Shear and Time-Dependent Changes in Mac-1, LFA-1, and ICAM-3 Binding Regulate Neutrophil Homotypic Adhesion

Abstract

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s−1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s−1, where ∼40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of ∼100 s−1. Above 300 s−1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate ∼3-fold faster than that of Mac-1. The results suggest that the relative contributions of β2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.

Hydrodynamic Shear Regulates the Kinetics and Receptor Specificity of Polymorphonuclear Leukocyte-Colon Carcinoma Cell Adhesive Interactions

“…A recent study suggests that the capacity of CD11a to support adhesion decays with time at a much faster rate than that of CD11b, having a negligible role in PMN-PMN interactions after 5 min of fMLP stimulation (37). In light of these observations, we hypothesized that the lack of CD11a involvement in PMN-tumor cell interactions might be ascribed to the prolonged incubation times of the static adhesion assays (5-to 30-min incubation at 37°C) (10,11).…”

Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension

“…In some runs, to distinguish between the contributions of shear rate and shear stress, media viscosity was altered by the addition of dextran (MWt 2 ϫ 10 6 ) dissolved in HEPES buffer 29 to a final concentration of 1.5% wt/vol. For these runs, appropriate controls were performed through the addition of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer to the PRP-PPP mixture.…”

Endothelium-Neutrophil Interactions in ANCA-Associated Diseases

“…Moreover, TNF priming triggers the switching of the aMb2-integrin (Mac-1) to an active conformation, allowing its binding to intracellular adhesion molecule 3 (ICAM-3) on adjacent neutrophils. 23 It is thus reasonable to propose an initial step of low-grade activation of circulating neutrophils ( Figure 2B) promoted by TNF-a and complement, allowing neutrophil-neutrophil interactions through b2-integrin binding of iC3b and/or ICAM-3. This would result in a transient homotypic aggregation, similar to that observed in the Shwartzmann phenomenon 24 and to that recently described after in vivo injection of granulocyte-colony stimulating factor.…”

“…21 This releases the powerful agonist C5a, able to promote homotypic aggregation of circulating neutrophils, 22 via interactions of the TNFactivated aMb2 (Mac-1)-integrins with ICAM-3 or iC3b on bystander neutrophils. 23 TNF/ b2-integrin joint signals would promote sufficient ANCA antigen membrane expression to overcome the plasma inhibitory effect 27 and allow the access of ANCA Fab to their antigen, whereas their Fc portion interacts with FcgRIIa receptors. By doing so, ANCA give the final signals for the firm adhesion of neutrophils to the endothelium.…”

“…and increased cell rigidity 28 and homotypic aggregation, mediated by TNF-activated b2-integrins interacting with ICAM-3 on bystander neutrophils. 23 (B) Glomerular leukocyte adhesion induced by anti-GBM in mice and involving platelets. After anti-GBM antibody 68 or immune complex 136 deposition, platelets adhere to the glomerular endothelial lesions via platelet glycoprotein VI, fibrinogen being a potential ligand.…”