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 2

Hemolysis induced heme-dependent complement deposition in kidney in vivo.

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Hemolysis induced heme-dependent complement deposition in kidney in vivo...
(A) C3b/iC3b (false green color) staining of frozen kidney sections of mice injected with PBS or PHZ and sacrificed at 6 hours. Quantification of C3 fragment staining in glomeruli (n ≥ 5 mice per group). (B) Double staining of PHZ-injected mouse kidney sections for C3b/iC3b (false color green) and endothelial marker vWF (false color red). The merge image indicates colocalization (white arrows) in orange. (C–E) Evaluation of the renal injury in WT and C3–/– mice (n = between 4 and 12 mice per group) injected with PBS or PHZ by analyzing gene expression by RTqPCR. (C) NGAL. (D) Kim-1. (E) HO-1. (F) Comparison of the quantification of the C3 fragment staining in glomeruli of mice injected with PBS, PHZ, heme, or hemoglobin (Hb) (n ≥ 10 mice per group). (G) C3b/iC3b (false green color) staining and quantification in glomeruli of frozen kidney sections of mice injected with PBS or PHZ, with or without hemopexin (Hx) at 24 hours after treatment. (H) C3b/iC3b (false green color) staining and quantification in glomeruli of frozen kidney sections of mice injected with PBS or PHZ, with or without haptoglobin (Hp) at 24 hours after treatment (n ≥ 4 mice per group). (I and J) C3b/iC3b staining (false color green) of kidney sections of mice injected with heme (I) or Hb (J), with or without Hx. Quantification of C3 staining in glomeruli (n ≥ 4 mice per group). * P < 0.05, ** P < 0.005, Mann-Whitney test (A, I, and J); Kruskal-Wallis with Dunn’s test for multiple pairwise comparisons (F–H); 2-way ANOVA with Tukey’s test for multiple comparisons (C–E). Values are shown as box plots with median and minimum/maximum points. Scale bar: 50 μm.