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Complement is involved in sickle red cell adhesion
endothelial cells:2,50-55 (ii) P-selectin has been reported to bind C3b present on the membranes of circulating cells such as platelets;56 and (iii) Mac-1 is a well-known receptor for iC3b, and potentially C3dg associated with the cell membrane.25 In addition, P-selectin has been reported to bind RBC, targeting red cell plasma-membrane sialic acid, whereas the presence of a Mac-1 binding site on RBC membranes is still under investigation.29,48,51,57 In our model, the expression of P-selectin and Mac-1 was significantly increased on TNF-a-activated vascular endothelium when compared to the expression on vehicle-treated cells (Online Supplementary Figure S4A). Both anti-P-selectin and anti-Mac1 antibodies prevented the adhesion of sickle RBC to the activated vascular endothelium (Figure 5A). Whereas the effect of the anti-P-selectin antibody on RBC adhesion may be mediated through interference with two different targets (i.e., iC3b and/or sialic acid), the interplay between Mac-1 and iC3b deposited on sickle RBC is con- sidered to be more selective.25,58 The anti-adhesive effect of the anti-Mac-1 antibody was more pronounced than that of the anti-P-selectin antibody, but the difference was not statistically significant. No effect on the adhesion of sickle RBC was documented in the presence of a control anti- body (Online Supplementary Figure S4B). Collectively, these findings indicate that C3b/iC3b is deposited on sickle RBC membranes as a new ligand, bridging sickle RBC to the activated vascular endothelial surface through binding to the pro-adhesive molecules P-selectin and/or Mac-1.
Discussion
In this study, we confirmed activation of the AP in SCD and demonstrated, for the first time, cutaneous vascular deposition of C5b-9, supporting the involvement of com- plement in the microvascular injury associated with SCD.
AB
We then showed that activation of the AP results in C3 split-fragments being bound to the sickle RBC surface, thereby contributing to the adhesion of RBC to the inflammatory activated vascular endothelial surface. Notably, decoration of erythrocytes with C3 opsonins was more evident in patients undergoing vaso-occlusive crises, further supporting the involvement of complement in microvascular injury in SCD. Since sickle RBC, in con- trast to paroxysmal nocturnal hemoglobinuria RBC, still contain the functional complement regulators CD55 and CD59, phosphatidylserine-mediated complement activa- tion may allow for a certain degree of opsonization with- out inducing hemolysis. This situation would lead to the observed accumulation of C3d-containing opsonins (i.e., C3b, iC3b, C3dg), which have all been associated with cell interactions and signaling functions.59 Thus, we pro- pose that C3 split-fragments on RBC might favor cell-cell interactions, supporting previous observations of reduced RBC-cell interactions in mice genetically lacking the C3 complement fraction.51,60
We also demonstrated here that FH prevents the adhe- sion of sickle RBC and normalizes their trajectory and transverse velocity on TNF-a-activated vascular endothe- lial surfaces through a mechanism involving P-selectin and/or Mac-1 as pro-adhesion molecule(s). FH binds to C3b/iC3b molecules present on cell surfaces and inhibits the AP. The algorithm developed in the present study allowed us to go further in describing RBC adhesion to the endothelial surface. Indeed, we showed that SCD RBC adhere to endothelial cells with a peculiar dynamic behav- ior not shown by healthy RBC. The stop-and-go motion of sickle RBC contributes to the irregular trajectory of these cells during their transit on the vascular endothelial surface. This slow and irregular movement clearly affects the speed transition time of sickle RBC when compared to healthy control RBC, possibly contributing to the reduc-
Figure 5. The anti-adhesive effect of factor H involves P-selectin and Mac-1 pro-adhesive molecules. (A) Adhesion of sickle cell disease red blood cells (SCD RBC) after 6 min of perfusion on activated or non-activated endothelium (± TNF-a) pre-coated with either anti-P-selectin antibody or anti-Mac1 (CD11b/CD18) antibody. Data shown are representative of six other independent assays with similar results. Wilcoxon test: *indicates the corresponding significance. Adhesion is expressed as median values with a minimum-maximum range and illustrated by box plots. A value of P<0.05 is considered statistically significant. Statistical analysis as in Figure 2A. (B) Schematic model of the beneficial action of factor H in reducing adhesion of C3-derived opsonins on sickle RBC to the TNF-a-activated vascular endothelium. C3 split-fragments on erythrocytes might favor cell-cell interactions through P-selectin and Mac-1. P-selectin might bind RBC through two different tar- gets, iC3b and/or sialic acid; in contrast, Mac-1 targets only iC3b deposits on sickle RBC as a more selective interaction. FH and FH19-20 segment normalized the transit of sickle RBC across the TNF-a-activated vascular endothelial surface, abolishing the “stop-and-go” behavior of the sickle RBC. This effect positively affected (shortened) the transit time of sickle RBC, thereby reducing the likelihood of the RBC sickling during their transit through the microcirculation AP: alternative com- plement pathway; SCD: sickle cell disease; PS: phosphatidylserine; FH: factor H.
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