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

Subhadip Raychaudhuri,Aaron F.H Lum,Scott I Simon,Donald E Staunton,Melissa R Sarantos

doi:10.1074/jbc.m501662200

Abstract

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(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.

Highlights

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
Addition of mAb 240Q, which is associated with allosterically stabilizing CD18 into a ligand binding conformation [1, 6, 15, 16], did not itself induce activation of LFA-1 on beads but in conjunction with Mg2ϩ augmented intercellular adhesion molecule (ICAM)-1 binding by 50% above stimulation with Mg2ϩ alone
Addition of the allosteric small molecule IC487475, which binds with high affinity to the I domain (i.e. ϳ10 nM), abrogated ICAM-1 binding stimulated by Mg2ϩ (Fig. 1b)

Summary

Introduction

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. 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. Further evidence linking allosteric shifts in I domain conformation to ICAM-1 binding is the activity of a class of allosteric small molecule antagonists engineered to inhibit LFA-1 function (6 –9). We present here a new small molecule allosteric inhibitor that targets the IDAS and downshifts LFA-1 from a high to intermediate affinity. This small molecule is similar to the diaryl sulfide cinnamide antagonists [13]. Buffered saline; MIDAS, metal ion-dependent adhesion site; IDAS, I domain allosteric site; MES, 4-morpholineethanesulfonic acid; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MFI, mean fluorescent intensities; IL, interleukin

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Conclusion