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G B Nash

Publications and source records attributed to G B Nash.

At least 37 records · Page 2Linked to original sources
PubMed1999

Neutrophils sense flow-generated stress and direct their migration through alphaVbeta3-integrin.

During inflammation neutrophils are recruited from the blood onto the surface of microvascular endothelial cells. In this milieu the presence of soluble chemotactic gradients is disallowed by blood flow. However, directional cues are still required for neutrophils to migrate to the junctions of endothelial cells where extravasation occurs. Shear forces generated by flowing blood provide a potential alternative guide. In our flow-based adhesion assay neutrophils preferentially migrated in the direction of flow when activated after attachment to platelet monolayers. Neutralizing alphaVbeta3-integrin with monoclonal antibodies or turning the flow off randomized the direction of migration without affecting migration velocity. Purified, immobilized alphaVbeta3-integrin ligands, CD31 and fibronectin, could both support flow-directed neutrophil migration in a concentration-dependent manner. Migration could be randomized by neutralizing alphaVbeta3-integrin interactions with the substrate using antibodies or Arg-Gly-Asp-containing peptide. These results exemplify mechanical signal transduction through integrin-ligand interactions and reveal a guidance system that was hitherto unknown in neutrophils. In more general terms, it demonstrates that cells can use integrin molecules to "sample" their physical microenvironment through adhesion and use this information to modulate their behavior.

Blood Circulation↗
PubMed1999

Conditions under which immobilized platelets activate as well as capture flowing neutrophils.

Immobilized activated platelets present P-selectin and efficiently capture flowing neutrophils. We investigated how the treatment of the platelets affected whether adherent neutrophils rolled continuously or became immobilized. Washed platelets were maintained in a 'resting' state by Ca++ chelation, prostacyclin and theophylline. When these platelets were adhered to glass that had been coated with aminopropyltriethoxysilane (APES) they retained discoidal morphology. Compared to a confluent surface of spread platelets prepared by allowing heparinized platelet-rich plasma to settle on APES-glass, 'resting' platelets captured far fewer flowing neutrophils, which rolled rapidly. However, if neutrophils were perfused along with thrombin (>/= 0.2 U/ml), then the resting platelets rapidly changed shape, neutrophil binding increased markedly, rolling velocity decreased, and 40-70% of the neutrophils were immobilized via beta2-integrins. Similar effects could be induced using ADP perfused with the neutrophils. Thrombin did not itself activate neutrophils, and stationary adhesion could also be induced if platelets were treated with thrombin before addition of neutrophils. After thrombin treatment of platelets, rolling adhesion was only fully re-established after a prolonged period of washout. Thus, platelets presented a stable surface-bound agent able to activate neutrophils. Blockade of platelet-activating factor receptor, leukotriene B4 receptor, or CXC-chemokine receptor 1 (CXCR1) on neutrophils did not inhibit conversion from rolling to stationary adhesion, but blockade of CXCR2 maintained a higher proportion of rolling adhesion. Thus, platelets attached to damaged vessels may capture flowing neutrophils, but the stability of neutrophil deposition will depend on the scale of the local generation of platelet agonists such as thrombin and ADP.

Blood Platelets↗
PubMed1999

Measurement of leukocyte rheology in vascular disease: clinical rationale and methodology. International Society of Clinical Hemorheology.

The measurement of leukocyte rheology in vascular disease is a recent development with a wide range of new opportunities. The International Society of Clinical Hemorheology has asked an expert panel to propose guidelines for the investigation of leukocyte rheology in clinical situations. This article first discusses the mechanical, adhesive and related functional properties of leukocytes (especially neutrophils) which influence their circulation, and establishes the rationale for clinically-related measurements of parameters which describe them. It is concluded that quantitation of leukocyte adhesion molecules, and of their endothelial receptors may assist understanding of leukocyte behaviour in vascular disease, along with measurements of flow resistance of leukocytes, free radical production, degranulation and gene expression. For instance, vascular cell adhesion molecule (VCAM-1) is abnormally present on endothelial cells in atherosclerosis, diabetes mellitus and inflammatory conditions. Soluble forms of intercellular adhesion molecule (ICAM-1) or VCAM can be found elevated in the blood of patients with rheumatoid arthritis or infections disease. In the second part of the article, possible technical approaches are presented and possible avenues for leukocyte rheological investigations are discussed.

Arteriosclerosis↗
PubMed1998

Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of different chemotactic agents.

Neutrophils must adhere to the vessel wall, migrate, and degranulate in an ordered manner to perform their protective function. Disruption of these processes may be pathogenic. Current knowledge of the degranulation process is derived almost exclusively from studies on neutrophils in suspension, in which priming with the nonphysiological agent cytochalasin B is necessary to obtain elastase release in response to activating agents. To avoid this, we have adopted a different approach. Using a novel flow-based adhesion system, we have been able to quantify the release of elastase from the primary granules of activated neutrophils adherent to immobilized platelets or purified receptors without priming. Comparing stimuli, formyl tripeptide (fMLP), interleukin-8 (IL-8), activated complement fragment C5a, and platelet-activating factor (PAF) all induced rapid conversion to CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over neutrophils already rolling on platelet monolayers or purified P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release of elastase from the adherent cells in minutes. Neutrophils stimulated in suspension showed little degranulation. Treatment of neutrophils with an inhibitor of 5-lipoxygenase-activating protein (MK886) and thus synthesis of leukotrienes (LTs) or with an antagonist of the LTB4 receptor (LY223982) blocked the release of elastase. This indicated that endogenous synthesis of 5-lipoxygenase products such as LTs and autocrine activation of neutrophils was required for fMLP-driven elastase release. We hypothesize that the differential ability of PAF and fMLP to induce elastase release from surface-adherent neutrophils could arise from differential ability to generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are not released until activated neutrophils have migrated into the extravascular tissues.

Blood Platelets↗
PubMed1998

Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin.

We investigated how disruption of the actin cytoskeleton with cytochalasins modified adhesion of neutrophils rolling on a platelet monolayer in vitro at 37 degrees C. When perfused at a wall shear stress of 0.1 Pa over rolling cells, cytochalasin B, cytochalasin D and dihydro-cytochalasin B each induced dose-dependent (approximately 1-10 microg/ml) conversion to stationary attachment over minutes. Stopping was associated with cell elongation to a teardrop shape. Increased deformability of cytochalasin-treated cells was independently evidenced by more rapid entry into a micropipette. Spherical shape and rolling were reestablished concurrently on washout of the cytochalasins, while increasing the shear stress in the range 0.2 to 1.0 Pa induced tear-drop-shaped cells to restart rolling even in the continued presence of cytochalasin. When cells were pretreated with cytochalasin B, they attached efficiently at 0.1 Pa, rolled initially and only stopped after approximately 30 seconds when elongation had been established. Adhesion was selectin-mediated in the presence or absence of cytochalasin B, as judged by inhibition of attachment by antibody against P-selectin and failure of antibody against beta2-integrin CD18 to influence adhesion. Cessation of rolling is unlikely to have arisen from an increase in adhesive contact area induced by deformation because stopped cells were found to be attached only at their pointed end. Failure of adhesive bonds to peel may have arisen because selectin ligands freed of cytoskeletal restraint were dragged into this tethered region and clustered there, and because force applied to bonds was influenced by the change in cell shape. These results suggest that cytoskeletal structure is an important modulator of dynamic adhesive responses of leukocytes, via effects on adhesion receptors and cellular mechanics.

Cell Adhesion↗
PubMed1998

Sparsely adherent platelets support capture and immobilization of flowing neutrophils.

Continuous monolayers of activated platelets support selectin-mediated rolling and integrin-mediated immobilization of flowing neutrophils. Because platelets attached to the vessel wall in vivo may not be confluent, we investigated the ability of sparsely adherent platelets to capture flowing neutrophils that were unstimulated or activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide (10(-7) mol/L). Different degrees of deposition of platelets were obtained by perfusing heparinized blood for varying periods through glass capillaries coated with collagen type III. Perfusion for 10 to 180 seconds gave approximately 1% to approximately 10% coverage of the surface with platelets. When purified neutrophils were subsequently flowed over the platelets at a wall shear stress of 0.1 Pa, they formed repeated short-lived attachments to individual or small groups of platelets. Increasing surface coverage with platelets was associated with increasing numbers of neutrophils adherent at any time, decreasing the overall rate of motion of adherent neutrophils and increasing the stability of adhesion (eg, proportion remaining with time). The net effect was to actually decrease the flux of adherent cells (number of adhesive interactions seen per unit area per unit time), indicating that parameters based on through-flow of adhesive cells must be interpreted with caution. When neutrophils were preactivated with fMLP they formed stable, stationary attachments. The flux of adherent activated cells was low, although the number of cells adherent remained high, reinforcing the conclusion that assays of the adhesion of flowing cells must carefully characterize the different types of adhesive behavior. Overall, small numbers of platelets can support short-lived attachments of flowing neutrophils and allow immobilization of activated cells and may amplify inflammatory processes thereby.

Blood Platelets↗
PubMed1998

Multiple adhesive phenotypes linked to rosetting binding of erythrocytes in Plasmodium falciparum malaria.

The cerebral form of severe malaria is associated with excessive intravascular sequestration of Plasmodium falciparum-infected erythrocytes (PRBC). Retention and accumulation of PRBC may lead to occlusion of brain microvessels and direct the triggering of acute pathologic changes. Here we report that by selection, cloning, and subcloning, we have identified rare P. falciparum parasites expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. Rosetting PRBC not only bound uninfected erythrocytes but also formed autoagglutinates, adhered to endothelial cells, and bound to CD36, immunoglobulins, and the blood group A antigen. The linkage of rosetting, autoagglutination, and cytoadherence involved the coexpression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor for P. falciparum-infected erythrocytes. Limited proteolysis that differentially cleaved the rosetting ligand PfEMP1 from the PRBC surface abrogated all the binding phenotypes of these parasites, implicating the variant antigen PfEMP1 as a carrier of multiple ligand specificities. The results encourage the further study of pan-adhesion as a potentially important parasite phenotype in the pathogenesis of severe P. falciparum malaria.

Animals↗
PubMed1998

Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha.

Migration of circulating neutrophils occurs in several steps: capture and rolling adhesion are followed by activation of beta 2-integrins and immobilisation, and then neutrophils move over and through the endothelium. However, it is not clear how the underlying mechanisms and completion of each step depend on the concentration of stimulatory cytokines such as tumour necrosis factor-alpha (TNF). We therefore perfused neutrophils over human umbilical vein endothelial cells (HUVEC) which had been cultured with varying concentration of TNF (1-1000 U/ml) for 4 h, and recorded adhesion and migration by videomicroscopy. The number of adherent neutrophils increased with increasing TNF up to 5 U/ml, but changed little at higher concentrations. Interestingly, rolling adhesion at first predominated, but an increasing proportion of adherent cells became immobilised and migrated through the HUVEC monolayer over the complete TNF range. Immobilisation was inhibited by treating neutrophils with antibody against CD18, so that the major change in adhesive behaviour at higher levels of TNF occurred because the surface of the HUVEC presented agent(s) able to activate neutrophil beta 2-integrins. It was also evident that the selectins initiating capture of flowing neutrophils varied with concentration of TNF. At 100 U/ml TNF, both E-selectin and P-selectin supported capture and rolling adhesion, and antibody blockade of both receptors was required to inhibit adhesion. At lower dose (10 U/ml TNF), stable adhesion was blocked by antibody against E-selectin, although short-lived attachments could still be seen which were inhibited by antibody against P-selectin. Expression of sclectins increased with increasing concentration of TNF, judging from surface ELISA and reduction in the velocity of rolling adherent cells. Thus the efficiency of capture, the selectins mediating capture and the proportion of captured cells immobilised and migrating all depend on the concentration of TNF to which endothelial cells are exposed. These results suggest a model in which highly localised and efficient migration of neutrophils is achieved if a concentration gradient of TNF exists around an inflammatory locus.

Cell Movement↗
PubMed1998

Neutrophils rolling on immobilised platelets migrate into homotypic aggregates after activation.

Interactions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10(-7) M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of approximately 500 microm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/CD18 and ICAM-3, between alpha(v)beta3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.

Antibodies, Monoclonal↗
PubMed1997

Actin polymerisation regulates integrin-mediated adhesion as well as rigidity of neutrophils.

Activation of adherent neutrophils causes them to convert from selectin-mediated rolling to integrin-mediated immobilisation and migration. Migration is known to depend on formation and redistribution of filamentous (F) actin, but although immobilisation in seconds parallels early cortical actin polymerisation, no link has been proven. We tested the effect of the actin-polymerising agent jasplakinolide (10 microM) on adhesive and mechanical properties of neutrophils. Pretreated cells were able to adhere and roll on immobilised platelets in a flow-based adhesion assay, but whereas untreated rolling cells became immobilised in seconds when chemotactic formyl peptide (fMLP, 0.1 microM) was superfused over them, the cells treated with jasplakinolide continued rolling. Pretreatment with jasplakinolide also blocked de novo expression of integrin CD11b and shape change which otherwise occurred in minutes after treatment with fMLP. Jasplakinolide directly caused actin polymerisation within neutrophils, evidenced by a marked increase in rigidity (resistance to aspiration into a 5 microm micropipette) and increase in association of actin with the Triton-insoluble cytoskeleton. These results indicate that rearrangement of the actin cytoskeleton regulates integrin-mediated adhesion of activated neutrophils, as well as their migration and mechanical properties.

Actins↗
PubMed1997

Cross-talk between cell adhesion molecules regulates the migration velocity of neutrophils.

BACKGROUND: Although the adhesive mechanisms underlying the capture and immobilization of circulating neutrophils in inflamed blood vessels have been well described, factors controlling the subsequent migration of neutrophils over and through the blood vessel endothelium are poorly understood. Directional rearrangement of the actin cytoskeleton within the neutrophil, along with modulation of integrin-mediated adhesion, are necessary for neutrophil migration. Signals from chemotactic agents and from the adhesive substrate may regulate these processes, but little is known about their relative importance or their mode of integration. RESULTS: We examined the kinetics of neutrophil migration after formyl tripeptide or platelet-activating factor was perfused over neutrophils that were already rolling on the adhesion molecule P-selectin, which was presented either on the surface of immobilized platelets or in purified form coated on glass capillaries. Upon activation, neutrophils stopped rolling, spread and began to migrate; each of these processes was dependent on beta2 integrin (CD11b/CD18). The rate of migration increased over a period of about 8 minutes and was modulated directly by both the P-selectin and the CD31 surface receptors. Antibody blockade of either CD31 or P-selectin on platelets resulted in a reduction in the velocity of migration, and simultaneous blockade of both receptors reduced velocity further. Purified CD31 and P-selectin (but not a control adhesion molecule, ICAM-1) increased migration velocity in a concentration-dependent and additive manner that reconstituted the migratory behaviour observed on platelets. CONCLUSIONS: These studies show that binding of ligands to CD31 and/or P-selectin modifies the rate of integrin-supported neutrophil migration. This novel example of 'cross-talk' between surface receptors suggests that cell adhesion molecules might generally transduce accessory signals between adjacent cells to modify their migratory responses to chemotactic signals.

Animals↗
PubMed1997

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.

Animals↗
PubMed1997

Rheological analysis of the formation of rosettes by red blood cells parasitized by Plasmodium falciparum.

Red blood cells infected by mature malarial parasites of the species Plasmodium falciparum can adhere to non-parasitized red cells (rosetting) and also to endothelial cells (cytoadhesion). To investigate how the circulation might influence rosetting, we studied formation of rosettes in cell suspensions sheared in a cone-and-plate viscometer, and the ability of flowing non-parasitized cells to bind to parasitized cells already adherent to a surface. After rosettes of strain R29 had been disrupted with fucoidan, they reformed slowly under stationary conditions but more rapidly in suspensions sheared at low stress (about 0.1-0.2 Pa). Strain Malayan Camp gave a lower rosetting frequency which actually increased at low shear. Increasing shear stress was associated with reduction in rosette formation, although rosetting occurred at >1 Pa, suggesting that rosettes could form in the systemic circulation. Rosetting inhibited adhesion of flowing parasitized cells to immobilized platelets (which express the cytoadhesion receptor CD36), as evidenced by increased adhesion after disruption of rosettes. The de-rosetted adherent cells parasitized by R29 supported only a low level of rosetting when non-parasitized cells were flowed over them at a wall shear of 0.1 Pa, with little increase if the stress was decreased to 0.05 Pa. Rosettes formed in the circulation might obstruct microvessels and inhibit cytoadhesion if they reached venules. However, if cytoadhesion occurred before rosetting, then adherent cells should not efficiently form rosettes.

Animals↗
PubMed1997

Endothelial-borne platelet-activating factor and interleukin-8 rapidly immobilize rolling neutrophils.

The kinetics of the response of integrins to activating signal(s) must be rapid to ensure that rolling neutrophils are localized at the sites of inflammation. From video records, we analyzed the adhesion of individual neutrophils in a flow-based in vitro model of endothelial hypoxia and reoxygenation. There were numerous rolling interactions between flowing neutrophils and P-selectin on human umbilical vein endothelial cells after hypoxia, but 90% lasted for < 1 s, with approximately 30% converted to stationary attachment via beta 2-integrin(s). Interleukin-8 (IL-8) and platelet-activating factor (PAF) were responsible for neutrophil activation in this model [G. E Rainger, A. Fisher, C. Shearman, and G. B. Nash. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1398-H1406, 1995]. In the presence of a PAF-receptor antagonist, IL-8 acting alone induced conversion of rolling to stationary adhesion in as little as 80 ms after the initial attachment of a neutrophil, with a median response time of 240 ms. In the presence of a monoclonal antibody that neutralized IL-8 activity, PAF acting alone required a minimum duration of rolling of 560 ms to promote stationary adhesion, with a significantly longer median duration of 720 ms. In a reconstituted model, treatment of endothelial cells with hydrogen peroxide induced short-lived rolling of neutrophils supported by P-selectin. Exogenously added IL-8 and/or PAF bound to the endothelial surface and successfully induced the immobilization of neutrophils. Rapid and distinct kinetics of the conversion to stationary adhesion were observed again for IL-8 or PAF. Thus although endothelial-presented signals differed in their rate of action, neutrophils could be localized within one or two endothelial cell diameters of their initial adhesive contact point.

Cell Adhesion↗
PubMed1996

Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets.

In an in vitro flow model, unstimulated neutrophils rolled steadily over a surface coated with platelets, until superfusion of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) caused a dose-dependent (10(-11) to 10(-7) mol/L) transition from rolling to stationary attachment in seconds, followed more slowly by neutrophil shape change and spreading on the surface, However, at low concentrations of Ca2+ and Mg2+ (0.1 mmol/L and 0.05 mmol/L, respectively, rather than physiologic 1 mmol/L and 0.5 mmol/L), neutrophils first halted but then started to roll again and to detach from the surface over 5 to 10 minutes. At the low cation concentration, stopping was largely inhibited by antibodies to the neutrophil integrins CD18 or CD11b, but not CD11a. When neutrophils were pretreated with antibodies to CD11b or CD18 in 1 mmol/L Ca2+ 0.5 mmol/L Mg2+, stopping was not prevented but delayed. However, if antibodies were also included with the superfused fMLP, stopping was inhibited, and detachment followed. This indicates that CD11b/CD18 was newly expressed during shape change and mediated the second phase of neutrophil immobilization and spreading in a cation-dependent manner. Prestimulated neutrophils also bound to platelets and spread, but immobilization was blocked if they were perfused with antibody to CD18 or CD11b or with low Ca2+ and Mg2+. Examining the cation-dependence further, it was evident that the presence of Mg2+ was essential for integrin-mediated adhesion and that the Mg2+ concentration determined whether immobilization could be maintained or was transient. Continuous superfusion of fMLP was also essential for maintenance of stable adhesion and spreading. Thus, activation of constitutive CD11b/CD18 rapidly and reversibly converted rolling to stationary attachment, whereas maintenance of adhesion and neutrophil spreading required continual expression of additional CD11b/CD18 that was only functional at physiologic Mg2+. Continual activation and deactivation of CD11b/CD18 during de novo expression could mediate immobilization and onward migration of neutrophils in vivo, and activated platelets appear capable of supporting this process as well as endothelial cells.

Blood Platelets↗
PubMed1996

Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells. Studies by partitioning in aqueous phase systems.

Counter-current distribution (CCD) of red blood cells (RBC) from individuals with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.

Cell Separation↗
PubMed1996

Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells.

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-isopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the "middle" cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37 degrees C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4 degrees C. On the other hand, incubation at 37 degrees C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction.

Adult↗