J. Pediatr. (Rio J.) vol.91 número4

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www.jped.com.br

ORIGINAL

ARTICLE

Association

of

neopterin

as

a

marker

of

immune

system

activation

and

juvenile

rheumatoid

arthritis

activity

夽

Mones

M. Abu

Shady

a

_,

_Hanan

_A.

_Fathy

b

_,

_Alaa

_Ali

a

_,

_Eman

_R.

_Youness

c,∗

,

Gihan

A. Fathy

a

a_Child_Health_Department,_Medical_Division,_National_Research_Centre,_Cairo,_Egypt

b_Research_Department,_National_Centre_for_Radiation_Research_and_Technology,_Atomic_Energy_Authority,_Cairo,_Egypt c_Medical_Biochemistry_Division,_National_Research_Centre,_Cairo,_Egypt

Received13March2014;accepted10September2014 Availableonline26February2015

KEYWORDS

MCP-1; TNF-␣;

Rheumatoidarthritis

Abstract

Objective: Toevaluateneopterinplasmaconcentrationsinpatientswithactivejuvenile idio-pathicarthritis(JIA)andcorrelatethemwithdiseaseactivity.

Methods: Sixty patients diagnosed as activeJIA, as well as another 60 apparently healthy age- and gender-matched children as controls, were recruited from the Pediatrics Allergy andImmunologyClinic, AinShams University. Diseaseactivitywas assessed bythe Juvenile ArthritisDiseaseActivityScore27(JADAS-27).Laboratoryinvestigationswereperformedforall patients,includingdeterminationofhemoglobinconcentration(Hgb),erythrocyte sedimenta-tionrate(ESR),andC-reactiveprotein.Serumconcentrationsoftumornecrosisfactor-alpha (TNF-␣),interleukin-6(IL-6),monocytechemoattractantprotein-1(MCP-1),andneopterinwere measured.

Results: SignificantdifferenceswerefoundbetweenJIApatientsandcontrolswithregardto themeanlevelsofHgb,ESR,TNF-␣,IL-6,andMCP-1(p<0.05).Astatisticallysignificanthigher meanlevelserumneopterin concentration(p<0.05)wasfound inJIApatients(20.43±8.73 nmol/L)thanincontrols(6.88±2.87nmol/L)(p<0.05).Positivesignificantcorrelationswere detectedbetweenserumneopterinandESR,TNF-␣,IL-6,MCP-1,andJADAS-27(p<0.05).No correlation was found between serum neopterin andCRP (p>0.05).Multiple linear regres-sionanalysisshowedthatJADAS-27andESRwerethemainvariablesassociatedwithserum neopterininJIApatients(p<0.05).

Conclusion: TheelevationofplasmaneopterinconcentrationsinearlyJIApatientsmayindicate stimulationofimmuneresponse.Serumneopterincanbeusedasasensitivemarkerforassaying backgroundinflammationanddiseaseactivityscoreinJIApatients.

夽

Pleasecitethisarticleas:AbuShadyMM,FathyHA,AliA,YounessER,FathyGA.Associationofneopterinasamarkerofimmunesystem activationandjuvenilerheumatoidarthritisactivity.JPediatr(RioJ).2015;91:352---7.

∗_{Corresponding}_author.

E-mail:hoctober2000@yahoo.com(E.R.Youness).

http://dx.doi.org/10.1016/j.jped.2014.09.007

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PALAVRAS-CHAVE

MCP-1; FNT-␣;

Artritereumatoide

Associac¸ãodeneopterinacomomarcadordeativac¸ãodosistemaimunológicoe

atividadedaartritereumatoidejuvenil

Resumo

Objetivo: avaliar as concentrac¸ões plasmáticas de neopterina em pacientes com artrite idiopáticajuvenil(AIJ)ativaecorrelacioná-lascomaatividadedadoenc¸a.

Métodos: Sessenta pacientes diagnosticados com AIJ ativa, bem como outras 60 crianças aparentementesaudáveiscomamesmaidadeesexonogrupodecontrole,foramrecrutados daclínicadeAlergiaeImunologiaInfantildaUniversidadeAinShams.Aatividadedadoençafoi avaliadapeloEscoredeAtividadedaDoençadaArtriteJuvenilem27Articulações(JADAS-27). Foramrealizadasinvestigaçõeslaboratoriaisemtodosospacientes,incluindoadeterminação daconcentraçãodehemoglobinas,ataxadesedimentaçãodeeritrócitoseaproteínaC-reativa. Forammensuradasasconcentraçõesséricasdofatordenecrosetumoralalfa,interleucina-6e proteínaquimiotáticademonócitos-1eneopterina.

Resultados: FoiencontradaumadiferençasignificativaentreospacientescomAIJeos con-trolesquantoàsmédiasdeHb,TSE,FNT-␣,IL-6eMCP-1(p<0,05).Foiencontradoumnível estatísticaesignificativamentemaiordeconcentraçãomédiadeneopterinasérica(p<0,05) em pacientescomAIJ(valormédio de20,43±8,73nmol/L)queem controles(valormédio de6,88±2,87nmol/L)(p<0,05).Foramdetectadascorrelaçõespositivassignificativasentrea neopterinaséricaeaTSE,FNT-␣,IL-6,MCP-1eJADAS-27(p<0,05).Nãofoiencontrada nen-huma correlaçãoentreaneopterinaséricaeaPCR (p>0,05).A análisederegressão linear múltiplamostrouqueoJADAS-27eaTSEforamasprincipaisvariáveisassociadasàneopterina séricaempacientescomAIJ(p<0,05).

Conclusão: A elevação das concentrações plasmáticasde neopterina em pacientescomAIJ precocepodeindicarumestímuloderespostaimune.Aneopterinaséricapodeserusadacomo umindicadorsensívelparaanalisarohistóricodeinflamaçõeseoescoredeatividadedadoença empacientescomAIJ.

Introduction

Juvenileidiopathicarthritis(JIA)isgenerallyconsidered a clinical syndrome involving several disease subsets, with a number of inflammatory flows, leading to an eventual commonpathwayinwhichpersistentsynovialinflammation andassociateddamage toarticular cartilageand underly-ing bone arepresent.1 _One _main _inflammatory _process_in the pathophysiology of the JIAconsists of overproduction of tumor necrosis factor that leads to overproduction of many cytokines such as interleukin-6, which causes per-sistent inflammationand jointdestruction.2---4 _The _disease arises in a genetically susceptible individual due to envi-ronmentalfactors.5_Moreover,_it_has_been_proposed_that_an antigen-drivenautoimmuneprocessmediatesthe inflamma-torypathologyinsomecasesofarthritis(e.g.,oligoarthritis, polyarthritis).Incontrast,therearenosignsof lymphocyte-mediated,antigen-specificimmuneresponsesinindividuals with systemic onset disease. Recent investigations in the pathophysiology of systemic onset disease have indicated that this disorder is due to an uncontrolled activation of theinnate immunesystem.6_Regardless _of_the _differences intheunderlyingpathogenesisofthevarioustypes ofJIA, pro-inflammatory cytokinesareconsistently overproduced andarerelatedtotheclinicalmanifestationsinalltypesof JIA.7

The International League of Associations for Rheuma-tology (ILAR) classification system divides JIA into seven clinical subgroups.8 _The _few _population _based _estimates available indicate that the prevalence of JIA is approxi-mately one totwo per 1,000 children, andthe incidence is11to14newcasesper100,000children.9

Neopterin, a pyrazino-pyrimidine compound, is syn-thesized by monocytes and macrophages in response to interferon-␥ (IFN-␥) produced by activatedT-cells. It is a markerofcellularimmuneresponse,andlevelsareelevated inconditionsofT-cellormacrophagesactivation,including autoimmunediseasessuchassystemiclupuserythematosus andrheumatoidarthritis.10,11

Thecurrent studywasundertakentoassessthe associ-ationof plasma levelof neopterin with inflammatoryand diseaseactivityinJIApatients.

Subjects

and

methods

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Table1 Demographic,biochemical,anddiseasecharacteristicsofallsubjects.

JIA Control p

Mean±SD Mean±SD

Age(years) 12.20±2.80 12.84±2.46 >0.05

Sex(No.M/F) 30/30 30/30

Hgb(g/dL) 9.24±2.82 12.66±3.66 <0.001a

ESR(mm/h) 50.64±13.43 16.82±4.35 <0.001a

CRP(mg/L) 10.42±3.20

TNF-␣(pg/mL) 9.34±1.44 2.4±0.58 <0.001a

IL-6(pg/mL) 30.76±8.28 5.26±2.44 <0.001 MCP-1(pg/mL) 300.33±78.25 100.56±40.34 <0.001a

Neopterin(nmol/L) 20.43±8.73 6.88±2.87 <0.001a

JADAS-27 40.34±8.24

JIA,juvenileidiopathicarthritis;Hgb,hemoglobinconcentration;ESR,erythrocytesedimentationrate;CRP,C-reactiveprotein;

TNF-␣,tumornecrosisfactor-alpha;IL-6,interleukin-6;MCP-1,monocytechemoattractantprotein-1;JADAS-27,JuvenileArthritisDisease ActivityScore-27.

a_p_<_0.001_is_highly_significant.

corresponding documents were approved by Ethical and ResearchCommitteeof theNationalResearchCenter.Age ranged from 5 to 15 years with a mean age 12.20±2.8 years.PatientswereeligibleiftheymettheEdmonton Inter-national Leagueof Associations for Rheumatology criteria (second revision) for a diagnosis of JIA.8 _Disease activ-ity was measured using a validated score, the JADAS-27 (Juvenile ArthritisDiseaseActivity Score-27).12 _This _score includesfourmeasures:physicianglobalassessmentof dis-easeactivityusingavisualanalogscale(VAS),parentglobal assessmentofchild’swell-beingdeterminedbyaVAS,count of joints with active disease (evaluating 27 joints), and erythrocytesedimentationrate(ESR).ESRisnormalizedto ascorerangingfrom0to10,by theformula(ESR-20)/10. JADAS-27iscalculatedasthesimplelinearsumofthescores ofitsfourcomponents,whichyieldsatotalscoreof0-57, withhigherscores associated withworse disease activity. Thirty-eightpatients(63.3%)hadpolyarticularonsetJIA,15 patients(25%)hadpauciarticularonset,andsevenpatients (11.7%)hadsystemiconset.

All patients had full history takenand were subjected to clinical examination. Laboratory investigations were performed for all patients, including determination of hemoglobin concentration (Hgb in g/dL), ESR in mm/h by Westergren method, and C-reactive protein (CRP in mg/L) detection by the latex agglutination slide test. Serumconcentrationsoftumornecrosisfactor-alpha (TNF-␣), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were measured by multiplex enzyme-linked immunosorbent assay (ELISA) (Millipore®, Merck KgaA,Darmstadt,Germany).

Assay

of

serum

neopterin

(nmol/L)

Serumneopterin levels wereexamined usingan ELISA kit (Neopterin ELISA,IBL, Hamburg, Germany). The assay is based on the basic principle of competition between a peroxide-conjugatedandnon-conjugatedantigenforafixed number of antibody binding sites. The peroxidase conju-gated,antigen-antibodycomplexesbindtothewellsofthe microfilterstrips,whicharecoatedwithagoodanti-rabbit

antibody.Unboundedantigenis thenremovedbywashing. Afterthesubstratereaction,theopticaldensityismeasured at450nm.Astandardcurveisplottedandneopterin concen-tration inthe sample isdetermined byinterpolationfrom thestandardcurve.

Statistical

analysis

StatisticalanalysiswasperformedusingStatisticalPackage for Social Sciences (SPSS) version 16 for Windows (SPSS Inc.,Chicago,IL,USA).Continuousdatawereexpressedas mean±standarddeviationandwerecomparedbyStudent’s

t-test.Pearson’scorrelationanalysiswasconductedto eval-uate the association between continuous exposure and continuous covariates. Multiple linear regression analysis wasperformedtoidentifytheinfluenceof multiple varia-bles(age,sex,ESR,CRP,TNF-␣,IL-6,MCP-1,andJADAS-27) on a dependent variable (serum neopterin). p<0.05 was consideredasstatisticallysignificant.

Results

The clinical characteristics of patients with JIA and con-trolsubjectsareshowninTable1.Nosignificantdifference wasdetected between patients and controls asregard to theirmeanages(p>0.05).However,significantdifferences werefoundbetweenbothgroupswithregardtothemeans of theother variables(Hgb,ESR, TNF-␣,IL-6, MCP-1,and neopterin;p<0.05).

Demographicandbiochemicalcharacteristicwere com-paredbetweenmalesandfemalesinthepatientsgroupin

Table2.MeanJADAS-27washigherinfemalesthaninmales andthe differencewassignificant (p<0.05),while no sig-nificant differences weredetected asregard totheother variables.

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Table2 Demographic,biochemical,anddiseasecharacteristicsinmaleandfemaleJIApatients.

Males Females p

Mean±SD Mean±SD

Age(years) 12.15±3.25 11.96±2.76 >0.05

Hgb(g/dL) 9.54±3.36 9.16±2.49 >0.05

ESR(mm/h) 48.54±11.63 51.47±10.33 >0.05

CRP(mg/L) 9.72±2.47 12.47±3.43

TNF-␣(pg/mL) 8.24±1.46 10.04±1.37 >0.05

IL-6(pg/mL) 29.46±6.46 31.46±4.67 >0.05

MCP-1(pg/mL) 295.33±78.25 304.74±38.23 >0.05

Neopterin(nmol/L) 19.84±7.49 20.16±2.42 >0.05

JADAS-27 38.56±8.46 49.38±10.36 <0.05a

JIA,juvenileidiopathicarthritis;Hgb,hemoglobinconcentration;ESR,erythrocytesedimentationrate;CRP,C-reactiveprotein;

TNF-␣,tumornecrosisfactor-alpha;IL-6,interleukin-6;MCP-1,monocytechemoattractantprotein-1;JADAS-27,JuvenileArthritisDisease ActivityScore-27.

a _p_<_0.05_is_significant.

Table 3 Correlations between neopterin, inflammatory markers,anddiseaseactivityinJIApatients.

Neopterin

r p

ESR 0.36 <0.001a

CRP 0.08 >0.05 TNF-␣ 0.46 <0.001a

IL-6 0.24 <0.05b

MCP-1 0.32 <0.001a

JADAS-27 0.33 <0.001a JIA, juvenile idiopathic arthritis; ESR, erythrocyte sedimen-tation rate; CRP, C-reactive protein; TNF-␣, tumor necrosis factor-alpha; IL-6, interleukin-6; MCP-1, monocyte chemoat-tractantprotein-1;JADAS-27,JuvenileArthritisDiseaseActivity Score-27.

a _p_<_0.001_is_highly_significant. b _p_<_0.05_is_significant.

MCP-1, and JADAS-27(p<0.05). No significant correlation wasfoundbetweenserumneopterinandCRP(p>0.05).

Multiplelinearregressionanalysisfortheassociationof the different variables with serum neopterin is shown in

Table 4.After adjustmentfor age, sex, andinflammatory markers,JADAS-27andESRwerethemainpredictorsforthe highneopterinlevelsseeninpatientswithJIA(p<0.05).The effectsofTNF-␣,IL-6,andMCP-1wereattenuatedbythe adjustmentofallthevariables.

Discussion

Thepresentstudyshowedahighlevelofserumneopterin, as a marker of macrophage activation, in patients with JIA.NeopterinandESRaresignificantpredictorsfordisease activityinsuchpatients.

Previous studies of neopterin in rheumatoid arthri-tis (RA) have been performed to define the relationship between neopterin and disease activity. Generally, they foundthatneopterincorrelatedwithdiseaseactivityinRA

Table4 Multiplelinearregression analysisfor neopterin anddifferentindependentvariables.

Variables Neopterin(r2₌_0.458)

Standardized␤coefficient t p

Age 0.065 0.710 >0.05

Sex -0.018 -0.203 >0.05

ESR 0.517 4.934 <0.001a

CRP 0.230 1.934 >0.05 TNF-␣ 0.164 1.842 >0.05 IL-6 0.315 1.872 >0.05 MCP-1 0.232 1.723 >0.05 JADAS-27 0.534 4.862 <0.001a JIA, juvenile idiopathic arthritis; ESR, erythrocyte sedimen-tation rate; CRP, C-reactive protein; TNF-␣, tumor necrosis factor-alpha; IL-6, interleukin-6; MCP-1, monocyte chemoat-tractantprotein-1;JADAS-27,JuvenileArthritisDiseaseActivity Score-27.

a _p_<_0.001_is_highly_significant.

anddecreasedwithtreatment.11,13,14_In_contrast_to_the cur-rentstudy,apreviousstudyinJapandetectedsignificantly increasedneopterininSLE(SystemicLupusErythematosus) patients(p<0.001)butnotinRApatients.However,patients withRAhadagreaterconcentrationofneopterininsynovial fluid.15

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kynurenine/tryptophan (r=0.486, p<0.05), erythrocyte sedimentation rate (r=0.472, p<0.05), and RF(r=0.478, p<0.05)inagroupofrheumatoidarthritispatients,which arein concordance withthe results of thepresent study. However,contrarytothepresent results,theyfound that neopterin and TNF-␣ levels did not showstatistically sig-nificant differences between patient and control groups. Thedifferencemaybeduetotheagerangeintheirstudy, asit was adult group. In 2013, D’agostino et al.18 evalu-atedneopterinplasmaconcentrationsinpatientswithearly Rheumatoid Arthritis (RA) and correlated them with dis-easeactivity.In agreementwiththepresent results, they detected a statistically significant elevation of neopterin mean concentration in early RA patients: mean value of 8.92±0.93 [3.94-28.3] nmol/L (p<0.001). Pearson prod-uct moment correlation suggested a correlation between neopterinconcentrations andDAS-28(r=0.208,p=0.065). Similar results were obtained by Arshadi et al.,19 _who detected significantly higher level of neopterin in RA patients compared to healthy controls. Moreover, plasma neopterinlevelwasincreasedinpatientswithactivedisease andalsowascorrelatedwithdisease activity parameters. The previous results are in concordance withthe present study.Butincontrastwiththepresentstudy,theyfounda higherneopterin level in male RApatients versusfemale patients and a significant correlation of plasma level of neopterinwithagein boththeRAandcontrol group.The differenceintheresultsmaybeattributedtotheadultage groupof their study comparedto theyoung age group in the present study. Also Fagerer etal., in 2013,20 _studied theinvolvementofspecificchemokinesintheinflammatory processofpatientswithRAandcardiovasculardiseasewith activatedproduction ofthepteridine neopterin.In agree-ment with the current study, they found high neopterin levelsin RApatientsandsignificantly elevated concentra-tionsof neopterin in patients withRA pluscardiovascular disease(CVD)comparedtoRAwithoutCVD(p<0.03).

Tothebestoftheauthors’knowledge,fewstudieshave includedbothneopterinasapro-inflammatorymarkerand JIA.TwostudiesfoundhighserumconcentrationsofIL-10, IL-18,IL-6,andneopterininpatientswithsystemicJIA com-plicated by macrophage activation syndrome. They found thatIL-10andIL-18remainedelevatedintheinactive dis-ease,whileotherpro-inflammatorycytokinesnormalizedin theinactivephaseofthedisease.21,22_In_2014,_Brunner_et_al. 23_evaluated_tryptophan_as_biomarker_in_JIA_and_correlated

itwithneopterininserumandsynovialfluid.Theydetected thatserumtryptophanshowednorelevantdifferenceinJIA patients vs. controls in contrast to neopterin, which was higherinpatientsthancontrols.

The presentstudy hasseverallimitations.It was cross-sectionalindesign,andthereforecausalinferencescannot be drawn. Also, the effects of the type of therapy on neopterinlevelswerenotfollowed,sotheiruseasamarker for therapy efficacy cannot be evaluated. The relatively smallsamplesizecouldberesponsibleforinadequate sta-tistical power. Larger sample sizes would have increased numberswithineachJIAcategory,allowingforgreater pre-cision inassessing the pro-inflammatorymarkers between categories.

In conclusion, the present study emphasized that macrophage activation, reflected by increased serum

neopterin, is a sensitive marker for assaying background inflammationanddiseaseactivityscoreinJIApatients.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

References

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ERRATUM

Erratum

on

‘‘Association

of

neopterin

as

a

marker

of

immune

system

activation

and

juvenile

rheumatoid

arthritis

activity’’

夽

Errata

de

‘‘Associac

¸ão

de

neopterina

como

marcador

de

ativac

¸ão

do

sistema

imunológico

e

atividade

da

artrite

reumatoide

juvenil’’

Mones

M. Abu

Shady

a

_,

_Hanan

_A.

_Fathy

b

_,

_Alaa

_Ali

a

_,

_Eman

_R.

_Youness

c,∗

,

Gihan

A. Fathy

a

b_Research_Department,_National_Centre_for_Radiation_Research_and_Technology,_Atomic_Energy_Authority,_Cairo,_Egypt

c_Medical_Biochemistry_Division,_National_Research_Centre,_Cairo,_Egypt

Intheaffiliationoftheauthorsoftheoriginalarticle‘‘Associationofneopterin asamarkerofimmunesystemactivation andjuvenilerheumatoidarthritisactivity’’(JPediatr(RioJ).2015;91(4):352-357),whereitreads:

MonesM.AbuShadya_,_Hanan_A._Fathyb_,_Alaa_Alia_,_Eman_R._Younessc_,_Gihan_A._Fathya

b_Health_Research_Department,_National_Research_Centre,_Cairo,_Egypt

c_Medical_Biochemistry_Division,_National_Center_for_Radiation_and_Technology,_Cairo,_Egypt

itshouldread

MonesM.AbuShadya_,_Hanan_A._Fathyb_,_Alaa_Alia_,_Eman_R._Younessc_,_Gihan_A._Fathya

b_Research_Department,_National_Centre_for_Radiation_Research_and_Technology,_Atomic_Energy_Authority,_Cairo,_Egypt