动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用
Summary
免疫细胞对血管壁的动态黏附是肠道归巢的前提。在这里, 我们提出了一个功能性的体外检测的协议, 以影响分析抗整合素抗体, 趋化因子或其他因素的细胞动态细胞黏附的人体细胞使用 addressin 涂层毛细血管。
Abstract
免疫细胞的肠道归巢对炎症性肠病 (IBD) 发病有重要意义。整合素依赖细胞黏附 addressins 是这一过程中的关键步骤, 并成功地建立了干扰粘连的治疗策略。anti-α4β7整合素抗体, vedolizumab, 用于临床治疗克罗恩病 (CD) 和溃疡性结肠炎 (UC) 和进一步的化合物可能会跟随。
由于该领域功能研究的有限可用技术, 抗整合素抗体的黏附程序和作用机制的细节在许多方面仍然不清楚。
在此, 我们提出了动态粘附试验的功能分析的人细胞黏附在流动条件下的影响, 抗整合素疗法的背景下, IBD。它的基础上, 通过 addressin 涂层超薄玻璃毛细血管灌注的原发性人类细胞和实时显微分析。该检测提供了各种改进和修改的机会, 并持有机械发现和转化应用的潜力。
Introduction
细胞运动是对多细胞生物体的发展和功能必不可少的严格调控的过程, 但也牵涉到多种疾病的发病机制1。近年来, 从血流到外周组织的免疫细胞的归巢过程越来越受到重视, 因为它有助于免疫介导疾病炎症组织中致病细胞的补充和扩张2 ,3。特别是, 在炎症性肠病 (IBD) 中, 归巢已被证明具有平移相关性。治疗 anti-α4β7整合素抗体 vedolizumab 干预肠道归巢已显示疗效的大型临床试验4,5 , 已成功地用于实际临床实践6,7,8. 进一步的化合物可能会跟随9,10。同样, 治疗 anti-α4整合素抗体, natalizumab, 用于治疗多发性硬化症 (MS)11。
然而, 我们对整个归巢过程的功能理解, 特别是这种治疗抗体的作用机制仍然是有限的。这是很好的确定, 归巢包括几个步骤, 包括细胞的结合和滚动的后续细胞黏附导致坚定的逮捕后, 反式内皮移植12,13。上述抗体中和整合在细胞表面防止与 addressins 的相互作用的血管壁内皮。这被认为阻碍牢固的细胞黏附力14,15。然而, 我们只是开始了解特定整合对细胞归一化的差异相关性。此外, 抗整合素抗体对不同细胞亚群和剂量反应关联的影响在很大程度上是未知的, 导致肠内归巢和抗粘连治疗在 IBD 领域的许多开放性问题。
因此, 迫切需要解决这些问题的方便工具。抗整合素抗体对整合蛋白-addressin 相互作用的影响迄今主要通过评估结合效能/束缚抑制与流式细胞术或通过静态黏附力测定16,17 ,18,19,20, 因而以明显的简单化和偏离从生理情况。我们最近建立了动态粘附试验研究整合素依赖性的人体细胞对 addressins 和抗整合酶抗体在剪切应力下的作用2。该技术的原理已被证明与小鼠细胞21,22。在这里, 它的适应和发展, 以解决上述的翻译问题, 开辟了新的途径, 以更好地了解治疗机制的抗整合素抗体在体内。
Protocol
以下各节所述的研究是根据埃尔兰根-亚历山大大学的道德委员会的批准进行的。 1. 毛细血管的制备 将矩形毛细血管连接到橡胶管材上, 用小剪刀将油管一侧拉伸, 并小心地将毛细管 (大约0.5 厘米) 插入管内。用塑料石蜡膜密封毛细管与管的连接。 毛细管与20µL 的 addressin (重组 Fc 嵌合体; 5 µg/毫升 addressin,如MAdCAM-1, 在涂层缓冲器 (150 毫米氯化钠 + 1 毫米 HEP…
Representative Results
本论文提出的方法旨在模拟人体细胞粘附到内皮壁的体内过程, 尽可能地对细胞黏附力和干扰抗体的作用进行功能性评估。因此, 超薄毛细血管涂上 addressins, 用灌注泵将荧光标记的人类细胞灌注。使用活细胞成像可以实时观察人体细胞与 addressins 的黏附力 (图 1)。 该方法特别适用于炎症性肠病的抗?…
Discussion
上述协议描述了一种有效的方法来研究人体免疫细胞对内皮配体的动态黏附力。通过涂层配体的变化, 灌注细胞类型或亚群, 孵化与附加刺激或不同中和抗体, 它有几乎无限的潜在应用。因此, 这种动态黏附力的检测可能有助于回答基本研究的基本问题, 以及翻译查询, 可以帮助开发和优化临床治疗与药物干扰粘连的过程。
这项试验的效用, 以调查临床抗粘连治疗的效果最近已经…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
CN、IA、最惠国和深圳的研究是由临床研究交叉学科中心 (IZKF) 和大学埃尔兰根-纽伦堡, 其他 Kröner 费森尤斯基金会, 弗里茨弯机基金会, 德国克罗恩和结肠炎基金会 (DCCV), 德国研究理事会的临床研究小组 CEDER (DFG), DFG 专题计划在微生物组, 新兴的领域主动性和 DFG 合作研究中心 643, 796 和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 |
Referenzen
- von Andrian, U. H., Mackay, C. R. T-Cell Function and Migration – Two Sides of the Same Coin. New England Journal of Medicine. 343 (14), 1020-1034 (2000).
- Zundler, S., et al. The α4β1 Homing Pathway Is Essential for Ileal Homing of Crohn’s Disease Effector T Cells In Vivo. Inflammatory Bowel Diseases. 23 (3), 379-391 (2017).
- Binder, M. -. T., et al. Similar inhibition of dynamic adhesion of lymphocytes from IBD patients to MAdCAM-1 by vedolizumab and etrolizumab-s. Inflammatory Bowel Diseases. , (2018).
- Feagan, B. G., et al. Vedolizumab as induction and maintenance therapy for ulcerative colitis. The New England Journal of Medicine. 369 (8), 699-710 (2013).
- Sandborn, W. J., et al. Vedolizumab as induction and maintenance therapy for Crohn’s disease. The New England Journal of Medicine. 369 (8), 711-721 (2013).
- Baumgart, D. C., Bokemeyer, B., Drabik, A., Stallmach, A., Schreiber, S. Vedolizumab Germany Consortium Vedolizumab induction therapy for inflammatory bowel disease in clinical practice–a nationwide consecutive German cohort study. Alimentary Pharmacology & Therapeutics. 43 (10), 1090-1102 (2016).
- Kopylov, U., et al. Efficacy and Safety of Vedolizumab for Induction of Remission in Inflammatory Bowel Disease-the Israeli Real-World Experience. Inflammatory Bowel Diseases. 23 (3), 404-408 (2017).
- Amiot, A., et al. Effectiveness and Safety of Vedolizumab Induction Therapy for Patients With Inflammatory Bowel Disease. Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association. 14 (11), 1593-1601 (2016).
- Vermeire, S., et al. Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial. Lancet. 384 (9940), 309-318 (2014).
- Vermeire, S., et al. Anti-MAdCAM antibody (PF-00547659) for ulcerative colitis (TURANDOT): a phase 2 randomised, double-blind, placebo-controlled trial. Lancet. 390 (10090), 135-144 (2017).
- Miller, D. H., et al. A Controlled Trial of Natalizumab for Relapsing Multiple Sclerosis. New England Journal of Medicine. 348 (1), 15-23 (2003).
- Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Reviews. Immunology. 7 (9), 678-689 (2007).
- Ley, K., Rivera-Nieves, J., Sandborn, W. J., Shattil, S. Integrin-based therapeutics: biological basis, clinical use and new drugs. Nature Reviews. Drug Discovery. 15 (3), 173-183 (2016).
- Wyant, T., Yang, L., Fedyk, E. In vitro assessment of the effects of vedolizumab binding on peripheral blood lymphocytes. mAbs. 5 (6), 842-850 (2013).
- Fischer, A., et al. Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo. Gut. 65 (10), 1642-1664 (2016).
- Wyant, T., Estevam, J., Yang, L., Rosario, M. Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab. Cytometry. Part B, Clinical Cytometry. 90 (2), 168-176 (2016).
- Tidswell, M., et al. Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1. Journal of Immunology. 159 (3), 1497-1505 (1997).
- Soler, D., Chapman, T., Yang, L. -. L., Wyant, T., Egan, R., Fedyk, E. R. The Binding Specificity and Selective Antagonism of Vedolizumab, an Anti-α4β7 Integrin Therapeutic Antibody in Development for Inflammatory Bowel Diseases. Journal of Pharmacology and Experimental Therapeutics. 330 (3), 864-875 (2009).
- Parikh, A., et al. Vedolizumab for the treatment of active ulcerative colitis: a randomized controlled phase 2 dose-ranging study. Inflammatory Bowel Diseases. 18 (8), 1470-1479 (2012).
- Wendt, E., White, G. E., Ferry, H., Huhn, M., Greaves, D. R., Keshav, S. Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells. The Journal of Immunology. 196 (9), 3910-3919 (2016).
- Nussbaum, C., et al. Sphingosine-1-phosphate receptor 3 promotes leukocyte rolling by mobilizing endothelial P-selectin. Nature Communications. 6, (2015).
- Pruenster, M., et al. Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion. Nature Communications. 6, (2015).
- Binder, M. -. T., et al. Similar Inhibition of Dynamic Adhesion of Lymphocytes From IBD Patients to MAdCAM-1 by Vedolizumab and Etrolizumab-s. Inflammatory Bowel Diseases. 24 (6), 1237-1250 (2018).
- Wang, L., et al. Vessel Sampling and Blood Flow Velocity Distribution With Vessel Diameter for Characterizing the Human Bulbar Conjunctival Microvasculature. Eye & Contact Lens. 42 (2), 135-140 (2016).
- House, S. D., Johnson, P. C. Diameter and blood flow of skeletal muscle venules during local flow regulation. The American Journal of Physiology. 250 (5 Pt 2), H828-H837 (1986).
- Zundler, S., Neurath, M. F. Pathogenic T cell subsets in allergic and chronic inflammatory bowel disorders. Immunological Reviews. 278 (1), 263-276 (2017).
- Zundler, S., Becker, E., Weidinger, C., Siegmund, B. Anti-Adhesion Therapies in Inflammatory Bowel Disease-Molecular and Clinical Aspects. Frontiers in Immunology. 8, (2017).
- Zhou, Y., Kucik, D. F., Szalai, A. J., Edberg, J. C. Human Neutrophil Flow Chamber Adhesion Assay. Journal of Visualized Experiments: JoVE. (89), (2014).
- Zundler, S., et al. Blockade of αEβ7 integrin suppresses accumulation of CD8(+) and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo. Gut. 66 (11), 1936-1948 (2017).
Diesen Artikel zitieren
Becker, E., Schramm, S., Binder, M., Allner, C., Wiendl, M., Neufert, C., Atreya, I., Neurath, M., Zundler, S. Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease. J. Vis. Exp. (139), e58210, doi:10.3791/58210 (2018).