&#x5BFC;&#x81F4;&#x5C11;&#x62DB;&#x52DF;&#x7684;&#x4F20;&#x67D3;&#x6027;&#x8179;&#x819C;&#x708E;&#x7684;&#x76F8;&#x5173;&#x6A21;&#x578B;&#x4E2D;&#x7684;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x3002;&#x663E;&#x7136;&#xFF0C;Vav1 &#x662F; LFA 1 &#x4E0E;&#x57FA;&#x7840;&#x7F16;&#x7A0B;&#x7684;&#x55DC;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x8840;&#x7BA1;&#x5185;&#x722C;&#x7F51;&#x7684; Mac-1 &#x4E4B;&#x95F4;&#x590D;&#x6742;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x5173;&#x952E;&#x3002;

Vav1 &#x662F; mechanotactic &#x722C;&#x884C;&#x548C;&#x8FC1;&#x79FB;&#x7684;&#x55DC;&#x4E2D;&#x6027;&#x7C92;&#x7EC6;&#x80DE;&#x4ECE;&#x7EA2;&#x80BF;&#x7684;&#x5FAE;&#x8840;&#x7BA1;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

Mac-1-dependent crawling is a new step in the leukocyte recruitment cascade that follows LFA-1-dependent adhesion and precedes emigration. Neutrophil adhesion via LFA-1 has been shown to induce cytoskeletal reorganization through Vav1-dependent signaling, and the current study investigates the role of Vav1 in the leukocyte recruitment process in vivo with particular attention to the events immediately downstream of LFA-1-dependent adhesion. Intravital and spinning-disk-confocal microscopy was used to investigate intravascular crawling in relation to endothelial junctions in vivo in wild-type and Vav1(-/-) mice. Adherent wild-type neutrophils almost immediately began crawling perpendicular to blood flow via Mac-1 until they reached an endothelial junction where they often changed direction. This pattern of perpendicular, mechanotactic crawling was recapitulated in vitro when shear was applied. In sharp contrast, the movement of Vav1(-/-) neutrophils was always in the direction of flow and appeared more passive as if the cells were dragged in the direction of flow in vivo and in vitro. More than 80% of Vav1(-/-) neutrophils moved independent of Mac-1 and could be detached with LFA-1 Abs. An inability to release the uropod was frequently noted for Vav1(-/-) neutrophils, leading to greatly elongated tails. The Vav1(-/-) neutrophils failed to stop or follow junctions and ultimately detached, leading to fewer emigrated neutrophils. The Vav1(-/-) phenotype resulted in fewer neutrophils recruited in a relevant model of infectious peritonitis. Clearly, Vav1 is critical for the complex interplay between LFA-1 and Mac-1 that underlies the programmed intravascular crawling of neutrophils.

Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature.

Mac-1-dependent rampant est une nouvelle &eacute;tape dans la cascade de recrutement des leucocytes qui suit l&#39;adh&eacute;rence d&eacute;pendante LFA-1 et qui pr&eacute;c&egrave;de l&#39;&eacute;migration. Adh&eacute;sion des neutrophiles par LFA-1 a &eacute;t&eacute; montr&eacute;e pour induire la r&eacute;organisation du cytosquelette par Vav1-d&eacute;pendante de signalisation, et la pr&eacute;sente &eacute;tude examine le r&ocirc;le de Vav1 dans le processus de recrutement des leucocytes in vivo avec une attention particuli&egrave;re aux &eacute;v&eacute;nements imm&eacute;diatement en aval de l&#39;adh&eacute;rence d&eacute;pendante LFA-1. Microscopie intravitale et filature-disque-confocale a permis d&#39;&eacute;tudier &agrave; ramper intravasculaire en ce qui concerne les jonctions endoth&eacute;liales in vivo chez le type sauvage et Vav1(-/-) souris. Adh&eacute;rentes sauvage neutrophiles presque imm&eacute;diatement commenc&egrave;rent &agrave; ramper perpendiculaire au flux sanguin via Mac-1 jusqu&#39;&agrave; ce qu&#39;ils ont atteint une jonction endoth&eacute;liale o&ugrave; ils changent souvent de direction. Cette tendance &agrave; la perpendiculaire, mechanotactic ramper a &eacute;t&eacute; r&eacute;expos&eacute;e in vitro lorsque l&#39;inclinaison est appliqu&eacute;e. Contraste, le mouvement des neutrophiles Vav1(-/-) &eacute;tait toujours dans le sens d&#39;&eacute;coulement et semblait plus passif, comme si les cellules ont &eacute;t&eacute; entra&icirc;n&eacute;s dans le sens d&#39;&eacute;coulement in vivo et in vitro. Plus de 80 % de polynucl&eacute;aires neutrophiles Vav1(-/-) d&eacute;plac&eacute; ind&eacute;pendamment de Mac-1 et pourrait &ecirc;tre d&eacute;tach&eacute; avec Abs de LFA-1. Une incapacit&eacute; &agrave; rel&acirc;cher l&#39;uropode a not&eacute; fr&eacute;quemment pour les neutrophiles Vav1(-/-), conduisant &agrave; des queues consid&eacute;rablement allong&eacute;es. Les neutrophiles Vav1(-/-) n&#39;a pas pu arr&ecirc;ter ou suivre des jonctions et finalement d&eacute;tach&eacute;, entra&icirc;nant moins de neutrophiles a &eacute;migr&eacute;. Le ph&eacute;notype Vav1(-/-) a entra&icirc;n&eacute; moins de neutrophiles recrut&eacute;s dans un mod&egrave;le pertinent de P&eacute;ritonite infectieuse. De toute &eacute;vidence, Vav1 est essentiel pour les interactions complexes entre les LFA-1 et Mac-1 qui sous-tend le rampant intravasculaire programm&eacute;e des neutrophiles.

Vav1 est essentiel pour mechanotactic rampant et la migration des neutrophiles hors de la microcirculation enflamm&eacute;e.

Mac-1-abh&auml;ngigen crawling wird ein neuer Schritt in der Leukozyten-Rekrutierung-Kaskade, die folgt LFA-1-abh&auml;ngige Adh&auml;sion und Auswanderung vorausgeht. Nachweislich Neutrophil Adh&auml;sion &uuml;ber LFA-1 Zytoskelett Reorganisation durch Vav1-abh&auml;ngige Signalisierung zu veranlassen und die aktuelle Studie untersucht die Rolle der Vav1 in die Leukozyten-Recruiting-Prozess, der in-vivo mit besonderem Augenmerk auf die Ereignisse unmittelbar hinter der LFA-1-abh&auml;ngige Adh&auml;sion. Intravital und Spinning-Disk-konfokale Mikroskopie wurde verwendet, um zu untersuchen, intravaskul&auml;re Crawlen in Bezug auf die in-vivo in Wildtyp und Vav1(-/-) M&auml;usen endothelialen Kreuzungen. Adh&auml;rente Wildtyp Neutrophilen begann fast sofort, &uuml;ber Mac-1 senkrecht zur Durchblutung kriechen, bis sie ein endothelial Knotenpunkt erreichte, wo sie oft die Richtung ge&auml;ndert. Dieses Muster der senkrecht, Mechanotactic, Crawlen war in-vitro rekapitulierte, wenn Scherung zugewiesen wurde. In scharfem Kontrast die Bewegung der Vav1(-/-) Neutrophilen war immer in Flie&szlig;richtung und erschien mehr passive, als ob die Zellen in Flie&szlig;richtung in-vivo und in vitro gezogen wurden. Mehr als 80 % der Vav1(-/-) Neutrophilen zog unabh&auml;ngig von Mac-1 und konnte mit LFA-1 Abs getrennt werden. Die Unf&auml;higkeit, die Uropod freigeben f&uuml;r Vav1(-/-) Neutrophilen, was zu erheblich verl&auml;ngerten Schwanz h&auml;ufig erw&auml;hnt. Die Vav1(-/-)-neutrophile nicht stoppen oder Kreuzungen zu folgen und letztlich getrennt, was zu weniger emigrierten Neutrophilen. Der Vav1(-/-)-Ph&auml;notyp ergab weniger neutrophile rekrutiert in einem entsprechenden Modell der infekti&ouml;se Peritonitis. Vav1 ist, entscheidend f&uuml;r das komplexe Zusammenspiel zwischen LFA-1 und Mac-1, die zugrunde liegt, die programmierten intravaskul&auml;re durchforsten von Neutrophilen.

Vav1 unbedingt Mechanotactic kriechen und Migration von Neutrophilen aus den entz&uuml;ndeten Microvasculature.

Mac-1-dipendente crawling &egrave; un nuovo passo nella cascata di reclutamento dei leucociti che segue l&#39;adesione LFA-1-dipendente e precede l&#39;emigrazione. Adesione dei neutrofili via LFA-1 &egrave; stato indicato per indurre la riorganizzazione del citoscheletro attraverso la segnalazione Vav1-dipendente, e l&#39;attuale studio indaga il ruolo di Vav1 nel processo di reclutamento dei leucociti in vivo con particolare attenzione agli eventi immediatamente a valle dell&#39;adesione LFA-1-dipendente. Intravital e filatura-disco-confocale microscopia &egrave; stata utilizzata per indagare intravascolare strisciando in relazione alle giunzioni endoteliali in vivo nel wild-type e topi Vav1(-/-). Aderenti selvaggio-tipo neutrofili iniziarono quasi immediatamente strisciando perpendicolare al flusso di sangue attraverso Mac-1 fino a quando hanno raggiunto una giunzione endoteliale dove hanno cambiato spesso direzione. Questo modello di mechanotactic perpendicolare, strisciando &egrave; stato ricapitolato in vitro quando il taglio &egrave; stato applicato. In netto contrasto, il movimento dei neutrofili Vav1(-/-) era sempre in direzione del flusso e apparso pi&ugrave; passivo, come se le celle sono state trascinate nella direzione del flusso in vivo e in vitro. Pi&ugrave; dell&#39;80% dei neutrofili Vav1(-/-) spostato indipendente di Mac-1 e poteva essere staccata con Abs di LFA-1. Un&#39;incapacit&agrave; di rilasciare i uropod frequentemente &egrave; stata notata per Vav1(-/-) i neutrofili, che conduce a coda molto allungate. I neutrofili Vav1(-/-) non &egrave; riuscito a fermare o seguire le giunzioni e infine staccata, portando a un minor numero di neutrofili emigrati. Il fenotipo di Vav1(-/-) ha provocato meno neutrofili reclutati in un modello pertinente di peritonite infettiva. Chiaramente, Vav1 &egrave; fondamentale per la complessa interazione tra LFA-1 e Mac-1 che sottende la programmata strisciando intravascolare dei neutrofili.

Vav1 &egrave; essenziale per la migrazione dei neutrofili fuori il microvasculature infiammato e mechanotactic strisciando.

Mac-1 &#x4F9D;&#x5B58;&#x3092;&#x30AF;&#x30ED;&#x30FC;&#x30EB; LFA 1 &#x4F9D;&#x5B58;&#x63A5;&#x7740;&#x306B;&#x7D9A;&#x304F;&#x3001;&#x79FB;&#x4F4F;&#x524D;&#x306B;&#x767D;&#x8840;&#x7403;&#x306E;&#x52DF;&#x96C6;&#x306E;&#x7FFC;&#x5217;&#x5185;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30B9;&#x30C6;&#x30C3;&#x30D7;&#x3067;&#x3059;&#x3002;&#x597D;&#x4E2D;&#x7403;&#x306E;&#x4ED8;&#x7740; LFA 1 &#x3092;&#x4ECB;&#x3057;&#x3066;&#x3092;&#x4ECB;&#x3057;&#x3066; Vav1 &#x4F9D;&#x5B58;&#x6027;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x3001;&#x7D30;&#x80DE;&#x9AA8;&#x683C;&#x306E;&#x518D;&#x7DE8;&#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x73FE;&#x5728;&#x306E;&#x7814;&#x7A76;&#x306B;&#x7279;&#x306B;&#x6CE8;&#x610F;&#x3092;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x3092;&#x751F;&#x4F53;&#x5185;&#x3067;&#x767D;&#x8840;&#x7403;&#x63A1;&#x7528;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B; Vav1 &#x306E;&#x5F79;&#x5272;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B; LFA-1 &#x4F9D;&#x5B58;&#x63A5;&#x7740;&#x306E;&#x4E0B;&#x6D41;&#x3067;&#x3059;&#x3050;&#x306B;&#x3002;&#x751F;&#x4F53;&#x3068;&#x56DE;&#x8EE2;&#x30C7;&#x30A3;&#x30B9;&#x30AF;&#x5171;&#x7126;&#x70B9;&#x9855;&#x5FAE;&#x93E1;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x63A5;&#x5408;&#x91CE;&#x751F;&#x578B;&#x304A;&#x3088;&#x3073; Vav1(-/-) &#x30DE;&#x30A6;&#x30B9;&#x306E;&#x751F;&#x4F53;&#x5185;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x4ED8;&#x7740;&#x6027;&#x306E;&#x91CE;&#x751F;&#x578B;&#x597D;&#x4E2D;&#x7403;&#x306F;&#x307B;&#x3068;&#x3093;&#x3069;&#x3059;&#x3050;&#x306B;&#x5F7C;&#x3089;&#x306F;&#x5F7C;&#x3089;&#x306F;&#x3057;&#x3070;&#x3057;&#x3070;&#x65B9;&#x5411;&#x3092;&#x5909;&#x3048;&#x305F;&#x306A;&#x5185;&#x76AE;&#x63A5;&#x5408;&#x306B;&#x9054;&#x3059;&#x308B;&#x307E;&#x3067; Mac-1 &#x3092;&#x4ECB;&#x3057;&#x3066;&#x8840;&#x6DB2;&#x306E;&#x6D41;&#x308C;&#x306B;&#x76F4;&#x89D2;&#x306A;&#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x3092;&#x59CB;&#x3081;&#x305F;&#x3002;&#x305B;&#x3093;&#x65AD;&#x304C;&#x9069;&#x7528;&#x3055;&#x308C;&#x305F;&#x3068;&#x304D;&#x3053;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x5782;&#x76F4;&#x3001;mechanotactic &#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x306E; in vitro &#x518D;&#x8868;&#x73FE;&#x3060;&#x3063;&#x305F;&#x3002;&#x92ED;&#x3044;&#x30B3;&#x30F3;&#x30C8;&#x30E9;&#x30B9;&#x30C8; Vav1(-/-) &#x597D;&#x4E2D;&#x7403;&#x306E;&#x904B;&#x52D5;&#x306E;&#x6D41;&#x308C;&#x306E;&#x65B9;&#x5411;&#x306B;&#x5E38;&#x306B;&#x3060;&#x3063;&#x305F;&#x3057;&#x3001;&#x7D30;&#x80DE; in vivo &#x304A;&#x3088;&#x3073; in vitro &#x3067;&#x306E;&#x6D41;&#x308C;&#x306E;&#x65B9;&#x5411;&#x306B;&#x30C9;&#x30E9;&#x30C3;&#x30B0;&#x3055;&#x308C;&#x305F;&#x5834;&#x5408;&#x3068;&#x3057;&#x3066;&#x30D1;&#x30C3;&#x30B7;&#x30D6;&#x767B;&#x5834;&#x3057;&#x307E;&#x3059;&#x3002;Vav1(-/-) &#x597D;&#x4E2D;&#x7403;&#x306E; 80 &#xFF05; &#x4EE5;&#x4E0A;&#x306F;&#x3001;Mac-1 &#x306E;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x79FB;&#x52D5;&#x3001;LFA 1 Abs &#x3092;&#x30C7;&#x30BF;&#x30C3;&#x30C1;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x5C3E;&#x6247;&#x80A2;&#x3092;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3067;&#x304D;&#x306A;&#x3044;&#x3053;&#x3068;&#x306F;&#x3001;&#x983B;&#x7E41;&#x306B;&#x5927;&#x304D;&#x304F;&#x7D30;&#x9577;&#x3044;&#x5C3E;&#x3092;&#x30EA;&#x30FC;&#x30C9;&#x3001;Vav1(-/-) &#x306E;&#x597D;&#x4E2D;&#x7403;&#x3092;&#x6307;&#x6458;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;Vav1(-/-) &#x597D;&#x4E2D;&#x7403;&#x3092;&#x505C;&#x6B62;&#x307E;&#x305F;&#x306F;&#x63A5;&#x5408;&#x306B;&#x5F93;&#x3046;&#x306B;&#x5931;&#x6557;&#x3057;&#x3001;&#x6700;&#x7D42;&#x7684;&#x306B;&#x30C7;&#x30BF;&#x30C3;&#x30C1;&#x5C11;&#x306A;&#x3044;&#x79FB;&#x4F4F;&#x3057;&#x305F;&#x597D;&#x4E2D;&#x7403;&#x306B; &#x3002;Vav1(-/-) &#x8868;&#x73FE;&#x578B;&#x4F1D;&#x67D3;&#x6027;&#x8179;&#x819C;&#x708E;&#x306E;&#x95A2;&#x9023;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30E2;&#x30C7;&#x30EB;&#x3067;&#x52DF;&#x96C6;&#x3057;&#x305F;&#x5C11;&#x6570;&#x306E;&#x597D;&#x4E2D;&#x7403;&#x306E;&#x7D50;&#x679C;&#x3002;&#x660E;&#x3089;&#x304B;&#x306B;&#x3001;Vav1 &#x306F; LFA 1 &#x3068;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3055;&#x308C;&#x305F;&#x8840;&#x7BA1;&#x5185;&#x597D;&#x4E2D;&#x7403;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x30DE;&#x30C3;&#x30AF; 1 &#x9593;&#x306E;&#x8907;&#x96D1;&#x306A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x3068;&#x3063;&#x3066;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;

Vav1 &#x306F; mechanotactic &#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x3068;&#x597D;&#x4E2D;&#x7403;&#x708E;&#x75C7;&#x8840;&#x7BA1;&#x304B;&#x3089;&#x79FB;&#x884C;&#x3059;&#x308B;&#x4E0A;&#x3067;&#x6B20;&#x304B;&#x305B;&#x307E;&#x305B;&#x3093;&#x3002;

&#xB9E5; 1 &#xC885;&#xC18D; &#xD06C;&#xB864;&#xB9C1; LFA 1 &#xC885;&#xC18D; &#xC811;&#xCC29; &#xB530;&#xB974;&#xACE0; &#xC55E;&#xC5D0; &#xC774;&#xC8FC; &#xD558;&#xB294; &#xBC31;&#xD608;&#xAD6C; &#xBAA8;&#xC9D1; &#xCE90;&#xC2A4;&#xCF00;&#xC774;&#xB4DC;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xB2E8;&#xACC4;&#xC785;&#xB2C8;&#xB2E4;. LFA-1&#xC744; &#xD1B5;&#xD574; &#xD638; &#xC811;&#xCC29; Vav1 &#xC885;&#xC18D; &#xC2E0;&#xD638;&#xB97C; &#xD1B5;&#xD574; cytoskeletal &#xAC1C;&#xD3B8;&#xC744; &#xC720;&#xB3C4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xD45C;&#xC2DC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC0F; &#xD604;&#xC7AC; &#xC5F0;&#xAD6C; &#xC870;&#xC0AC;&#xB294; &#xC774;&#xBCA4;&#xD2B8;&#xC5D0; &#xD2B9;&#xD788; &#xAD00;&#xC2EC;&#xC744; &#xAC00;&#xC9C4; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xBC31;&#xD608;&#xAD6C; &#xC2E0;&#xADDC; &#xBAA8;&#xC9D1; &#xACFC;&#xC815;&#xC5D0;&#xC11C; vav1&#xC758; &#xC5ED;&#xD560; &#xC989;&#xC2DC; LFA 1 &#xC885;&#xC18D; &#xC811;&#xCC29;&#xC758; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC;. Intravital &#xBC0F; &#xD68C;&#xC804; &#xB514;&#xC2A4;&#xD06C; confocal &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; intravascular &#xD06C;&#xB864;&#xB9C1; &#xB0B4; &#xD53C; &#xC811;&#xD569; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xBC0F; Vav1(-/-) &#xB9C8;&#xC6B0;&#xC2A4; &#xC0DD;&#xCCB4; &#xC870;&#xAC74;&#xC5D0; &#xAD00;&#xD558;&#xC5EC; &#xC870;&#xC0AC; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. &#xBD80;&#xCC29; &#xD55C; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xD638; &#xC911;&#xAD6C;&#xB294; &#xAC70;&#xC758; &#xC989;&#xC2DC; &#xADF8;&#xB4E4;&#xC774; &#xC5B4;&#xB514; &#xADF8;&#xB4E4;&#xC740; &#xC885;&#xC885; &#xBC29;&#xD5A5; &#xBCC0;&#xACBD; &#xB0B4; &#xD53C; &#xC811;&#xD569;&#xC5D0; &#xB3C4;&#xB2EC;&#xD560; &#xB54C;&#xAE4C;&#xC9C0; &#xB9E5; 1&#xC744; &#xD1B5;&#xD574; &#xD608;&#xC561;&#xC758; &#xD750;&#xB984;&#xC5D0; &#xC218;&#xC9C1;&#xC778; &#xD06C;&#xB864;&#xB9C1; &#xC2DC;&#xC791; &#xD588;&#xB2E4;. &#xD06C;&#xB864; &#xB9C1; &#xD558;&#xB294; &#xC218;&#xC9C1;, mechanotactic&#xC758;&#xC774; &#xD328;&#xD134; &#xC804;&#xB2E8; &#xC801;&#xC6A9;&#xD560; &#xB54C; &#xCCB4; &#xC678; recapitulated &#xD588;&#xB2E4;. &#xC608;&#xB9AC;&#xD55C; &#xB300;&#xC870;&#xC5D0;&#xC11C; Vav1(-/-) &#xD638; &#xC911;&#xAD6C;&#xC758; &#xC6C0;&#xC9C1;&#xC784; &#xD750;&#xB984; &#xBC29;&#xD5A5;&#xC5D0;&#xB294; &#xD56D;&#xC0C1; &#xACE0; &#xC140; &#xD750;&#xB984; in vivo&#xC640; &#xCCB4; &#xC678;&#xC758; &#xBC29;&#xD5A5;&#xC73C;&#xB85C; &#xB4DC;&#xB798;&#xADF8; &#xD588;&#xB2E4; &#xB9C8;&#xCE58; &#xC218;&#xB3D9; &#xB4F1;&#xC7A5;. Vav1(-/-) &#xD638; &#xC911;&#xAD6C;&#xC758; 80% &#xC774;&#xC0C1;&#xC774; &#xB9E5; 1&#xC758; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xC774;&#xB3D9; &#xD558; &#xACE0; LFA-1 &#xBCF5; &#xADFC;&#xC73C;&#xB85C; &#xBD84;&#xB9AC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Uropod&#xC744; &#xCD9C;&#xC2DC; &#xD558;&#xB294; &#xBB34; &#xB2A5;&#xB825;&#xC740; &#xC790;&#xC8FC; Vav1(-/-) &#xD638; &#xC911;&#xAD6C;, &#xD06C;&#xAC8C; &#xAE38;&#xCB49;&#xD55C; &#xAF2C;&#xB9AC;&#xB97C; &#xC120;&#xB3C4; &#xB300; &#xD55C; &#xC9C0;&#xC801; &#xD588;&#xB2E4;. Vav1(-/-) &#xD638; &#xC911;&#xAD6C; &#xC911;&#xC9C0; &#xB610;&#xB294; &#xAD50;&#xCC28;&#xC810;&#xC5D0; &#xB530;&#xB77C; &#xC2E4;&#xD328; &#xD558; &#xACE0; &#xAD81;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xBD84;&#xB9AC;, &#xC801;&#xC740; &#xC774;&#xBBFC;&#xB41C; &#xD638; &#xC911;&#xAD6C;&#xB85C; &#xC774;&#xC5B4;&#xC9C0;&#xB294;. Vav1(-/-) &#xD615; &#xC801;&#xC740; neutrophils &#xC804;&#xC5FC; &#xC131; &#xBCF5; &#xB9C9; &#xC5FC;&#xC758; &#xAD00;&#xB828; &#xBAA8;&#xB378;&#xC5D0;&#xC11C; &#xCC44;&#xC6A9; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. &#xBA85;&#xD655; &#xD558; &#xAC8C;, vav1&#xC740; LFA-1&#xACFC; &#xD638; &#xC911;&#xAD6C;&#xC758; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xB41C; intravascular &#xD06C;&#xB864; &#xB9C1;&#xC758; &#xAE30;&#xCD08;&#xAC00; &#xB418;&#xB294; &#xB9E5; 1 &#xC0AC;&#xC774;&#xC758; &#xBCF5;&#xC7A1; &#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

Vav1 mechanotactic&#xB97C; &#xD06C;&#xB864; &#xB9C1; &#xD558; &#xACE0; &#xC5FC;&#xC99D;&#xC774; microvasculature&#xC5D0;&#xC11C; &#xD638; &#xC911;&#xAD6C;&#xC758; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Mac-1-dependiente de arrastre es un nuevo hito en la cascada de reclutamiento de leucocitos que sigue adhesi&oacute;n LFA-1-dependiente y precede a la emigraci&oacute;n. Adherencia de neutr&oacute;fila v&iacute;a LFA-1 se ha demostrado para inducir la reorganizaci&oacute;n citoesquel&eacute;tica mediante se&ntilde;alizaci&oacute;n Vav1-dependiente, y el presente estudio investiga el papel de Vav1 en el proceso de reclutamiento de leucocitos in vivo con especial atenci&oacute;n a los acontecimientos inmediatamente aguas abajo de adhesi&oacute;n LFA-1-dependiente. Microscopia intravital y spinning-disco-confocal fue utilizada para investigar arrastr&aacute;ndose intravascular en relaci&oacute;n con uniones endoteliales in vivo en salvaje-tipo y ratones Vav1(-/-). Adherentes salvaje-tipo neutr&oacute;filos comenzaron casi inmediatamente arrastr&aacute;ndose perpendicular al flujo sangu&iacute;neo mediante Mac-1 hasta llegar a un cruce endotelial donde a menudo cambiaron de direcci&oacute;n. Este patr&oacute;n de mechanotactic perpendicular, arrastr&aacute;ndose fue recapitulado in vitro cuando se aplic&oacute; el esquileo. En contraste, el movimiento de neutr&oacute;filos Vav1(-/-) estaba siempre en la direcci&oacute;n del flujo y apareci&oacute; m&aacute;s pasivo como si las c&eacute;lulas fueron arrastradas en la direcci&oacute;n del flujo in vivo e in vitro. M&aacute;s del 80% de los neutr&oacute;filos Vav1(-/-) movido independiente de Mac-1 y podr&iacute;a ser separado con Abs de LFA-1. La incapacidad para liberar la uropod con frecuencia destac&oacute; por neutr&oacute;filos Vav1(-/-), hacia la cola muy alargada. Los neutr&oacute;filos Vav1(-/-) no pudo parar o seguir a las ensambladuras y separado en &uacute;ltima instancia, conduce a menos neutr&oacute;filos emigrados. El fenotipo de Vav1(-/-) result&oacute; en menos neutr&oacute;filos reclutados en un modelo relevante de peritonitis infecciosa. Claramente, Vav1 es cr&iacute;tico para la compleja interacci&oacute;n entre LFA-1 y Mac-1 que subyace a la programada arrastr&aacute;ndose intravascular de los neutr&oacute;filos.

Vav1 es esencial para mechanotactic arrastr&aacute;ndose y migraci&oacute;n de neutr&oacute;filos de la microvascularizaci&oacute;n inflamada.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.

During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

Article	Year
The International journal of developmental biology\| PubMed ID: 12653250	2003
Journal of leukocyte biology\| PubMed ID: 16959897	2006
The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation. Immunology\| PubMed ID: 17627769	2007
The Journal of membrane biology\| PubMed ID: 18273533	2008
Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19454683	2009
American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 20056898	2010
American journal of physiology. Cell physiology\| PubMed ID: 20702689	2010
Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo. Blood\| PubMed ID: 21030556	2011
Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 21217012	2011
Frontiers in neuroscience\| PubMed ID: 21833164	2011
Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration. European journal of immunology\| PubMed ID: 22095445	2012
American journal of physiology. Cell physiology\| PubMed ID: 22159087	2012

Sean Parsons

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