Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

Abstract

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Complement activation in sickle cell disease: Dependence on cell density, hemolysis and modulation by hydroxyurea therapy

“…Hemolysis‐derived products or anti‐endothelial antibodies present in the serum could explain this activation. This complement over activation is concordant with the complement‐mediated cytotoxicity from SCD patients' sera (modified Ham's test), the enhanced C3 deposits on ECs exposed to SCD RBC microvesicles, and the endothelial C3 deposits in tissues of SCD mice and biopsies of patients . This over activation is indeed at least in part heme‐dependent since it was inhibited by the heme scavenger hemopexin.…”

“…The mechanism of this complement activation is not yet fully elucidated. in vitro studies and mouse models have provided evidence that the activation of the cascade in SCD could proceed by different mechanisms: on ischemic tissue, via cell‐free heme/heme‐carrying RBC microvesicles, or directly on the surface of the SCD RBCs . The clinical relevance of the progression of the complement cascade to its terminal steps has not been investigated in detail in a large cohort of SCD patients.…”

Critical role of C5a in sickle cell disease

“…Recently, Merle et al found C3b/iC3b and C5b‐9 deposits in the kidneys of SCD patients and two mouse models of SCD, including Townes mice as confirmed here. They induced intravascular hemolysis or injection of heme or hemoglobin in WT mice to provide evidence that these complement deposits could be triggered by cell‐free heme . Heme and heme‐loaded microvesicles induced activation of the alternative complement pathway and heme triggered endothelial expression of P‐selectin, C3aR and C5aR and down regulation of CD46.…”

“…Ischemia‐reperfusion physiology underpins SCD biology when vessels are transiently occluded in organs throughout the body and subsequently reopened . In this ischemia‐reperfusion paradigm, the innate immune system plays a key role in promoting the inflammatory response, which includes TLR4 signaling and complement activation …”

“…Several studies suggested that complement activation takes place through the alternative pathway, which would be triggered by the membrane phospholipid changes that occur in SS red blood cells (RBCs) . Moreover, recent investigations have suggested that alternative pathway activation may be increased by a high blood concentration of phosphatidylserine‐rich microparticles as may occur in SCD; alternative pathway activation can also be demonstrated in stored RBCs and this pathway is activated due to heme released by lysed RBCs . Therefore, these studies of SCD patients showing evidence of complement activation suggest that complement, another major component of innate immunity, may play a fundamental role in the pathophysiology of SCD.…”