Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1

Abstract

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the ␤ 2 integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.Neutrophils circulate in the bloodstream to sites of inflammation where they adhere and transmigrate through the endothelium as the initial step in combating infection and to facilitate wound healing. Recruitment from the circulation involves a multistep process of cell rolling, activation, and arrest. The heterodimeric integrin receptor LFA-1 1 is composed of the ␣L (CD11a) and ␤ 2 (CD18) subunits and is constitutively expressed in a low affinity conformation on the plasma membrane of leukocytes (1-3). Neutrophils encountering chemokines on inflamed endothelium are activated to shift LFA-1 from the low to high affinity conformation, which supports tight binding to endothelial ICAM-1. Increases in integrin affinity correlate in time with adhesion function as recently demonstrated in aggregation of cells expressing ␣4␤1 and vascular cell adhesion molecule (4). ICAM-1 recognizes LFA-1 through an inserted (I) domain in the ␣ subunit. There is strong evidence correlating shifts in I domain conformation to affinity changes in binding ICAM-1. Mutations in I domain residues stabilized distinct structural conformations correlating to LFA-1 affinity. ICAM-1 equilibrium binding constants increase over 4 orders of magnitude ranging between low (i.e. 1600 M), intermediate (i.e. 9 M), and high...

Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium

“…37 BIRT is an allosteric small molecule antagonist that is structurally analogous to and functionally identical with BIRT377. 23,37,38 Lovastatin sodium was purchased from Calbiochem (La Jolla, CA). Monoclonal antibody 240Q binds CD18 and induces an active conformation, as previously described.…”

“…To determine the effect of allosteric inhibitors on the expression of the open I-domain, rLFA-1-derivatized beads were prepared as previously described. 37 Briefly, amino microsphere latex beads (6-m diameter) (Biosciences, Piscataway, NJ) were mixed with a sulfosuccinimidyl maleimide-N-hydroxysuccinimide ester cross-linker (Pierce, Rockford, IL), according to the manufacturer's protocol. Sulfhydrylated anti-leucine zipper mAb 324C-SH was prepared by incubating mAb 324C with a sulfhydryl linker (Pierce) according to the manufacturer's protocol.…”

“…IC487475 is a small molecule of the P-arylthiocinnamides series that targets the I-domain allosteric site of LFA-1. 37 BIRT is an allosteric small molecule antagonist that is structurally analogous to and functionally identical with BIRT377. 23,37,38 Lovastatin sodium was purchased from Calbiochem (La Jolla, CA).…”

Rolling on E- or P-selectin induces the extended but not high-affinity conformation of LFA-1 in neutrophils

“…[1][2][3] In Ficoll-isolated human neutrophils, this process leads to arrest from rolling, polarization, and firm adhesion to endothelial monolayers under flow. [4][5][6][7] The proximal signal transduction pathway leading from E-selectin binding to integrin-dependent adhesion is unknown. Whether neutrophil integrins assume the extended or high affinity conformation is also unknown.…”

Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo

“…4 Avidity denotes the stability of integrin-mediated cell adhesion and is primarily influenced by membrane rearrangement of integrin receptors from sparse to dense clusters that promote multivalent binding. 2,5,6 Chemokine binding to G protein-coupled receptors (GPCRs) rapidly triggers the high-affinity conformation of integrins. Once a sufficient number of integrins adopt a fully activated state, they can bind to their ligands, an important step in leukocyte recruitment.…”