Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles
Nicolas S. Merle, Anne Grunenwald, Helena Rajaratnam, Viviane Gnemmi, Marie Frimat, Marie-Lucile Figueres, Samantha Knockaert, Sanah Bouzekri, Dominique Charue, Remi Noe, Tania Robe-Rybkine, Marie Le-Hoang, Nathan Brinkman, Thomas Gentinetta, Monika Edler, Sara Petrillo, Emanuela Tolosano, Sylvia Miescher, Sylvain Le Jeune, Pascal Houillier, Sophie Chauvet, Marion Rabant, Jordan D. Dimitrov, Veronique Fremeaux-Bacchi, Olivier P. Blanc-Brude, Lubka T. Roumenina
Nicolas S. Merle, Anne Grunenwald, Helena Rajaratnam, Viviane Gnemmi, Marie Frimat, Marie-Lucile Figueres, Samantha Knockaert, Sanah Bouzekri, Dominique Charue, Remi Noe, Tania Robe-Rybkine, Marie Le-Hoang, Nathan Brinkman, Thomas Gentinetta, Monika Edler, Sara Petrillo, Emanuela Tolosano, Sylvia Miescher, Sylvain Le Jeune, Pascal Houillier, Sophie Chauvet, Marion Rabant, Jordan D. Dimitrov, Veronique Fremeaux-Bacchi, Olivier P. Blanc-Brude, Lubka T. Roumenina
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Research Article Inflammation Vascular biology

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.

Authors

Nicolas S. Merle, Anne Grunenwald, Helena Rajaratnam, Viviane Gnemmi, Marie Frimat, Marie-Lucile Figueres, Samantha Knockaert, Sanah Bouzekri, Dominique Charue, Remi Noe, Tania Robe-Rybkine, Marie Le-Hoang, Nathan Brinkman, Thomas Gentinetta, Monika Edler, Sara Petrillo, Emanuela Tolosano, Sylvia Miescher, Sylvain Le Jeune, Pascal Houillier, Sophie Chauvet, Marion Rabant, Jordan D. Dimitrov, Veronique Fremeaux-Bacchi, Olivier P. Blanc-Brude, Lubka T. Roumenina

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Figure 6

Schematic view of different nonexclusive mechanisms leading to complement activation during hemolysis.

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Schematic view of different nonexclusive mechanisms leading to complemen...
Extracellular heme can serve as an enhancer of the pathological process in SCD and other hemolytic diseases. (i) Heme activates EC by its proinflammatory and prooxidant effects. (ii) Heme induces hydrolysis of C3 into C3(H2O) and provokes direct complement activation in the serum. (iii) C3b generated by the fluid-phase C3 convertase will bind to the EC membrane and will form new AP C3 convertases that can locally autoamplify and generate C3b and C5b-9 deposition. (iv) Released C3a and C5a can bind to C3aR and C5aR to mediate proinflammatory processes. In the context of SCD, sickle RBCs will generate more heme-positive MVs. They carry heme, which will activate complement in serum and also on the EC surface. We hypothesize that this complement activation will contribute to the vascular injury and tissue damage in SCD. Hx was able to block, partially or completely, the hemolysis-induced complement activation in vitro and in vivo.