Background: Neutrophil recruitment to inflamed endothelium significantly contributes to the hypercoagulable state seen in sickle cell disease (SCD). We previously demonstrated that neutrophil binding on E-selectin was significantly enhanced under hypoxia in SCD. However, we observed considerable heterogeneity in adhesion profiles and clinical characteristics of SCD. Here, we interrogated if the differential interaction of neutrophils with E-selectin is mechanistically linked to clinical features and the course of SCD.

Aims: To investigate if profiles of differential neutrophil binding on E-selectin correlate with clinical characteristics of SCD.

Methods: Venous blood samples were collected from 35 adult patients with homozygous (HbSS) SCD in EDTA vacutainers during a non-crisis clinic visit. Samples were re-calcified with Hank’s buffer (1:1 v/v) and injected into E-selectin immobilized microchannels at typical shear stress values seen in post-capillary venules. Neutrophils bound on E-selectin under shear were quantified in a 32 cm² window (Fig 1A-B).

Results: Two groups of patients with distinct lactate dehydrogenase (LDH) levels and absolute reticulocyte counts (ARCs) were identified based on K-means cluttering analysis (Fig. 1C). Group 2 patients (N = 19) had significantly higher LDH levels and ARCs as well as lower number of neutrophils bound on E-selectin (Fig. 1D) and fetal hemoglobin (HbF) levels (Fig. 1E) compared to Group 1 patients (N = 16). Moreover, 79% (15/19) of Group 2 patients were transfusion-dependent compared to 31% (5/16) of Group 1 patients. Mechanistically, the degree of neutrophil activation, assessed by L-selectin shedding/blockade, was inversely related to neutrophil binding on E-selectin (Fig. 1F-H).

Conclusions: Our results show that SCD patients with a more severe hemolytic phenotype and greater transfusion dependency have constitutively less neutrophil binding on E-selectin. Further, profiling neutrophil adhesion may help predict response to anti-E-selectin therapy. Future experiments will focus on analyzing neutrophil adhesion on ICAM-1 or endothelial cells for assessing α_Mβ₂ upregulation levels.

Figure 1. Neutrophil binding on E-selectin correlates with clinical variables in SCD. (A) Macroscopic view of the 3-channel microfluidic device whereby 3 identical microchannels with separate inlets and outlets, each 50-µm tall, 4-mm wide, and 25-mm long, are shown. (B) Adherent cells were permeabilized and stained for neutrophil elastase. Scale: 50 µm. (C) Two sub-groups of patients with distinct LDH levels and ARCs were identified via K-means clustering analysis. The dashed rectangular regions represent normal ranges for the given clinical parameters. Group 2 patients (N = 19) had significantly lower (D) number of neutrophils bound on E-selectin (P = 0.003, Mann-Whitney) and (E) HbF levels (P = 0.043, Mann-Whitney) compared to Group 1 patients (N = 16). (F) Neutrophil activation by 25 µg/mL TNF-α or (G) L-selectin blockade by 10 µg/mL anti-L-selectin antibody led to significantly decreased neutrophils bound on E-selectin compared to the control (P = 0.030 or P = 0.026, pared t-test, N = 5 in each group). Neutrophil adhesion was normalized based on the mean adhesion value of control samples and shown in percentage. (G) L-selectin blockade had no reducing effect on TNF-α stimulated neutrophils binding on E-selectin (P > 0.05, N = 4 in each group). Neutrophil adhesion was normalized based on the mean adhesion value of TNF-α stimulated samples and shown in percentage. LDH: lactate dehydrogenase. ARCs: absolute reticulocyte counts. HbF: fetal hemoglobin. TNF-α: tumor necrosis factor-α. Data cross lines represent the mean. Error bars indicate the standard deviation.

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