Abstract
Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.
Highlights
Neutrophils are among the first cells to respond to acute inflammation through a multistep process of specific bond formation with adhesion molecules up-regulated on the surface of activated endothelium [1, 2]
We directly observed the dynamics of neutrophil adhesion to a monolayer of cells expressing intercellular adhesion molecule 1 (ICAM-1) in a parallel plate flow chamber at defined shear stress, and we quantitated these dynamics over the time course of activation with IL-8 and mAb 240Q
In the current study we investigated the following two modes of CD18 activation in order to elucidate the molecular dynamics of neutrophil capture in shear flow: stabilization of the active conformation through binding of mAb 240Q, and chemotactic signaling via the IL-8 receptors, CXCR1 and CXCR2
Summary
Isolation of Human Neutrophils and PBMC—Whole blood was obtained from healthy adult individuals by venipuncture into sterile syringes with heparin (10 units/ml of blood, Elkins-Sinn, Inc., Cherry Hill, NJ) using the University of California, Davis, approved human subjects protocol (Protocol Identification 993120). Preparation of Latex Beads Presenting ICAM-1 on Their Surface—To provide a quantitative measure of CD18-dependent capture of ICAM-1, fluorescent latex beads (1 m diameter, Fluospheres, Molecular Probes, Eugene, OR) coated with protein A were derivatized with recombinant human ICAM-1/Fc. Beads were washed three times with Dulbecco’s PBS without calcium and magnesium (Invitrogen), resuspended in HEPES buffer, and incubated in a sonic bath for 1 h with a saturating concentration (15 g/ml) of ICAM-1/Fc to allow for both dispersion and coating of beads. To determine the kinetics of CD18 activation, neutrophils and PBMC were mixed with 327C-Alexa and examined for 10 min with the flow cytometer under conditions of stimulation by soluble ICAM-1/Fc (150 g/ml), 240Q, or IL-8 using identical stimulation protocols as the bead adhesion assay. Neutrophils (2 ϫ 106 cells) were added to a 1 M Fluo-3-AM solution in HEPES buffer with 0.1% Me2SO with no calcium or HSA and incubated
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