Sickle erythrocytes inhibit human endothelial cell DNA synthesis

Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling. These results indicate that interaction between sickle erythrocytes and the endothelium may result in altered endothelial function. This altered endothelial function may contribute to the development of vascular occlusive phenomena in patients with sickle cell anemia.     

Inflammatory mediators promote strong sickle cell adherence to endothelium under venular flow conditions

Adherence of sickle erythrocytes to endothelium in venules is thought to initiate or propagate vaso-occlusive episodes. Because of blood shear forces with normal microvascular flow, adherence in post-capillary venules requires binding via high-affinity receptor-mediated pathways. Microvascular flow in sickle patients is episodic, even in asymptomatic patients, so adherence may also occur at low shear not requiring high-affinity binding. Sickle cell binding to endothelium was quantified under flow or static incubation with unusually large vWF, thrombospondin, alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/fibronectin (FN). Adherence under flow at 0.5 dyne/cm(2) shear stress leads to the greatest number of adherent sickle cells. Adherence under flow at 1.0 dyne/cm(2) leads to the strongest adherence. Static incubation conditions promote weak adherence of low numbers of sickle cells to endothelium. Following attachment at 1.0 dyne/cm(2), adherence strength was 2.5 +/- 0.1 or 2.6 +/- 0.2 dynes/cm(2) for alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/FN pathways, a level 50% greater than adherence strength mediated by thrombospondin or ULvWF (1.7 +/- 0.08 or 1.6 +/- 0.07 dynes/cm(2), respectively). Sickle cell adhesion promoted by simultaneous activation of alpha(4)beta(1)/VCAM-1 and alpha(4)beta(1)/FN pathways is the strongest at 6.2 +/- 0.2 dynes/cm(2) and adherent red cells resist detachment shear stresses up to 10 dynes/cm(2). These data demonstrate that sickle cell adhesion to endothelium is regulated both by receptor/ligand affinity and flow conditions. Thus, both microvascular flow conditions and receptor-ligand interactions may regulate sickle cell adherence in vivo.     

Histamine increases sickle erythrocyte adherence to endothelium

Complications of sickle cell anaemia include vascular occlusion triggered by the adherence of sickle erythrocytes to endothelium in the postcapillary venules. Adherence can be promoted by inflammatory mediators that induce endothelial cell adhesion molecule expression and arrest flowing erythrocytes. The present study characterised the effect of histamine stimulation on the kinetics of sickle cell adherence to large vessel and microvascular endothelium under physiological flow. Increased sickle cell adherence was observed within minutes of endothelial activation by histamine and reached a maximum value within 30 min. At steady state, sickle cell adherence to histamine-stimulated endothelium was 47 +/- 4 adherent cells/mm(2), 2.6-fold higher than sickle cell adherence to unstimulated endothelial cells. Histamine-induced sickle cell adherence occurred rapidly and transiently. Studies using histamine receptor agonists and antagonists suggest that histamine-induced sickle cell adhesion depends on simultaneous stimulation of the H(2) and H(4) histamine receptors and endothelial P-selectin expression. These data show that histamine release may promote sickle cell adherence and vaso-occlusion. In vivo histamine release should be studied to determine its role in sickle complications and whether blocking of specific histamine receptors may prevent clinical complications or adverse effects from histamine release stimulated by opiate analgesic treatment.     

Sickle cell vaso-occlusion: multistep and multicellular paradigm

Sickle cell disease is characterized by recurrent, painful episodes and organ damage resulting from microvascular occlusion. Seminal studies performed 20 years ago revealed increased adherence of sickle erythrocytes to vascular endothelial cells. Subsequent work showed that these interactions were mediated by multiple adhesion pathways, but the relevance of these interactions has not been evaluated in vivo. Clinical data suggest that leukocytes may play a role, because leukocytosis correlates with clinical severity and early death, and administration of myeloid growth factors to patients can precipitate sickle cell crises. In addition, recent experimental data using intravital microscopy indicate that sickle erythrocytes can interact with adherent leukocytes in inflamed postcapillary and collecting venules. A novel multistep model for sickle cell vaso-occlusion is proposed in which endothelial activation is induced by sickle cells or secondary inflammatory stimuli and leads to the recruitment of adherent leukocytes. The resulting adherent leukocytes interact with circulating sickle erythrocytes, and this interaction impedes microvascular blood flow. Finally, irregularly shaped sickle cells become nonspecifically trapped, resulting in vaso-occlusion. The molecular mechanisms and requirements for the heterotypic interactions between erythrocytes and leukocytes are currently unknown and may involve further activation of adherent leukocytes or circulating erythrocytes. This model offers exciting new opportunities for therapeutic intervention and suggests a critical participation of adherent leukocytes in sickle cell vaso-occlusion.     

Thrombospondin from activated platelets promotes sickle erythrocyte adherence to human microvascular endothelium under physiologic flow: a potential role for platelet activation in sickle cell vaso-occlusion

Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic vascular occlusion in patients with sickle cell disease (SCD). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes. To test the hypothesis that platelet activation contributes to sickle erythrocyte binding, we investigated whether factors released from activated sickle platelets promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions. Activated sickle platelet supernatant (ASPS) promoted high levels of sickle erythrocyte adherence to MEC (55.4 +/- 3.9 erythrocytes/mm2) but only moderate adherence of normal erythrocytes to MEC (14.1 +/- 0.7 erythrocytes/mm2). When MEC were incubated with an antibody (OKM5) against CD36 (a thrombospondin [TSP] receptor), platelet supernatant mediated sickle erythrocyte adherence was inhibited 86%, suggesting that TSP participated in the adherence. To further define the role of TSP in adherence, additional studies using purified TSP were performed. At a concentration of 0.2 micrograms/mL TSP in serum-free media (SFM), sickle erythrocyte adherence to MEC was 33.9 +/- 2.7 erythrocytes/mm2 and sixfold greater than either sickle erythrocyte adherence in the absence of TSP or normal erythrocyte adherence in the presence of TSP. Doubling the concentration of TSP to 0.4 micrograms/mL proportionally increased adherence of sickle erythrocytes. Incubation of MEC with OKM5 or anti-alpha v monoclonal antibodies inhibited TSP-mediated sickle erythrocyte adherence more than 95%. These data suggest that activated platelet release factors, including alpha-granule TSP, which promote receptor-mediated sickle erythrocyte adherence to microvascular endothelium. Such factors released during in vivo platelet activation could contribute to vaso-occlusive complications by promoting erythrocyte adherence and microvascular occlusion.     

Endothelial cell interactions with sickle cell, sickle trait, mechanically injured, and normal erythrocytes under controlled flow

Increased adhesive forces between sickle erythrocytes and endothelial cells (EC) have been hypothesized to play a role in the initiation of vasoocclusion in sickle cell anemia. Erythrocyte/human umbilical vein EC interactions were studied under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle cell trait (AS), mechanically injured normal, and "high-reticulocyte control" RBC by using video microscopy and digital image processing. The number of adherent RBC was determined at ten-minute intervals during a washout period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) AA RBC were also more adherent than control normal cells but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less- dense RBC were more adherent to EC than dense cells for normal, SS, and high-reticulocyte control RBC. The number of cells adherent at a given time during washout was a very strong function of wall shear rate. In addition, at all shear rates studied, the average velocity of individual SS RBC in the region near the EC surface was approximately half that of AA RBC at the same bulk volumetric flow rate through the flow chamber. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of less-dense RBC present. Increased adherence of the less-dense cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells.     

Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular- cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/- 17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu- treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.     

Sickle erythrocyte adherence to endothelium at low shear: role of shear stress in propagation of vaso-occlusion

Under venular flow conditions, sickle cell adherence to endothelium is mediated by cell adhesion molecules and adhesive proteins associated with inflammation, coagulation, and endothelial perturbation. Periodic and reduced blood flow are observed in sickle microcirculation during hematologic steady state, suggesting that blood flow is compromised in sickle microcirculation. We tested the hypothesis that low blood flow enhances adherence by quantifying sickle cell adhesion to endothelium under venular flow (1.0 dyne/cm(2) shear stress) and low flow (0.1 dyne/cm(2) shear stress), with and without addition of adhesion promoting agonists. Under low flow, sickle cell adherence to endothelium increases with contact time in the absence of endothelial activation or adhesive protein addition. In contrast, at venular shear stress, sickle cell adherence only occurs following endothelial activation with TNF-alpha or addition of thrombospondin. Analysis of these data with a mathematical model reveals that at low flow adherence is "transport-controlled," meaning that contact time between sickle cells and endothelium is a more important determinant of adherence than high-affinity receptor-ligand interactions. Low-affinity interactions are sufficient for adhesion at low flow. In contrast, at venular flow (1 dyne/cm(2) shear stress) adherence is "affinity-controlled," meaning that adherence requires induction of specific high-affinity receptor-ligand interactions. These findings demonstrate that in addition to activating factors and adherence proteins, microvascular shear stress is an important determinant of sickle cell adhesion to endothelium. This suggests that in vivo, erythrostasis is an important determinant of adhesion that can act either independently or concurrently with ongoing acute events to induce adhesive interactions and vaso-occlusion.     

P-selectin mediates the adhesion of sickle erythrocytes to the endothelium

The adherence of sickle red blood cells (RBCs) to the vascular endothelium may contribute to painful vaso-occlusion in sickle cell disease. Sickle cell adherence involves several receptor-mediated processes and may be potentiated by the up-regulated expression of adhesion molecules on activated endothelial cells. Recent results showed that thrombin rapidly increases the adhesivity of endothelial cells for sickle erythrocytes. The current report presents the first evidence for the novel adhesion of normal and, to a greater extent, sickle RBCs to endothelial P-selectin. Studies of the possible interaction of erythrocytes with P-selectin revealed that either P-selectin blocking monoclonal antibodies or sialyl Lewis tetrasaccharide inhibits the enhanced adherence of normal and sickle cells to thrombin-treated endothelial cells. Both RBC types also adhere to immobilized recombinant P-selectin. Pretreating erythrocytes with sialidase reduces their adherence to activated endothelial cells and to immobilized recombinant P-selectin. Herein the first evidence is presented for the binding of normal or sickle erythrocytes to P-selectin. This novel finding suggests that P-selectin inhibition be considered as a potential approach to therapy for the treatment of painful vaso-occlusion in sickle cell disease.     

Double-stranded RNA induces sickle erythrocyte adherence to endothelium: a potential role for viral infection in vaso-occlusive pain episodes in sickle cell anemia

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double- stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1- -dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double- stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.     

Adherence of sickle erythrocytes to vascular endothelial cells: requirement for both cell membrane changes and plasma factors

Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.     

Cytoadherence of Plasmodium falciparum-infected erythrocytes to human umbilical vein and human dermal microvascular endothelial cells under shear conditions

To investigate the role of hemodynamics in the adherence of Plasmodium falciparum-infected erythrocytes to cerebral endothelium in vivo, we investigated cytoadherence of parasitized erythrocytes to human umbilical vein endothelial cells (HUVEC) under shear conditions in vitro. At 1.0 dyne/cm2 shear stress, parasitized red blood cell (RBC) adherence to HUVEC ranged from 9.9 +/- 1.0 (+/- SEM) to 75.2 +/- 4.8 RBC/mm2 (mean +/- SEM: 35.1 +/- 2.8 RBC/mm2) and was 13-fold greater than uninfected erythrocyte adherence to HUVEC (range 0.1 +/- 0.1 to 6.7 +/- 1.6 RBC/mm2, mean +/- SEM 2.8 +/- 0.8 RBC/mm2). Only erythrocytes infected with trophozoites and schizonts adhered to HUVEC under shear conditions. Parasitized erythrocyte adherence to HUVEC decreased from 28.4 +/- 2.7 RBC/mm2 to 12.7 +/- 2.4 RBC/mm2 when shear stress was increased from 1.0 to 2.0 dynes/cm2. At 4.0 dynes/cm2, parasitized erythrocyte adherence decreased further to 2.0 +/- 1.3 RBC/mm2. In falciparum malaria patients, endothelial cytoadherence predominates in the microcirculation. Therefore, we also investigated adherence of parasitized erythrocytes to human dermal microvascular endothelial cells (MEC). At 1.0 dyne/cm2, cytoadherence of P. falciparum-infected erythrocytes to MEC ranged from 7.9 +/- 1.1 to 60.0 +/- 2.4 RBC/mm2 (mean +/- SEM: 23.0 +/- 1.7 RBC/mm2) and was 10-fold greater than uninfected erythrocyte cytoadherence to MEC (mean +/- SEM: 2.2 +/- 0.6 RBC/mm2). These data indicate that P. falciparum-infected erythrocytes adhere to human umbilical vein and microvascular endothelial cells under shear stress conditions typical of the postcapillary venules in vivo, and that cytoadherence is specific for parasitized erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications

To understand the role of sickle cell adherence to the vascular endothelium in the pathophysiology of sickle cell anemia (SS) vasoocclusion, we have carried out a microcirculatory study utilizing the ex vivo mesocecum vasculature of the rat. A single bolus of washed oxy-normal (AA) erythrocytes or oxy-SS cells (unseparated or density-defined SS cell classes) was infused. Hemodynamic monitoring and intravital microscopic observations of the microvascular flow revealed higher peripheral resistance for SS erythrocytes and adherence of these cells exclusively to the venular endothelium but rare or no adherence of AA cells. The extent of adhesion was inversely correlated with venular diameters (r = -0.812; P less than 0.00001). The adhesion of SS erythrocytes is density-class dependent: reticulocytes and young discocytes (SS1) greater than discocytes (SS2) greater than irreversible sickle cells and unsicklable dense discocytes (SS4). Selective secondary trapping of SS4 (dense cells) is found in postcapillary venules where deformable SS cells are preferentially adhered. We conclude that in the oxygenated condition, vasoocclusion can be induced by two events: (i) random precapillary obstruction by a small number of SS4 cells; (ii) increased adhesion of SS1 and SS2 cells in the immediate postcapillary venules. A combination of precapillary obstruction, adhesion in postcapillary venules, and secondary trapping of dense cells may induce local hypoxia, increased polymerization of hemoglobin S, and rigidity of SS erythrocytes, thereby extending obstruction to nearby vessels.     

Sickling-induced binding of immunoglobulin to sickle erythrocytes

Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.     

Exercise-induced hemolysis in sickle cell anemia: shear sensitivity and erythrocyte dehydration

We describe a steady-state patient with sickle cell anemia (SS disease) who developed sporadic hemoglobinuria, historically related to vigorous exercise. We studied him and four other patients with SS disease and demonstrated exercise-induced hemoglobinemia. To see if SS erythrocytes were abnormally fragile when exposed to shear forces that could be generated in small vessels of exercising muscles, we exposed them to physiologic shear rates in a cone-plate viscometer. We show that SS erythrocytes are more shear sensitive than normal erythrocytes. This phenomenon is directly related to the presence of dehydrated cells as demonstrated by the increasing shear sensitivity of increasingly dehydrated cells separated on Stractan density gradients. Normal shear sensitivity could be restored to dehydrated layers by restoring normal hydration. Restoration of shear stability was complete in all layers except for the most dense ISC layer. A control group of patients with SC disease exhibited no exercise-induced hemoglobinemia, no abnormal shear sensitivity of whole blood, and only rare dehydrated ISCs. These studies suggest that the exercise-induced hemolysis in SS patients is related to the lysis of dehydrated, shear-sensitive cells. This same process may also contribute to the chronic hemolysis of SS disease--a phenomenon known to correlate with the numbers of dehydrated ISCs.     

Autologous IgM, IgA, and complement binding to sickle erythrocytes in vivo. Evidence for the existence of dense sickle cell subsets

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities > 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high- density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high- density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.     

Nitric oxide attenuates normal and sickle red blood cell adherence to pulmonary endothelium

Increased adherence of sickle red blood cells (RBC) to endothelium is implicated as an initiating event of vaso-occlusion in sickle cell disease. Although much is known about the humoral influences of this interaction, there has been little investigation regarding endothelial contributions. Endothelial derived nitric oxide (NO) inhibits adhesion of platelets and leukocytes to endothelium and decreases expression of VCAM-1, an endothelial adhesion site implicated in sickle RBC/endothelial adherence. However, whether NO inhibits RBC adherence to endothelium is unexplored. We tested this hypothesis with endothelial monolayers exposed to RBC from normal (Hb AA) and sickle cell (Hb SS) volunteers in a parallel plate flow chamber. To decrease NO production, endothelial monolayers were exposed to 100 microM nitro-L-arginine (NLA), an inhibitor of nitric oxide synthase, resulting in an 87% increase in normal RBC adherence (P = 0.002). Because adherence of normal RBC to endothelium was low, the effect of DETA-NO, an NO donor, was tested after activation of endothelium with TNF-alpha increased adherence by 130% (P < 0.001). Subsequent addition of 2 mM DETA-NO produced a 75% decrease in adherence of normal RBC to endothelium (P = 0.03). At baseline, sickle RBC were significantly more adherent than normal RBC (P < 0.001) and DETA-NO decreased sickle RBC adherence by 54% (P = 0.04). Thus, NO inhibits both normal and sickle RBC adherence to endothelium. Strategies that enhance NO activity may be therapeutic in sickle cell disease.     

Cell-bound autologous immunoglobulin in erythrocyte subpopulations from patients with sickle cell disease

Previously, we have demonstrated a parallel between most-dense (bouyant density) sickle erythrocyte subpopulations and most-dense aged normal red cells in the organization of membrane components in the intact cell. The present study has addressed the possibility that a corresponding similarity may exist between most-dense sickled red cell subpopulations and aged normal erythrocytes in the development of membrane protein components that function as receptors for autologous immunoglobulin (Ig). Autologous IgG retained by density-fractionated erythrocytes has been estimated by a nonequilibrium 125I-protein A (Staphylococcus aureus) binding assay. Results show that most-dense sickle cell fractions contain more (2.7-fold and 1.8-fold, P less than .005) cell-bound IgG in comparison to younger sickle erythrocyte fractions sedimenting at low density. Parallel findings were obtained after similar analyses of normal (homozygous-A) erythrocyte fractions. Detection of the presence of specific IgG was also carried out by direct binding of fluorescein isothiocyanate-conjugated anti-human IgG to density-separated red cell fractions followed by analyses of the fluorescent cell populations by flow cytometry. Results showed significantly higher levels of IgG bound to most-dense (12.1% +/- 2.5% and 8.8% +/- 0.5%-) sickle red cell subpopulations (P less than .005) in comparison to younger sickle erythrocyte fractions sedimenting at low densities (3.8% +/- 0.32% and 4.7% +/- 1.6% IgG-positive red cell subpopulation). These results indicate that some of the same membrane changes that occur at about 120 days in normal red cells are also apparent in the chronologically younger (life span in vivo, ten to 40 days) sickle erythrocyte. The increased retention of IgG by most-dense irreversibly sickled cell-enriched fractions in comparison to least- dense reversibly sickled cells or pre-irreversibly sickled erythrocyte fractions, suggests that alterations in the topography of the sickle cell membrane during the transformation in vivo to the most-dense irreversibly sickled cell morphology may produce the unmasking of cryptic antigenic sites. In addition, these findings may indicate that opsonization of specific erythrocyte subpopulations may play a role in the pathophysiology of sickle cell disease.     

Perfusion with sickle erythrocytes up-regulates ICAM-1 and VCAM-1 gene expression in cultured human endothelial cells

Sickle cell anemia is characterized by periodic vasoocclusive crises. Increased adhesion of sickle erythrocytes to vascular endothelium is a possible contributing factor to vasoocclusion. This study determined the effect of sickle erythrocyte perfusion at a venous shear stress level (1 dyne/cm(2)) on endothelial cell (EC) monolayers. Sickle erythrocytes up-regulated intercellular adhesion molecule-1 (ICAM-1) gene expression in cultured human endothelial cells. This was accompanied by increased cell surface expression of ICAM-1 and also elevated release of soluble ICAM-1 molecules. Expression of vascular cell adhesion molecule-1 (VCAM-1) messenger RNA (mRNA) was also strikingly elevated in cultured ECs after exposure to sickle cell perfusion, although increases in membrane-bound and soluble VCAM-1 levels were small. The presence of cytokine interleukin-1beta in the perfusion system enhanced the production of ICAM-1 and VCAM-1 mRNA, cell surface expression, and the concentrations of circulating forms. This is the first demonstration that sickle erythrocytes have direct effects on gene regulation in cultured human ECs under well-defined flow environments. The results suggest that perfusion with sickle erythrocytes increases the expression of cell adhesion molecules on ECs and stimulates the release of soluble cell adhesion molecules, which may serve as indicators of injury and/or activation of endothelial cells. The interactions between sickle red blood flow, inflammatory cytokines, and vascular adhesion events may render sickle cell disease patients vulnerable to vasoocclusive crises.     

Erythrocyte-active agents and treatment of sickle cell disease

The sickle hemoglobin (HbS)-containing erythrocyte and its membrane represent a logical target for sickle cell disease therapy. Several antisickling agents which interfere with HbS polymerization have been studied over the last 30 years, but none has overcome the challenge of delivering high concentrations inside the sickle red blood cell without toxicity. The sickle erythrocyte membrane has also been targeted for therapeutic developments. Prevention of sickle cell dehydration by use of specific blockers of ion transport pathways mediating potassium loss from the sickle erythrocyte has been shown to be a feasible strategy in vitro, in vivo in transgenic sickle mice, and in patients. Other approaches have focused on improving the hemorheology of sickle erythrocytes and reducing their abnormal adhesion to endothelial cells. These potential treatments could be used alone or in combination with other approved therapies, such as hydroxyurea.