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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Introduction

Lymphocyte migration from the blood into tissues is crucial for immune surveillance. A sequence of specific molecular interactions ensures site specific extravasation into small intestine, skin, lymph nodes, the central nervous system (CNS), and other tissues1. Alterations in lymphocyte migration are involved in the pathophysiology of a number of wide spread diseases2. Migration into the immune-privileged CNS is tightly regulated and accordingly alterations of this process are involved in CNS-related diseases like encephalomyelitis3, neuromyelitis optica, stroke, and multiple sclerosis (MS)2,4,5,6,7. Therefore, it is important to study lymphocyte extravasation to better understand disease pathophysiology and to develop tools for amelioration of disease burden8,9,10,11,12.

Lymphocytes migrate into the CNS via distinct routes. Extravasation through postcapillary venules into the subarachnoid space via the blood-cerebrospinal fluid barrier within the choroid plexus and across the blood-brain barrier have been described1,13,14,15. Migration across the blood-brain barrier is conducted by the interaction of lymphocytes with endothelial cells14. In contrast to endothelial cells in the periphery, endothelial cells of the CNS express high amounts of tight junction molecules, thereby strictly limiting the amount of cells and proteins capable of crossing the blood-brain barrier16. Inflammation results in loosening of tight junctions and induces the expression of adhesion molecules; thus, enhancing lymphocyte migration into the CNS1,17,18.

Extravasation via the blood-brain barrier is a multistep process. Lymphocytes tether to the endothelial cells and then roll along the endothelium in a process mainly mediated by selectins1,15. Subsequently, interactions between chemokines secreted by the endothelium and the respective chemokine receptors expressed on lymphocytes induce conformational changes of integrins, thereby promoting firm adhesion to the endothelial cells1. Finally, lymphocytes either crawl along the endothelial barrier against the blood flow before transmigrating into the perivascular space, or stall immediately and directly transmigrate at the site of firm adhesion1,19,20. All these steps of lymphocyte extravasation can be analyzed in vitro using distinct techniques21. Time-lapse video microscopy is used to study the initial tethering and rolling15. Adhesion assays provide detailed information about firm arrest to endothelial barriers22. Transmigration assays as demonstrated here allow analysis of immune-cell transmigration21,23,24,25,26,27,28,29.

Using the human in vitro blood brain barrier model, we could recently show that a higher migratory capacity of CD56brightCD16dim/- NK cells compared to their CD56dimCD16+ counterparts was reflected by a predominance of this NK cell subset in the intrathecal compartment21. Thus, our experimental setup seems to be suitable to mimic the in vivo situation.

Protocol

1. Cell Culture of Human Brain Microvascular Endothelial Cells (HBMEC)

  1. Coating of cell culture flasks
    1. To prepare the fibronectin solution, add 10 mL PBS to a 15 mL centrifuge tube. Add 150 µL fibronectin and mix well.
    2. To cover the bottom a T-25 cell culture flask add 2 mL of the fibronectin solution. Incubate the cell culture flask for at least 3 h at 37 °C in the incubator. Fibronectin coated flasks can be stored for 2 weeks at 37 °C / 5% CO2.
  2. Seeding and cell culture of HBMEC
    1. Aspirate fibronectin solution from the bottom of the cell culture flask. Add 7.2 x 104 HBMEC/cm² suspended in 6 mL ECM-b medium (= ECM-b supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement). Incubate at 37 °C / 5% CO2. Check cell growth daily using a microscope.
    2. Change the medium every 3 days. Harvest or split cells, when HBMEC reach approximately 80% confluence. HBMEC should be used between passage 1 and 15 to avoid loss of physiological properties.
  3. Harvest HBMEC.
    1. Prepare accutase solution by mixing accutase (1x) with PBS at a ratio of 1:1. Keep accutase solution at 37 °C in a water bath until further use.
    2. Transfer ECM-b medium from the cell culture flask to a 15 mL centrifuge tube. Wash HBMEC by adding 5 mL PBS to the bottom of the cell culture flask. Aspirate PBS and repeat the washing step two more times.
    3. Add 2 mL pre-warmed accutase solution. Incubate at 37 °C for 2 min. Afterwards, HBMEC are re-suspended by firmly tapping the cell culture flask several times. Cell detachment is controlled using a microscope
    4. The ECM-b-medium previously stored in a 15 mL tube is added back to the cell culture flask as soon as HBMEC start to detach. Rinse the bottom of the flask repeatedly until most HBMEC are re-suspended.
    5. Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 300 x g for 10 min at room temperature. Discard supernatant and re-suspend cells in 1 mL ECM-b medium. Count cells and dilute the cell suspension to achieve a final concentration of 3 x 105 HBMEC per mL ECM-b medium.

2. Preparation of the Cell Culture Inserts

  1. Coating of cell culture inserts
    Important note: Avoid touching the membrane of the cell culture inserts.
    1. Add 100 µL fibronectin solution (see 1.1.1) to each cell culture insert (Figure 1A) and one well of a 96-well flat bottom plate (optical control well). Incubate for at least 3 h at 37 °C. After incubation aspirate fibronectin solution.
    2. Add 100 µL HBMEC suspension to the cell culture inserts and the optical control well. Add 600 µl ECM-b medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 °C / 5% CO2 until barrier integrity (Figure 1B) is reached, check cell growth by microscopic evaluation of the HBMEC in the optical control well. Note: Cell growth beyond four days is not recommended.
    3. Optional: To mimic inflammatory conditions aspirate the medium from the lower compartment and replace it with ECM-b medium supplemented with 500 U/mL IFN-γ/TNF-α 24 h prior the migration assay.

3. Quality Control with Evans Blue on the Day of the Transmigration Assay

  1. Preparation of evans blue solution
    1. To prepare PBS/B27 solution mix 10 mL PBS with 200 µl B27 supplement using a 15 mL centrifuge tube. Dilute evans blue stock solution (20 mg/mL PBS) 1:1,000 with PBS/B27.
  2. Evans blue permeability assay
    1. Aspirate the medium from the lower compartment followed by the upper compartment of one cell culture insert containing a confluent HBMEC monolayer. Add 100 µL evans blue solution to the cell culture insert.
    2. Add 600 µL PBS/B27 to the lower compartment and incubate for 60 min at 37 °C / 5% CO2. Carefully remove the cell culture insert using forceps.
  3. Evans blue measurement
    1. Remove PBS/B27 from the lower compartment and transfer 100 µL each to two wells of a black polystyrol 96-well flat bottom plate. Insert plate in a Tecan Infinite M200 Pro plate reader and determine optimal z-position.
    2. Measure excitation of evans blue using respective settings (for example: excitation: 620 nm, emission: 680 nm, excitation bandwidth: 9 nm, emission bandwidth: 20 nm, 175x enhancement, 25 flashes, time of integration: 20 µs).
    3. To determine HBMEC barrier functions compare acquired data to a standard curve depicting evans blue permeation across HBMEC at different time points after seeding cells (Figure 1B, right).

4. Migration Assay

  1. Preparation of peripheral blood mononuclear cells (PBMC).
    1. Add 10 mL RPMI into a 15 mL centrifuge tube and add 200 µL B27 supplement. Count PBMC and centrifuge cells at 300 x g for 5 min. Re-suspend PBMC to a final concentration of 5 x 106 cells/mL RPMI/B27.
  2. Set-up of the migration assay
    1. Aspirate medium from the lower compartment followed by the upper compartment of cell culture inserts containing confluent HBMEC monolayers (Figure 1A). Per donor add 100 µL PBMC suspension each to the cell culture inserts and also to one well of a 24-well plate per (in vitro control).
    2. Add 600 µL RPMI/B27 to the lower compartment of the cell culture inserts and 500 µL to the PBMC of the in vitro control and incubate 6 h at 37 °C / 5% CO2.
  3. Harvesting of migrated PBMC
    1. Take out the cell culture insert using forceps and carefully rinse the bottom with 400 µL PBS without touching the membrane. Discard the cell culture insert.
    2. Add 20 µL flow count fluorospheres (approximately 1,000 beads/µL) to the lower compartment of the cell culture insert as well as to the in vitro control and mix well. Transfer 1 mL of resulting PBMC suspension to flow cytometry tubes.

5. Flow Cytometry

  1. Sample preparation
    1. Centrifuge PBMC at 300 x g for 5 min at room temperature.
    2. Prepare antibody solution by adding fluorochrome-conjugated antibodies to 100 µL flow cytometry buffer (PBS/1% BSA/2 mM EDTA) per sample. For the results presented below 1 µL CD4-FITC, 1 µLCD3-PerCP/Cy5.5, 1 µL CD56-PC7, 1 µL CD8-A700, and 1 µL CD16-A750 were used per sample.
    3. Re-suspend PBMC in 100 µL of the antibody solution and incubate for 30 min at 4 °C.
    4. Add 250 µL flow cytometry buffer and centrifuge at 300 x g for 5 min.
  2. Sample acquisition
    1. Re-suspend PBMC in the required amount (varies depending on the flow cytometer used) of flow cytometry buffer.
    2. Acquire stained PBMC using a flow cytometer with an active detector between 525 and 700 nm wavelength to detect flow count fluorospheres (excitation 488 nm, emission 525 - 700 nm).
      (The following steps are an example if a Gallios flow cytometer operated with Kaluza G software is used: (1) Start the computer. (2) When the operating system is fully loaded, start the flow cytometer by pressing the "cytometer on" button. (3) Load the respective acquisition protocol by pressing "open protocol" button. (4) Choose the required protocol and select "open". (5) Duplicate the protocol for every sample by clicking with the right mouse button on the protocol visible in the virtual carousel and a left click on the field "duplicate". (6) Label each sample in the sample list. (7) Transfer the samples to the indicated positions of the carousel and start the acquisition.)
  3. Sample analysis
    1. Open resulting flow cytometry data using the respective software. Determine number of subpopulations of interest for transmigrated PBMC as well as cells from in vitro control wells and flow count fluorospheres using the respective analysis software.
      (An example of the gating strategy is given in the results part (Figure 1 C: To analyze the transmigration of NK-cell subsets, first select lymphocytes in a sideward scatter channel (SSC) versus forward scatter channel (FSC) plot. Lymphocytes are then displayed in a CD3 versus CD56 plot and CD56+CD3- NK cells are selected. To distinguish between NK-cell subsets, NK cells are displayed in a CD56 versus CD16 plot and CD56brightCD16dim/- as well as CD56dimCD16+ NK cells are selected. In addition, flow count fluorospheres are selected from a FSC versus SSC plot and subsequently displayed in a plot of a channel with an emission between 525 and 700 nm versus time to determine their number.)
    2. To calculate the total cell number of each sample, normalize the detected number of cells using flow count fluorospheres:
      figure-protocol-9489
    3. Determine percentage of migrated cells as ratio between total migrated cells and total cells in the in vitro control.

Results

Representative results showing transmigration of NK-cell and T-cell subsets using the human blood-brain barrier model (Figure 1A) are shown. The integrity of the HBMEC monolayer was validated by staining of the tight junction molecule ZO-1, transendothelial electrical resistance (TEER) measurements, and evans blue permeation (Figure 1B). Following 3 - 4 days culture HBMEC expressed the tight junction molecule ZO-1 (Figure 1B, left). Furth...

Discussion

Here we present a technique to investigate the transmigration of lymphocytes across the human blood-brain barrier. In vitro analysis of lymphocyte migration to the CNS is important to study basic processes of lymphocyte extravasation, potential disease-related alterations, and new therapeutic approaches.

Several modifications of the blood-brain barrier model are possible. For example, cells from the upper compartment could be analyzed to investigate the composition of the non-migrated...

Disclosures

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

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

NameCompanyCatalog NumberComments
PBSGibco14190-094without CaCl2 or MgCl2
Fibronectin 1 mg/mLSigmaF1141-5MGfrom bovine plasma
T-25 cell culture flaskGreiner BioOne690160
HBMECScienCell1000
PelobiotechPB-H-6023
AccutaseSigmaA6964-100ML
ECM-bScienCell1001-b
FBSScienCell1001-b
Penicillin/StreptomycinScienCell1001-b
Endothelial cell growth supplementScienCell1001-b
TranswellCorning3472clear, 6.5 mm diameter, 3.0 µm pore size
96-well flat bottom plateCorning3596
Evans blueSigmaE2129-10Gstock solution: 1 g/50 mL PBS
B27Gibco17504-04450x concentrated
Infinite M200ProTecan
96-well black flat bottom plateGreiner BioOne675086
48-well plateCorning3526
RPMI 1640Gibco61870-010
Flow Count FluorospheresBeckman Coulter7547053
Na-EDTASigmaE5134
BSASigmaA2153
Gallios 10-color flow cytometerBeckman Coulter
Kaluza 1.5aBeckman Coulter
TNF-αPeprotech300-01A
IFN-γPeprotech300-02
CD3-PerCP/Cy5.5Biolegend300430clone UCHT1
CD56-PC7Beckman CoulterA21692clone N901
CD16-A750Beckman CoulterA66330clone 3G8
CD4-FITCBiolegend300506clone RPA-T4
CD8-A700Beckman CoulterA66332clone B9.11

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Lymphocyte ExtravasationIn Vitro ModelBlood brain BarrierInflammatory DiseasesCentral Nervous SystemTransmigration AssayEvans Blue PermeationHBMECFibronectinAccutaseCell Culture Inserts

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