Activated polymorphonuclear leukocytes affect red blood cell aggregability

Transcription

1 Activated polymorphonuclear leukocytes affect red blood cell aggregability Oguz K. Baskurt and Herbert J. Meiselman Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles Abstract: Activated leukocytes can affect adjacent cells by generating oxygen free radicals and secreting proteolytic enzymes, and red blood cells (RBC) exposed to such agents should be susceptible to their effects. This study was thus designed to investigate the effects of activated polymorphonuclear leukocytes (PMN) on RBC aggregability (i.e., on intrinsic RBC aggregation characteristics). PMN were isolated from human blood by density separation and suspended in glucose-enriched buffer with RBC isolated from the same blood sample (RBC/PMN ratio of 150:1). PMN were then activated in this suspension by adding 1 ng/ml tumor necrosis factor (TNF- ) and 10 7 M N-formyl-methionyl-leucylphenylalanine (fmlp) or fmlp alone. After incubation for 2hat37 C, RBC aggregation behavior in autologous plasma was assessed; RBC deformability and partition coefficients were also measured. RBC aggregation was significantly increased after incubation and deformability and partitioning were decreased; these effects were prevented by phenylmethylsulfonyl fluoride (1 mm) or superoxide dismutase (20 g/ml) plus catalase (40 g/ml). TNF- and fmlp alone had no effect on RBC aggregation if PMN were not present. Activated PMN can thus markedly affect RBC aggregability, apparently via both proteolytic enzymes and oxygen free radicals; enhanced aggregation seen in clinical states associated with PMN activation or observed during in vivo RBC aging may also involve such PMN-RBC interactions. J. Leukoc. Biol. 63: 89 93; Key Words: aggregation leukocyte activation antioxidant enzymes erythrocyte INTRODUCTION Previous literature reports have documented that polymorphonuclear leukocyte (PMN or neutrophil) activation is associated with the following structural and functional alterations within the cell: (1) activated leukocytes exhibit morphological transformation (e.g., pseudopod formation) and cellular mechanical alterations [1, 2] that have been shown to be associated with actin polymerization [3]; (2) the surface expression of adhesion molecules on PMN increases consequent to activation [4, 5]; and (3) PMN activation processes are associated with an increased level of secretory activity, resulting in a massive production and release of chemotactic agents, oxygen free radicals, and proteolytic enzymes [6 8]. These agents released by activated PMN can affect neighboring cells/tissues (e.g., vascular endothelial cells), as well as areas more distant to the release site, and are known to be involved in the inflammatory response [7]. They may thus contribute to tissue damage, with this inflammatory tissue injury being important in the pathogenesis of clinical disorders such as ischemia-reperfusion injury, systemic inflammatory response syndrome, and acute respiratory distress syndrome [7]. Activated PMN may also affect other blood cells, and it has been previously reported that activated leukocytes induce several structural and functional changes in neighboring red blood cells (RBC). These alterations include increased membrane lipid peroxidation and cell lysis [9 12], with changes of RBC membrane skeletal proteins (e.g., cross-linking between spectrin and hemoglobin), which were associated with decreased RBC deformability [13]; oxygen free radicals have been suggested to play an important role in such leukocyte-mediated changes of RBC properties [13]. Activated PMN have also been shown to be cytotoxic to rat hepatic parenchymal cells, with cathepsin G and elastase proposed as being responsible for this cytotoxicity [14]. Recent findings from our laboratory have indicated that the intrinsic aggregation characteristics of RBC (i.e., RBC aggregation tendency related to cellular factors or RBC aggregability) are enhanced by oxygen free radicals when such free radicals are generated via addition of chemical agents (i.e., hypoxanthinexanthine oxidase) to RBC suspensions [15]. Furthermore, proteolytic treatment is known to increase RBC aggregability [16, 17], thereby suggesting another mechanism for possible effects of activated leukocytes on RBC aggregation. Clinical states known to be associated with leukocyte activation are also generally associated with enhanced RBC aggregation, and it has recently been shown that enhanced red cell aggregation in such cases can, at least in part, be explained by altered RBC factors [18]. This study was therefore designed to examine the effects of activated PMN on red blood cell factors that affect RBC aggregation (i.e., Abbreviations: RBC, red blood cells; PMN, polymorphonuclear leukocytes; TNF-, tumor necrosis factor ; fmlp, N-formyl-methionyl-leucyl-phenylalanine; PBS, phosphate-buffered saline; SOD, superoxide dismutase; CAT, catalase; PMSF, phenylmethylsulfonyl fluoride. Correspondence: Dr. H. J. Meiselman, Department of Physiology and Biophysics, USC School of Medicine, 1333 San Pablo Street, MMR 626, Los Angeles, CA meiselma@hsc.usc.edu. Present address of Oguz K. Baskurt: Department of Physiology, Akdeniz University Faculty of Medicine, Antalya, Turkey. Received May 27, 1997; revised August 27, 1997; accepted September 10, Journal of Leukocyte Biology Volume 63, January

2 RBC aggregability), and thus to explore the possibility that, solely by affecting RBC properties, PMN-RBC interactions can result in enhanced RBC aggregation. MATERIALS AND METHODS Blood samples, isolation of RBC and PMN Venous blood samples (20 ml) were collected via syringe from healthy adult laboratory personnel and anticoagulated with Na-heparin (10 IU/mL). PMN isolation was carried out as previously described [3] using sterile and endotoxinfree conditions. The blood was diluted by 30% with isotonic phosphate-buffered saline (PBS, ph 7.4, 290 mosmol/kg), then carefully layered on 5 ml of 60% Percoll (Sigma Chemical Co., St. Louis, MO) and centrifuged at 400 g for 20 min. The supernatant was removed down to 2 mm above the RBC layer and the remaining Percoll and the upper 1.5 ml of the RBC layer was added to 40 ml of sterile water for hypotonic lysis of the RBC. After 30 s, 5 ml of 10 PBS was added to reestablish isotonicity. The PMN were then harvested and washed twice with PBS. Red blood cells were collected from the RBC layer remaining after the Percoll separation procedure and were also washed twice with PBS. PMN and RBC were separately resuspended in glucose-enriched (5 mm) PBS and cell counts adjusted to cells/ml for PMN and 10 9 /ml for RBC using an electronic hematology analyzer (Helios, Roche Diagnostic Systems, Branchburg, NJ). Incubations The above-mentioned RBC (10 9 /ml) and PMN ( ) suspensions were mixed at a ratio of 3:1 to obtain a RBC/PMN ratio of 150:1. This RBC-PMN suspension was then divided into five aliquots that were treated as follows: (1) antioxidant enzymes superoxide dismutase (SOD, 20 µg/ml, Sigma) plus catalase (CAT, 40 µg/ml, Sigma) were added to one aliquot and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 1 mm, Sigma) was added to another; (2) tumor necrosis factor (TNF-, 1 ng/ml, Sigma) was added to each of these two aliquots plus an additional aliquot; (3) the suspensions were allowed to incubate at 37 C for 10 min; (4) N-formyl-methionyl-leucyl-phenylalanine (fmlp, 10 7 M, Sigma) was added to these three aliquots plus an additional aliquot. Thus, five separate PMN-RBC aliquots were prepared: SOD CAT TNF- fmlp; PMSF TNF- fmlp; TNF- fmlp; fmlp; and control to which no chemicals were added. The aliquots were well mixed, centrifuged at 50 g for 3 min to assure cell-cell contact [11], and then incubated at 37 C for 2 h without agitation. In a separate set of experiments, RBC suspensions prepared as described above were incubated without the addition of PMN but with TNF- and/or fmlp at the concentrations mentioned above in order to test the direct effects of these agents. After the 2-h incubation, the suspensions were washed twice in PBS with the PMN removed as part of each wash cycle. The RBC were then resuspended in autologous plasma at a hematocrit of 40% for aggregation studies or at other values as appropriate for specific tests (see below). All preparation procedures and measurements were carried out within 6 h after venipuncture. Determination of RBC aggregation indexes RBC aggregation was quantified using a photometric rheoscope (Myrenne Aggregometer, Myrenne GmbH, Roetgen, Germany) interfaced to a digital computer. RBC aggregation was measured, after a brief shearing period at 600 s 1, at stasis (M), and at 3 s 1 (M1) by integrating the light transmission through the sample for 10 s as previously described [19]. The disaggregation shear rate ( Tmin) was measured by shearing RBC suspensions at 11 separate levels of shear between 7.5 and 500 s 1, then fitting a third order polynomial to the resulting light transmission-shear rate data to calculate the shear rate at minimal light transmission. Note that increases of M, M1, or Tmin indicate enhanced RBC aggregation: M reflects aggregation at stasis, M1 reflects aggregation promoted by low shear, and Tmin represents the minimal shear rate necessary to fully disperse RBC aggregates [19]. All aggregation measurements were carried out at room temperature (22 1 C). Ektacytometry RBC deformability was determined at various fluid shear stresses by laser diffraction analysis using an ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands). The system has been described elsewhere in detail [20]. Briefly, a dilute suspension of RBC in an isotonic, viscous medium is sheared in a Couette system made of glass (0.3 mm gap between the cylinders) and a laser light beam is projected perpendicular to the flow field. RBC deform and align with the fluid streamlines, the resulting laser diffraction pattern is analyzed by microcomputer, and elongation indexes (EI) are calculated for shear rates between 0.5 and 50 Pascal (Pa); an increased EI indicates greater cell deformation. For these studies, RBC were suspended in a PBS-dextran 70 solution (viscosity 24.8 mpa.s; osmolality 293 mosmol/kg) at a cell count of /ml. All measurements were carried out at 25 C. Determination of RBC partition coefficient [21] RBC partition coefficients were determined at room temperature (22 1 C) using a two-phase aqueous system containing 5% dextran 500 (mol. wt. 500,000, Pharmacia Fine Chemicals AB, Uppsala, Sweden) and 4.3% polyethyleneglycol (PEG, mol. wt. 8000, Sigma) in isotonic phosphate buffer (ph 6.8). The two polymers were dissolved in the phosphate buffer, the phases allowed to equilibrate for 24 h, and then each phase (dextran-rich bottom, PEG-rich top) was harvested and frozen until use. PBS-washed, packed RBC (30 µl) were added to tubes containing 1.5 ml of each phase, mixed well, and allowed to distribute (i.e., partition) between the phases for 20 min. The hemoglobin concentration in the top phase was measured spectrophotometrically at 540 nm after lysing the RBC in Drabkin s reagent, and the partition coefficient calculated as the ratio of hemoglobin content of the top phase to the total hemoglobin in the 30 µl of packed RBC. Note that because the two-phase system used for these measurements is charge-sensitive (i.e., a small voltage difference exists between the two phases with the top phase positive [21]), a lower partition coefficient indicates a decreased RBC surface charge. Statistics Results are presented as mean SEM and between-group comparisons were performed by paired t-tests. Differences with P values smaller than 0.05 were deemed to be statistically significant. RESULTS Effect of activated PMN on RBC aggregability The RBC aggregation indexes M and M1 were both significantly increased after incubation with activated PMN (Fig. 1), with this increase more pronounced if the PMN were pretreated with TNF- before the addition of fmlp. The minimal shear rate necessary to disperse RBC aggregates ( Tmin) was also significantly increased after incubation with activated PMN (Fig. 2), and as observed for M and M1 (Fig. 1), this increase was greater if the PMN were pretreated with TNF- before the addition of fmlp (i.e., 60% increase with both TNF- and fmlp). Both the protease inhibitor (PMSF) and the antioxidant enzymes (SOD and CAT) were effective in preventing the effect of activated PMN on RBC aggregability, although the approximately 9% increase of M1 with PMSF did reach significance (Figs. 1 and 2). Effect of activated PMN on RBC two-phase partition coefficients Incubation of RBC with activated PMN resulted in significant decreases of the two-phase partition coefficient, with the greatest decrease observed if the PMN were pretreated with TNF- before the addition of fmlp (Fig. 3). The protease inhibitor PMSF and the antioxidant enzymes SOD CAT partially prevented this decrease yet were unable to fully counter the combined effects of TNF- plus fmlp. 90 Journal of Leukocyte Biology Volume 63, January 1998

3 Fig. 1. RBC aggregation indexes M (left) and M1 (right) after incubation with PMN activated by TNF- and/or fmlp, with or without a protease inhibitor (PMSF) or antioxidant enzymes (SOD CAT). Data are mean SEM of six experiments. Difference from control: *P Effect of activated PMN on RBC deformability The presence of activated PMN in the RBC-PMN suspensions resulted in a reduction of RBC deformability as indicated by decreased EI values (Fig. 4). Again, as for the aggregation indexes (Figs. 1 and 2) and the partition coefficients (Fig. 3), the greatest change was noted if the PMN were pretreated with TNF- before the addition of fmlp. The protease inhibitor PMSF was ineffective in preventing the effects of activated PMN on RBC deformability; the antioxidant enzymes SOD CAT partially offset this effect (Fig. 4). Specific effects of TNF- and/or fmlp When PMN were not present in the RBC suspensions, no significant alterations of M, M1, Tmin, partition coefficients, or RBC deformability were observed for red cells incubated with TNF- and/or fmlp. DISCUSSION The results of this study clearly indicate that PMN activated in vitro by TNF- and/or fmlp can induce alterations of RBC properties, which result in hyper-aggregability when these red cells are tested in autologous plasma: this enhanced tendency for aggregate formation is reflected by higher aggregation indexes (both at stasis and at low shear rate) and by increased disaggregation shear rates ( Tmin). Previous reports [e.g., ref. 22] have shown that TNF- and fmlp have additive effects on PMN activation, with TNF- playing a priming role for the enhanced response. This additive effect of these two agents on PMN activation was evident in this study, since the changes of RBC aggregability, two-phase partition coefficient, and RBC deformability were more prominent if PMN were pre-incubated with TNF- before stimulation with fmlp (Figs. 1 4). This additive effect on the parameters measured also implies that the effect of PMN activation on neighboring RBC is related to the degree of activation (i.e., greater activation by fmlp when the PMN are primed by TNF- ). These results thus suggest the merit of future studies in which PMN activation is achieved via various concentrations of TNF- or fmlp, or via the use of other agents such as phorbol myristate acetate or zymosan-activated plasma; previous reports [e.g., refs. 1 and 3] have indicated marked, dose-related effects of such agents on PMN deformability, thereby implying graded activation. Note that the effects of PMN activation seen herein are clearly PMN-mediated: without PMN in the RBC suspension, neither fmlp alone nor in combination with TNF- had any significant effects on the measured RBC properties. When activated, PMN produce and secrete oxygen free radicals and proteolytic enzymes that can attack biological Fig. 2. Disaggregation shear rate ( Tmin, s 1 ) after incubation with PMN activated by TNF- and/or fmlp, with or without a protease inhibitor (PMSF) or antioxidant enzymes (SOD CAT). Data are mean SEM of six experiments. Difference from control: *P Fig. 3. RBC two-phase partition coefficients (PC) after incubation with PMN activated by TNF- and/or fmlp, with or without a protease inhibitor (PMSF) or antioxidant enzymes (SOD CAT). Data are mean SEM of six experiments. Difference from control: *P 0.05; P Baskurt and Meiselman PMN effects on RBC aggregation 91

4 Fig. 4. RBC deformation index EI at a shear stress of 5 Pa after incubation with PMN activated by TNF- and/or fmlp, with or without a protease inhibitor (PMSF) or antioxidant enzymes (SOD CAT). Data are mean SEM of six experiments. Difference from control: P 0.01; P material in the environment of the activated PMN [e.g., ref. 23]. Oxygen free radicals are well known to affect RBC mechanical properties, most probably via their effects on the RBC membrane cytoskeleton [9, 13, 15, 24, 25]. This effect on RBC mechanical behavior was confirmed in this study, as the impairment in RBC deformability consequent to PMN stimulation by TNF- fmlp was substantially inhibited by antioxidant enzymes (SOD and CAT), but essentially unaffected by the protease inhibitor PMSF (Fig. 4). In contrast, the protease inhibitor and antioxidant enzymes were found to be equally effective in preventing the effect of activated PMN on the M aggregation index and the disaggregation shear rate (Figs. 1 and 2). The effects of proteolytic treatment on RBC aggregability have been previously reported [16, 17] and have been related to the alterations of the RBC surface charge as assessed by two-phase partitioning [16]. RBC partition coefficients decreased significantly after incubation with PMN activated by TNF- and/or fmlp, thereby suggesting a decreased surface charge; this effect was partially prevented by both the protease inhibitor and the antioxidant enzymes (Fig. 3). It is now generally accepted that RBC aggregation behavior is affected by both the composition of the suspending medium and the properties of the RBC [26, 27]. Macromolecules in the suspending medium induce red cell aggregation either by forming bridges between the membranes of adjacent RBC (i.e., the bridging model) or by generating an osmotic force between adjacent RBC (i.e., the depletion model) [26]. In the current study, RBC aggregation after various treatments was measured in the same suspending medium (i.e., autologous plasma), thereby excluding possible artifacts due to variations of medium composition: the differences of RBC aggregability observed herein therefore must solely reflect altered RBC properties. As detailed by Chien [28] and others, RBC aggregation depends on an energy balance between aggregating and disaggregating forces: (1) aggregating forces are mainly related to the concentration and physicochemical properties of macromolecules (e.g., fibrinogen, dextran) in the suspending medium and their interactions with the RBC surface; (2) disaggregating forces include electrostatic repulsive forces and membrane strain energy in addition to fluid shear stresses. Electrostatic repulsive forces originate from the negative surface charge of RBC, and therefore a decrease in surface charge shifts the energy balance in favor of aggregation [29]. Membrane strain energy is related to the status of RBC membrane cytoskeleton, increasing with stiffening of the membrane, which is also reflected by decreased RBC deformability. With regard to the above factors known to affect RBC aggregation, our results indicate that activated PMN induce opposite alterations of two membrane properties that are related to aggregability: (1) decreased surface charge (Fig. 3), which tends to promote aggregation; (2) decreased RBC deformability (Fig. 4), which is expected to reduce aggregation. Thus, an increased tendency for aggregation as evidenced by increased aggregation indexes and disaggregation shear rate (Figs. 1 and 2) seems to imply that decreased surface charge (Fig. 3) was the dominant factor. However, changes in aggregating forces may also be involved because factors generated by activated PMN may have qualitatively or quantitatively affected the interactions between plasma proteins and the RBC surface. Although neither the existence of membrane binding site(s) nor their chemical nature have been fully established, certain RBC membrane proteins have been proposed to be involved in this process [30]. It is therefore possible that proteolytic enzymes and oxygen free radicals may cause certain structural alterations, thereby altering the adsorption of macromolecules to such sites. An enhanced RBC aggregation tendency consequent to exposure to activated PMN (Figs. 1 and 2) may also be relevant to changes of RBC aggregability that occur during in vivo red cell aging in the circulation. Using density separation to obtain relatively young (i.e., less dense) and relatively old (i.e., more dense) red blood cells, Meiselman and co-workers [26, 27, 31, 32] have shown the following: (1) for erythrocytes from adult donors, older RBC aggregate at least twofold more strongly and to a much greater extent than younger cells when suspended in either autologous plasma or in defined solutions of aggregating polymers (e.g., mol. wt. 70,000 dextran); (2) RBC from full-term neonates also exhibit this density-associated difference in aggregability (i.e., more dense less dense), although the difference in aggregation tendency between more dense and less dense neonatal RBC is less than for density-separated red cells from adults; (3) membrane viscosity, and hence the time constant for the viscoelastic shape response of the cell, increases with cell density for RBC from adults. Given the nominal 120-day circulatory lifespan for adult RBC, it is tempting to speculate that, during this time period, repetitive exposure of RBC to products of PMN activation may lead to an age-associated increase of RBC aggregability and changes of membrane mechanical behavior. The shorter lifespan of neonatal RBC (approximately 60 days [33, 34]), and thus their more limited duration of exposure to the in vivo circulatory environment, may play a role in their smaller age-associated change in aggregability; such smaller age-associated changes of aggregability are in contrast to the accelerated in vivo aging [34] of other neonatal RBC properties (e.g., cell volume, intracellular hemoglobin concentration, age-dependent enzymes). Although the data obtained in this study do not explicitly support such hypotheses regarding in vivo RBC aging, they are consistent with such concepts and thus suggest avenues for future studies of red cell aging [e.g., 35 and 36]. RBC aggregation is an important determinant of blood viscosity, especially during low-flow and thus low-shear conditions, 92 Journal of Leukocyte Biology Volume 63, January 1998

5 with the formation of RBC aggregates tending to increase viscosity by increasing particle size and hence energy dissipation in the suspension [28, 37]. Aggregation is usually not observed on the arterial side of the circulation owing to the high flow and shear conditions [37]. However, RBC aggregation can be evident, even under normal physiological conditions, when shearing forces are low enough to permit RBC aggregate formation (i.e., in post-capillary venules and small veins). An increased tendency for RBC aggregate formation, originating from either alterations of plasma composition or from changes of RBC cellular properties, may have profound effects on in vivo blood flow and RBC flux [28]; a recent report by Cabel et al. [38] clearly indicates the contribution of RBC aggregation to venous vascular resistance. These hemorheological consequences should thus be considered in the therapeutic approach to tissue perfusion problems encountered in severe clinical conditions (e.g., sepsis) that are known to be associated with leukocyte activation [39]. ACKNOWLEDGMENTS This work was supported by an International Scholar Award from the J. William Fulbright Foreign Scholarship Board (O. K. B) and by NIH Research Grants HL15722 and HL (H. J. M.). We wish to thank Dr. Mahmoud Razavian for his help with the aqueous two-phase polymer partitioning studies of the various RBC populations. In addition, we wish to extend our appreciation to E. Baskurt and D. Meiselman for their continuing support of our efforts. REFERENCES 1. Buttrum, S. M., Drost, E. M., Macnee, W., Goffin, E., Lockwood, C. M., Hatton, R., Nash, G. B. (1994) Rheological response of neutrophils to different types of stimulation. J. Appl. Physiol. 77, Buttrum, S. M., Nash, G. B., Hatton, R. (1996) Changes in neutrophil rheology after acute ischemia and reperfusion in the rat hindlimb. J. Lab. Clin. Med. 128, Pecsvarady, Z., Fisher, T. C., Fabok, A., Coates, T. D., Meiselman, H. J. (1992) Kinetics of granulocyte deformability following exposure to chemotactic stimuli. Blood Cells 18, Ley, K. (1996) Molecular mechanisms of leukocyte recruitment in the inflammatory process. Cardiovasc. Res. 32, Ali, H., Haribabu, B., Richardson, R. M., Snyderman, R. (1997) Mechanisms of inflammation and leukocyte activation. Med. Clin. North. Am. 81, Balazovich, K. J., Suchard, S. J., Remick, D. G., Boxer, L. A. (1996) Tumor necrosis factor-alpha and FMLP receptors are functionally linked during FMLP-stimulated activation of adherent human neutrophils. Blood 88, Downey, G. P., Fukushima, T., Fialkow, L., Waddell, T. K. (1995) Intracellular signaling in neutrophil priming and activation. Semin. Cell Biol. 6, Ceriana, P. (1992) Effect of myocardial ischemia-reperfusion on granulocyte elastase release. Anaest. Intensive Care 20, Baskurt, O. K., Edremitlioglu, M., Temiz, A. (1994) In vitro effects of in vivo activated leukocytes on RBC filterability and lipid peroxidation. Clin. Hemorheol. 14, Claster, S., Chiu, D. T. Y., Quintanilha, A., Lubin, B. (1984) Neutrophils mediate lipid peroxidation in human red cells. Blood 64, Weiss, S. J. (1980) The role of superoxide in the destruction of erythrocyte targets by human neutrophils. J. Biol. Chem. 255, Van Kessel, K. P., Van Strijp, J. A., Van Kats-Renaud, H. J., Miltenburg, L. A., Fluit, A. C., Verhoef, J. (1990) Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediated cytotoxicity: use of cytoplasts and cells from chronoic grannulomatous disease patient. J. Leukoc. Biol. 48, Baskurt, O. K. (1996) Activated granulocyte induced alterations in red blood cells and protection by antioxidant enzymes. Clin. Hemorheol. 16, Ho, J. S., Buchweitz, J. P., Roth, R. A., Ganey, P. E. (1996) Identification of factors from rat neutrophils responsible for toxicity to isolated hepatocytes. J. Leukoc. Biol. 59, Baskurt, O. K., Temiz, A., Meiselman, H. J. (1997) Effect of superoxide anions on red blood cell rheologic properties. Free Rad. Biol. Med., in press. 16. Bohler, T., Linderkamp, O. (1993) Effect of neurominidase and trypsin on surface charge and aggregation of red blood cells. Clin. Hemorheol. 13, Rampling, M. W., Pearson, M. J. (1994) Enzymatic degradation of the red cell surface and its effect on rouleaux formation. Clin. Hemorheol. 14, Baskurt, O. K., Temiz, A., Meiselman, H. J. (1997) Red blood cell aggregation in experimental sepsis. J. Lab. Clin. Med., 130, Bauersachs, R. M., Wenby, R. B., Meiselman, H. J. (1989) Determination of specific red blood cell aggregation indices via an automated system. Clin. Hemorheol. 9, Hardeman, M. R., Goedhart, P. T., Dobbe, J. G. G., Lettinga, K. P. (1994) Laser-assisted optical rotational cell analyzer (LORCA). 1. A new instrument for measurement of various structural hemorheological parameters. Clin. Hemorheol. 14, Walter, H. (1985) Surface properties of cells reflected by partitioning: red blood cells as a model. In Partitioning in Aqueus Two-Phase Systems (H. Walter, D. E. Brooks, and D. Fisher, eds.) Orlando, FL: Academic Press, Weiss, M., Buhl, R., Medve, M., Schneider, E. M. (1997) Tumor necrosis factor-alpha modulates the selective interference of hypnotics and sedatives to suppress N-formyl-methionyl-leucyl-phenylalanine-induced oxidative burst formation in neutrophils. Crit. Care Med. 25, Mazzoni, M. C., Schmid-Schonbein, G. W. (1996) Mechanisms and consequences of cell activation in the microcirculation. Cardiovasc. Res. 32, Snyder, L. M., Fortier, N., Trainor, J., Jacobs, J., Leb, L., Lubin, S., Chiu, D., Shohet, S., Mohandas, N. (1985) Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. J. Clin. Invest. 76, Uyesaka, N., Hasegawa, S., Ishioka, N., Ishioka, R., Shio, H., Schechter, A. N. (1992) Effect of superoxide anions on red cell deformability and membrane proteins. Biorheology 29, Meiselman, H. J. (1993) Red blood cell role in RBC aggregation: and beyond. Clin. Hemorheol. 12, Nash, G. B., Wenby, R., Sowemimo-Coker, S. O., Meiselman, H. J. (1987) Influence of cellular properties of red cell aggregation. Clin. Hemorheol. 7, Chien, S. (1975) Biophysical behavior of red cells in suspension. In The Red Blood Cell (D. M. Surgenor, ed.) New York: Academic Press, Chien, S., Sung, L. A. (1990) Molecular basis of red cell membrane rheology. Biorheology 27, Fabry, T. L. (1987) Mechanism of erythrocyte aggregation and sedimentation. Blood 70, Sowemimo-Coker, S. O., Whittingstall, P., Pietsch, L., Bauersachs, R. M., Wenby, R. B., Meiselman, H. J. (1989) Effects of cellular factors on the aggregation behavior of human, rat and bovine erythrocytes. Clin. Hemorheol. 9, Nash, G. B., Meiselman, H. J. (1983) Red cell and ghost viscoelasticity: effects of hemoglobin concentration and in vivo ageing. Biophys. J. 43, Linderkamp, O., Nash, G. B., Wu, P. Y. K., Meiselman, H. J. (1986) Deformability and intrinsic material properties of neonatal red blood cells. Blood 67, Linderkamp, O., Wu, P. Y. K., Meiselman, H. J. (1982) Deformability of density-separated red blood cells in normal newborn infants and adults. Pediatr. Res. 16, Clark, M. R. (1988) Senescence of red blood cells: progress and problems. Physiol. Rev. 68, Sorette, M. P., Clark, M. R. (1991) Specificity of autologous antibody binding to phenylhydrazine-treated human erythrocytes: implications for models of red cell aging. J. Lab. Clin. Med. 117, Schmid-Schonbein, H. (1988) Fluid dynamics and hemorheology in vivo: the interactions of hemodynamic parameters and hemorheological properties in determining the flow behavior of blood in microvascular networks. In Clinical Blood Rheology. (G. D. O. Lowe, ed.) Boca Raton, FL: CRC Press, Cabel, M., Meiselman, H. J., Popel, A. S., Johnson, P. C. (1997) Contribution of red blood cell aggregation to venous vascular resistance in skeletal muscle. Am. J. Physiol. 272, H1020 H Welbourn, C. R. B., Goldman, G., Paterson, I. S., Valeri, C. R., Shepro, D., Hechtman, H. B. (1991) Pathophysiology of ischaemia repersion injury: central role of the neutrophil. Br. J. Surg. 78, Baskurt and Meiselman PMN effects on RBC aggregation 93