Immunology and Infection
Published: September 20th, 2018
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 ....
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
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 (
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.......
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.....
|Adhesion buffer: 150mM NaCl + 1mM HEPES + 1mM MgCl2 + 1mM CaCl2
|Blocking solution: 1x PBS in ddH2O + 5 % BSA
|Bovine Serum albumin (BSA)
|Capillaries: Rectangle Boro Tubing 0,20x2.00 mm ID, 50 mm length
|CellTrace™ CFSE Cell Proliferation Kit
|Centrifuge (Rotixa 50 RS)
|Coating buffer: 150 mM NaCl + 1 mM HEPES
|Confocal Microscope (TCS SP8)
|EDTA KE/9 ml Monovette
|Falcons (50 mL)
|Fc chimera isotype control
|Flow Rates Peristaltic Pump (LabV1)
|Baoding Shenchen Precision Pump Company
|Human IgG Isotype Control
|Intercellular Adhesion Molecule 1 (ICAM-1) Fc chimera
|mouse IgG isotype control
|Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MAdCAM-1) Fc chimera
|Neubauer Counting chamber
|Phosphate Buffered Saline (PBS)
|w/o Mg and Ca
|Plastic paraffin film: Parafilm (PM-996)
|purified anti-human CD18
|RPMI Medium 1640
|Gibco Life Technologies
|Rubber tubing: SC0059T 3-Stop LMT-55 Tubing, 1.02mm ID, 406.4 mm length
|Vascular Cell Adhesion Molecule 1 (VCAM-1) Fc chimera
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