high-throughput screening of compounds using neutrophils isolated from human blood in a 384-well plate format

人中性粒细胞中 β2 整合素激活抑制剂的筛选

Many inflammatory diseases are characterized by the infiltration of neutrophils at the site of swelling or injury1. To infiltrate these tissues, neutrophils must complete the neutrophil recruitment cascade, which involves arrest to the endothelium, extravasation across the vessel wall, and recruitment into the tissue2. Circulating neutrophils need &#946;2 integrin activation to complete this cascade, especially for the arrest phase. Thus, integrin-inhibiting drugs that reduce neutrophil adhesion, extravasation, and recruitment may effectively treat inflammatory diseases3,4.
&#946;2 integrins have been targeted for inflammatory diseases before. Efalizumab, a monoclonal antibody directly targeting integrin &#945;L&#946;2, was developed to treat psoriasis5. However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7. New anti-inflammatory integrin-based therapies should consider maintaining the anti-infection functions of leukocytes to minimize side effects. The side effects of efalizumab might be due to the prolonged circulation of monoclonal antibodies in the bloodstream, which could inhibit immune functions in the long term8. A recent study shows that efalizumab mediates &#945;L&#946;2 crosslinking and the unwanted internalization of &#945;4 integrins, providing an alternative explanation for the side effects9. Thus, short-lived, small-molecule antagonists might avoid this problem.
A high-throughput method to screen small-molecule &#946;2 integrin antagonists using human neutrophils is presented here. &#946;2 integrin activation requires conformational changes of the integrin ectodomain to gain access to and increase its binding affinity to its ligand. In the canonical switchblade model, the bent-closed integrin ectodomain first extends to an extended-closed conformation and then opens its headpiece to a fully activated extended-open conformation10,11,12,13. There is also an alternative pathway that starts from the bent-closed to bent-open and extended-open, eventually14,15,16,17,18,19. The conformation-specific antibody mAb24 binds to an epitope in the human &#946;2-I-like domain when the headpiece of the ectodomain is open20,21,22,23.
Here, mAb24-APC is used to determine whether the &#946;2 integrins are activated. To activate neutrophils and integrin, N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial-derived short chemotactic peptide that can activate neutrophil &#946;2 integrins24, is used as a stimulus in this protocol. When fMLP binds to the Fpr1 on neutrophils, downstream signaling cascades involving G-proteins, phospholipase C&#946;, and phosphoinositide 3-kinase &#947; are activated. These signaling events ultimately result in integrin activation via the inside-out signaling pathway18,25. Besides small molecule antagonists that directly bind to &#946;2 integrins and prevent conformational changes of integrin activation26, compounds that can inhibit components in the &#946;2 integrin inside-out activation signaling pathway would also be detected with this method. Automated flow cytometers enable high-throughput screening. Identifying new antagonists may not only deepen our understanding of integrin physiology but also provide translational insight into integrin-based anti-inflammation therapy.

作者聚焦：鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

一种基于流式细胞术的高通量技术筛选整合素抑制药物

Our research is focused on neutrophil integrin, and in this study we aim to establish a method to find small molecular antagonists of Beta-2 integrin activation. We believe with this method we can find new antagonists that may deepen our understanding in integrin physiology, and also provide translational insight into integrin-based anti-inflammatory therapy. In the future, we will focus on discovering new anti-inflammatory drugs and dissecting their mechanisms of action.

Watch this Scientific Journal Video about High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format at JoVE.com

High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format  

使用从人血液中分离的中性粒细胞以 384 孔板形式对化合物进行高通量筛选

使用血清移液管，在 15 毫升离心管中小心地将 4 毫升肝素化的人血液涂在 8 毫升密度梯度培养基上。将血液在 550 摄氏度下离心 30 至 50 分钟。然后使用一毫升移液管去除含有血浆和单核细胞的上层。将中性粒细胞从下部混浊带和透明液体下方约三到四毫升收集到含有 10 毫升 PBS 的 15 毫升离心管中。轻轻地将试管倒置两到三次，以混合中性粒细胞悬浮液。将中性粒细胞悬浮液在400G下在20摄氏度下离心10分钟，然后小心地从管中倒出上清液，并将沉淀重悬于5毫升PBS中。再次，将悬浮液在 300 摄氏度下离心 10 分钟。除去上清液后，用真空抽吸消除管壁和管口周围残留的上清液。将沉淀重悬于一毫升中性粒细胞培养基中。将 1 毫升 1.2 微克/毫升抗体溶液与 0.2 毫升中性粒细胞培养基混合在 1.5 毫升管中。将 25 微升抗体溶液加入 384 孔板中的阴性对照孔中。在 15 毫升试管中，混合 9 毫升抗体溶液、21.6 微升 FMLP 溶液和 1.7784 毫升中性粒细胞培养基。将 25 微升该混合物加入阳性对照中，并在 384 孔板中测试孔。使用多通道移液器，将 5 微升化合物溶液从化合物库板转移到 384 孔板。将悬浮液以 500G 离心一分钟。使用16通道移液器向每个孔中加入20微升中性粒细胞悬浮液。将板在振荡器上以 300 RPM 孵育 10 分钟。然后为了固定细胞，向每个孔中加入三微升 16% 多聚甲醛，并在冰上孵育 10 分钟。

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Research

JoVE Journal

Immunology and Infection

High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format

194 次观看.  UConn Health. 使用血清移液管，在 15 毫升离心管中小心地将 4 毫升肝素化的人血液涂在 8 毫升密度梯度培养基上。将血液在 550 摄氏度下离心 30 至 50 分钟。然后使用一毫升移液管去除含有血浆和单核细胞的上层。将中性粒细胞从下部混浊带和透明液体下方约三到四毫升收集到含有 10 毫升 PBS 的 15 毫升离心管中。轻轻地将试管倒置两到三次，以混合中性粒细胞悬浮液。将中性粒细胞?...

视频: High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format  

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.