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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Published: September 20th, 2018

DOI:

10.3791/58210

1Department of Medicine 1, University of Erlangen-Nuremberg

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-α4β7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn's disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.

The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.

Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Cell motion is a tightly regulated process indispensable for the development and function of multi-cellular organisms, but is also implicated in the pathogenesis of a multitude of diseases1. Recently, the homing process of immune cells from the blood stream to the peripheral tissues has gained increasing attention, since it contributes to replenishment and expansion of pathogenic cells in inflamed tissues in immunologically mediated diseases2,3. In particular, homing has been shown to have translational relevance in inflammatory bowel diseases (IBD). The therapeutic anti-α4β7 ....

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The studies described in the following sections have been performed according to approval of the Ethics committee of the Friedrich-Alexander University Erlangen-Nuremberg.

1. Preparation of Capillaries

  1. Connect rectangular capillaries to the rubber tubing by stretching the tubing on one side with small scissors and carefully inserting the capillary (approximately 0.5 cm) into the tube. Seal the connection between capillary and tube with plastic paraffin film.
  2. Coat the capi.......

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The method presented in this manuscript aims to simulate the in vivo process of human cell adhesion to the endothelial wall as closely as possible to functionally assess cell adhesion and the role of interfering antibodies. Therefore, ultrathin capillaries are coated with addressins and perfused with fluorescently labeled human cells of interest using a perfusion pump. Using live cell imaging the adhesion of human cells to the addressins can be observed in real time (

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The above protocol describes a useful technique to study dynamic adhesion of human immune cells to endothelial ligands. Through variation of the coated ligands, the perfused cell types or subsets, incubation with additional stimuli or different neutralizing antibodies, it has almost unlimited potential applications. Therefore, such dynamic adhesion assays may be useful to answer both fundamental questions of basic research as well as translational queries that might help to develop and optimize clinical therapy with drug.......

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The research of CN, IA, MFN and SZ was supported by the Interdisciplinary Center for Clinical Research (IZKF) and the ELAN program of the University Erlangen-Nuremberg, the Else Kröner-Fresenius-Stiftung, the Fritz-Bender-Stiftung, the German Crohn's and Colitis Foundation (DCCV), the Clinical Research Group CEDER of the German Research Council (DFG), the DFG topic program on Microbiota, the Emerging Field Initiative and the DFG Collaborative Research Centers 643, 796 and 1181.

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Name Company Catalog Number Comments
48-Well plate Sarstedt 833,923
Adhesion buffer: 150mM NaCl + 1mM HEPES + 1mM MgCl2 + 1mM CaCl2
Blocking solution: 1x PBS in ddH2O + 5 % BSA
Bovine Serum albumin (BSA) Applichem A1391,0100
CaCl2 Merck 2382
Capillaries: Rectangle Boro Tubing 0,20x2.00 mm ID, 50 mm length CM Scientific 3520-050
CCL-2, human Immunotools 11343384
CD4-Microbeads, human Miltenyi Biotec 130-045-101
CellTrace™ CFSE Cell Proliferation Kit ThermoFischer Scientific C34554
Centrifuge (Rotixa 50 RS) Hettrich
Coating buffer: 150 mM NaCl + 1 mM HEPES
Confocal Microscope (TCS SP8) Leica
CXCL-10, human Immunotools 11343884
Dextran 500 Roth 9219.3
EDTA KE/9 ml Monovette Sarstedt
Falcons (50 mL) Sarstedt 62,547,004
Fc chimera isotype control R&D Systems 110-HG
Flow Rates Peristaltic Pump (LabV1) Baoding Shenchen Precision Pump Company
HEPES VWR J848-100ML
Human IgG Isotype Control ThermoFischer Scientific 31154
Intercellular Adhesion Molecule 1 (ICAM-1) Fc chimera R&D Systems 720-IC-050
LS-Columns Miltenyi Biotec 130-042-401
MgCl2 Roth
MnCl2 Roth
mouse IgG isotype control Miltenyi Biotec 130-106-545
Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MAdCAM-1) Fc chimera R&D Systems 6056-MC
NaCl Roth 3957.3
Natalizumab Biogen
Neubauer Counting chamber Roth T729.1
Pancoll, human PAN Biotech P04-601000
Phosphate Buffered Saline (PBS)  Biochrom L 182-10 w/o Mg and Ca
Plastic paraffin film: Parafilm (PM-996) VWR 52858-000
purified anti-human CD18 Biolegend 302102
RPMI Medium 1640 Gibco Life Technologies 61870-010
Rubber tubing: SC0059T 3-Stop LMT-55 Tubing, 1.02mm ID, 406.4 mm length Ismatec SC0059
Serological Pipetts Sarstedt 861,254,025
Trypan blue Roth CN76.1
Vascular Cell Adhesion Molecule 1 (VCAM-1) Fc chimera Biolegend 553706
Vedolizumab (Entyvio) Takeda

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