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E. Lombardi et al.
adhesion of SCD RBC to TNF-a-activated endothelium when compared to normal RBC (Figure 2A, Online Supplementary Video S1). We next investigated the effect of FH on the adhesion of healthy or SCD RBC to activated vascular endothelium. The dose-response curve with FH showed a significant reduction in RBC adhesion at con- centrations ≥9 nM (Figure 2B). This was more pronounced for SCD RBC than for healthy RBC (Figure 2B). We, there- fore, expanded the tests to include a larger number of SCD patients using FH concentrations of 9 or 18 nM; as expect- ed, reduced adhesion of SCD RBC to the vascular endothelium was observed in the presence of either 9 nM or 18 nM FH (Figure 3A). Previous studies have identified two major regions on FH (i.e., domains 6-8 and 19-20) as the putative sites of interaction with cell surface con-
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stituents such as glycosaminoglycans; furthermore, domains 19-20 are also responsible for binding to sialic acids or C3d-containing deposits.29 We, therefore, evaluat- ed whether any of these segments had activity similar to that of full-length FH.
Figure 1. The adhesion of C3d-positive sickle red blood cells is prevented by factor H. (A) Plasma samples from healthy controls (AA) and patients with sickle cell disease (SCD) were tested by enzyme-linked immunosorbent assay (see Methods section); *P<0.05 AA versus SCD; n=10 in each group. (B) Deposition of C5b-9 (orange fluorescence) assessed by immunofluorescent staining involving the abluminal aspect of the microvasculature in apparently normal skin of a patient with SCD (direct immunofluorescence; original magnification x100). Inset. Detail of the vessels showing intense granular deposition of C5b-9; direct immunofluorescence; original magnification: x400. Nuclei were stained with Prolong Gold antifade reagent with DAPI (blue fluorescence). The image shown is one representative image of 16 others with similar results. Lower panel. The percentage of vessels positive (+) for C5b9 granular deposition in skin biopsies from healthy subjects (AA) and sickle cell patients (SCD). Data are shown as means ± SD **P<0.002 AA versus SCD. (C) Left panel. Representative immunohistochemical image of a normal skin biopsy from a SCD patient showing a small vessel in the superficial dermis (arrow) characterized by co-expression of C5b9 (brown) and CD31 (red); for comparison a C5b9- negative vessel (circle) only decorated with CD31 staining (red) is highlighted. One representative image is shown; all 16 gave similar results (n=16). Right panel. Quantification of C5b9-positive vessels in skin biopsies from healthy subjects (see Online Supplementary Figure S2) and SCD patients. Data are shown as means ± SD; **P<0.01 AA versus SCD. (D) Percentages of C3d+ red blood cells (RBC) in healthy donors (AA) and in sickle cell subjects (n=16 AA; n=16 SCD). The dashed line indicates the threshold of normality, corresponding to C3d+ <0.5% of RBC; *P<0.05 AA versus SCD; **P<0.01 AA versus SCD. (E) Percentages of phosphatidylser- ine-positive (PS+) RBC in healthy donors (AA) and in sickle cell subjects (n=20 AA; n=32 SCD). The dashed line indicates the threshold of normality, corresponding to PS+ <0.5 % of RBC; **P<0.01 AA versus SCD.
Perfusion experiments performed on TNF-a-activated endothelium demonstrated that, like intact FH, FH19-20 (18 nM) strongly prevented the adhesion of sickle RBC to the surface of TNF-a-activated vascular endothelium. Conversely, FH6-8 showed a trend, which did not reach statistical significance, toward a reduction in RBC adhe- sion (Figure 3B). Our data indicate that FH prevents sickle cell adhesion to the activated endothelium through its interaction with cell-surface sialic acids and C3b/iC3b found on the surface of pathological RBC (Figure 3B).
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haematologica | 2019; 104(5)