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 5

Erythrocyte MPs from SCD patients activated complement in a heme-dependent, Hx-sensitive manner.

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Erythrocyte MPs from SCD patients activated complement in a heme-depende...
(A and B) NHS was incubated with 1,000 MV/μl RBC MVs from HDs (n = 12) or SCD patients (n = 12) diluted in TBS. After 30 minutes at 37°C, the levels of released Ba (A) and sC5b-9 (B) were measured by ELISA. **P < 0.005, Mann-Whitney test. (C) Linear correlation between measured Ba and sC5b-9 levels with RBC MPs from SCD (n = 12) in tested samples. R2 = 0.4811; P = 0.0085. (D and E) NHS was incubated with 1,000 MV/μl MVs from SCD patients (n = 12) diluted in TBS with or without 25 μM Hx. After 30 minutes at 37°C, the levels of released Ba (D) and sC5b-9 (E) were measured by ELISA. *P < 0.05, ***P < 0.001, Wilcoxon test after a Shapiro-Wilk test for normality. (F and G) HUVECs were treated with 1,000 MV/μl RBC MPs from HDs (n = 4) or SCD patient (n = 7) diluted in M199 medium for 30 minutes at 37°C. Without removing the supernatant, HUVECs were exposed to NHS (final concentration at 33% diluted in M199 FCS-free) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition by flow cytometry. (F) Flow cytometry histograms. (G) Quantitative analyses. *P < 0.05, Mann-Whitney test. (H) HUVECs were treated with 1,000 MV/μl RBC MPs from 1 SCD patient with or without 5 μM Hx. As above, NHS was added and cells were stained for C3 deposition by flow cytometry. Values are shown as box plots with median and minimum/maximum points.