Calcium Flux in Neutrophils Synchronizes β2 Integrin Adhesive and Signaling Events that Guide Inflammatory Recruitment

Abstract

Abstract-Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity b 2 integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner.

Enhancement of bacterial virulence by antibody neutralization of immune-activating toxins

“…Our results went beyond questioning the value of anti-staphylococcal antibodies in prevention of infections by S. aureus (as some studies have found that high titers of antibodies to S. aureus are not effective in preventing re-infection [16][17][18][19][20] ), but supported the hypothesis that the manner by which PVL plays a role in CA-MRSA pathogenesis is by antibody-mediated neutralization of the host's innate response activated by sub-lytic amounts of PVL produced early in the course of infection. Mechanistically, we confirmed that sub-lytic concentrations of PVL induced the opening of calcium ion channels in PMNs, 21,22 a hallmark of PMN activation, 23 and showed that antibodies directed to PVL inhibited this activation (Fig. 2B).…”

Antibody-mediated enhancement of community-acquired methicillin-resistant Staphylococcus aureus infection

“…Previous studies have found that the induction of proinflammatory cytokine release by PMNs exposed to sublytic levels of PVL results from the opening of calcium ion (Ca 2+ ) channels, which is a necessary step in PMN activation (16). We next explored whether antibodies to PVL affected PVL-mediated Ca 2+ influx into PMNs by measuring the emission of fluorescence by cells preloaded with a calcium indicator.…”

The Role of CD14 in Neutrophil Recruitment within the Liver Microcirculation during Endotoxemia

“…To assay adhesive strength of LPS-treated WT and CD14 2/2 neutrophils in vitro, vascular mimetic microfluidic chambers (a generous gift from Scott Simon, Department of Biomedical Engineering, University of California, Davis, Davis, CA) were used. As previously described (19), the chamber consists of a single channel 600 mm in width and 70 mm in height and uses a network of vacuum channels to reversibly bind to a layer of adhesion molecules. Coverslips were coated with 10% heat-inactivated mouse serum at room temperature for 3 to 4 h to allow for adhesion.…”