Neutrophil recruitment limited by high affinity bent β2 integrin binding ligand in cis

Abstract

Abstract Neutrophils are essential in innate immunity and inflammation. Many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here, by using high-resolution quantitative dynamic foot printing (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, we show that a substantial fraction of β2 integrins on human neutrophils acquires an unexpected E−H+ conformation. Beside the observations consistent with the canonical switchblade model, we discovered a new alternative model that some β2 integrins transition from E−H− to E−H+ to E+H+ during activation. Using flow cytometry based Förster resonance energy transfer (FRET) assay, we find out that E−H+ β2 integrin binds its ligand intercellular adhesion molecule 1 (ICAM-1) in cis. And this cis binding significantly inhibits the conformational transitioning of β2 integrin from E−H+ to E+H+ and the neutrophil adhesion under flow, which indicates a novel endogenous anti-inflammatory mechanism inhibits neutrophil accumulation and inflammation.