Interaction forces between red cells agglutinated by antibody. III. Micromanipulation.

In the flow studies described in two previous papers (Tha, S. P., and H. L. Goldsmith, 1986, Biophys. J. 50:1109-1116; Tha, S. P., J. Shuster, and H. L. Goldsmith, 1986, Biophys. J. 50:1117-1126), hydrodynamic forces of the order of 10(-11) N (mu dyn) were applied to measure the force of separation of doublets of hardened, sphered human red blood cells cross-linked by anti-B antibody. The same cell preparation and hyperimmune antiserum has here been used to carry out experiments with micropipet aspiration techniques. One cell of a doublet was aspirated onto a holding pipet, and a second aspiration pipet was brought into proximity of the other cell so that the two pipets and the doublet were colinear. Suction was then raised until the two cells separated. Some doublets were assembled by aspiration of a singlet, bringing a second singlet into apposition with the first, and releasing it from the pipet which was then withdrawn. Cells could be repeatedly assembled and separated. At 3.56% vol/vol antiserum, the mean normal force of separation was 0.45 +/- 0.11 nN in phosphate-buffered saline suspensions containing 2.5 x 10(4) cells/microliter; at 1.22% vol/vol antiserum, the value was 0.22 +/- 0.11 nN. The above values of the force were approximately 2.5 x greater than those from the flow studies. The data could be fitted to a Poisson distribution with 0.05 nN as the force needed to break a single cross-bridge (c.f. 0.024 nN from the previous hydrodynamic data). The forces of separation of randomly assembled doublets were lower than those of preexisting doublets. Repeated assembly and separation of doublets showed that the cell surfaces are nonuniform in adhesion strength both over the local scale less than 0.25 micron2 and the cell population.     

Interaction forces between red cells agglutinated by antibody. I. Theoretical.

A general method of calculating forces, torques, and translational and rotational velocities of rigid, neutrally buoyant spheres suspended in viscous liquids undergoing a uniform shear flow has been given by Arp and Mason (1977). The method is based on the matrix formulation of hydrodynamic resistances in creeping flow by Brenner and O'Neill (1972). We describe the solution of the Brenner-O'Neill force-torque vector equation in terms of the particle and external flow field coordinates and derive expressions for the normal force acting along, and the shear force acting perpendicular to, the axis of the doublet of spheres, the latter explicitly given for the first time. The equations consist of a term comprising force and torque coefficients obtained from the matrices of the hydrodynamic resistances (functions of the distance h between sphere surfaces which have been computed), and terms comprising the orientation of the doublet axis relative to the coordinates of the external flow field and the shear stress (which can be experimentally determined). We have applied the theory to a system of doublets of sphered, hardened human red cells of group A or B antigenic type cross-linked by the corresponding antibody at a fixed interparticle distance. Working from studies of the breakup of doublets of red cells in an accelerating Poiseuille flow, given in the succeeding paper, we are able to compute the hydrodynamic force required to separate the two spheres. Previous work has shown that the theory can be applied to doublets in a variable shear, Poiseuille flow, provided the ratio of particle to tube diameter is small. In calculating the force-torque coefficients it was assumed that the cells are crosslinked by antibody with h = 20 nm.     

Interaction of bacteria and red blood cells

The interaction of staphylococci, streptococci, meningococci, enterobacteria, leptospires and other microorganisms with red blood cells is considered. Three forms of the interaction of bacteria and red blood cells are discussed: adhesion, the influence of secretory factors on red blood cells, the action of pathogenic bacteria on hemoglobin. The applied aspects of the interaction of bacteria and red blood cells in the human body are presented in accordance with the results of clinical and laboratory studies.     

Interaction forces between red cells agglutinated by antibody. II. Measurement of hydrodynamic force of breakup.

The expressions derived in the previous paper for the respective normal, F3, and shear forces, Fshear, acting along and perpendicular to the axis of a doublet of rigid spheres, were used to determine the hydrodynamic forces required to separate two red cell spheres of antigenic type B crosslinked by the corresponding antibody. Cells were sphered and swollen in isotonic buffered glycerol containing 8 X 10(-5) M sodium dodecyl sulfate, fixed in 0.085% glutaraldehyde, and suspended in aqueous glycerol (viscosity: 15-34 mPa s), containing 0.15 M NaCl and anti-B antibody from human hyperimmune antiserum at concentrations from 0.73 to 3.56 vol%. After incubating and mixing for 12 h, doublets were observed through a microscope flowing in a 178-micron tube by gravity feed between two reservoirs. Using a traveling microtube apparatus, the doublets were tracked in a constantly accelerating flow and the translational and rotational motions were recorded on videotape until breakup occurred. From a frame by frame replay of the tape, the radial position, velocity and orientation of the doublet were obtained and the normal and shear forces of separation at breakup computed. Both forces increased significantly with increasing antiserum concentration, the mean values of F3 increasing from 0.060 to 0.197 nN, and Fshear from 0.023 to 0.072 nN. There was no significant effect of glycerol viscosity on the forces of separation. It was not possible to determine whether the shear or normal force was responsible for doublet separation. Measurements of the mean dimensionless period of rotation, TG, of doublets in suspensions containing 0.73 and 2.40% antiserum undergoing steady flow were also made to test whether the spheres were rigidly linked or capable of some independent rotation. A fairly narrow distribution in TG about the value 15.64, predicted for rigidly-linked doublets, was obtained at both antiserum concentrations.     

Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up.

We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-microns-diameter tubes, and the hydrodynamic normal force at break-up, Fn, is computed from the measured doublet velocity and radial position. Previous results showed a large range of Fn, the mean of which increased with [antiserum], and an absence of clustering at discrete values of Fn. Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of Fn. The results showed that the data are as scattered as ever, with Fn varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in Fn could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (Fn from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91% survived into the constant force region; 16% of these broke up after time intervals, tP, of 2-30s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, tc, from < 1 to 10 s. Thus, doublets do indeed break up under a constant shear stress, if given time. The average time to break-up decreased significantly with increasing force, while the fraction of doublets broken up increased. At a given Fn, the fraction of break-ups decreased with increasing [IgM], suggesting that the average number of bonds had also increased.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Interaction of acidic liposomes with red blood cells: Induction of endocytosis and shedding of particles

Calcium ions induced tight binding of massive amounts of liposomes containing cardiolipin, phosphatidylethanolamine and phosphatidylcholine to erythrocytes. Initially, liposome-liposome fusion occurred and only subsequently the resulting large structures adhered to cells. Large clumps composed of liposomes and cells were formed. Upon prolonged incubation, the clumps were dissipated spontaneously and excess liposomes were released. A constant amount of phospholipid (15–25 nmol/10⁸ cells) were incorporated into the cell membranes. Upon disaggregation, the cells shed erythrocyte particles. The latter were isolated and shown to contain lipids from both cellular and liposomal origin. The particles lacked spectrins and contained variable amounts of band 3 content. Liposomes induced endocytosis in reticulocytes but not in mature erythrocytes. In most cases, the liposomes themselves were not engulfed by the cells and remained attached to their surface. The relevance of this phenomenon to delivery of liposome contents into cells is discussed.     

Interaction of doxorubicin and idarubicin with red blood cells from acute myeloid leukaemia patients

Doxorubicin (DOX) and idarubicin (IDA) are anthracycline antibiotics, widely used in human cancer treatment. The present study addressed the effects of these two drugs on lipid bilayer fluidity, protein conformation and microviscosity in erythrocytes from acute myeloid leukaemia patients, using electron spin resonance (ESR) spectroscopy and fluorescence measurements. Only DOX caused statistically significant changes in the parameters examined. Within 30 min of drug injection, changes were observed in the fluidity of the hydrophobic parts of the lipid bilayer and erythrocyte membrane protein conformation. These changes persisted for up to 24h. Analysis of the EPR Tempamine spectrum also showed that the microviscosity of the erythrocyte interior increased during the early stages of the drug effect. Idarubicin, in contrast, caused no identifiable change in any of the parameters studied and therefore seems to be safe for erythrocytes. We conclude that IDA is markedly less toxic than DOX to erythrocytes from acute myeloid leukaemia patients.     

Hypotonic hemolysis of human red blood cells: a two-phase process.

Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.     

Loading red blood cells with trehalose: a step towards biostabilization

A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. As a first step toward RBC preservation, we present a method for loading RBCs with trehalose. The method is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37 degrees C in a time span of 7 h. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, resulting in intracellular trehalose concentrations of about 40 mM. During the loading period, the levels of ATP and 2,3-DPG are maintained close to the levels of fresh RBCs. Increasing the membrane fluidity through the use of a benzyl alcohol results in a higher concentration of intracellular trehalose, suggesting the importance of the membrane physical state for the uptake of the sugar. Osmotic fragility data show that trehalose exerts osmotic protection on RBCs. Flow cytometry data demonstrate that incubation of RBCs in a hypertonic trehalose solution results in a fraction of cells with different complexity and that it can be removed by washing and resuspending the RBCs in an iso-osmotic medium. The data provide an important first step in long-term preservation of RBCs.     

Desferrioxamine decreases NAD redox potential of intact red blood cells: evidence for desferrioxamine as an inducer of oxidant stress in red blood cells

Background  

Desferrioxamine (DFO) is an important iron chelating agent. It has also been thought of as an agent with anti-oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBC) by inducing intracellular oxidant stress. To further understand the mechanism of DFO's interaction with RBC, we conducted a study to determine the effect of DFO upon RBC's redox status.     

Evaluation of intermolecular forces in a circulating system

Intercellular interactions, which are mediated by a variety of complex intercellular molecules through the processes of formation and dissociation of molecular bonds, play a critical role in regulating cellular functions in biological systems. Various approaches are applied to evaluate intercellular or molecular bonding forces. To quantify the intermolecular interaction forces, flow chamber has become a meaningful technique as it can ultimately mimic the cellular microenvironment in vivo under physiological flow conditions. Hydrodynamic forces are usually used to predict the intercellular forces down to the single molecular level. However, results show that only using hydrodynamic force will overestimate up to 30% of the receptor-ligand strength when the non-specific forces such as Derjaguin-Landau-Verway-Overbeek (DLVO) forces become un-neglected. Due to the nature of high ion concentration in the physiological condition, electrostatic force is largely screened which will cause DLVO force unbalanced. In this study, we propose to take account of the DLVO force, including van der Waals (VDW) force and electrostatic force, to predict the intermolecular forces of a cell doublet and cell-substrate model in a circulating system. Results also show that the DLVO force has a nonlinear effect as the cell-cell or cell-substrate distance changes. In addition, we used the framework of high accuracy hydrodynamic theories proved in colloidal systems. It is concluded that DLVO force could not be ignored in quantitative studies of molecular interaction forces in circulating system. More accurate prediction of intercellular forces needs to take account of both hydrodynamic force and DLVO force.     

Guanine ribonucleotide metabolism in human red blood cells: evidence for a high rate of GMP dephosphorylation

The flux rates through the metabolic pathways affecting the maintenance of GuRN pool in intact human RBC were studied. Normal RBC, incubated in KRBB, exhibited a markedly higher accumulation in nucleotides of Gu than of Hx. Addition of 8-AGuo, a potent inhibitor of PNP, resulted in a marked increase in the accumulation of label in the nucleosides, in Ino following incubation with Hx, and in Guo following incubation with Gu, indicating a very high rate of IMP and GMP degradation to bases through their respective nucleosides. Most of the degradation of GMP is by dephosphorylation to Guo, rather than through reductive deamination to IMP. The ultimate fate of IMP in RBC is its degradation to Ino and consequently to Hx. The contribution of AdRN or of IMP to the GuRN pool is negligible. The results indicate that concerning IMP and GMP, human RBC contain very active futile cycles, nucleotide----nucleoside----base----nucleotide, catalyzed by 5'-nucleotidase, PNP, and HGPRT. The operation of the complete cycles is essential for the maintenance of GuRN and the IMP pool size. These results may explain the finding of reduced GTP content in RBC from patients with an inborn deficiency of PNP or of HGPRT.     

Determination of Fc function with frozen red blood cells.

The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility.