| Summary: | On arrested neutrophils a focal adhesive cluster of ~200 high affinity (HA) β<sub>2</sub>-integrin bonds under tension is sufficient to trigger Ca<sup>2+</sup> flux that signals an increase in activation in direct proportion to increments in shear stress. We reasoned that a threshold tension acting on individual β<sub>2</sub>-integrin bonds provides a mechanical means of transducing the magnitude of fluid drag force into signals that enhance the efficiency of neutrophil recruitment and effector function. Tension gauge tethers (TGT) are a duplex of DNA nucleotides that rupture at a precise shear force, which increases with the extent of nucleotide overlap, ranging from a tolerance of 54pN to 12pN. TGT annealed to a substrate captures neutrophils via allosteric antibodies that stabilize LFA-1 in a high- or low-affinity conformation. Neutrophils sheared on TGT substrates were recorded in real time to form HA β<sub>2</sub>-integrin bonds and flux cytosolic Ca<sup>2+</sup>, which elicited shape change and downstream production of reactive oxygen species. A threshold force of 33pN triggered consolidation of HA β<sub>2</sub>-integrin bonds and triggered membrane influx of Ca<sup>2+</sup>, whereas an optimum tension of 54pN efficiently transduced activation at a level equivalent to chemotactic stimulation on ICAM-1. We conclude that neutrophils sense the level of fluid drag transduced through individual β<sub>2</sub>-integrin bonds, providing an intrinsic means to modulate inflammatory response in the microcirculation.
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