Questo lavoro è stato supportato finanziariamente da NIH (1 R01 AI095338-01A1).

Gli autori dichiarano di non avere interessi finanziari concorrenti.

La migrazione neutrofila attraverso le superfici epiteliali mucose è una caratteristica comune nella patologia della malattia a seguito dell'infezione da agenti patogenibatterici 3. La metodologia descritta nel presente documento offre un approccio rapido e diretto per isolare sperimentalmente questo evento discreto utilizzando un sistema di saggi di cocoltura in vitro derivato da cellule umane che modella una caratteristica del processo infiammatorio innescato da infezioni batteriche. Questo sistema...

Le superfici mucose fungono da barriere fisiche e immunologiche che forniscono protezione contro le minacce esterne pervasivenell'ambiente 1,2. Questa barriera epiteliale protettiva può essere compromessa quando gli organismi patogeni invadono2. Nel caso di un agente patogeno batterico, questo incontro spesso istiga un processo infiammatorio attivando il sistema immunitario innato e innescando una rapida mobilitazione dei granulociti del primo soccorritore noti come neutrofili2-4. Gli agenti chemiotattici che facilitano il reclutamento dei neutrofili sono prodotti in parte dalle cellule epiteliali mucose che cercano di liberare l'ospite dall'agente patogeno ofaoffendente 2-4. Un'infiltrazione neutrofila eccessiva o irrisolta della superficie epiteliale mucosa può causare una patologiasignificativa 1,5. Questa è una conseguenza del danno tissutale non specifico causato dall'arsenale neutrofilo antibatterico5-7. In questi casi, la capacità di clearance batterica dei neutrofili è oscurata dalla distruzione del tessuto ospite durante un insulto infettivo.  L'interruzione della funzione protettiva di barriera epiteliale può portare a una maggiore esposizione del tessuto sottostante a microrganismi e / o tossine, esacerbando ulteriormente la patologiadella malattia 8,9. Queste conseguenze possono essere osservate in più sistemi di organi tra cui il polmone e il trattodigestivo 1,5. Inoltre, condizioni infiammatorie non infettive come gravi attacchi di asma, broncopneumopatia cronica ostruttiva (BPCO), sindrome da distress respiratorio acuto (ARDS) e malattia infiammatoria intestinale (IBD) sono contrassegnate dalla violazione patologica della barriera epiteliale mucosa da un'eccessiva risposta neutrofila4,5,10-12.
Il complesso processo di reclutamento dei neutrofili a seguito di infezione della mucosa comporta diversi passaggicompartimentati 1,5,13,14. In primo luogo, i neutrofili devono partire dalla circolazione attraverso una serie di interazioni cellula-cellula che facilitano la migrazione trans-endoteliale1,13. I neutrofili navigano quindi nello spazio interstiziale esistente contenente matriceextracellulare 1,14. Per raggiungere il lume della mucosa infetta, i neutrofili devono quindi migrare attraverso la barriera epiteliale1,4,5. Questo intricato fenomeno multistep è spesso studiato in aggregato utilizzando modelli animali in vivo di infezione15. Tali modelli sono utili per stabilire la necessità di fattori specifici, come chemiochine, molecole di adesione o percorsi di segnalazione che partecipano al processo complessivo ma sono in gran parte inadeguati per risolvere i contributi molecolari critici per ogni fasecompartimentata distinta 16. I sistemi in vitro coculati che modellano la migrazione trans-endoteliale, trans-matriciale o transepiteliale dei neutrofili sono stati particolarmente utili aquesto proposito 1,14,16,17.
È stato sviluppato un solido sistema di dosaggio della cocoltura allo scopo di decifrare i meccanismi responsabili della migrazione transeteliale neutrofila in risposta all'infezionepatogena 18-22. Questo modello prevede di infettare la superficie apicale degli strati cellulari epiteliali umani polarizzati con un agente patogeno batterico seguito dall'applicazione di neutrofili umani appena isolati sulla superficie basolaterale18-22. I neutrofili migrano attraverso la barriera epiteliale in risposta a prodotti chemiotattici derivati epiteliali secreti a seguito di infezionepatogena 18,21-23. Questo sistema modello è stato utilizzato utilizzando colture epiteliali intestinali e polmonari esposte a patogeni batterici specifici del tessuto appropriati e ha svelato nuovi meccanismi molecolari probabilmente importanti per il processo di reclutamento dei neutrofili durante l'infezione dellamucosa 3,8,19,24-28. La forza di questo modello di cocultura in vitro è che un approccio riduzionista consente allo sperimentatore di manipolare sperimentalmente l'agente patogeno, la barriera epiteliale e / o il neutrofilo in un sistema ben controllato, altamente riproducibile, abbastanza economico. Le informazioni raccolte da questo approccio possono essere efficacemente sfruttate per condurre analisi mirate degli eventi compartimentati durante il reclutamento di neutrofili utilizzando modelli di infezione in vivo 22,29,30.
Questo articolo illustra i molteplici passaggi necessari per la corretta istituzione di questo modello riproducibile per esplorare la migrazione transeteliale neutrofila indotta da agenti patogeni. Le barriere epiteliali polmonari infettate dall'agente patogeno Pseudomonas aeruginosa sono presenti in questo articolo; tuttavia, altri epiteli tissutali e agenti patogeni possono essere sostituiti con piccole modifiche. La semina e la valutazione di strati di cellule epiteliali polmonari polarizzate su filtri transwell permeabili rivestiti di collagene invertito è dettagliata nel presente documento, così come la crescita della P. aeruginosa patogena e l'isolamento dei neutrofili dal sangue intero. Come questi componenti sono combinati per osservare la migrazione transeteliale neutrofila indotta da agenti patogeni viene presentato insieme ad opportuni controlli positivi e negativi per stabilire un saggio riproducibile. La versatilità di questo approccio per esaminare vari aspetti della migrazione transeteliale neutrofila indotta da agenti patogeni viene discussa con riferimento a studi specifici in letteratura.

<table border="0" cellpadding="0" cellspacing="0" width="1470">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">NCl-H292 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:137px;">CRL-1848</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">RPMI-1640 medium</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:137px;">30-2001</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Pseudomonas aeruginosa PAO1</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:137px;">#47085</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Escherichia coli MC1000</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:137px;">#39531</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">D-PBS (1x) liquid</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">14190-144</td>
			<td style="width:667px;">without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Heat Inactivated Fetal bovine serum</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">10082-147</td>
			<td style="width:667px;">10% added to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Penicillin-Streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:667px;">100x: 10,000 units of penicillin and 10,000 &#181;g of streptomycin per ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">25300-062</td>
			<td style="width:667px;">50 ml aliquots are stored frozen at -20 &#186;C.&#160; Aliquot in use can be stored at 4 &#186;C short-term.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Hank&#39;s Balanced Salt Solution - HBSS(-)</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">14175-079</td>
			<td style="width:667px;">Sterile, without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Trypan Blue Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">15250-061.</td>
			<td style="width:667px;">Stock = 0.4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Collagen, Rat Tail</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:137px;">A10483-01</td>
			<td style="width:667px;">Can also be isolated in the laboratory directly from the tails of rats using standard protocols</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Citric acid</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">C1909-500G</td>
			<td style="width:667px;">Component of 1 M citrate buffer and acid citrate dextrose (ACD) solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Sodium Citrate</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">S4641-500G</td>
			<td style="width:667px;">Component of 1 M citrate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Dextrose anhydrous</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">D8066-250G</td>
			<td style="width:667px;">Component of acid citrate dextrose (ACD) solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Ammonium Chloride</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">213330-500G</td>
			<td style="width:667px;">Component of red blood cell (RBC) lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Sodium bicarbonate</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">S6014-500G</td>
			<td style="width:667px;">Component of red blood cell (RBC) lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">EDTA</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">ED-100G</td>
			<td style="width:667px;">Component of red blood cell (RBC) lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">HBSS(+) powder</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">H1387-10L</td>
			<td style="width:667px;">Key component of HBSS+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">HEPES</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">H3375-500G</td>
			<td style="width:667px;">Component of HBSS+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Sigmacote</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">SL2-25ML</td>
			<td style="width:667px;">Follow vendor instructions to coat glass pipette tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Triton X-100</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">T-9284</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">2,2&#39;-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">A9941-50TAB</td>
			<td style="width:667px;">Key component of ABTS substrate solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">30% Hydrogen Peroxide Solution</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">H1009-100ML</td>
			<td style="width:667px;">Component of ABTS substrate solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">N-Formyl-Met-Leu-Phe (fMLP or fMLF)</td>
			<td style="width:147px;">Sigma-Aldrich&#160;</td>
			<td style="width:137px;">F-3506</td>
			<td style="width:667px;">A Stock solution of 10 mM in DMSO should be prepared and aliquots stored at -20 &#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Gelatin Type B</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">M-12026</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Pseudomonas isolation agar&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">DF0927-17-1</td>
			<td style="width:667px;">Follow manufacturer&#8217;s instructions to make PIA plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Ficoll-Paque PLUS&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">45-001-749</td>
			<td style="width:667px;">Optional, can improve neutrophil purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;"></td>
			<td style="width:147px;"></td>
			<td style="width:137px;"></td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;"></td>
			<td style="width:147px;"></td>
			<td style="width:137px;"></td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;"></td>
			<td style="width:147px;"></td>
			<td style="width:137px;"></td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;"></td>
			<td style="width:147px;"></td>
			<td style="width:137px;"></td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;"></td>
			<td style="width:147px;"></td>
			<td style="width:137px;"></td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Name of Material / Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:667px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">24-well migration plate</td>
			<td style="width:147px;">Corning Incorporated</td>
			<td style="width:137px;">#3524</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">24-well wash plate</td>
			<td style="width:147px;">Falcon</td>
			<td style="width:137px;">35-1147</td>
			<td style="width:667px;">Can be reused if soaked in 70% ethanol and washed thoroughly prior to reuse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">96-well plate</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">#12565501</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:520px;">Transwell Permeable Supports&#160;</td>
			<td style="width:147px;">Corning Incorporated</td>
			<td style="width:137px;">#3415</td>
			<td style="width:667px;">Polycarbonate; Diameter: 6.5 mm; Growth area: 0.33 cm2; Dish style: 24-well plate; Pore size: 3.0 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Petri dish</td>
			<td style="width:147px;">Falcon</td>
			<td style="width:137px;">35-1013</td>
			<td style="width:667px;">Each Petri dish holds 24 inverted 0.33 cm2 Transwells.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">500 ml 0.2 &#956;m filter / flask</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">09-740-25A</td>
			<td style="width:667px;">To sterilize acid citrate dextrose (ACD) solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">5-3/4 in glass Pasteur pipette</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">13-678-20A</td>
			<td style="width:667px;">Coat tips with Sigmacote prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Hemostat</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:137px;">13-812-14</td>
			<td style="width:667px;">Curved, Serrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Invertoskop Inverted Microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:137px;">#342222</td>
			<td style="width:667px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:520px;">Versa-Max Microplate Reader</td>
			<td style="width:147px;">Molecular Devices</td>
			<td style="width:137px;">#432789</td>
			<td style="width:667px;"></td>
		</tr>
	</tbody>
</table>

in vitro coculture assay to assess pathogen induced neutrophil trans-epithelial migration

Le superfici mucose fungono da barriere protettive contro gli organismi patogeni. Le risposte immunitarie innate vengono attivate al momento del rilevamento dell'agente patogeno che porta all'infiltrazione di tessuti con cellule infiammatorie migratorie, principalmente neutrofili. Questo processo ha il potenziale per essere distruttivo per i tessuti se eccessivo o tenuto in uno stato irrisolto.  I modelli in vitro coculati possono essere utilizzati per studiare i meccanismi molecolari unici coinvolti nella migrazione transeteliale neutrofila indotta da agenti patogeni. Questo tipo di modello offre versatilità nella progettazione sperimentale con l'opportunità di manipolazione controllata dell'agente patogeno, della barriera epiteliale o del neutrofilo. L'infezione patogena della superficie apicale dei monostrati epiteliali polarizzati coltivati su filtri transwell permeabili istiga la migrazione basolaterale ad apicale transeteliale dei neutrofili applicata alla superficie basolaterale. Il modello in vitro descritto nel presente documento dimostra i molteplici passaggi necessari per dimostrare la migrazione neutrofila attraverso un monostrato epiteliale polmonare polarizzato che è stato infettato da P. aeruginosa patogena (PAO1). Viene descritta la semina e la 155azione di transmeabili con cellule epiteliali polmonari derivate dall'uomo, insieme all'isolamento dei neutrofili da tutto il sangue umano e alla ristrutturazione di PAO1 e K12 E. coli nonpathogenico (MC1000).  Il processo di emigrazione e l'analisi quantitativa dei neutrofili migrati con successo che sono stati mobilitati in risposta all'infezione patogena sono mostrati con dati rappresentativi, compresi i controlli positivi e negativi. Questo sistema di modelli in vitro può essere manipolato e applicato ad altre superfici mucose. Le risposte infiammatorie che comportano un'eccessiva infiltrazione di neutrofili possono essere distruttive per ospitare i tessuti e possono verificarsi in assenza di infezioni patogene. Una migliore comprensione dei meccanismi molecolari che promuovono la migrazione transeteliale neutrofila attraverso la manipolazione sperimentale del sistema di saggi di cocultura in vitro descritto nel presente documento ha un potenziale significativo per identificare nuovi obiettivi terapeutici per una serie di malattie infettive e infiammatorie mucose.

Diversi studi hanno dimostrato che gli strati epiteliali infetti da agenti patogeni facilitano la migrazione transetelialeneutrofila 3,8,19,24-28,31,32. Ciò avviene attraverso un'induzione specifica patogena di un gradiente chemiotattico neutrofilo derivato da cellule epiteliali3,23. Ad esempio, la P. aeruginosa patogena che interagisce con la superficie apicale delle cellule epiteliali polmonari fa sì che un numero sostanziale di neutrofili migri attraverso lo strato epiteliale18,22...

Watch this Scientific Journal Video about In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration at JoVE.com

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

La migrazione transeteliale neutrofila in risposta all'infezione batterica della mucosa contribuisce alla lesione epiteliale e alla malattia clinica. È stato sviluppato un modello in vitro che combina agenti patogeni, neutrofili umani e strati cellulari epiteliali umani polarizzati coltivati su filtri transwell per facilitare le indagini verso lo svelamento dei meccanismi molecolari che orchestrano questo fenomeno.

In vitro Test di cocultura per valutare la migrazione transeteliale neutrofila indotta da agenti patogeni

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

in-vitro-coculture-assay-to-assess-pathogen-induced-neutrophil-trans

Research

JoVE Journal

Immunology and Infection

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

17.4K Views.  Harvard Medical School. La migrazione transeteliale neutrofila in risposta all'infezione batterica della mucosa contribuisce alla lesione epiteliale e alla malattia clinica. È stato sviluppato un modello in vitro che combina agenti patogeni, neutrofili umani e strati cellulari epiteliali umani polarizzati coltivati su filtri transwell per facilitare le indagini verso lo svelamento dei meccanismi molecolari che orchestrano questo fenomeno.

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

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

Test per patogeni associati modello molecolare (PAMP)-trigger Immunità (PTI) nelle piante

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

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Immunologia e infezioni

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Monitoraggio Crawling neutrofili intraluminale, migrazione e chemiotassi transendoteliale in tessuto al microscopio video intravitale

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

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

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

La valutazione quantitativa della migrazione dei neutrofili Umana Attraverso una coltivate vescica Epitelio

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

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.

Umano camera di flusso neutrofili saggio

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Un romanzo In Vitro ferita guarigione test per valutare la migrazione cellulare

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Un'analisi di High-throughput per valutare e quantificare la trappola extracellulari del neutrofilo formazione

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...