Nitric oxide from polymorphonuclear leukocytes modulates red blood cell deformability in vitro

European Journal of Pharmacology, 234 (1993) 17-22

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© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 52992

Nitric oxide from polymorphonuclear leukocytes modulates red blood cell deformability in vitro R y s z a r d K o r b u t a n d R y s z a r d J. G r y g l e w s k i Department of Pharmacology, Nicolaus Copernicus University School of Medicine in Cracow, 16 Grzegorzecka, 31-531 Cracow, Poland

Received 27 July 1992, revised MS received 5 January 1993, accepted 12 January 1993

We confirmed that iloprost is very potent in preserving the deformability of rabbit red blood cells (RBC). Incubation of RBC with a small number (up to 1.2 x 10 6 cells/ml) of polymorphonuclear leukocytes (PMNs) caused a gradual decline in RBC deformability. The addition of PMNs up to 2.8 x 10 6 cells/ml increased RBC deformability but, at higher concentrations, both in the presence and absence of a neutrophil activating cytokine (interleukin-8; IL-8), PMNs reduced the deformability of RBCs. In the presence of a small number of PMNs, the deformability of RBC was increased by nitric oxide (NO) donors, such as sydnonimine (SIN-I) or sodium nitroprusside, and reduced by the NO synthase inhibitor, N%monomethyl-L-arginine. We suggest that the deformability of RBC is modulated by PMNs via the release of NO and that the NO concentration is of critical importance in this modulatory mechanism. NO seems to preserve or enhance RBC deformability within a certain range of concentrations, but these effects are reversed or eliminated at both too low and too high concentrations. Nitric oxide (NO); Polymorphonuclear leukocytes; Red blood cells deformability

I. Introduction

2. Materials and methods

Many diseases of the heart and circulatory system have been linked with an insufficient deformability of red blood cells (RBC) (Forman et al., 1973; Jutte et al., 1980; Belch et al., 1985). Interestingly, RBC deformability influences the function of other blood cells such as platelets (Turitto and Weiss, 1980) and their adherence to the vessel wall (Aartas et al., 1983). Activated Polymorphonuclear leukocytes (PMNs), PMN supernatant, and synthetic platelet-activating factor (PAF) increase the rigidity of RBC in suspension in vitro (Petitfrere et al., 1991). However, PMNs can also inhibit platelet aggregation (Salvemini et al., 1989) by releasing an antiaggregatory factor identified as nitric oxide (NO) (Wright et al., 1989), which seems to play an important role in cell to cell interactions. We studied whether NO from non-activated PMNs or from NO-donating agents affects the deformability of native RBCs in vitro.

2.1. Methods

Correspondence to: R. Korbut, Department of Pharmacology, Nicolaus Copernicus University School of Medicine in Cracow, 16 Grzegorzecka, 31-531 Cracow, Poland. Tel. 48.12.211168, fax 48.12.217217.

Erythrocyte detormability was measured by the erythrocyte flow rate method of Reid et al. (1976), as modified by Heilmann and Schmid-Schonbein (1981). Under standard conditions erythrocytes were passed through a membrane filter using a negative pressure of 20 cm water. The deformability of RBCs was determined by the speed of flow, the pore diameter of 5/xm being less than that of RBCs and thus limiting flow according to the flexibility of the RBC membranes. The time was recorded for the passage of 0.5 ml of a suspension of RBC. The results are expressed as the number of RBC filtered per minute (RBC n u m b e r / min), which we termed the 'deformability index' (Di). The assay system contained two identical filters, so that control and treated samples were assessed simultaneously. Erythrocytes were isolated from rabbit arterial blood, withdrawn from the left ventricle under general anaesthesia with sodium pentobarbitone (Vegetal, Polfa, 2030 m g / k g i.v.) and collected into tri-sodium citrate (3.15% w / v ) in a ratio of 9 : 1. Immediately after withdrawal, the blood was centrifuged at 1400 × g (4000 r.p.m.) for 10 rain, and the supernatant and the buffy

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coat were removed by aspiration. The number of erythrocytes was determined and then the RBC were resuspended in their native platelet-poor plasma to give a haematocrit 0.39-0.4 in each experiment. Microscopic examination revealed that the suspension consisted mainly of red blood cells and a small number of neutrophils (never more than 1.2 × 106 PMNs/ml). Only single platelets were seen.

in suspension were viable, as judged by Trypan blue exclusion. PMNs were resuspended in phosphate buffered saline (PBS) containing 10/xM indomethacin and the volume was adjusted to give a final concentration of 108 ceils per ml. PMNs at various concentrations (1-7 × 106 cells/ml) in a volume 5-100 /~1 were incubated with a standard number of erythrocytes at room temperature. In some experiments PMNs were pretreated with superoxide dismutase (20 U/ml) for 15 min before adding them to the RBC suspensions. In other experiments PMNs were activated with interleukin-8 (IL-8) (50 ng/ml) for 30 min before the addition of erythrocytes. To assess the activation of PMNs during preparation or by IL-8, cell-free supernatants were assayed for /3-glucuronidase activity, an enzyme localized in the azurophilic granules of PMNs and released during cell activation (Patterson et al., 1990).

2.2. Manipulation of red cell deformability Changes in red cell deformability were induced in vitro by treating the RBC with various compounds a n d / o r by enrichment of RBC suspensions with increasing numbers of native, isolated PMNs. At time 0, vehicle, various compounds or isolated native PMNs were added to plastic tubes containing RBC suspensions and incubated at room temperature (22-25°C) for 120 min with gentle agitation. Samples (2 ml) were taken at time 0 and after 30, 60 and 120 rain of incubation. At each time interval, red cell deformability was compared to that of control samples treated with vehicle. Readings were taken in duplicate, using a new filter for each measurement. All studies were performed within 150 min of blood withdrawal.

2.4. Materials and reagents The membrane filters had pores of 5 ~m in diameter, with 4 × l0 s pores per cm 2 and a thickness of 10 /zm (Nucleopore filters, GSI Darmstadt, Germany). Chemicals used were sodium nitroprusside (NaNP, Sigma Chem. Co., Poole, UK), iloprost (gift from Schering AG, Germany), sydnonimine (SIN-l), a metabolite of molsidomine (gift from Dr. Nitz, Cassella, Germany), L-arginine HC1 (Sigma, Germany), Na-monomethyl-L-arginine citrate (MeArg, Ultrafine Chemicals (Manchester, U.K.), superoxide dismutase

2.3. Isolation of PMNs PMNs were prepared from citrated plasma as described by Boyum (1968). More than 95% of the cells

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Fig. 1. The effect of iloprost (open diamonds, 3/xM, three experiments) or sydnonimine (SIN-l; solid squares, 100/zM, six experiments) on the deformability of red blood cells (RBC) incubated at 22°C. Each point: mean of n experiments; * * * P < 0.001 and n.s. = not significant as compared to corresponding point of the control curve (open squares, 19 experiments),

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that of control samples at 30 and 60 min for SIN-1 and at 0, 30, 60 and 120 min for iloprost (fig. 1). These effects were dose-dependent (fig. 2). Iloprost was the most active and the potencies of SIN-1 and NaNP were one order of magnitude lower than that of iloprost with less steep dose-response curves (fig. 2). The gradual decline in RBC deformability observed during the course of the experiment was speeded up during the first 60 rain of the treatment of with MeArg (30 /zM) (fig. 3). Microscopic observation of the RBC showed that they did not change shape. This activity of MeArg was prevented by co-administration of MeArg with L-arginine (but not D-arginine) at a concentration of 100/~M (fig. 3).

(SOD, Sigma, Germany), interleukin-8 (IL-8, Calbiochem, UK) and phenolphthalein glucuronic acid (Sigma Chem. Co., Poole,U.K.).

2.5. Statistical analysis In each experiment the permutation test and experiment-related error probability for first-order error was calculated. The results are expressed as the means +_ S.D. of n experiments, and were assessed by a two-way analysis of variance and by a least-significance procedure to determine the nature of the response. A 'P' value of less than 0.05 was considered statistically significant.

3.2. Addition of PMNs 3. Results

When PMNs were added at a low concentration (2.3 x 106 cells/ml) to the RBC suspensions and incubated for 30 min, RBC deformability was unchanged (fig. 4). At a higher concentration (2.8 x 10 6 of cells/ml), PMNs significantly increased RBC deformability. However, further increases in the concentration of PMNs reduced deformability (fig. 4), so that PMNs at 6.3 x 10 6 P M N s / m l significantly reduced the deformability index below the corresponding control level (fig. 4). The change in RBC deformability caused by increasing numbers of PMNs (2.8 or 6.3 x 10 6 cells/ml) was not influenced by pretreatment of the PMNs with SOD (20 U / m l , n = 6), but was reduced by pretreatment of the cells with MeArg. In the presence of 2.8 X 10 6 P M N s / m l MeArg decreased RBC deforma-

3.1. Pharmacological regulation of deformability The RBC suspension was always contaminated with PMNs but never by more than 1.2 X 10 6 cells/ml. Incubation of R B C / P M N s suspensions for 120 min at room temperature caused a significant, gradual decline in the RBC D i. The D i value at time O in control experiments was 300 _+ 37 RBC X 106/min (mean _+ S.D.; n = 1 9 ) and after 120 min 135_+21 R B C x 106/min; n = 19 (fig. 1). Incubation of erythrocyte suspensions with the stable analogue of prostacyclin, iloprost (3 /zM), or with NO donors, such as SIN-1 (100 /zM, active metabolite of molsidomine) or NaNP (100 /.~M), significantly increased deformability compared to

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Fig. 2. Dose-response curves for the effects of iloprost (open diamonds), sydnonimine (SIN-l; solid squares) or sodium nitroprusside (open squares) on the deformabilty of red blood cells (RBC) after a 60 min incubation at 22°C. Each point: mean of three experiments.

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Fig. 3. Decrease in red blood cell (RBC) deformability by the NO synthase inhibitor NG-monomethyl-L-arginine (MeArg; solid squares, 30 #M, six experiments) and its reversal by L-arginine (open diamonds, 100/zM, three experiments). Each point: mean of n experiments; *** P < 0.001 and n.s. = not significant as compared to corresponding point of the control curve (open squares, nine experiments).

bility by 63 + 9 % (n = 4, P < 0.05 as c o m p a r e d to nont r e a t e d cells) b u t in t h e p r e s e n c e 3.4 x 106 o f P M N s / m l by only 43 _+ 7 % (n = 4; P < 0.05). In 3 e x p e r i m e n t s P M N s w e r e p r e t r e a t e d with 50 n g / m l of IL-8 for 30 min b e f o r e a d d i n g t h e m to the R B C (fig. 4). IL-8 d i d n o t i n f l u e n c e R B C d e f o r m a b i l i t y in t h e s a m p l e s with few P M N s (up to 1.2 X 106 c e l l s / m l ) b u t it significantly i n c r e a s e d R B C d e f o r m a b i l i t y in s a m p l e s with 2.3 x 106

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PMNs/ml. At higher concentrations of PMNs treated with IL-8, t h e R B C d e f o r m a b i l i t y index was d e c r e a s e d m o r e t h a n that for n o n - t r e a t e d s a m p l e s (fig. 4). In t h r e e e x p e r i m e n t s t h e i n c r e a s e in R B C d e f o r m a b i l i t y c a u s e d by P M N s (2.3 × 106 c e l l s / m l ) a c t i v a t e d with IL-8 was i n h i b i t e d 61 + 11% (P < 0.05) by M e A r g (30 /xM) a d d e d to P M N s 10 min b e f o r e IL-8. M e A r g also r e d u c e d R B C d e f o r m a b i l i t y in the e x p e r i m e n t s with

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Fig. 4. Relative effect of polymorphonuclear leukocytes (PMNs) incubated with (open squares, three experiments) or without (solid squares, four experiments) interleukin-8 (IL-8; 50 ng/ml) on the deformability of red blood cells (RBC) after a 30 min incubation at 22°C. Each point: mean of n experiments; * P < 0.05 as compared to corresponding point without IL-8; ** P < 0.01 as compared to the first point of this curve.

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2.8 × 106 P M N s / m l (39 + 7%; n = 3) but had no effect on RBC deformability in experiments with 4.3 × 10 6 PMNs/ml. Determination of /3-glucuronidase released from PMNs treated with IL-8 revealed that the 50% increase in its activity correlated with the increase in RBC deformability. However, the further increase in /3-glucuronidase activity was associated with a decreases in deformability. The flow time for 0.5 ml of leukocytes alone at a concentrations from 1 to 7 x 10 ~' cells/ml, suspended in the native platelet poor plasma, was 6 _+ 2 s (three experiments) and did not depend upon the number of cells.

4. Discussion

In our study, RBC deformability was influenced by PMNs via a mechanism involving NO release. This conclusion is based on the observation that incubation at room temperature of rabbit RBC with a small number of PMNs (up to 1.2 × 10 6 cells/ml) caused a gradual decline in the deformability of RBC, a decline that was potentiated by treatment of the R B C / P M N s suspension with MeArg, a NO synthesis inhibitor (Rees et al., 1989) and reversed by co-administration of Larginine. Moreover, this decrease in RBC deformability was inhibited by NO donors such as SIN-1 and NaNP. These results may simply indicate that when a small number of PMNs are present in RBC suspensions, the amount of NO continuously generated by PMNs does not protect RBC sufficiently against a loss of deformability. Under such conditions, the preservation of RBC deformability requires additional NO, which can be achieved by increasing the number of PMNs or by administering NO donors. Indeed, when the number of PMNs was increased sufficiently (within a narrow range between 2.3 and 2.8 x 106 of cells/ml) or when PMNs were activated (up to 50% increase in /3-glucuronidase release) by IL-8 (Thelen et al., 1988) or when we administered an NO donor (Katsuki et al., 1977), RBC deformability increased. The involvement of mediators formed by the cyclooxygenase pathway (e.g. prostacyclin) was excluded by pretreatment of PMNs with indomethacin. The mechanism by which NO regulates these effects requires further elucidation, in that increasing the NO concentration above a certain threshold resulted in a significant reduction in RBC deformability. This inhibition of RBC deformability caused by increasing the concentration of PMNs was reversed by the addition of MeArg, leading us to speculate that it is a consequence of higher concentrations of NO. Interestingly, the percentage reduction caused by MeArg was inversely proportional to the number of PMNs, so that the more PMNs and the

more intense the generation of NO, the less damaging MeArg was for RBC deformability. The biological significance of NO production by leukocytes (Rimele et al., 1988) is obscure. It has been suggested that the simultaneous release of NO and Superoxide anions ( 0 2) (McCall et al., 1989) by activated neutrophils is an autoregulatory mechanism (Gryglewski et al., 1986) modulating the interaction between PMNs and other blood cells such as platelets (Salvemini et al., 1989). In our experiments, however, SOD, a scavenger of O 2 , did not influence the changes in RBC deformability induced by increasing the number of PMNs. This observation excludes the possibility that these effects are related to the decreased concentration and half life of NO or to any potential direct effect of 0 2 on the RBC themselves. We believe that NO produced by PMNs is an important factor in the regulation of RBC deformability under non-activated conditions and that the amount of NO available to RBC is of critical importance in this mechanism. Below a certain concentration, NO enhances RBC deformability; however, above this concentration a toxic effect is observed. An increased concentration of NO as a result of PMN activation occurs in a number of clinical conditions including septic shock and myocardial infarction, thereby disturbing the hydrodynamic properties of flowing blood and diminishing peripheral flow. The administration of NO synthase inhibitors in these conditions would be hazardous, because a too great a reduction in NO concentration could result in a decrease in RBC deformability and in an increase in blood viscosity. RBC deformability was significantly enhanced by iloprost, which increased the deformability of RBC in the presence of both low and high (not shown) numbers of PMNs. Iloprost was not only the most potent agent in preserving RBC deformability but also the most rapidly acting as it was able to improve RBC deformability immediately after administration. This effect of prostacyclin or its stable analogue iloprost, has been reported by many investigators (Neri Serneri, 1981; Galanti et al., 1983; Kovacs and O-Grady, 1984a). An impairment of RBC deformability has also been observed after oral administration of aspirin in humans (Kovacs and O-Grady, 1984b). Despite numerous studies of clinical conditions (e.g. Raynaud's disease, systemic sclerosis) in which prostaglandin (PG)I2 production is reduced (Gaylarde, 1981; Belch et al., 1985) the mechanism of the effects of PGI 2 is still unknown. In the experiments reported here, RBC and PMNs interacted freely with each other and it is not improbable that the effects of PGI 2 are due to the direct protection of PMN functions by PGI 2 (Korbut et al., 1989). However, further studies are required to confirm this idea. The interaction between PMNs and RBC in the regulation of deformability is a complex phenomenon

22 and studies on flow through the microcirculation and on interaction with endothelial cells may explain the functional behaviour of RBC.

Acknowledgements Authors wish to thank Mrs A. Starosciak for technical assistance. This study was supported by a grant from KBN Poland.

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