Sickle Cell Vaso-Occlusion Is Triggered by E-Selectin Ligand Signaling and Propagated by the Leukocyte Integrin Mac-1.
Authors: Hidalgo Andres , Chang Jungshan , Peired Anna J. , Chiang Elaine Y. , Frenette Paul S. 
Published: April 2020

Abstract
Vasoocclusion (VOC) is the leading cause of morbidity and mortality in patients with sickle cell disease (SCD). Intravital microscopy studies in a murine model of SCD have revealed that capture of sickle red blood cells (RBC) by intravascular adherent leukocytes (WBC) plays an important role in VOC, and that deficiency in both P-and E-selectins protect from VOC. Here, we have investigated the cellular and molecular mechanisms leading to sickle RBCs interactions with adherent WBCs. Intravital microscopy analyses of the individual role of P- or E-selectin revealed, unexpectedly, a profound reduction in RBC-WBC interactions in Berkeley sickle mice lacking E-selectin (Sele−/−; &gt;97% reduction), whereas the protection was only partial in the absence of endothelial P-selectin. Since E-selectin is expressed exclusively on the endothelium, and its deficiency does not alter WBC recruitment, we hypothesized that E-selectin might provide activation signals to neutrophils that allow them to capture RBCs. During our studies, we observed that RBC-WBC interactions are not exclusive of sickle animals but are also present in wild-type B6 mice treated with TNF-α, suggesting that this phenomenon accompanies a physiological inflammatory response. We found that RBC-WBC interactions in B6 mice occur at a lower frequency than in sickle mice and that these interactions are also reduced in Sele−/− mice (60% reduction; p&lt;0.05). We thus reasoned that B6 mice might provide a convenient model to gain molecular insight into RBC-WBC interactions in vivo. Since PSGL-1, CD44 and ESL-1 harbor the entire E-selectin ligand activity on neutrophils in vivo (Hidalgo et al., Immunity 2007), we investigated which of these glycoproteins mediates the signals allowing RBC capture. High speed digital multichannel fluorescence intravital microscopy analyses revealed that RBC-WBC interactions were only markedly reduced in the absence of ESL-1 (63% reduction, p&lt;0.001), but not in the absence of PSGL-1 or CD44. Further detailed image analyses mapped RBC captures at the leading edge of adherent neutrophils, an area where chemokine receptors and integrins may accumulate. Since selectin-mediated signaling is known to activate β2 integrins, we tested the role of Mac-1, whose expression and affinity are elevated in neutrophils from SCD patients. We found that RBC-WBC interactions were virtually absent in mice deficient in Mac-1 (97% reduction; p&lt;0.0001). To determine whether E-selectin/ESL-1-mediated signaling promoted Mac-1 activation, we developed an assay to assess Mac-1 activity in real time on adherent WBCs in vivo. Albumin-coated fluorobeads bound to subsets of adherent leukocytes in TNF-α-stimulated venules of B6 mice. These interactions were Mac-1-dependent since they were ablated in Itgam−/− mice. Absence of E-selectin or ESL-1, but not P-selectin, PSGL-1 or CD44, significantly reduced Mac-1 activity (by 36% and 52%, respectively; p&lt;0.05). In contrast, Mac-1 activation on adherent leukocytes was dramatically increased in inflamed venules of sickle mice (2.5-fold; p&lt;0.001) and was restricted to a subset of adherent neutrophils. Preliminary experiments in which Mac-1 function is blocked in sickle animals with a monoclonal antibody revealed a reduction of RBC-WBC interactions (by 57%) compared to an isotype control antibody. Our results indicate that the binding of E-selectin to neutrophil ESL-1 promotes Mac-1 activation, which in turn mediates the capture of sickle RBCs. These findings provide attractive therapeutic targets to alleviate this devastating disease.