neutrophils screening assay using flow cytometry

ヒト好中球におけるβ2インテグリン活性化阻害剤のスクリーニング

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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.

著者スポットライト:β2インテグリン活性化の低分子アンタゴニストを同定する方法の開発 

インテグリン阻害薬スクリーニングのためのフローサイトメトリーベースのハイスループット技術

Watch this Scientific Journal Video about Neutrophils Screening Assay Using Flow Cytometry at JoVE.com

Neutrophils Screening Assay Using Flow Cytometry  

フローサイトメトリーを用いた好中球スクリーニングアッセイ

フローサイトメーターの電源を入れた後、化合物処理した好中球懸濁液を含む384ウェルプレートをサンプルホルダーにロードします。ソフトウェアを開き、新しい実験を開始します。384 Wellを選択し、目的のフローラ蛍光体を選択します。次に、プレート設定をクリックし、ドラッグしてすべてのウェルを選択し、サンプルをクリックします。高スループット スイッチをオンにし、レート パネルで [高] を選択します。volumeボックスに50と入力し、奇数列のサンプルを選択して、攪拌スイッチをアクティブにします。最後に、右側のパネルでサンプルに名前を付けます。試験した化合物の平均蛍光密度値はカットオフラインを上回っており、どの化合物もベータ2インテグリン拮抗薬として同定されなかったことを示しています。

neutrophils-screening-assay-using-flow-cytometry

Research

JoVE Journal

Immunology and Infection

Neutrophils Screening Assay Using Flow Cytometry

284 ビュー. フローサイトメーターの電源を入れた後、化合物処理した好中球懸濁液を含む384ウェルプレートをサンプルホルダーにロードします。ソフトウェアを開き、新しい実験を開始します。384 Wellを選択し、目的のフローラ蛍光体を選択します。次に、プレート設定をクリックし、ドラッグしてすべてのウェルを選択し、サンプルをクリックします。高スループット スイ?...

ビデオ: Neutrophils Screening Assay Using Flow Cytometry  

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.