5. Mechanisms of leukocyte rolling and arrest during inflammation
Inflammatory response following bacterial infection involves neutrophil adhesion to the inflamed endothelium of blood vessels with high wall shear stress (t > 6 dyn/cm2). Neutrophil-endothelial adhesion starts with rolling along the vessel wall mediated by P-selectin on the endothelium binding to P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils, followed by firm arrest which is mediated by activated β2-integrins (LFA-1 and Mac-1) on the neutrophil binding to inter-cellular-adhesion-molecule-1 (ICAM-1) on endothelium. In October 2010 (Sundd, P. et al. Nature Methods, 2010), I introduced quantitative Dynamic Footprinting (qDF) microscopy which is an adaptation of TIRF microscopy and allows estimation of z-distances in the footprints (cell-substrate contact zone) of rolling neutrophils. This study revealed that neutrophils rolling at high shear stress (> 6 dyn/cm2) deform creating a four-fold larger footprint with the P-selectin substrate than that predicted by computational models and low resolution in vivo images, and that the rolling is further facilitated by three to four long membrane tethers which can extend up to 16 µm behind the rolling cell. In the most recent study (Sundd P. et al, Nature, 2012), I have discovered ‘sling’, an autonomous adhesive structure made by rolling neutrophils. I have shown that long tethers made by neutrophils rolling at high shear stress (6-10 dyn/cm2) do not retract as postulated, but instead persist and appear as ‘slings’ at the front of rolling neutrophils (Movie 1). Slings are made by rolling neutrophils in vitro and in a model of acute inflammation in vivo. Selectin ligand PSGL-1 is presented as discrete sticky patches while integrin LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling neutrophils and undergo a step-wise peeling from the P-selectin substrate which is enabled by the tandem failure of PSGL-1 patches under hydrodynamic forces (Movie 2). Currently, we are conducting experiments in an in vitro microfluidic flow chamber to elucidate the cytoskeletal organization responsible for the ability of slings to withstand hydrodynamic forces at high shear stresses.
We are also conducting qDF experiments to study the nature of sling formation by different mouse circulating monocyte subsets (Gr-1+/Ly6Chi and Gr-1-/Ly6Clow) and their role in monocyte adhesion during inflammation.
During bacterial infection neutrophils leave the blood stream and enter the infected tissue to resolve infection. In order to leave the blood stream, neutrophils have to first roll along the walls of blood vessels. In some blood vessels, the blood flow is very fast, however, neutrophils still manage to roll along the vessel wall and enter the infected tissue. We discovered that neutrophils form long tube like structures known as ‘slings’ that help them to roll in presence of fast blood flow. Top panel-animation showing the side view of a rolling neutrophil. The rolling neutrophil forms a ‘sling’ in the front and then wraps it around. Bottom panel-experimental movie showing a mouse neutrophil forming a sling in the front. The cell is rolling on a P-selectin coated cover glass in a microfluidic device and the movie was recorded using qDF. The view is from the bottom of the cell. The animation in the top panel is inspired by the experimental movie shown in the bottom panel. Sundd P. et al. Nature 488:399-403,2012.
Step-wise peeling of a sling. PSGL-1 patches (red spots) visible on sling (green). TIRF excitation 561 and 488 nm, incidence angle θ = 70⁰. P-selectin 20 molecules/µm2. Wall shear stress 10 dyn/cm2. View from the bottom. Frame rate 5 s-1. Isolated mouse bone marrow neutrophils were stained with membrane dye DiO and AlexaFluor568-conjugated nonblocking Ab against PSGL-1. Cells were allowed to roll on P-selectin coated cover glass in a microfluidic device and images were recorded using dual color qDF (DqDF). Sundd P. et al. Nature 488:399-403,2012.