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Abstract
Neutrophils are essential for innate immunity and inflammation and 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 we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.
Highlights
Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are b2 integrin-dependent
The ‘switchblade’ model of integrin activation (Supplementary Fig. 1a)[25] suggests a two-step activation process in which integrin extension (E þ ) is followed by a rearrangement in the ligand-binding site leading to high-affinity (H þ ). b2 integrin extension is detected by monoclonal antibody KIM127, which recognizes a neoepitope[27] that is hidden in the bent knee of human b2
Since E À H þ integrin is not expected to bind ligand in trans, we considered whether E À H þ integrin may bind ligand in cis, that is, intercellular adhesion molecules (ICAMs)-1 expressed on the neutrophil
Summary
B2 integrin activation on rolling human neutrophils. Microfluidic chambers[15] were coated with recombinant human. Since LFA-1 binds both ICAM-1 and ICAM-3, we tested the contribution of both ligands and found that blocking ICAM-3 alone had a stronger effect on the number of arrested neutrophils than blocking ICAM-1 alone This is consistent with the higher expression of ICAM-3 than ICAM-1 on human neutrophils (Supplementary Fig. 11). After injecting the chemokine CXCL1, the number of arrested ICAM-1/2-double-knockout leukocytes was twice that of wild-type neutrophils (Fig. 7p) These data show that the binding of E À H þ b2 integrin to ICAM-1 and 2 in cis strongly inhibits leukocyte adhesion in vivo. Our data support a new model (Supplementary Fig. 1b) where resting E À H À LFA-1 and Mac-1 are stimulated by chemokine to assume the E À H þ conformation that binds mAb24, but not KIM127 This conformation is stabilized by interaction with ICAM-1 on the neutrophil in cis. This converts E À H þ to E þ H þ integrin, which is able to bind ICAM-1 in trans (on the substrate, endothelium or other leukocytes) and promote arrest
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