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

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 β2 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 diseases^3,4.

β2 integrins have been targeted for inflammatory diseases before. Efalizumab, a monoclonal antibody directly targeting integrin αLβ2, was developed to treat psoriasis⁵. However, efalizumab was withdrawn due to its lethal side effect – progressive multifocal leukoencephalopathy resulting from JC virus reactivation^6,7. 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 αLβ2 crosslinking and the unwanted internalization of α4 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 β2 integrin antagonists using human neutrophils is presented here. β2 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 conformation^10,11,12,13. There is also an alternative pathway that starts from the bent-closed to bent-open and extended-open, eventually^{14,15,16,17,18,19}. The conformation-specific antibody mAb24 binds to an epitope in the human β2-I-like domain when the headpiece of the ectodomain is open^20,21,22,23.

Here, mAb24-APC is used to determine whether the β2 integrins are activated. To activate neutrophils and integrin, N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial-derived short chemotactic peptide that can activate neutrophil β2 integrins²⁴, is used as a stimulus in this protocol. When fMLP binds to the Fpr1 on neutrophils, downstream signaling cascades involving G-proteins, phospholipase Cβ, and phosphoinositide 3-kinase γ are activated. These signaling events ultimately result in integrin activation via the inside-out signaling pathway^18,25. Besides small molecule antagonists that directly bind to β2 integrins and prevent conformational changes of integrin activation²⁶, compounds that can inhibit components in the β2 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.