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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Pediatrics, Division of Research
Hematology, Jefferson Medical College, Thomas Jefferson University;
Division of Hematology, St Christopher's Hospital for Children,
MCP-Hahnemann University School of Medicine; and The Marian Anderson
Comprehensive Sickle Cell Center; all of Philadelphia, PA.
To assess whether fetal hemoglobin (HbF) modulates the
adhesion of sickle erythrocytes to endothelium, children with
homozygous sickle cell anemia (SS disease) were studied, using
this physiologically crucial period to evaluate the relationships
between HbF and the major erythrocyte adhesion markers. The mean level
of CD36+ erythrocytes was 2.59% ± 2.15% (± SD,
n = 40) with an inverse relationship between CD36 positivity and F
cells (R = Although the polymerization of hemoglobin S
(HbS) in the deoxygenated state in sickle cell disease (SCD) is the
watershed event leading to vaso-occlusion, factors that retard the
transit time of sickle erythrocytes in the microcirculation, including the cell's adhesive properties, also play a crucial role in this process.1 Since the initial observation by Hebbel et al
that sickle red cell-endothelial adherence is correlated with clinical disease severity,2 the last 2 decades have seen a stepwise elucidation of the surface adhesion-related molecules and markers present on the erythrocyte and endothelium that play a role in this
process.3,4 Important adhesion-related receptors on the
sickle erythrocyte include the integrin
A high level of fetal hemoglobin (HbF) in SCD correlates with
increased life expectancy and a decrease in the incidence of vaso-occlusive crises.24 The clinical benefits of
hydroxyurea therapy in decreasing the rate of painful crises are
attributed, for the most part, to its ability to increase the level of
HbF. However, a variety of other potential red cell-related modes of action have been postulated, including effects on erythrocyte hydration, cation transport, cell deformability, a decrease in erythrocyte-endothelial adhesion, and a decrease in the expression of
erythrocyte adhesion-related markers, including VLA4 and
CD36.25-30 Although some of these mechanisms appear to be
related to HbF (eg, cell hydration effects),25 in other
instances the changes seemingly have occurred prior to a substantial
increase in HbF levels (eg, adhesion marker-related
events).29,30 Because infants with SCD manifest high
levels of HbF, we have used this physiologically important period to
evaluate whether potentially important relationships do indeed exist
between HbF, the major adhesion-related markers CD36 and VLA4, and the
adhesion process. Such relationships might serve to extend our
knowledge regarding the protective effects of HbF in the
pathophysiology of SCD.
Materials
Collection of blood
Flow cytometric analysis of F cells F cells were analyzed as previously described.31,32 In brief, a sample of packed red cells (20-50 µL) was fixed with 4% formaldehyde (wt/vol) in Dulbecco phosphate-buffered saline (DPBS) for 45 minutes at room temperature. The fixed cells were then permeabilized by treating sequentially at 20°C with 1 mL acetone:water (1:1,
vol/vol), acetone, and acetone:water (1:1, vol/vol). One million fixed
and permeabilized red cells (in a total volume of 100 µL) were
incubated with 5 µL FITC-labeled anti-HbF for 30 minutes at room
temperature in the dark, washed, and suspended in 1 mL DPBS. The cells
were then analyzed immediately in a Becton Dickinson flow cytometer
(Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with
a 15 mW, 488 nm, air-cooled argon-ion laser and formatted for one-color
analysis at a flow rate of 300 to 500 cells per second. Data from
50 000 events were collected and analyzed using CellQuest software
(Becton Dickinson). Nonspecific membrane immunofluorescence was
determined using fixed, permeabilized red cells stained with
FITC-labeled negative isotypic control antibody.
Flow cytometric analysis of adhesive receptors on erythrocytes One million washed red cells (suspended in a total volume of 100 µL DPBS containing 2% fetal calf serum and 0.1% sodium azide) were incubated with 20 µL PE-labeled antiglycophorin A and 20 µL of either FITC-labeled anti-CD36, anti-CD49d, anti-CD71, or negative isotypic control antibody for 30 minutes at room temperature. The incubation mixture was diluted with 1 mL DPBS, and cells were pelleted at 300g for 10 minutes. Following an additional wash in DPBS, cells were suspended in 1 mL DPBS and analyzed immediately in a Becton Dickinson flow cytometer formatted for 2-color analysis. Fluorescence compensation settings were established using appropriate controls, including cells stained with PE- and FITC-labeled isotopic-negative control antibodies, antiglycophorin A-PE alone, anti-CD36-FITC alone, anti-CD49d-FITC alone, or anti-CD71-FITC alone. Data from 50 000 events were collected for analysis. Percent CD36+, CD49d+, or CD71+ red cells, defined as antiglycophorin A+ events simultaneously stained for CD36, CD49d, or CD71, were determined by setting quadrants on the FL1 (CD36, CD49d, or CD71) and FL2 (glycophorin A) dot plot. Events not staining for glycophorin A did not exceed 0.5%. For the calculation of percent receptor-positive red cells, these latter events were gated out by analysis. Nonspecific membrane immunofluorescence in the double-positive area was determined using cells stained with PE-labeled antiglycophorin A and FITC-labeled negative isotypic control antibody, and these values were subtracted from the respective sample fluorescence.Flow cytometric analysis of adhesive receptors on F cells and non-F cells Anti-CD71-PE-, anti-CD49d-PE-, or anti-CD36 plus antimouse IgG- PE-labeled washed red cells were fixed, permeabilized, and stained with anti-HbF-FITC as described above and analyzed in a flow cytometer formatted for 2-color analysis. Non-red cell-associated events were gated out as described above, and the marker-positive F cells and non-F cells in the erythrocyte region were then determined using dot plots of FITC and PE fluorescence.Red cell adhesion assay Adherence of sickle red cells to endothelial cell monolayers was evaluated using 51Cr-labeled sickle red cells and human retinal capillary endothelial cells (HRCECs) and/or fetal bovine aortic endothelial cells (FBAECs) in the adhesion assay of Hebbel et al.2 51Cr-labeled sickle erythrocytes were prepared as previously described.33 HRCECs were obtained from human retinas by collagenase digestion, identified, and cultured in minimal essential medium supplemented with 10% fetal calf serum as previously described.34 FBAECs were isolated, identified, and cultured in minimal essential medium supplemented with 10% fetal calf serum as previously described.35 Endothelial cells were plated at a density of 200 000 cells per well into wells of 12-well plates, grown to confluence, and then coincubated with 51Cr-labeled sickle red blood cells. Adhesion assays were conducted in the absence of plasma (basal adhesion) and in the presence of 10% autologous plasma (plasma-induced adhesion) at 37°C for 45 minutes, and the nonadherent red blood cells were removed. The monolayers were then washed, and adherent red blood cells were determined by 51Cr release following cell lysis. 51Cr-labeled control red cells were concomitantly evaluated in adhesion assays in the absence of plasma. The adhesinogenic potential of the patient's red cells was expressed as an adhesion ratio, which was determined by dividing the number of adherent sickle red cells by adherent control red cells.Analysis of hemoglobin, white blood cells, and platelets Hemoglobin values, WBC counts, and platelet counts were obtained by routine measurements in a Coulter Counter, model Stk S.Data analysis Statistical evaluation was performed using the Sigmastat Statistical Package (Jandel Scientific, San Rafael, CA). All results are presented as the mean ± SD. Both Pearson and Spearman correlation tests were employed to determine the relationship between 2 variables. Both forward and backward stepwise regression analyses were performed in multiple regression models to determine the dependency of the expression of sickle erythrocyte adhesion markers and adhesion on a variety of hematologic parameters.
CD36+ erythrocytes and their relationships CD36+ red cells were absent or present at a very low concentration in blood from control donors, with values remaining at approximately the same level for all ages evaluated (0.05% ± 0.05%, n = 14, mean ± SD). In contrast, in patients with SS disease, a positive correlation was noted between age and CD36 positivity (Figure 1A, R = 0.56, P < .0002, n = 40). Although levels of CD36 positivity in infants with SS disease younger than 5 months of age were comparable to control values, significant increases were noted in older children (2.59% ± 2.15%). A striking inverse relationship was also noted between the levels of CD36+ sickle red cells and F-cell numbers (Figure 1B, R =0.76,
P < .000 00 002). No such relationship was observed
with control red cells (R = 0.03, P > .90,
n = 14). In univariate analyses, correlations were noted between the
levels of CD36+ sickle erythrocytes and various other
hematologic parameters, including hemoglobin (R = 0.71,
P < .000 0005), WBC count (R = 0.58,
P < .0001), and platelet count (R = 0.37,
P < .02), as assessed using the Pearson test. Multiple
regression analyses were therefore performed using both forward and
backward regression models to determine the parameter(s) that modulated
the levels of CD36+ red cells. In these regression models,
percent CD36+ erythrocytes was entered as the dependent
variable and F-cell number, age, hemoglobin, WBC count, and platelet
count as independent variables. A significant relationship was noted
with F cells (power of the test with  at 0.05 = 1.00), which was
the only independent variable that stayed in both models. Additional
studies (n = 17) were conducted to assess CD36 positivity associated
with the F-cell versus non-F-cell fractions. In these studies,
although a striking positive correlation was noted with the non-F-cell
fraction (Figure 1D, R = 0.84,
P < .000 03), an inverse correlation was observed between the F-cell fraction and CD36+ F-cell numbers
(Figure 1C, R = 0.64, P < .006).
VLA4+ erythrocytes and their relationships VLA4+ red cells were almost absent in the control donors evaluated in our study (0.05 ± 0.04%, n = 14) with no relationships observed with either age or F-cell number (R = 0.20, P > .50, and R =0.13,
P > .60, respectively). The mean level for
VLA4+ red cells measured in patients with SS disease was
0.31% ± 0.45% (n = 40). Relationships similar to those seen with
CD36+ sickle erythrocytes were also noted when the VLA4
data were analyzed for both age and F-cell-related correlates. A
positive relationship was noted between age and VLA4 positivity (Figure
2A, R = 0.43, P < .006), and an inverse correlation was observed with
F-cell number (Figure 2B, R = 0.63,
P < .000 02). In univariate analyses, correlations were
also noted between the levels of VLA4+ sickle erythrocytes
and various other hematologic parameters, including hemoglobin
(R = 0.58, P < .0001) and WBC count
(R = 0.49, P < .002), as assessed using the
Pearson test (n = 40). Multiple regression analyses were therefore
performed to determine the parameter(s) that modulated the levels of
VLA4+ red cells. In these regression models, percent
VLA4+ erythrocytes was entered as the dependent variable
and F-cell number, age, hemoglobin, and WBC count as independent
variables. A significant relationship was noted with F cells (power of
the test with at 0.05 = 0.995), which was the only independent
variable that stayed in both regression models. In a subgroup of SS
patients (n = 33), we additionally assessed VLA4 positivity
associated with both F cells and non-F cells. Although a significant
positive correlation was noted with the non-F-cell fraction (Figure
2D, R = 0.85, P < .000 0001), no
relationship was noted between VLA4+ F cells and the F-cell
fraction (Figure 2C, R = 0.27, P > .10).
CD71+ erythrocytes and their relationships Because stress reticulocytes express the highest levels of adhesive markers, in parallel studies, we assessed this subpopulation using CD71 as the marker. Minimal levels of CD71+ red cells were measured in control donors (0.04% ± 0.03%, n = 14), with no correlation observed either with age or with F-cell number (R =0.14, P > .60, and
R = 0.02, P > .90, respectively). Correlations similar to those seen with CD36+ and
VLA4+ sickle red cells were also noted when the data from
stress reticulocytes were analyzed for age and F-cell-related
correlates. Although values in infants with SS disease were not
significantly different from control values, levels of
CD71+ red cells were increased in the older child
(5.81% ± 4.21%), and a significant positive correlation was noted
with age (Figure 3A,
R = 0.67, P < .000 003, n = 40). A
striking inverse correlation was observed between the levels of
CD71+ sickle erythrocytes and F-cell number (Figure 3B,
R = 0.79, P < .000 00 001). In
univariate analyses, correlations were also noted between the levels of
stress reticulocytes and various other hematologic parameters,
including hemoglobin (R = 0.74,
P < .000 00 006), WBC count (R = 0.60,
P < .000 05), and platelet count (R = 0.32, P < .05), as assessed using the
Pearson test. Multiple regression analyses were therefore performed
using both forward and backward regression models to determine the
parameter(s) that modulated the levels of stress reticulocytes. In
these models, percent CD71+ red cells was entered as the
dependent variable and F-cell number, age, hemoglobin, WBC count, and
platelet count as independent variables. Although both F-cell number
and hemoglobin stayed in the model (R = 0.82,
R2 = 0.67, power of the test with at
0.05 = 1.00), forward regression analyses demonstrated that the
predominant variable that influenced circulating levels of stress
reticulocytes was F-cell number (R = 0.79,
R2 = 0.63, power of the test with at
0.05 = 1.00), with hemoglobin contributing only minimally to the
model ( R2 = 0.04). In a subgroup of
patients (n = 34), we additionally assessed CD71 positivity
associated with both the F-cell and non-F-cell fractions. Although a
significant positive correlation was noted with the non-F-cell
fraction (Figure 3D, R = 0.82,
P < .000 0001), no relationship was noted with the
F-cell fraction (Figure 3C, R = 0.24,
P > .10).
Sickle erythrocyte adhesion and its relationships We have used both HRCECs and FBAECs to evaluate the adhesinogenic potential of sickle red cells. As depicted in Figure 4, using the red cells from 20 patients with SS disease, we assessed both basal and plasma-induced adhesion in simultaneous assays using human retinal capillary endothelium and bovine aortic endothelium and noted a remarkable congruency between the 2 in vitro cell systems (R = 0.77, P < .000 00 001). Because most of the adhesion data from the patients in this study were acquired using bovine endothelial cells, adhesion ratios obtained with the latter cell system were analyzed for relationships with various hematologic parameters. Mean basal and plasma-induced adhesion ratios in the SS patient group were 2.33 ± 2.95 and 6.30 ± 8.42, respectively. Correlations similar to those seen with adhesion markers and stress reticulocytes were also observed when the adhesion data were analyzed for F-cell-related correlates. As shown in Figure 5, a significant inverse relationship was observed between F-cell number and both basal (R =0.54,
P < .0005, n = 39, Figure 5A) and plasma-induced adhesion (R = 0.53, P < .0006, n = 39,
Figure 5B). Similar relationships with F cells were also noted when the
adhesion data from HRCECs were analyzed (R = 0.73,
P < .004, n = 20 for basal adhesion and
R = 0.83, P < .0003, n = 20 for
plasma-induced adhesion). In univariate analyses, correlations were
also noted between adhesion and both adhesion markers (n = 31) and
various other hematologic parameters (n = 39) as assessed using the
Pearson test. Basal adhesion correlated with CD36+ sickle
erythrocytes (R = 0.52, P < .003), stress
reticulocytes (R = 0.54, P < .002),
hemoglobin (R = 0.37, P < .03), WBC count (R = 0.32, P < .05), and age
(R = 0.42, P < .008). Multiple regression analyses were therefore performed using both forward and backward regression models to determine the parameter(s) that modulated basal
adhesion. In the first model, basal adhesion ratios were entered as the
dependent variable and F-cell number, age, hemoglobin, and WBC count as
independent variables; in the second, CD36+ sickle red
cells, stress reticulocytes, and F-cell number were entered as the
independent variables. A significant relationship was noted only with
F-cell numbers in both models (R = 0.54,
R2 = 0.29, power of the test with at
0.05 = 0.95; R = 0.59,
R2 = 0.35, power of the test with at
0.05 = 0.946, respectively).
Plasma-induced adhesion also correlated in univariate analyses with
CD36+ sickle red cells (R = 0.40,
P < .03), VLA4+ sickle erythrocytes
(R = 0.42, P < .02), stress reticulocytes (R = 0.54, P < .002), hemoglobin
(R =
Clinical observations have long suggested that increased levels of
HbF provide beneficial effects in patients with SCD24 and
that the cornerstone of this effect is based on the inhibition of
sickle hemoglobin polymerization by the glutamine residue at We have approached this issue by alternatively studying a homozygous SS
patient cohort with high HbF levels, ie, infants and young children. We
have evaluated subjects during this physiologically important period to
assess whether HbF may indeed modulate the adhesion process. Our
results demonstrate striking inverse correlations between 2 important
adhesion-related receptors on the sickle erythrocyte, namely VLA4
( In concomitant studies evaluating adhesion, we have demonstrated, in keeping with the findings discussed above, that a striking inverse correlation was also noted between both basal and plasma-induced adhesion and F-cell numbers (Figure 5). In multivariate analyses with basal or plasma-induced adhesion as the dependent variable and independent variables that included age and the hematologic parameters F cells, hemoglobin levels, WBC counts, and platelet counts, a significant relationship was noted solely with F-cell numbers. Additionally, in analyses where basal or plasma-induced adhesion was the dependent variable and the independent variables included such adhesion-related parameters as CD36, VLA4, and CD71 positivity and F-cell numbers, a significant relationship was noted solely with F cells. These findings demonstrate that SS patients with high levels of F cells not only have a decrease in adhesion marker-positive erythrocytes but that this decrease does indeed translate into erythrocytes that are qualitatively less adhesive in their interaction with endothelium. Because red cell-endothelial adherence was shown by Hebbel in his static adhesion assay to correlate with clinical disease severity,2,3 our observations suggest that besides its inhibition of sickle hemoglobin polymerization, the protective effect of HbF in ameliorating vaso-occlusive symptomatology is in part due to its modulation of the adhesive process. In summary, our evaluation of infants and children with SS disease has provided data suggesting that HbF may indeed modulate adhesion and the adhesion-related markers VLA4 and CD36. In addition, levels of stress reticulocytes that have the most marked adhesive potential appeared to be similarly modulated by F-cell numbers. The amelioration of clinical symptoms related to vaso-occlusion has previously been noted by 8 weeks after initiation of hydroxyurea therapy in adults with SS disease, but peak increases in F-cell numbers did not occur until approximately 16 to 24 weeks in this study.28 Additionally, the linear decrease in VLA4 expression reported by Styles and coworkers continued for 10 weeks after treatment initiation, with the nadir of VLA4 being reached by 20 weeks, whereas changes in HbF levels occurred at a relatively slower rate.30 These spatially juxtaposed observations have been interpreted to signify that adhesion marker expression was not influenced by HbF levels. However, in the multicenter trial of 152 men and women with SS disease treated with hydroxyurea, F-cell measurements did indeed demonstrate a significant increase by 8 weeks after initiation of therapy,28 including a documented range of F-cell values at which, in our study, lower adhesion ratios were noted (Figure 5). Therefore, an amelioration of vaso-occlusive symptomatology in the individual patient that occurred by 2 to 3 months after hydroxyurea could indeed have been due to changes in the levels of F cells via effects on the adhesion process. The present observations in the arena of red cell-endothelial adhesion together with the recently reported demonstration of a protective effect of HbF on erythrocyte microvesicle formation, membrane flip-flop and subsequent phosphatidylserine exposure, and concomitant coagulation activation39 serve to extend the known effects and relationships of fetal hemoglobin. Our data should, in addition, provide the impetus for a thorough evaluation of such biologic markers in the planned future clinical trials of hydroxyurea in infancy and early childhood such that further insights into disease pathophysiology and treatment can be established.
Submitted September 25, 2000; accepted January 4, 2001.
Supported by grants HL51497 and 1P60HL62148 from the National Heart, Lung, and Blood Institute of the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: B. N. Yamaja Setty, Dept of Pediatrics, Thomas Jefferson University, 1025 Walnut St, Suite 727, Philadelphia PA 19107.
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© 2001 by The American Society of Hematology.
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  B. N. Y. Setty and S. G. Betal Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes Blood, January 15, 2008; 111(2): 905 - 914. [Abstract] [Full Text] [PDF]
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 J. Haynes Jr., B. Obiako, R. B. Hester, B. S. Baliga, and T. Stevens Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H379 - H385. [Abstract] [Full Text] [PDF]
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B. N. Y. Setty, S. Kulkarni, and M. J. Stuart Role of erythrocyte phosphatidylserine in sickle red cell-endothelial adhesion Blood, March 1, 2002; 99(5): 1564 - 1571. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1 (very late activation antigen-4
[VLA4])
) and in the
presence of plasma (
). The solid line represents the
linear-regression fit to the data, and the dotted lines represent the
99% confidence interval curves. The R and P
values for the overall relationship, which included both basal and
plasma-induced adhesion, are presented in the figure. Similar
R and P values were also noted when the basal and
plasma-induced adhesion ratios from these 2 cell systems were analyzed
separately (R = 0.652, P < .002 for basal
and R = 0.812, P < .000 02 for
plasma-induced adhesion).
-87 of
the HbF molecule.