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E. Lombardi et al.
impaired blood flow. Vaso-occlusive events in the micro- circulation result from a complex and only partially under- stood scenario involving interactions between different cell types. These cells include dense, dehydrated sickle cells, reticulocytes, abnormally activated endothelial cells, leukocytes and platelets.1-4 Plasma factors such as coagula- tion system cytokines and oxidized pro-inflammatory lipids may also be involved. In addition, cyclic polymer- ization-depolymerization promotes RBC membrane oxi- dation and reduces RBC survival in the peripheral circula- tion.1,5,6 The resulting increase in free hemoglobin and free heme, a consequence of the saturation of the physiologi- cal system and local reduction of nitric oxide bioavailabil- ity, leads to a pro-coagulant state with increased risk of thrombotic events.2,3,7-10 All this evidence indicates that sickle cell vasculopathy is a crucial player in RBC adhesion and in the development of acute vaso-occlusion in SCD patients.
Although progress has been made in recent decades in understanding the pathogenesis of SCD, the molecular events involved in these processes are still only partially delineated. Whereas a key role for complement activation has been highlighted in chronic inflammatory processes characterized by hemolysis and inflammatory vasculopa- thy such as atypical hemolytic uremic syndromes and paroxysmal nocturnal hemoglobinuria11-14 the involvement of complement in SCD has been less extensively explored. Previous studies have revealed: (i) an activation of the alternative complement pathway (AP) in SCD patients; (ii) a reduction in the activating proteases factors B and D, modulating complement activation; (iii) a decrease in the plasma levels of factor H (FH), the major soluble regulator of AP activation; and (iv) increased deposition of the com- plement opsonin C3b on RBC exposing phosphatidylser- ine.15-22 Preliminary data from a mouse model of SCD sug- gest a possible role for complement activation in the gen- eration of vaso-occlusive crises, as an additional disease mechanism contributing to the severity of acute clinical manifestations related to SCD.23,24
Because of its potential detrimental effects on host cells, the AP is finely regulated by membrane-bound and solu- ble regulators. Circulating FH plays a particularly impor- tant role, since this regulator not only binds to C3b and prevents the formation of C3b convertases, but it is also able to recognize self-associated molecular patterns such as sialic acid and glycosaminoglycans present on the membranes of most healthy cells.25-27 Any interference with this recognition process, resulting from either poly- morphisms or blocking antibodies against FH, may have severe pathological consequences as described for atypical hemolytic uremic syndromes and other complement- mediated disorders.28
Here, we found that sickle RBC are characterized by membrane deposition of C3b, which acts as a marker for the activation of the AP on sickle RBC. We sought to determine whether C3b deposition on RBC might possi- bly stimulate vaso-occlusive crises by favoring cell-cell interactions. Indeed, we now demonstrate for the first time a peculiar ex vivo motion profile (“stop-and-go” behavior) of SCD red cells during their transit on vascular endothelial surfaces, a motion that prolongs their transit on the vascular endothelial surface and promotes the adhesion of sickle RBC. We show that FH and its 19-20 domain,29,30 which primarily targets C3b, prevent the adhesion of sickle RBC to the endothelium. We further
document that FH acts by preventing the adhesion of sick- le RBC to P-selectin and/or the receptor Mac-1 (CD11b/CD18). Our data provide a rationale for further investigation of FH and other modulators of the AP as novel disease-modifying molecules with potential impli- cations for the treatment of the clinical manifestations of SCD.
Methods
Study design
We studied SCD subjects (n=29; 26 SS and 3 Sβ0) referred to the Department of Medicine, University of Verona and Azienda Ospedaliera Integrata of Verona (Italy) between January 2012 and January 2017. SCD patients were evaluated at steady state, and none of them had been on either hydroxyurea or a transfusion reg- imen during the 6 months immediately prior to our analysis. Healthy controls were matched by age, sex and ethnic back- ground. The study was approved by the Ethical Committee of the Azienda Ospedaliera Integrata of Verona (Italy) and informed con- sent was obtained from patients and healthy controls (ethical approval FGRF13IT). Table 1 shows the demographic characteris- tics of both the healthy subjects and SCD patients studied. Biochemical and hematologic parameters as well as plasma levels of C3 and C4 were determined according to clinical and laborato- ry standards at the Laboratory of Medicine, University of Verona and Azienda Ospedaliera Integrata of Verona (Italy). Plasma C5a (EIA Quidel Corp., San Diego, CA, USA), plasma vascular cell adhesion molecule-1 (Invitrogen, Carlsbad, CA, USA) and serum FH (Hycult Biotech, Uden, the Netherlands) were determined by enzyme-linked immunosorbent assays, according to the manufac- turers’ protocols.31
Evaluation of C5b-9 complement deposition on fixed skin biopsies
Skin punch biopsies were carried out on the volar surface of the left arm on apparently normal skin. The samples were paraffin- embedded and stained with hematoxylin and eosin before exam- ination; they were also used to estimate the presence of C5b-9 complement deposition within the microvasculature.32 Details on the immunofluorescent and immunohistochemical staining assays are reported in the Online Supplementary Methods.32-34
Measurements of phosphatidylserine-positive and C3d-positive red blood cells
Phosphatidylserine-positive cells were detected as previously reported.35-37 Details are given in the Online Supplementary Methods.
Red blood cell adhesion assay
The real-time adhesion of RBC collected from healthy and SCD subjects on inactive or activated endothelium [in the absence or presence of tumor necrosis factor-alpha (TNF-a), respectively] was determined as previously reported38-40 with or without FH or the FH19-20 or FH68 domains.41 Details are provided in the Online Supplementary Methods.
Development of an algorithm to determine red blood cell transit and flux trajectory
The algorithm to determine RBC transit and flux trajectory is described in the Online Supplementary Methods.
P-selectin and Mac-1 expression in vitro in vascular endothelial cells
Details on immunoblotting42 and the cytofluorimetric analysis
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haematologica | 2019; 104(5)