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 diseases2,3. In particular, homing has been shown to have translational relevance in inflammatory bowel diseases (IBD). The therapeutic anti-α4β7 integrin antibody vedolizumab interfering with gut homing has shown efficacy in large clinical trials4,5 and has been successfully used in real-world clinical practice6,7,8. Further compounds are likely to follow9,10. Similarly, the therapeutic anti-α4 integrin antibody, natalizumab, is used for the treatment of multiple sclerosis (MS)11.
However, our functional understanding of the homing process in general and the mechanism of action of such therapeutic antibodies in particular is still limited. It is well established that homing consists of several steps including cell tethering and rolling with subsequent cell adhesion leading to firm arrest followed by trans endothelial migration12,13. The above-mentioned antibodies neutralize integrins on the cell surface preventing interaction with addressins on the endothelium of the vessel wall. This is thought to impede firm cell adhesion14,15. Yet, we are only beginning to understand the differential relevance of specific integrins for cell homing of distinct cell subsets. Moreover, the effects of anti-integrin antibodies on different cell subsets and dose-response associations are largely unknown leading to lots of open questions in the field of gut homing and anti-adhesion therapies in IBD.
Therefore, convenient tools to address such questions are desperately needed. The effect of anti-integrin antibodies on integrin-addressin interaction has so far predominantly been evaluated by assessing binding efficacy/binding inhibition with flow cytometry or through static adhesion assays16,17,18,19,20, thus with apparent simplification and deviation from the physiological situation. We recently established a dynamic adhesion assay to study integrin-dependent adhesion of human cells to addressins and the effects of anti-integrin antibodies under shear stress2. The principle of the technique has earlier been demonstrated with mouse cells21,22. Here, it was adapted and developed to address the above mentioned translational questions, opening novel avenues to better understand the mechanisms of therapy with anti-integrin antibodies in vivo.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 0,20×2.00 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, 1.02mm 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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