Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation

Increased leukocyte adhesion to vascular endothelium contributesto vaso-occlusion in sickle cell disease. Since nitric oxidebioavailability is decreased in sickle cell disease and nitricoxide may inhibit leukocyte adhesion, we investigated whetherstimulation of NO-signaling pathways can reduce the adhesiveproperties of neutrophils from sickle cell disease individuals(sickle cell diseaseneu). sickle cell diseaseneu presented greateradhesion in vitro to both fibronectin and ICAM-1 than controlneutrophils. Co-incubation of sickle cell diseaseneu with thenitric oxide-donor agents, sodium nitroprusside and dietheylamineNONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272,all significantly reduced the increased adhesion to fibronectin/ICAM-1.Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor,reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin,implicating cGMP-dependent signaling in this mechanism. Interestingly,intracellular cGMP was significantly higher in neutrophils fromsickle cell disease individuals on hydroxyurea (sickle celldiseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesionto fibronectin/ICAM-1 was significantly lower than that of sicklecell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependentpathway may have beneficial effects on leukocyte function ifused in these subjects.

Key words: adhesion, leukocyte, nitric oxide, sickle cell disease, vaso-occlusion.

Introduction

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Introduction

Leukocytes play a central role in sickle cell disease (SCD)pathophysiology. Sickle cell crises are often associated withinfection, neutrophil counts are higher in SCD individuals,and polymorphonuclear leukocytosis has been correlated withan increased rate of early death, acute chest syndrome and stroke.¹There is growing evidence to suggest that leukocytes participatein the initiation and propagation of vaso-occlusive processesby which the recruitment of large, less deformable, adherentwhite cells to the vascular endothelium and their interactionwith circulating erythrocytes may impair blood flow.²

Neutrophils of SCD individuals are more able to adhere to fibronectinand to endothelial monolayers than healthy individual neutrophils.³Neutrophils express a variety of adhesion molecules on theirsurface that are required for transendothelial migration. TheL- and P- selectins mediate tethering and rolling on the endothelium,while firm adhesion is mediated by the β2 integrins Mac-1(CD11b/CD18) and LFA-1 (CD11a/CD18).⁴ Mac-1 expression has beenshown to be increased on stimulated SCD neutrophils.^5,6 Therefore,pharmacological approaches to inhibit increased leukocyte adhesiveinteractions may represent important strategies for the preventionof SCD vaso-occlusion.

Recent reports suggest that nitric oxide (NO) bioavailabilitymay be decreased in SCD.⁷ Vascular cell-free hemoglobin, releasedduring hemolysis, can consume the NO produced by endothelialcells thus facilitating vasoconstriction and platelet activation.⁷NO is also known to affect adhesion mechanisms. inhibiting endothelialadhesion molecule expression⁸ and reducing the adhesive propertiesof leukocytes.⁹

At present, hydroxyurea (HU) is the only widely-used therapyfor the treatment of SCD, reducing the frequency of vaso-occlusivecrisis, acute chest syndrome and transfusion, and increasingfetal hemoglobin (HbF) production in erythroid cells.¹⁰ Whileit has been proposed that HU induces HbF production via tyrosylradical activation on ribonucleotide reductase,¹¹ there is evidenceto suggest that HU may induce ${gamma}$ -globin gene expression in erythroleukemiccells and primary human erythroblasts via a NO-guanylate cyclasedependent pathway.^12,13 Furthermore, data suggest that HU maybe oxidized by haem groups to produce NO in vitro and in vivo,¹⁴suggesting that HU may act as a donor of NO in vivo.

The aim of this study was to compare the adhesive propertiesof neutrophils from SCD individuals with those on HU therapy.The study also aimed to determine whether the altered NO bioavailabilitythat is characteristic of SCD may cause changes in the intracellularlevels of NO metabolites and the principal NO second messenger,cyclic guanosine monophosphate (cGMP, produced by the activationof the guanylate cyclase enzyme). Finally, we determined whetherthe adhesive properties of SCD neutrophils may be abrogatedby pharmacological donation of NO in vitro.

Design and Methods

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Design and Methods

A total of 52 homozygous HbS/S patients were studied (19 ofwhom were on HU therapy). Six patients were also heterozygousfor ${alpha}$ -thalassaemia (2 patients on HU and 4 patients not on HU).Patients were not in crisis and had not received blood transfusionsin the previous three months. Patients on HU therapy had beentaking 20–30 mg/Kg for at least three months. Twenty-fourhealthy individuals were used as controls (age, 20–50).Informed consent was obtained from all patients and controls,and the study was approved by the local ethics committee. Forclinical characteristics of all patients and controls, see OnlineSupplementary Table 1.

Neutrophils were isolated from peripheral blood¹⁵ and staticassays¹⁵ were performed to determine their adhesion (2x10⁶ neutrophils/mLRPMI medium) to immobilised fibronectin (20 µg/mL) orrecombinant ICAM-1 (10 µg/mL) (30 mins., 37°C, 5%CO₂). The adhesion assay and materials used in this study aredescribed in Supplementary Information. For NO-donor/drug co-incubation,cells were co-incubated during the adhesion assay.

Total NO metabolites (NOx; nitrite plus nitrate) were quantifiedin lysed neutrophil (5x10⁶ cells/mL) ultrafiltrates using anitrate/nitrite colorimetric assay kit (Cayman, Ann Arbor, MI,USA) (see Supplementary Information). For intracellular cGMPdetermination, isolated neutrophils (1x10⁷ cells/ml PBS) wereincubated with a phosphodiesterase inhibitor and cyclic nucleotideswere extracted with perchloric acid (see Supplementary Information)before quantifying cGMP using commercially available ELISA kits(Cayman Laboratories, Ann Arbor, MI, USA).

For flow cytometry, isolated neutrophils (5x10⁶ cells/mL) wereincubated with anti-CD11a FITC, anti-CD11b Alexa Fluor and/oranti-CD49d PE monoclonal antibody (20 mins., RT, in the dark)and processed as described in Supplementary Information. Resultsare expressed as geometric mean cell fluorescence intensityvalues compared to that of isotype controls.

Results are expressed as means ± S.E.M. Statisticallysignificant differences between groups were determined usingthe Mann-Whitney non-parametric test. The Student’s pairedt-test was used to compare groups before and after treatmentwith specified drugs. Statistical significance was establishedas p<0.05.

Results and Discussion

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Results and Discussion

Leukocyte adhesion to the endothelium of the micro-circulationplays a key role in SCD vaso-occlusion.² Therefore, therapeuticapproaches to inhibit this adhesion may be important for theprevention of vaso-occlusion. Figure 1 confirms the increasedability of neutrophils from SCD individuals (SCDneu) to adhereto fibronectin, an extracellular matrix component, and furtherdemonstrates an increased ability of these cells to adhere torecombinant ICAM-1, an adhesion molecule expressed on activatedendothelial cells. Heterozygosity for ${alpha}$ -thalassemia did not significantlyalter SCDneu adhesive properties (data not shown).

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Figure 1. Adhesion of neutrophils isolated from control individuals, steady-state SCD individuals (SCD) and steady-state SCD individuals on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day) to fibronectin (A) and recombinant ICAM-1 (B). Results are expressed as percentage of adherent cells ± S.E.M. N $≥$ 16 and N $≥$ 6, for (A) and (B) respectively. *, p<0.05; ***, p<0.001, compared to control adhesion; #, p<0.05, compared to SCD adhesion.

Decreased NO bioavailability may contribute to the pathophysiologyof SCD.⁷ Intracellular levels of NOx were significantly lowerin SCDneu compared to levels in healthy control neutrophils(3.68±0.35 vs. 7.04±1.38 µM/1x10⁶ cells;p<0.05; n=9, respectively), indicating that reduced NO bioavailabilitymay result in decreased NO signaling in SCD leukocytes. Intracellularlevels of the NO second messenger, cGMP, however, were not significantlydifferent in SCD and control neutrophils (0.104±0.023vs. 0.114±0.021 pMol/1x10⁷ cells; p>0.05; n=19; n=11,respectively). Whether a reduction in intracellular NO-dependentsignaling contributes to the increase in leukocyte adhesiveproperties in SCD must still be determined. There is, however,some evidence that the up regulation of cyclic adenosine monophosphate(cAMP)-dependent signaling may contribute to increase the adhesiveproperties of SCD neutrophils.^15,16 This could be due to augmentedserum inflammatory factors in SCD.

Pharmacological stimulation of the NO-dependent pathway withthe NO donors Diethylamine NONOate (DEANO; 1 µM) and sodiumnitroprusside (SNP; 10 µM) significantly abrogated theadhesion of SCD, but not control neutrophils (CONneu), to bothfibronectin and ICAM-1 (Figure 2). Furthermore, the NO-independentguanylate cyclase activator BAY 41-2272 (150 nM) also significantlydecreased SCDneu, but not CONneu, adhesion to fibronectin andICAM-1 (Figure 2). Co-incubation of SCDneu with the NO donorsDEANO (1 µM) or SNP (10 µM), and with the inhibitorof guanylate cyclase 1H-[1,2,4] Oxadiazolo [4,3-a]quinoxalin-1-one(ODQ; 10 µM), prevented the reduction in SCDneu adhesionto fibronectin that is induced by these NO donors (Figure 2c),suggesting that NO donors may reduce SCD neutrophil adhesionproperties via a cGMP-dependent pathway.

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Figure 2. Adhesion of neutrophils isolated from control and SCD individuals to fibronectin (A) and ICAM-1 (B) after or without co-incubation with 1 µM DEANO, 10 µM SNP or 150 nM BAY 41-2272. (C) Basal adhesion of SCD neutrophils to fibronectin and adhesion following co-incubation with 1 µM DEANO, 10 µM SNP or 150 nM BAY 41-2272 in the presence or absence of the guanylate cyclase activator, ODQ (10 µM). Results are expressed as percentage of adherent cells ± S.E.M. N $≥$ 5, N $≥$ 3 and N $≥$ 8 for (A), (B) and (C) respectively. (A) and (B): **, p<0.01; ***, p<0.001, compared to basal control neutrophil adhesion; #, p<0.05; ##, p<0.01, compared to basal SCD neutrophil adhesion. (C): **, p<0.01, compared to basal SCD neutrophil adhesion; #, p<0.05, compared to incubation with DEANO alone; ${otimes}$ , p<0.05, compared to incubation with SNP alone.

The surface expressions of the integrin ${alpha}$ -subunits, CD11a andCD11b, were determined on control and SCD neutrophils by flowcytometry (Table 1). Data suggest that the LFA-1 and Mac-1 integrinsubunits, CD11a and CD11b, are not expressed at significantlydifferent levels on the surface of SCDneu, compared with CONneu.Some studies have reported an increased CD11b/CD18 expressionon unstimulated SCD neutrophils,⁵ while others have detectedsignificantly increased CD11b expression only after cell stimulation.⁶Integrins can mediate alterations in adhesive properties bothfollowing the mobilization of integrins to the cell surfaceand also as a consequence of changes in integrin affinity andavidity (mediated largely by integrin clustering).^17,18 Theaffinity of Mac-1 for its ligands, for example, is dynamicallyregulated by inside-out signaling, producing a form of the integrinthat binds to its substrate with a much greater affinity.¹⁷It is, therefore, possible that the increased adhesive propertiesof SCD neutrophils observed may be mediated by alterations inintegrin affinity rather than expression. The surface expressionof the VLA-4 integrin subunit, CD49d, was also determined onSCDneu, since the involvement of β1 integrins has beenimplicated in neutrophil recruitment during chronic inflammation¹⁹and NO signaling may have a role in controlling VLA-4 integrinexpression on the neutrophil cell surface.⁹ No significant alterationin CD49d expression was found, however, on the surface of SCDneu.Interestingly, following incubation with 10 µM SNP (30mins., 37°C), the surface expression of the CD11a subunitwas significantly decreased on both SCD and SCDHU neutrophilsto levels similar to those of CONneu (Table 1). However, incubationwith a NO donor did not decrease the surface expression of themajor neutrophil integrin Mac-1, as demonstrated by the unalteredCD11b expression following incubation with SNP.

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Table 1. Expressions of integrin a-subunits on the surface of neutrophils isolated from control individuals, steady-state SCD individuals (SCD) and steady-state SCD individuals on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day).

Data indicate that a reduction in adhesion molecule expressionmay not contribute significantly to the reduction in SCD neutrophiladhesion mediated by NO donors. Instead, these agents may abrogateSCD neutrophil adhesion via alterations in the function (viaaffinity or avidity alterations) of the adhesion molecules alreadyexpressed on the cell surface. Similarly, NO-dependent signalinghas been shown to alter platelet GPIIb/IIIa integrin function,as well as neutrophil Mac-1 integrin affinity or avidity.^9,20

Interestingly, the adhesion of neutrophils from SCD individualson HU therapy (SCDHUneu) demonstrated a significantly loweradhesion to fibronectin and ICAM-1 that approached that of CONneuadhesion (Figure 1). NOx in these cells were not found to besignificantly higher than in SCDneu (SCDHUneu; 4.22±0.92µM/1x10⁶ cells; p>0.05; n=11). However, intracellularcGMP levels were found to be approximately doubled in SCDHUneucompared with control and SCD neutrophils (0.241±0.034pMol/1x10⁷ cells; p<0.001; n=9).

The surface expressions of the CD11a, CD11b and CD49d integrinsubunits were not found to be altered significantly on SCDHUneu,indicating that HU therapy may reduce neutrophil adhesion viaalterations in integrin affinity / avidity, as also observedwhen SCD neutrophils were treated with NO donors. Alternatively,the possibility that SCD neutrophil adhesion may be mediatedby receptors other than those studied here should not be ruledout. Since activation of cGMP-dependent signaling was foundto reduce SCDneu adhesive properties, it is possible that theincreased intracellular cGMP levels observed in SCDHUneu maycontribute to the observed reduction in adhesion. These resultsprovide further evidence to support the hypothesis that HU mayexert some of its effects via up regulation of NO-cGMP signaling.Indeed, HU has been reported to induce NO-cGMP signaling inendothelial cells¹⁴ and cGMP levels have been found to be increasedin the plasma and red cells of SCD individuals on HU.^21,22 Furthermore,HU may increase NO bioavailability by decreasing hemolysis and,in turn, decreasing NO scavenging by cell-free plasma hemoglobinand arginine scavenging by cell-free arginase.⁷ The correlationof SCD/SCDHU neutrophil adhesiveness to markers of the hemolyticrate, hemoglobin levels and reticulocyte counts did not, however,reveal any significant associations with hemolytic rate (resultsnot shown). While HU may mediate a reduction in leukocyte adhesionby increasing intracellular cGMP levels, other mechanisms mayalso participate in the HU-mediated effect. In accordance withevidence that intracellular cAMP may participate in increasedSCD neutrophil adhesive properties,¹⁵ cAMP levels were foundto be reduced in SCD patients on HU therapy¹⁶ and it is possiblethat cross talk between the cAMP and cGMP pathways may resultin alterations in the cyclic nucleotide balance with a consequentreduction in SCD neutrophil adhesive function.

Our data suggest that although decreased NO bioavailabilitymay not play a major role in the increased adhesive propertiesof SCD leukocytes, pharmacological stimulation of the cGMP-dependentpathway may be a potential approach for reducing neutrophiladhesion to the vascular endothelium. A number of NO-donatingagents and drugs that stimulate cGMP accumulation show potentialfor use in vivo. Guanylate cyclase inhibitors, such as BAY 41-2272,have been successfully used to treat animal models of cardiovasculardisease, pulmonary hypertension and inflammation.^23–25Alternatively, drugs that have a more cell-specific effect,such as inhibitors of phosphodiesterases (PDE) may prove moreeffective in specifically reversing altered SCD neutrophil function.For example, sildenafil, a PDE5-specific inhibitor, is beingtested clinically in SCD patients with pulmonary hypertension²⁶and should it be found that this or any other PDE is expressedin leukocytes, such PDE inhibitors may also have the potentialfor abrogating altered SCD neutrophil adhesion.

Acknowledgments

we thank F. Pereira and Prof. I.L. Metze for flow cytometryanalysis and Prof. E. Antunes for providing BAY 41-2272. Funding:this study was supported by grants from CNPq and FAPESP, Brazil

Footnotes

The online version of this article contains a supplemental appendix.

Authorship and Disclosures

AAC and NC conceived the project, designed and performed experiments,analyzed and interpreted data, and wrote the manuscript. CFPperformed experiments. CFP and STOS assisted in data interpretationand approved the final manuscript. FFC contributed to projectdesign, data interpretation and the final revision of the paper.

The authors report no potential conflicts of interest.

Received for publication August 13, 2007. Revision received October 4, 2007. Accepted for publication October 30, 2007.

References

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