The Endothelin Receptor Etb Plays a Crucial Role for Recruitment of Neutrophils to the Vascular Wall in Sickle Cell Disease

Introduction: Although Sickle Cell Disease (SCD) is due to abnormal hemoglobin, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), play a key role in the pathophysiology of the disease, particularly in the vaso-occlusive events. Adhesion of PMNs to activated endothelium is critical in SCD and might contribute to vaso-occlusion; thus targeting PMN interactions with the endothelium may represent a good opportunity for new therapeutics. We looked for candidate mediators that may be involved in PMN activation and recruitment in tissues. We focused on endothelin-1 (ET-1) since (i) high levels of cytokines have been reported in SCD patients; (ii) we previously demonstrated that ET-1 receptors blockade in the SAD mouse model of SCD leads to reversal or prevention of vaso-occlusive events, nitrative stress, kidney and lung damage, and even death. While demonstrating that ET receptor (ETR) antagonism inhibited a tonic ET-1-dependent vasoconstriction during experimental vaso-occlusive crisis, we observed unexpected inhibition of PMN recruitment in lungs and kidneys. We postulated that activation of ETRs might stimulate a pathogenic proinflammatory role for PMNs in SCD.

Methods: In the present study, we combinedintravital videomicroscopy of the cremaster muscle microcirculation in SAD mice and quantitative microfluidic fluorescence microscopy of human blood to investigate the involvement of the ETRs in the interaction between neutrophils and endothelial cells.

Results: Experiments performed both on SAD mice and SCD patients indicate that blocking ETRs hinders PMN recruitment to endothelial cells in inflammatory conditions (Fig.1). In SAD mice, we showed that the ETRs are involved at several steps of PMN microvascular recruitment. Rolling adhesion involves the ETB receptor only; firm adhesion and post-adhesive dynamic events with transmigration involve both the ETA and the ETB receptors. Inhibition experiments performed with the highly selective BQ788 antagonist (specific for ETB receptor) provide potent anti-inflammatory action in SAD mice. In SCD patients and healthy volunteers, we evidenced that human PMNs display functional ETB receptors that trigger a downstream calcium signaling leading to enhanced adhesion to endothelial cells. Furthermore, we investigated the expression of preproET-1 mRNA and the secretion of ET-1 in human PMNs from SCD patients and healthy controls and we highlighted the ability of PMNs to produce and secrete ET-1. We also found an unexpected stimulatory role for the ETB receptor in PMN adhesion to endothelial cells in laminar flow conditions. Last, we showed that this abnormal adhesion involves ETB receptors on both endothelial cells and PMNs.

Conclusion: SAD mice and SCD patients both provide consistent evidence for a powerful anti-adhesion role of ET_B blockade. We emphasize that our work is the first to unravel the impact of the ET receptors on the different phases of PMN-endothelial interaction, further complementing early evidence that endothelins are chemoattractants for neutrophils in vitro. Our findings involving a potent contribution of the ET_B receptor to vascular inflammation are novel as well as the fact that this pathogenic phenomenon is found in sickle but not in normal mice. Overall, the aforementioned findings indicated that human neutrophils display functional ET_B receptors with calcium signaling capability, and we confirmed that human PMNs synthesize ET-1 that may be involved in autocrine and paracrine pathophysiological actions. Thus, the ET-1- ET_B axis should be considered a cytokine-like potent proinflammatory pathway in SCD. If endothelin receptors antagonists prove safe and effective for preventing or treating acute vasoocclusive events in the clinical setting, they should include anti- ET_B potency and may provide major benefits for lung and renal integrity, quality of life, and survival of SCD patients.

Figure 1

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Effect of BQ123 and BQ788 on neutrophils recruitment. (A) Representative images of cremasteric venules from SAD mice after local TNFa stimulation associated or not with specific blocking of ET_A (BQ123) or ET_B (BQ788) receptors. (B) Representative images of labeled PMNs (in green) that have gradually adhered to the endothelial cells. The three microfluidic channels were infused with the same batch of whole blood from a SCD patient without treatment (NT) or preincubated with BQ123 or BQ788

Figure 1

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Effect of BQ123 and BQ788 on neutrophils recruitment. (A) Representative images of cremasteric venules from SAD mice after local TNFa stimulation associated or not with specific blocking of ET_A (BQ123) or ET_B (BQ788) receptors. (B) Representative images of labeled PMNs (in green) that have gradually adhered to the endothelial cells. The three microfluidic channels were infused with the same batch of whole blood from a SCD patient without treatment (NT) or preincubated with BQ123 or BQ788

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