Page 75 - 2019_05-HaematologicaMondo-web

Complement is involved in sickle red cell adhesion
to determine P-selectin and Mac-1 expression are given in the Online Supplementary Methods.
Statistical analysis
All calculations were performed using the IBM SPSS 20.0 statis- tical package (IBM Inc., Armonk, NY, USA). The level of aggrega- tion was expressed as median values with the minimum-maxi- mum range and shown by means of either dot or box plots. Data were analyzed with non-parametric tests, the Mann-Whitney U test for unpaired samples and the Wilcoxon signed-rank test for paired samples. A P value <0.05 was considered statistically signif- icant.
Results
Patients with sickle cell disease show activation
of the alternative complement pathway and increased C3d-positive red blood cells
Untreated SCD subjects at steady state were studied. As shown in Table 1, reduced hemoglobin, increased reticu- locyte counts and raised levels of plasma lactate dehydro- genase, all signs of chronic hemolytic anemia, were observed. In SCD patients, we also found significant increases of the levels of plasma C-reactive protein and surface vascular cell adhesion molecule-1, indicating the presence of a chronic inflammatory vasculopathy in agree- ment with previous reports.2,3,9,43 Serum C3 and C4 levels were similar in healthy and SCD subjects (data not shown); whereas levels of the complement activation fragment C5a were significantly elevated in SCD patients when compared to the levels in healthy individuals (Figure 1A); statistical analyses were performed to exclude the possible contribution of confounding factors such as gender or smoking status. Our finding is consistent with previous reports, and confirms the substantial complement activa- tion in SCD patients, likely via the AP.15-22
Since studies in atypical hemolytic uremic syndromes have shown that skin biopsy might be a feasible method for documenting AP activation by C5b-9 vascular deposi- tion,32 we obtained skin biopsies from SCD patients at steady state. We considered skin to be an interesting win- dow of observation in SCD, since (i) it might be involved in the clinical manifestations of SCD, such as leg ulcers; and (ii) it has been widely used in SCD mouse models to functionally characterize the microvasculature.44-47 As shown in Figure 1B, we observed microvascular intense, focal granular deposition of C5b-9 in small vessels throughout the dermis of SCD patients. This pattern is similar to that reported in skin biopsies from patients with atypical hemolytic uremic syndrome, which is a throm- botic microangiopathy related to complement dysfunc- tion.32 No deposition of C5b9 was observed in skin from healthy controls (Online Supplementary Figure S1A,B). Co- localization of C59b deposits and CD31+ skin vessels was confirmed by immunohistochemical staining only in the skin biopsies from SCD patients (Figure 1C, Online Supplementary Figure S2A,B).
Taken together, our data indicate an activation of the AP in SCD, with possible involvement of complement in SCD vasculopathy and in related cellular adhesion events.
To understand whether AP activation occurs directly on sickle RBC, we measured the amounts of circulating RBC carrying C3-derived opsonins by detecting the presence of the common C3d fragment.11,12 As shown in Figure 1D,
Table 1. Demographic, hematologic and biochemical data for healthy subjects and patients with sickle cell disease.
Parameters
Age (years)
Gender (male/female) (n) Smokers (%) (n)
Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Hemoglobin (g/dL)
C-reactive protein (mg/L) sVCAM-1 (pg/mL)
Healthy subjects (n=29)
30.0 (25.0-42.5)
11/18
10.3% (3)
120 (110-130)
65 (58-77)
13.4 (12.5)
2 (1.8-2.1)
44.6 ±12
4.5 (3.8-5.2)
0.8 (0.7-1.0)
325.0 (261.5-413.0)
42.3 (39.2-46.7)
0.9 (0.2-12.4)
280 ±14
SCD patients (n=29)
20.0 (17.0-47.0)* 10/19
6.8% (2)
120 (112-130)
64 (60-76)
8.5 (8-9.5)*
4.5 (2.5-7.6)* 250 ±27*
10.2 (8.8-11.9)* 0.7 (0.3-1.5) 484.0 (310.2-1104.2)* 44.3 (35.6-49.7)
3.0 (1.0-15.8)* 820±36*
HbF (%)
Reticulocytes (cells * 103/mL) 9
White blood cells (10 cells/mL) Creatinine (mg/dL)
Lactate dehydrogenase (U/L) Albumin (g/L)
HbF: fetal hemoglobin; sVCAM-1: serum vascular adhesion molecule-1. Ranges are shown in parentheses. Bold * P<0.05 compared to healthy subjects.
higher numbers of C3d+ RBC were found in SCD patients in the steady state than in healthy subjects (representative scatter-plots are shown in Online Supplementary Figure S3). This fraction further increased in a subgroup of SCD patients during acute pain crises (SS steady state 2.5±0.8% versus SS pain crisis: 6.1±0.7% P<0.02; n=10), in agree- ment with an observation by Mold et al.18 Since a previous report linked activation of the AP with deposition of C3 opsonins to the exposure of phosphatidylserine on SCD RBC surfaces, we evaluated the percentage of phos- phatidylserine-positive RBC in our SCD patients17 and did indeed find higher percentages of phosphatidylserine-pos- itive RBC in SCD patients than in healthy subjects in agreement with previous studies35,36 (Figure 1E). In contrast to paroxysmal nocturnal hemoglobinuria, in which opsonization leads to rapid lysis of affected RBC.11,12 the presence of functional membrane regulators on SCD ery- throcytes allowed a low level of C3-fragment deposition, which might also be considered as a marker of comple- ment activation on sickle RBC. We, therefore, reasoned that the presence of C3b, iC3b or C3dg may function as a site of adhesion of SCD RBC to activated vascular endothelial surfaces, which may carry ligands for C3 frag- ments, such as Mac-1 and P-selectin.48,49 To study this pos- sibility, we used the C3b and iC3b binding plasma protein FH as an inhibitor25-27 and saw no significant differences in FH serum levels between healthy subjects and SCD patients [AA median: 841 (range 616-1528) mg/mL versus SCD median: 980 (741-1301) mg/mL; P=NS].
Factor H prevents the adhesion of sickle red blood cells to tumor necrosis factor-a-activated vascular endothelium
We performed an ex vivo adhesion assay using TNF-a- activated vascular endothelial cells and monitored, in real time, the adhesion of RBC collected from healthy and SCD subjects to inactive (no TNF-a) or activated endothe- lium (plus TNF-a) as previously described.38-40 Consistent with the literature, we observed significantly increased
haematologica | 2019; 104(5)
921