(37d) Impact of Red Blood Cell Rigidity on the Vascular Wall Adhesion of Neutrophils: Implication in the Pathology of Sickle Cell Disease

Introduction: Blood flow properties are influenced by multiphysics factors, specifically cell to cell interactions, shear rate of flow, red blood cell (RBC) deformability and vessel geometry. The RBC core of blood flow develops due to wall-induced migration of highly deformable RBCs. Hemodynamic, heterogeneous collisions between cellular components promote margination of WBCs and platelets to the vascular wall. The symptoms of many blood related diseases can be attributed to irregularities in cellular dynamics that arise due to abnormalities in blood cells, particularly RBCs. Contingent on the disease and disease severity, RBCs can be afflicted with severe membrane rigidity. Extensive experimental research exists on characterizing the chemistry and biology of rigid RBCs in many diseases. However, little experimental work has been conducted towards isolating and investigating the effect of RBC rigidity on cellular dynamics, specifically on the segregation behavior known as margination and its effect on the binding functionality of other types of blood cells, i.e. white blood cells (WBCs), and vascular targeted carriers (VTCs) while in flow. We utilized an in vitro model to examine how different degrees of RBC rigidity and volume fraction of rigid RBCs, i.e. rigid RBC concentration, impact how cell and particle marginate and adhere to the inflamed endothelium. Additionally, we utilized confocal microscopy to gain a greater understanding of the effect of rigid RBCs on RBC core and cell margination. This work is physiologically relevant in the many diseases that manifest in rigidified RBCs, as WBC transport can determine immune responsiveness in vivo.

Materials and Methods: Fresh human blood used in all assays was obtained via venipuncture. RBCs were isolated from whole blood. Separated RBCs were treated with distinct concentrations of tert-Butyl hydroperoxide (tBHP ) and reconstituted with healthy RBCs and plasma+WBCs at 40% final hematocrit for utilization in parallel plate flow chamber (PPFC) assays. RBC deformability was analyzed using a laser-assisted optical rotational cell analyzer (LORRCA; Mechatronics, Hoorn, The Netherlands). Carboxylated 500 nm and 2 µm PS and hydrogel particles had NeutrAvidin covalently attached to the carboxylic acid. Biotinylated Sialyl Lewis A (sLe^A, Glycotech, Inc.) was then coupled and calibrated to a site density of 1,000 sites/µm². WBC and particle adhesion in blood flow with rigid RBCs is compared, i.e. normalized, to WBC and/or particle adhesion of healthy controls, i.e. no rigid RBCs present. Flow distribution images were taking using confocal microscopy with a 20x water immersive objective. For all studies, all data points were included in the analyses and no outliers were excluded in calculations of means or statistical significance. Data are plotted with standard error bars and analyzed as indicated in figure legends. Asterisks indicate p values of *<0.05, **<0.01, ***<0.001 and ****<0.0001 as calculated by 2-way-ANOVA.

Results and Discussion: The TBHP treatment of RBCs only changed RBC membrane rigidity, and did not alter natural disk-like shape. Rigid RBCs are shown to reduce WBC adhesion by up to 80%, contingent on the degree of rigidity and concentration of treated RBCs as seen on Figure 1. WBC adhesion was reduced, although not always significantly lower than healthy control, in every iteration of the model, i.e. distinct wall shear rate (WSR) and RBC rigidities. Higher WSRs with rigid RBCs rendered a greater reduction in WBC adhesion. Particles did not follow similar adhesion trends of WBCs. Contingent on the particle deformability and size, particle adhesion either significantly reduced or improved with the presence of rigid RBCs. To compare RBC core distributions of different conditions to the healthy control with no rigid RBCs present, we used interquartile range (IQR) analysis and normalized to control. RBC core distribution IQR results show that the RBC core is expanded by up to 30% in size when rigid RBCs are present, Figure 2.

Conclusions: WBC adhesion is reduced more drastically by increasing degree of rigidity and WSR. Interestingly, more rigid RBCs does not always translate to worse WBC adhesion. These results hint that rigidity alone can largely disrupt normal hemodynamics and functionality of other blood cells and VTCs.