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

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 β2 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

Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response.

We describe the development, validation, and application of a novel PDMS-based microfluidic device for imaging leukocyte interaction with a biological substrate at defined shear force employing a parallel plate geometry that optimizes experimental throughput while decreasing reagent consumption. The device is vacuum bonded above a standard 6-well tissue culture plate that accommodates a monolayer of endothelial cells, thereby providing a channel to directly observe the kinetics of leukocyte adhesion under defined shear flow. Computational fluid dynamics (CFD) was applied to model the shear stress and the trajectory of leukocytes within the flow channels at a micron length scale. In order to test this model, neutrophil capture, rolling, and deceleration to arrest as a function of time and position was imaged in the transparent channels. Neutrophil recruitment to the substrate proved to be highly sensitive to disturbances in flow streamlines, which enhanced the rate of neutrophil-surface collisions at the entrance to the channels. Downstream from these disturbances, the relationship between receptor mediated deceleration of rolling neutrophils and dose response of stimulation by the chemokine IL-8 was found to provide a functional readout of integrin activation. This microfluidic technique allows detailed kinetic studies of cell adhesion and reveals neutrophil activation within seconds to chemotactic molecules at concentrations in the picoMolar range

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

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and β2-integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of β2-integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. An allosteric small molecule inhibitor targeted to the I-domain stabilized LFA-1 in an intermediate-affinity conformation, which supported neutrophil rolling but inhibited cell polarization and abrogated transmigration. We conclude that a shift in LFA-1 from intermediate to high affinity during the transition from rolling to arrest provides the contact-mediated signaling and guidance necessary for PMN transmigration on inflamed endothelium

Spatial Regulation of Inflammation by Human Aortic Endothelial Cells in a Linear Gradient of Shear Stress

Objective: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. Methods: Human aortic endothelial cells were stimulated with TNF‐α and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFκ B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. Results: VCAM‐1 and E‐selectin upregulation was greatest between 2–4 dyne/cm2 (6 and 4‐fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM‐1 expression and NFκ B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E‐selectin and VCAM‐1 expression was greatest. Conclusions: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of ∼ 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation

Clinical Evaluation of the Torq Zero Delay Centrifuge System for Decentralized Blood Collection and Stabilization

Blood sample collection and rapid separation—critical preanalytical steps in clinical chemistry—can be challenging in decentralized collection settings. To address this gap, the Torq™ zero delay centrifuge system includes a lightweight, hand-portable centrifuge (ZDrive™) and a disc-shaped blood collection device (ZDisc™) enabling immediate sample centrifugation at the point of collection. Here, we report results from clinical validation studies comparing performance of the Torq System with a conventional plasma separation tube (PST). Blood specimens from 134 subjects were collected and processed across three independent sites to compare ZDisc and PST performance in the assessment of 14 analytes (K, Na, Cl, Ca, BUN, creatinine, AST, ALT, ALP, total bilirubin, albumin, total protein, cholesterol, and triglycerides). A 31-subject precision study was performed to evaluate reproducibility of plasma test results from ZDiscs, and plasma quality was assessed by measuring hemolysis and blood cells from 10 subject specimens. The ZDisc successfully collected and processed samples from 134 subjects. ZDisc results agreed with reference PSTs for all 14 analytes with mean % biases well below clinically significant levels. Results were reproducible across different operators and ZDisc production lots, and plasma blood cell counts and hemolysis levels fell well below clinical acceptance thresholds. ZDiscs produce plasma samples equivalent to reference PSTs. Results support the suitability of the Torq System for remotely collecting and processing blood samples in decentralized settings