Name: High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format
Uploaded: 2024-02-02T12:00:00
Description: 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

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

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  

384ウェルプレートフォーマットでヒト血液から単離された好中球を用いた化合物のハイスループットスクリーニング(英語)

血清ピペットを使用して、15 mmリットルの遠心チューブ内で、4 ミリリットルのヘパリン処理ヒト血液を 8 ミリリットルの密度勾配培地に慎重に重ねます。550Gで20°Cで30〜50分間血液を遠心分離します。次に、1ミリリットルのピペットを使用して、血漿細胞と単核細胞を含む上層を取り除きます。好中球を下部の濁ったバンドから採取し、透明な液体の約3〜4ミリリットル下から、10ミリリットルのPBSを含む15ミリリットルの遠心分離管に集めます。チューブを2〜3回静かに反転させて、好中球懸濁液を混合します。好中球懸濁液を400gで20°Cで10分間遠心分離し、上清をチューブから慎重にデカントし、ペレットを5ミリリットルのPBSに再懸濁します。再度、懸濁液を300Gで20°Cで10分間遠心分離します。上澄み液を除去した後、真空吸引を使用して、チューブ壁とチューブ口の周りの残留上澄み液を除去します。ペレットを1ミリリットルの好中球培地に再懸濁します。1.5ミリリットルのチューブに、1ミリリットルあたり1.2マイクログラムの抗体溶液と0.2ミリリットルの好中球培地を混ぜ合わせます。25マイクロリットルの抗体溶液を384ウェルプレートのネガティブコントロールウェルに加えます。15ミリリットルのチューブに、9ミリリットルの抗体溶液、21.6マイクロリットルのFMLP溶液、および1.7784ミリリットルの好中球培地を混ぜ合わせます。この混合物を25マイクロリットルをポジティブコントロールに加え、384ウェルプレート内のテストウェルに入れます。マルチチャンネルピペットを使用して、5マイクロリットルの化合物溶液を化合物ライブラリプレートから384ウェルプレートに移します。懸濁液を500Gで1分間遠心分離します。16チャンネルピペットを使用して、20マイクロリットルの好中球懸濁液を各ウェルに加えます。プレートをシェーカー上で300RPMで10分間インキュベートします。次に、細胞を固定するために、各ウェルに16%パラホルムアルデヒドを3マイクロリットル添加し、氷上で10分間インキュベートします。

high-throughput-screening-compounds-using-neutrophils-isolated-from

Research

JoVE Journal

Immunology and Infection

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.