neutrophils screening assay using flow cytometry

Cribado de inhibidores de la activación de integrinas β2 en neutrófilos humanos

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

Autor destacado: Desarrollo de un método para identificar pequeños antagonistas moleculares de la activación de la integrina β2 

Una técnica de alto rendimiento basada en citometría de flujo para el cribado de fármacos inhibidores de integrinas

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 Neutrophils Screening Assay Using Flow Cytometry at JoVE.com

Neutrophils Screening Assay Using Flow Cytometry  

Ensayo de cribado de neutrófilos mediante citometría de flujo

Después de encender el citómetro de flujo, cargue la placa de 384 pocillos que contiene la suspensión de neutrófilos tratada con compuesto en el portamuestras. Abra el software, inicie un nuevo experimento. Seleccione 384 Pozo y elija los fluores de flora deseados.A continuación, haga clic en configuración de placa, arrastre para seleccionar todos los pocillos y haga clic en muestra. Encienda el interruptor de alto rendimiento y, en el panel de velocidad, seleccione alto. En el cuadro de volumen, introduzca 50, seleccione las muestras en columnas impares y active el interruptor de agitación.Por último, asigne un nombre a las muestras en el panel derecho. Los valores medios de densidad fluorescente de los compuestos ensayados estuvieron por encima de la línea de corte, lo que indica que ninguno de los compuestos se identificó como antagonistas de la integrina beta dos.

neutrophils-screening-assay-using-flow-cytometry

Research

JoVE Journal

Immunology and Infection

Neutrophils Screening Assay Using Flow Cytometry

278 vistas.  UConn Health. Después de encender el citómetro de flujo, cargue la placa de 384 pocillos que contiene la suspensión de neutrófilos tratada con compuesto en el portamuestras. Abra el software, inicie un nuevo experimento. Seleccione 384 Pozo y elija los fluores de flora deseados.A continuación, haga clic en configuración de placa, arrastre para seleccionar todos los pocillos y haga clic en muestra. Encienda el interruptor de alto rendimiento y, en el panel de velocidad, seleccione alto. En el c...

Video: 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.