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Introduction
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Immunology and Infection

Analyse af lymfocyt Ekstravasation Ved anvendelse af en<em> In vitro</em> Model af humant blod-hjernebarrieren

Published: April 05, 2017
doi:
10.3791/55390
, , , , ,
1Department of Neurology with Institute of Translational Neurology,University Hospital Münster

Summary

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Abstract

Lymfocyt ekstravasation i centralnervesystemet (CNS) er afgørende for immun overvågning. Sygdomsrelaterede ændringer af lymfocyt ekstravasation kan resultere i patofysiologiske forandringer i CNS. Således undersøgelse af lymfocytmigration i CNS er vigtigt at forstå inflammatoriske CNS-sygdomme og udvikle nye terapi tilgange. Vi præsenterer her en in vitro model af den menneskelige blod-hjerne-barrieren for at studere lymfocyt ekstravasation. Human hjerne mikrovaskulære endotelceller (HBMEC) er confluently dyrkes på et porøst polyethylenterephthalat Transwell indsætte at efterligne endotel af blod-hjerne-barrieren. Barrierefunktion er valideret af zonula occludens immunhistokemi, transendotele elektrisk modstand (TEER) målinger samt analyse af Evans blue permeation. Denne model giver mulighed for undersøgelse af diapedesis af sjældne lymfocytundergrupper såsom CD56 lyse CD16 dim / – NK-celler. Furthermmalm, virkningerne af andre celler, cytokiner og chemokiner, sygdomsrelaterede ændringer og distinkte behandlingsregimer på vandrende kapacitet af lymfocytter kan undersøges. Endelig kan analyseres virkningen af ​​inflammatoriske stimuli samt forskellige behandlingsregimer på endotelbarrieren.

Introduction

Lymfocytmigration fra blodet ind i væv er afgørende for immun overvågning. En sekvens af specifikke molekylære vekselvirkninger sikrer stedspecifik ekstravasation i tyndtarmen, hud, lymfeknuder, centralnervesystemet (CNS), og andre væv 1. Ændringer i lymfocytmigration er involveret i patofysiologien af en række bred spredning sygdomme 2. Migration i immun-priviligerede CNS reguleres stramt og følgelig ændringer af denne proces er involveret i CNS-relaterede sygdomme som encephalomyelitis 3, neuromyelitis optica, slagtilfælde og dissemineret sklerose (MS) 2, 4, 5, 6, 7. Derfor er det vigtigt at studere lymfocyt ekstravasation til bedre at forstå sygdommen patofysiologi og udvikle værktøjer til en melioration af sygdomstilfælde 8, 9, 10, 11, 12.

Lymfocytter migrerer i CNS via distinkte veje. Ekstravasation gennem postkapillære venuler i det subarachnoide rum via blod-cerebrospinalvæske barriere inden choroid plexus og over blod-hjerne-barrieren er blevet beskrevet 1, 13, 14, 15. Migration over blod-hjerne-barrieren udføres ved interaktionen af lymfocytter med endothelceller 14. I modsætning til endotelceller i periferien, endotelceller i CNS udtrykker høje mængder af tight junction-molekyler, hvorved strengt at begrænse mængden af ​​celler og proteiner i stand til at krydse blod-hjerne-barrierenlass = "xref"> 16. Inflammation resulterer i løsning af tight junctions og inducerer ekspressionen af ​​adhæsionsmolekyler; således, øge lymfocytmigrering i CNS 1, 17, 18.

Ekstravasation via blod-hjerne-barrieren er en flertrinsproces. Lymfocytter tøjr til endotelcellerne og derefter rulle langs endotel i en proces hovedsagelig medieret af selectiner 1, 15. Efterfølgende interaktioner mellem kemokiner udskilt af endothelet og de respektive chemokinreceptorer udtrykt på lymfocytter inducerer konformationelle ændringer af integriner og dermed fremme fast adhæsion til endotelcellerne 1. Endelig lymfocytter enten kravle langs endotel barriere mod blodstrømmen inden transmigrating ind i det perivaskulære rum eller stall umiddelbart og direkte transmigrate på stedet for fast adhæsion 1, 19, 20. Alle disse trin af lymfocyt ekstravasation kan analyseres in vitro ved anvendelse distinkte teknikker 21. Time-lapse video mikroskopi bruges til at studere den indledende tethering og rullende 15. Adhæsionsanalyser give detaljerede oplysninger om fast anholdelse at endotelbarrierer 22. Transmigration assays som påvist her tillader analyse af immun-celle transmigration 21, 23, 24, 25, 26, 27, 28, 29.

Anvendelse af det humane in vitro blodhjernebarrieren model, kunne vi for nylig viser, at en højere migrDELSE kapacitet CD56 lyse CD16 dim / – NK-celler i forhold til deres CD56 dim CD16 + modparter blev afspejlet ved en overvægt af dette NK-undergruppe i det intratekale rum 21. Således er vores forsøgsopstilling synes at være egnet til at efterligne in vivo situationen.

Protocol

1. Cell Culture of Human Brain mikrovaskulære endotelceller (HBMEC) Coating af cellekulturkolber Til fremstilling fibronectinopløsningen tilsættes 10 ml PBS til en 15 ml centrifugerør. Tilsæt 150 uL fibronektin og bland godt. At dække bunden en T-25 cellekulturkolbe tilsæt 2 ml fibronectinopløsningen. Inkubér cellekulturkolbe i mindst 3 timer ved 37 ° C i inkubatoren. Fibronectin coatede kolber kan opbevares i 2 uger ved 37 ° C / 5% CO2. Såning og …

Representative Results

Repræsentative resultater, der viser transmigration af NK-celle- og T-celle delmængder ved hjælp af blod-hjerne-barrieren model humane (figur 1A) er vist. Integritet HBMEC monolaget blev valideret ved farvning af tight junction-molekylet ZO-1, transendotele elektrisk modstand (TEER) målinger, og Evans blå permeation (figur 1B). Efter 3 – 4 dage kultur HBMEC udtrykte tight junction molekyle ZO-1 (figur 1B, venstre). Endvidere HBMEC v…

Discussion

Her præsenterer vi en teknik til at undersøge transmigration af lymfocytter på tværs af humant blod-hjerne-barrieren. In vitro analyse af lymfocyt migration til CNS er vigtigt at studere grundlæggende processer af lymfocyt ekstravasation, potentielle sygdomsrelaterede ændringer, og nye terapeutiske fremgangsmåder.

Adskillige modifikationer af blod-hjerne-barrieren model er mulige. For eksempel kunne celler fra det øvre rum analyseres for at undersøge sammensætningen af ​…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study has been supported by the Collaborative Research Centre CRC TR128 “Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease” (Project A9 to H.W. and C.C.G., project B1 to N.S.).

Materials

PBS Gibco 14190-094 without CaCl2 or MgCl2
Fibronectin 1mg/mL Sigma F1141-5MG from bovine plasma
T-25 cell culture flask Greiner BioOne 690160
HBMEC ScienCell 1000
Pelobiotech PB-H-6023
Accutase Sigma A6964-100ML
ECM-b ScienCell 1001-b
FBS ScienCell 1001-b
Penicillin/Streptomycin ScienCell 1001-b
Endothelial cell growth supplement ScienCell 1001-b
Transwell Corning 3472 clear, 6.5mm diameter, 3.0µm pore size
96-well flat bottom plate Corning 3596
Evans blue Sigma E2129-10G stock solution: 1 g/50 mL PBS
B27 Gibco 17504-044 50x concentrated
Infinite M200Pro Tecan
96-well black flat bottom plate Greiner BioOne 675086
48-well plate Corning 3526
RPMI 1640 Gibco 61870-010
Flow Count Fluorospheres Beckman Coulter 7547053
Na-EDTA Sigma E5134
BSA Sigma A2153
Gallios 10-color flow cytometer Beckman Coulter
Kaluza 1.5a Beckman Coulter
TNF-α Peprotech 300-01A
IFN-γ Peprotech 300-02
CD3-PerCP/Cy5.5 Biolegend 300430 clone UCHT1
CD56-PC7 Beckman Coulter A21692 clone N901
CD16-A750 Beckman Coulter A66330 clone 3G8
CD4-FITC Biolegend 300506 clone RPA-T4
CD8-A700 Beckman Coulter A66332 clone B9.11
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Tags

Lymphocyte ExtravasationIn Vitro ModelBlood-brain BarrierInflammatory DiseasesCentral Nervous SystemTransmigration AssayEvans Blue PermeationHBMECFibronectinAccutaseCell Culture Inserts
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Schulte-Mecklenbeck, A., Bhatia, U., Schneider-Hohendorf, T., Schwab, N., Wiendl, H., Gross, C. C. Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. (122), e55390, doi:10.3791/55390 (2017).
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