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

Summary

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.

Abstract

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.

Introduction

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 diseases^2,3. In particular, homing has been shown to have translational relevance in inflammatory bowel diseases (IBD). The therapeutic anti-α4β7 ....

Protocol

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 capillary with 20 µL of addressin (recombinant Fc chimera; 5 µg/mL addressin, e.g. MAdCAM-1, in coating buffer (150 mM NaCl + 1 mM HEPE....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
48-well plate	Sarstedt	833,923
Adhesion buffer: 150 mM NaCl + 1 mM HEPES + 1 mM MgCl2 + 1 mM CaCl2
Blocking solution: 1x PBS in ddH2O + 5 % BSA
Bovine Serum albumin (BSA)	Applichem	A1391,0100
CaCl2	Merck	2382
Capillaries: Rectangle Boro Tubing 0.20 mm x 2.00 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, 1.02 mm 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