Cooperation of kindlin-3 and talin-1 in integrin activation during neutrophil arrest and inflammation

Neutrophil arrest requires the activation of beta2 integrins. Two FERM domain proteins, kindlin-3 and talin-1, are necessary for neutrophil arrest. In this project, we explored the molecular mechanisms of talin-1 and kindlin-3 cooperation during neutrophil adhesion. Flow chamber experiments showed that talin-1 and kindlin-3 are simultaneously recruited to the plasma membrane of HL-60 cells during the transition from rolling to arrest. Using primary neutrophils expressing fluorescent fusion proteins of talin-1 or kindlin-3, we confirmed that both proteins are recruited to the plasma membrane at the time of arrest, supporting their coordinated role in neutrophil integrin activation. In a mouse cremaster muscle ischemia-reperfusion model, beta2 integrin activation was detected in neutrophils attached to the endothelium after ischemia-reperfusion. These findings support that kindlin-3 and talin-1 work together to induce high-affinity integrin activation during inflammation.

Methods
HL-60 cells were differentiated for 5-7 days in 1.3 % DMSO and flowed on P-selectin and ICAM-1 substrate in a microfluidic device. Mouse neutrophils were isolated by flushing mouse tibia or femur. Cells were perfused at 6 dyn/cm2 wall shear stress through a microfluidic flow chamber and examined by total internal reflection fluorescence microscopy. Confocal microscopy was utilized to examine the antibody labelling in the cremaster muscle microcirculation of a male mouse before and after cremaster ischemia and reperfusion.