Neutrophils form elongated shear‐derived particles (SDP) via shedding tethers and slings
Authors: Marki Alex , Buscher Konrad , Fan Zhichao , Yeh Yi‐Ting , Hartmann Nadine , Dan Jennifer , Bui Jack , Winkels Holger , Ehinger Erik , McArdle Sarah , Mikulski Zbigniew , Altman Yoav , Kronenberg Mitchell , Chien Shu , Ley Klaus 
Published: June 2021

Neutrophils are innate immune cells greatly contributing to the host defense against bacterial and fungal infection and to various sterile inflammatory conditions. To reach their site of action, circulating neutrophils initially roll, then arrest on the activated vessel wall and then migrate out from the vessel lumen. During rolling at high shear stress, neutrophil microvilli anchored to the endothelium by selectin bonds are pulled out behind the cell into several micron long, thin membrane tubes called tethers. As the anchoring bonds break, the tethers turn into slings and may land in front of the rolling cell and provide adhesive substrate for rolling. Here, we show that both tethers and slings can detach and form elongated neutrophil derived particles, which we call shear‐derived particles (SDP).Cremaster intravital microscopy experiments of Ly6G‐AF647 injected male mice showed the presence of SDPs with or without intrascrotal pretreatment with tumor necrosis factor (TNF), suggesting that in mice the cremaster preparation induced trauma is sufficient to induce SDP formation. In 27 vessels of 8 TNF‐pretreated mice, 234 SDPs were observed with median length of 6.5 μm (range 1.9–112 μm). SDPs appeared when rolling neutrophils left tethers behind, but some were also generated from detached slings. While most SDP appeared arrested, 18% “sneaked” along the vessel wall with the blood flow at a median velocity of 1.13 μm/s (range 0.23–4.95 μm/s), which was significantly lower than the neutrophil rolling velocity of 3.47 μm/s (range 0.7–9.76 μm/s). Free‐floating SDPs were visible in the blood harvested from these mice.SDP production by tether and sling detachment was reproduced in flow chamber experiments using CD16‐AF647 labeled human neutrophils. 11.5 (range 0–42) SDPs per 0.1 μl were found in plasma of 20 healthy donors by a CD16 staining and a standardized confocal microscopy scanning protocol. In 24 septic donors, significantly more (median count 70.5, range 0–140) SDPs were found, which were also longer (median length 5.4 μm vs. 4 μm, range 1.7–23 μm and 1.7–12.8 μm). Image stream (Amnis) analysis confirmed the increase of SDP count in septic plasma and showed that the ratio between CD16+ SDPs and CD16+ vesicles is on the order of 1:1000. We speculate that some SDPs may fragment into spherical vesicles. All significances: p<0.0001 with Student's T‐test.We conclude that rolling or arrested neutrophils lose their tethers and slings. These form a new class of elongated microparticles in the blood, which are elevated in sepsis. Further work is required to better characterize the conditions when SDP form and to characterize the composition and function of these structures.Support or Funding InformationK.Ley: NIH grant HL 78784, A.Marki: AHA postdoctoral fellowship 17POST33410940This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.