A New Model of Acute Sickle Cell Vaso-Occlusion Elicited by IgGMediated Hemolytic Transfusion Reactions
Authors: Jang Jung-Eun , Frenette Paul S. 
Published: April 2020

Sickle cell disease (SCD) is caused by a single amino acid substitution in the β-globin chain of hemoglobin. Acute vaso-occlusive crisis (VOC) is an important complication of SCD and is a major cause of morbidity and mortality for these patients. Red blood cell (RBC) transfusion therapy is widely used to manage SCD. However, IgG-mediated delayed hemolytic transfusion reaction (HTR) represent a serious side effect of transfusion therapy, and HTR can frequently trigger VOC in SCD patients. To understand HTR pathophysiology in SCD, we established a model of alloimmune IgG-mediated HTRs using a well-characterized humanized murine model of SCD (Paszty et al., Science 1997). We previously showed that VOC in this murine model of SCD is caused by interactions in the microvasculature between sickle RBCs and adherent white blood cells (WBCs) (Turhan et al, P.N.A.S. 2002). In addition, because IgG-mediated HTRs in wild-type (WT) mice are associated with a cytokine storm (Hod et al., Blood 2008), we hypothesized that the circulating pro-inflammatory cytokines induced by HTRs would lead to VOC in the murine model of SCD. To this end, fluorescently labeled RBCs from human glycophorin A transgenic (hGPA-Tg) or WT mice were transfused into SCD mice that had been passively immunized with an IgG monoclonal anti-hGPA antibody. Serial flow cytometry analyses revealed that the survival of incompatible hGPA-Tg RBCs in passively immunized SCD mice was 42 ± 10% 2 hours after transfusion, whereas the survival of compatible WT RBCs was 98 ± 2%. Using intravital microscopy, we examined leukocyte recruitment in post-capillary and collecting venules, and evaluated the interactions between RBCs and WBCs in passively immunized mice that were not previously treated with inflammatory cytokines. As compared to the transfusion of compatible WT RBCs, transfusion of incompatible hGPA-Tg RBCs significantly increased leukocyte adhesion to the endothelium by 1.6 fold (2,669 ± 186 vs 1,717 ± 107 adherent WBCs per mm2; p&lt;0.001) and sickle RBC-leukocyte interactions by 3.9 fold between 91 and 120 minutes (1.1 ± 0.2 vs 0.3 ± 0.2 RBC-WBC interactions per minute; p=0.01), leading to acute VOC in post-capillary venules by the 2 hour time point. Moreover, the survival of SCD mice transfused with incompatible RBCs was significantly shorter (by 3 hours) than control mice transfused with compatible RBCs (p=0.04, Log rank test). The time course of these complications correlated with increased levels of circulating pro-inflammatory cytokines (e.g. MCP-1, MIP-1β, KC) at 2 hours following incompatible transfusion, suggesting that endogenously produced cytokines may play an important role in the pathophysiology of VOC in this model. In conclusion, these in vivo results indicate that this IgG-mediated HTR model in SCD mice reproduces the VOC seen in SCD patients experiencing delayed HTRs. This model will be useful to identify the key inflammatory cytokine(s) that can trigger VOC and design novel therapies to alleviate this major manisfestation of the disease.