Contributions of Immune Cells to Vascular Occlusion in Sickle Cell Disease.
Authors: Frenette Paul S. , Hidalgo Andres , Chang Jungshan , Peired Anna 
Published: October 2019

Sickle cell disease (SCD) results from a single missense mutation in the β-globin gene, making the gene product susceptible to polymerize in conditions of low O2 tension. The resulting polymers can disrupt the normal architecture of sickle red blood cells (sRBC), altering their membrane and promoting adherence. sRBC adhesion events within the vasculature produces the painful vaso-occlusive episodes that account for most of the morbidity and mortality of this disease.1 The mechanisms mediating vaso-occlusion (VOC) in SCD are not well understood. Several studies have shown that sRBC, most notably the immature reticulocytes, can bind to endothelial cells (EC) though multiple overlapping adhesion mechanisms. However, whether these interactions are directly involved in VOC is controversial. sRBC can clearly interact with other blood cells inside the vasculature. For example, in a humanized mouse model of SCD, sRBCs interact prominently with leukocytes that are adherent in inflamed venules.2 A role for leukocytes in VOC is consistent with clinical data that have linked leukocyte counts, but not reticulocyte counts, with clinical manifestations of SCD. High-speed multichannel fluorescence intravital microscopy (MFIM) using low doses of lineage-specific antibodies has shown that sRBCs interact specifically with adherent neutrophils.3 Most adherent neutrophils are not immobile but rather crawl along inflamed venules, and virtually all of them exhibit a polarized appearance in vivo with clustered L-selectin at the uropod. Inhibition of or deficiency in the endothelial selectins (E- and P-selectins) protects SCD mice from VOC. Recent studies into the individual role of each selectin have revealed, unexpectedly, a major function for E-selectin. Whereas E-selectin inhibition or deficiency does not significantly affect neutrophil adhesion in inflamed venules, it dramatically blocks the interactions of RBC with adherent neutrophils, suggesting that E-selectin signaling into an adherent leukocyte may induce RBC capture. We have found that RBCs carrying normal hemoglobin (nRBCs) also interact with adherent neutrophils in inflamed venules of wildtype mice. In the mouse, three glycoproteins, P-selectin glycoprotein ligand-1 (PSGL-1, encoded by the Selplg gene), CD44 (encoded by Cd44), and E-selectin ligand-1 (ESL-1, encoded by Glg1) mediate all E-selectin binding activity of leukocytes.4 Detailed analyses of Selplg−/− mice, Cd44−/− mice, or chimeric mice, in which Glg1 is knocked-down by RNA interference, revealed that E-selectin-induced signals are mediated specifically by Glg1. Using MFIM analyses, we have mapped the location of RBC captures on the leading edge of polarized neutrophils. Further investigations have revealed that RBC capture was mediated by the β2 integrin Mac-1 (αMβ2). We have developed a new method to assay Mac-1 activation on adherent leukocytes in live mice. These analyses have shown that Glg1-mediated signals activate αMβ2 regionally at the leading edge, allowing RBC capture. These results suggest a new pathway for the development of targeted therapies for VOC. The fact that genetically normal RBCs are captured by neutrophils through mechanisms similar to sRBCs also suggests broad functions for this paradigm in other thrombo-inflammatory diseases.