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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Division of Research Hematology, Department of
Pediatrics, Jefferson Medical College, Thomas Jefferson University and
the Marian Anderson Comprehensive Sickle Cell Center, Philadelphia, PA.
Phosphatidlyserine (PS) exposure on the erythrocyte surface endows
the cell with the propensity of adhering to vascular endothelium. Because individuals with sickle cell disease (SCD) manifest loss of
erythrocyte membrane asymmetry with PS exposure, we have assessed the
contribution of this marker to the process of sickle
erythrocyte-microendothelial adhesion. Assays for plasma-induced
adhesion were conducted on unactivated endothelium, in the absence of
immobilized ligands, such that PS was compared to the erythrocyte
adhesion receptor CD36. Blocking studies with erythrocytes pretreated
with annexin V (to cloak PS) or anti-CD36 or both revealed an
inhibitory effect on adhesion of 36% ± 10% and 23% ± 8% with
blocking of both sites suggestive of an additive effect. We next
evaluated 87 blood samples from patients with SCD and grouped them into
4 categories based on adhesion marker (CD36 and PS) levels. Results
revealed a striking correlation between erythrocyte PS positivity and
adhesion. Analyses of the individual patient data demonstrated a
positive correlation between PS and adhesion (R = 0.52,
P < .000 001), whereas none was noted between adhesion
and CD36 (R = 0.2, P > .07). The effect of PS on
adhesion appears to be related to the quantitative differences in
erythrocyte markers in SCD, with PS the predominant marker when
compared to CD36 both in the total erythrocyte population, and when the
adherence-prone erythrocyte, the CD71+ stress reticulocyte,
was evaluated. Our study signals the entrance of an important new
contributor to the field of sickle erythrocyte-endothelial adhesion.
The implications of erythrocyte PS exposure in relation to the vascular
pathology of SCD need to be assessed.
(Blood. 2002;99:1564-1571) Since the initial observation by Hebbel and
coworkers that sickle red cell-endothelial adherence is correlated
with clinical disease severity,1 the last 2 decades have
seen a large body of work related to the surface adhesion molecules and
markers present on the erythrocyte and endothelium that play a role in the adhesion process.2,3 Receptors on the red blood cell (RBC) include the integrin Although the asymmetrical distribution of phospholipids appears to be
well conserved throughout the lifespan of the cell, studies using
annexin V (a calcium-dependent phospholipid-binding protein) have
demonstrated that in patients with sickle cell disease (SCD), the
anionic phospholipid phosphatidylserine (PS) is present on the RBC
surface.18-20 Potential consequences of such pathologic PS
exposure include an exacerbation of the anemia due to enhanced reticuloendothelial clearance and activation of
coagulation.21 Recent reports have demonstrated that PS
exposing human erythrocytes (produced by the action of ionophore on
control RBCs) adhere to vascular endothelium and the endothelial matrix
protein TSP.22,23 In the present study, we have sought to
extend these in vitro observations (on treated control erythrocytes) to
the clinical arena by performing studies in patients with SCD and
assessing the relative roles of some of the important erythrocyte
adhesion markers described to date. We demonstrate by in vitro blocking experiments and in the patient-related studies that erythrocyte PS
exposure appears to be a critical determinant in the adhesion process.
Materials
Collection of blood
RBC adhesion assay Adhesion of sickle RBCs to endothelial cell monolayers was evaluated in a static adhesion assay1 using 51Cr-labeled sickle RBCs and human retinal capillary microvascular endothelial cell (HRCMEC) monolayers. The 51Cr-labeled sickle RBCs were prepared as previously described.25 HRCMECs were isolated, identified, and cultured in minimal essential medium supplemented with 10% fetal calf serum.26 Cells from passages 8 to 16 were used in the adhesion experiments, each passage representing 2-cell doublings. For adhesion assay, 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 RBCs. Adhesion assays were conducted in the presence of 10% autologous plasma at 37°C for 45 minutes, and the nonadherent RBCs were removed. The monolayers were then washed and adherent RBCs were determined by 51Cr release following cell lysis. 51Cr-labeled control HbAA RBCs were concomitantly evaluated in adhesion assays in the presence and the absence of 10% autologous plasma. The adhesion potential of the test RBC was expressed as an adhesion ratio, which was determined by dividing the number of adherent erythrocytes by control HbAA RBCs assayed in the absence of plasma.Flow cytometric analysis of adhesive receptors and other surface markers on erythrocytes One million washed RBCs (suspended in a final volume of 100 µL Hanks balanced salt solution [HBSS]-HEPES buffer) were incubated for 30 minutes at room temperature with 20 µL PE-labeled anti-CD235a and 10 µL FITC-labeled annexin V in the presence of either 2.5 mM CaCl2 or 2.5 mM EDTA.19,20,27 Incubation mixtures were then diluted with 1 mL HBSS-HEPES buffer containing either 2.5 mM CaCl2 or 2.5 mM EDTA and analyzed in a Becton Dickinson Flow Cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) formatted for 2-color analysis. Fluorescence compensation settings were established using cells stained with anti-CD235a-PE alone, annexin V-FITC alone, PE-labeled isotopic negative control antibody, and annexin V-FITC in the presence of EDTA. Data from 50 000 events were collected for analysis. Using size scatter properties, RBC-associated events were separated from non-RBC-associated events (which did not exceed 0.5%) and were gated out from the analysis. As shown in Figure 1B, PS+ RBCs (events in quadrant Q2), defined as anti-CD235a+ events simultaneously stained for annexin V were determined by setting quadrants on the FL1 (annexin V) and FL2 (CD235a) dot plot. Nonspecific membrane immunofluorescence was determined using cells stained with anti-CD235a-PE and annexin V-FITC in the presence of EDTA, and these values (events in quadrant Q2, Figure 1A) were subtracted from the respective sample fluorescence. As shown in panels C and D of Figure 1, analysis of the data using the respective histograms of the annexin V-FITC fluorescence provided identical results. Data were expressed as percent PS+ RBCs.
The RBC adhesion receptors including CD36 and VLA-4, and CD71 were
analyzed by 2-color flow cytometry using erythrocytes stained with
anti-CD235a-PE and one of the FITC-labeled antibodies: anti-CD36, anti-CD49d, anti-CD71, or an isotypic negative control antibody as
previously described.24 Adhesion receptor-positive and
CD71+ erythrocytes were analyzed using the steps outlined
as above for PS+ RBCs. A representative analysis for
CD36+ RBCs is depicted in panels E to H of Figure 1.
Marker-positive stress reticulocytes were analyzed by flow cytometry
using 2-color analysis as detailed above using erythrocytes stained for
the transferrin receptor (anti-CD71-PE) and one of the FITC-labeled reagents: anti-CD36 or an isotypic negative control IgG, or annexin V
in the presence of 2.5 mM CaCl2 or 2.5 mM EDTA.
PS+ and CD36+ stress reticulocytes were
analyzed using the dot plots of anti-CD71-PE fluorescence and either
annexin V-FITC fluorescence (Figure
2A,B) or anti-CD36-FITC fluorescence
(Figure 2C,D), respectively. For analyses of RBCs positive for both PS
and CD36, anti-CD36-pure plus anti-mouse IgG-TC-labeled washed
sickle RBCs were stained with annexin V-FITC in the presence of
CaCl2 or EDTA and analyzed as shown in panels E and F of
Figure 2.
Treatment of sickle erythrocytes with anti-CD36 and annexin V The 51Cr-labeled and washed sickle red cells (2.5% hematocrit) from 6 donors were pretreated for 45 minutes at 37°C with 40 µg/mL anti-CD36 (to cloak cell surface CD36) or 40 µg/mL annexin V (to cloak cell surface PS) or both and then simultaneously evaluated for their residual adhesion potential and surface adhesion markers. Concentrations of anti-CD36 and annexin V for the blocking studies described above were selected from preliminary dose-response experiments (n = 3) performed using 1 to 100 µg/mL blocking agent. Both anti-CD36 and annexin V at a concentration of 40 µg/mL blocked 70% to 90% of RBC surface CD36 and PS in these preliminary experiments. To assess whether blocking one marker affected surface expression of the other, in additional preliminary experiments, RBCs were pretreated with either anti-CD36 (40 µg/mL) or annexin V (40 µg/mL), and then analyzed for both CD36 and PS by flow cytometry. Anti-CD36 treatment (n = 6) had no significant effect on PS exposure (4.9% ± 2.6% on untreated versus 4.4% ± 2.5% PS positivity on anti-CD36-treated RBCs, P > .25, paired t test). Similarly, annexin V treatment (n = 7) had no effect on CD36 exposure (2.63% ± 2.32% on untreated versus 2.53% ± 2.16% CD36 positivity on annexin V-treated RBCs, P > .45).Statistical analysis Statistical evaluation was performed using the Sigmastat Statistical Package (Jandel Scientific, San Rafael, CA). All results are presented as the mean ± SD. Because analyses of the adhesion marker data showed a nonparametric distribution, significant differences between control and the patient groups were analyzed using the Kruskal-Wallis test. If the P value for this overall comparison was significant at P < .05, group-wise comparisons were performed using the Mann-Whitney test. Statistical significance between 2 paired variables was analyzed using the paired Student t test. Both Pearson and Spearman correlation tests were used to determine the relationship between 2 variables. Both tests yielded similar results for the same pair of variables analyzed. R and P values presented for adhesion markers (except for VLA-4 related) were obtained by Pearson tests on log-transformed data. Because no VLA-4+ RBCs or reticulocytes were detected in many donors including patients with SCD, Pearson tests on VLA-4-related data were performed using nontransformed data.
Relationship between erythrocyte PS, CD36, VLA-4, and adhesion To assess the relative roles of adhesion markers in sickle erythrocyte-endothelial adhesion, we grouped patients into 4 arbitrary categories based on their adhesion marker values as depicted in Table 1. The surface levels of the main adhesion-related markers (PS, CD36, and VLA-4) and the adhesive properties of each patient group as compared to HbAA controls are shown in Table 2. Minimal levels of adhesion marker-positive RBCs were measured in controls. Both PS+ and CD36+ erythrocytes were elevated in all 4 SCD patient groups when compared to controls (P < .002 to <.000 001). Although the mean levels of VLA-4+ RBCs in SCD groups 1 and 4 were identical to control values, increased VLA-4 positivity was seen in SCD groups 2 and 3, with significant changes noted in group 2 (P < .05). The elevations in erythrocyte adhesion markers noted in the SCD patient groups 1 through 4 when compared to controls was accompanied by an increase in the adhesion of these RBCs to microvessel endothelium.
The relative role of erythrocyte PS versus CD36 in adhesion is demonstrated in the results presented in Table 2. Comparison of SCD groups 1 and 2 revealed that the mean levels of both PS+ and CD36+ erythrocytes in group 2 were significantly increased when compared to group 1 (5.6% versus 1.4% for PS and 2.2% versus 0.2% for CD36; P < .0001), with a concomitant increase in RBC-endothelial adhesion (6.5 versus 2.2; P < .0001). To further assess whether the increased adhesion noted in group 2 patients was due to the elevation in erythrocyte PS, RBC CD36, or a combination of both these adhesive markers, 2 further SCD patient groups were evaluated. Although group 3 demonstrated a significant elevation in erythrocyte CD36 levels when compared to group 1 (2.1% versus 0.2%, P < .0001) with the mean CD36 level in this patient group approximating that previously noted in group 2, mean adhesion values in group 3 were similar to those noted in group 1, and were markedly decreased when compared to group 2 (2.1% in group 3 versus 6.5% in group 2, P < .0001). This observation that the enhanced adhesion in group 2 individuals did not appear to be due to elevations in the levels of erythrocyte CD36 was further confirmed by the data we have obtained in group 4. Although levels of CD36 in this patient group were no different from those in group 1 (0.3% versus 0.23%, P > .2), adhesion was significantly increased (6.4% in group 4 versus 2.2% in group 1, P < .0001) and appeared to be related to the elevated levels of RBC PS in this group (4.4% versus 1.4% in group 1) with values for both erythrocyte PS positivity and adhesion not significantly different from those observed in group 2. When the group data from VLA-4+ erythrocytes was evaluated for a potential association with adhesion, no relationship was noted. Groups 1 and 4, in which the mean values of VLA-4+ erythrocytes were no different from each other or from the control population (0.05%), demonstrated significant differences in adhesion (2.2 versus 6.4, P < .0001), which thus could not be attributed to VLA-4 positivity. In additional analyses of the clinically related data, correlations
between adhesion and the individual levels for all adhesion-related markers in the patient groups were sought. Although a striking positive
correlation was noted between erythrocyte PS positivity and adhesion
(R = 0.52, P < .000 001, Figure
3B), no correlation was noted either with
CD36+ RBCs (R = 0.2, P > .07, Figure 3A) or
VLA-4 positivity (R = 0.09, P > .35).
Effects of blocking erythrocyte CD36 and PS on adhesion To further test the hypothesis that erythrocyte PS is a crucial mediator of RBC-endothelial adhesion, sickle RBCs were pretreated with anti-CD36 or annexin V or both and assessed for both cell surface markers and adhesion potential. For these studies we used sickle erythrocytes with the highest levels of both CD36 and PS (2.5% ± 1.2% and 9.5% ± 1.9%, respectively; n = 6). Following anti-CD36 and annexin V treatment, CD36 and PS sites on the RBC surface were blocked by 75% ± 5% and 87% ± 2%, respectively, with similar cloaking results noted in both single and double cloaking experiments. In concomitant functional adhesion assays, although anti-CD36-treated erythrocytes decreased adhesion by 23% ± 8%, annexin V-treated (PS cloaked) cells demonstrated a significantly greater inhibitory effect on adhesion of 36% ± 10% (P < .05). The inhibitory response observed with RBCs that were pretreated with both annexin V and anti-CD36 (50% ± 16%) was significantly different when compared to those noted with RBCs treated with either blocking agent alone (Figure 4, P < .05 and <.007 respectively), and was suggestive of an additive effect. One of the explanations for the observed partial inhibition of RBC adhesion (23%-35%) by anti-CD36 or annexin V treatment, despite blocking surface CD36 or PS by 75% to 85%, is that the same erythrocyte might be carrying multiple adhesion markers on its surface, which could therefore participate in adhesion reactions with endothelium even when one of the other adhesion markers/receptors is blocked. We have therefore analyzed the marker data for cells positive for both CD36 and PS. As shown in Table 2, approximately 50% of CD36+ RBCs in all groups were also positive for PS, whereas the levels of concomitant PS positivity for these dual positive erythrocytes varied between the groups, from a low of 2% to high of 54%.
Relationship between circulating levels of CD71+ stress reticulocytes and adhesion Because stress reticulocytes are known to express the highest levels of adhesive markers and have marked adhesion properties, we analyzed our adhesion data for potential correlates with CD71 positivity. As depicted in Figure 5A, significant positive correlations were noted between the levels of stress reticulocytes and adhesion (R = 0.561, P < .0002, n = 41). Additionally, when these stress reticulocytes were assessed for adhesion marker positivity (Figure 6), PS+ stress reticulocytes were found to be the most abundant cells present in this RBC fraction (24.4% ± 8.5% and 9.3% ± 8.2%, for PS and CD36 positivity, respectively). Levels of PS+ stress reticulocytes reported here are comparable to those reported in the literature.28 A positive correlation was also observed between PS+ stress reticulocytes and adhesion (Figure 5B, R = 0.553, P < .0003, n = 39) with correlation parameters almost identical to those obtained with stress reticulocytes (Figure 5A). No correlations were noted with CD36+ stress reticulocytes and adhesion (R = 0.166, P > .34, n = 39).
The microvascular occlusive event of SCD has a multifactorial
etiology dependent primarily on whether the rate of sickle hemoglobin polymer formation is within the range of the capillary transit time.29 Events that therefore slow the transit of sickle
RBCs in the microcirculation, such as factors enhancing
RBC-endothelial adhesion, can play a critical role in this
process. Vaso-occlusion appears to be initiated by the adhesion of the
least dense and highly adhesinogenic sickle reticulocyte to the
endothelium of the postcapillary venule, followed by the secondary
accumulation of poorly deformable dense cells leading to vascular
occlusion.2 To date, the adhesive relationships delineated
between the sickle RBC and the endothelium have been, for the most
part, receptor-mediated events, as depicted in Figure
7. One of the main mechanisms described is the bridging role of the plasma ligand TSP14,15 between the RBC receptor CD36 on the one hand and several constitutively present endothelial receptors on the other. Because TSP is comprised of
a number of heterogeneously distinct domains, vascular adhesion to TSP
depends on several endothelial sites, including the vitronectin receptor (
Because adhesion as described above involves interactions of some complexity, in this study we have only attempted a delineation of the relative roles of the adhesion markers erythrocyte PS when compared to CD36. Our in vitro system, which used unactivated endothelium for the plasma-induced adhesion assays we performed, effectively eliminated VLA-4/VCAM-1-mediated adhesive mechanisms because VCAM-1 is not constitutively expressed on unactivated endothelial cells (a finding that was confirmed by flow cytometric analyses of our microvascular retinal capillary endothelial cells; data not shown). The blocking experiments using anti-CD36 and annexin V unequivocally demonstrated that cloaking of the PS sites led to a significantly greater degree of inhibition of adhesion as compared to blocking of the erythrocyte CD36 receptor. In addition, when both these RBC adhesion markers were unavailable for interaction with plasma-soluble ligands and endothelial adhesive receptors, the inhibitory effect appeared to be additive, suggesting that the interactions with TSP or endothelial receptors occurred via distinct adhesive sites or domains. Our patient related data (Table 2 and Figure 3) was unexpected. It suggests that under the conditions of our study, that is, in unactivated endothelium, PS appears more critical to the adhesive process than mechanisms involving erythrocyte CD36. Because the endothelial receptor sites involved in RBC CD36 binding via TSP are constitutively expressed, the study undertaken appears to be a valid assessment of the comparative role of RBC PS versus CD36 in the adhesive process. Previous investigations and our present study do not address the quality or affinity of the adhesive interaction of PS versus CD36 with soluble ligands, subendothelial matrix proteins, or endothelial receptors. However, evaluation of the number of PS+ and CD36+ erythrocytes (Table 2) suggests that part of the dominant PS effect on adhesion appears to be due to the quantitative differences noted which favor PS positivity 2 to 3 times over CD36+ RBCs in SCD. In keeping with this finding is our additional observation that stress reticulocytes (which hitherto have had their strong adherent properties ascribed to their CD36 positivity8) are significantly more positive for PS than for the adhesion marker CD36 (Figure 6), and that the adhesion potential of these cells correlates exclusively with their PS positivity (Figure 5). Additionally, the coexpression marker data also favors PS in that 50% of the CD36+ cells also expressed PS. An alternative explanation for the results we obtained in our patient-related studies, as has been suggested by other investigators, is that erythrocyte CD36-TSP binding may not play such a central role in the sickle erythrocyte adhesion process as previously believed. A recent patient-related study demonstrating that the presence or absence of CD36 on sickle reticulocytes and RBCs had no effect on the clinical severity of sickle cell anemia also supports this latter conclusion.33 TSP binding to PS or other RBC markers not addressed in our study, such as erythrocyte sulfated glycolipids, band-3 peptide, or the integrin-associated protein, may be of more relevance.10,15,23,31,32,34,35 We used 10% autologous plasma for our adhesion studies. Although the individual patient with SCD exhibits quantitative differences in plasma adhesion-related ligands, these values are usually relatively high in all patients due to the SCD-related chronic proinflammatory and procoagulant phenotype.36 Thus, although the differences in adhesion we have noted in the patient groups appear from our composite data to be PS related, we cannot rule out some minimal effect on adhesion ratios in all patient groups evaluated due to the fact that we used diluted plasma rather than an undiluted preparation for these experiments. However, a previous study has demonstrated that the maximum plasma effect on sickle RBC-endothelial adhesion occurs in vitro at a 10% plasma dilution.37 A static adhesion assay was performed for the erythrocyte-microendothelial adhesion experiments and relationships described. Although it has been suggested that RBC adhesion mechanisms identified under conditions of flow are of more pathophysiologic relevance, it has been also noted that this view does not give consideration to the fact that microvessel blood flow can be intermittent,38 and that stasis may be enhanced by slow-moving and relatively large granulocytes or cellular aggregates,39 thus simulating more closely adhesion assays where flow is absent. On the other hand, in vivo data demonstrate that sickle erythrocyte adherence occurring in capillaries during periods of incipient flow stasis can be dislodged on removal of the pressure cuff and restoration of blood flow.40,41 However, because the initial correlation described between sickle erythrocyte adhesion to endothelium and microvascular complications in patients with SCD was observed using the static adhesion assay, Hebbel has suggested that low-affinity adhesion mechanisms (as reflected by static assays) are of major relevance to SCD pathophysiology.1,42 Several adhesive surface receptors on macrophages have been identified
to mediate the recognition and removal of PS+ cells in
vitro during apoptosis or programmed cell death. These include CD68,
CD36, CD14, and the class B scavenger proteins SR-BI that do not
discriminate between PS and other anionic phospholipids.43 Most recently, a receptor on macrophages and fibroblasts for
PS-specific clearance of apoptotic cells has also been
identified.44 In addition, plasma proteins such as
Submitted June 29, 2001; accepted October 23, 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, Department of Pediatrics, Thomas Jefferson University, 1025 Walnut St, Suite 727, Philadelphia, PA 19107; e-mail: yamaja.setty{at}mail.tju.edu.
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L. S. Kean, E. A. Manci, J. Perry, C. Balkan, S. Coley, D. Holtzclaw, A. B. Adams, C. P. Larsen, L. L. Hsu, and D. R. Archer Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease Blood, December 15, 2003; 102(13): 4582 - 4593. [Abstract] [Full Text] [PDF]
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P. G. Gallagher, S. H. Chang, M. P. Rettig, J. E. Neely, C. A. Hillery, B. D. Smith, and P. S. Low Altered erythrocyte endothelial adherence and membrane phospholipid asymmetry in hereditary hydrocytosis Blood, June 1, 2003; 101(11): 4625 - 4627. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
4
1 also known
as the very late activation antigen 4 (VLA-4),
(quadrant Q1) and PS+
(quadrant Q2) dot plot regions were set using the erythrocyte sample
stained in the presence of EDTA (A). Corresponding histogram profiles
of annexin V-FITC fluorescence are presented in panels C and D,
respectively. PS
