Braz. j. . vol.83 número1

www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Distinct

characteristics

of

nasal

polyps

with

and

without

eosinophilia

夽

Changzhi

Sun

,

Hong

Ouyang

,

Renzhong

Luo

a_{GuangzhouMedicalCollege,GuangzhouWomenandChildren’sMedicalCenter,DepartmentofOtolaryngology,Guangzhou,China} b_{ChinaThreeGorgesUniversity,RenHeHospital,DepartmentofOtolaryngology,Yichang,China}

Received3August2015;accepted20January2016 Availableonline22April2016

KEYWORDS

Nasalpolyps; Th2cells; Th17cells

Abstract

Introduction:EosinophilicandnoneosinophilicNasalpolyps(NPs)aredifferentsubtypesofNPs andrequiredifferenttreatmentmethods.

Objective:Tocomparethehistologiccharacteristics,mRNAandproteinexpressionbetween NasalPolypswithandwithouteosinophilia.

Methods:NPstissueswereobtainedfromeighty-sixNPspatientsduringsurgery.Eosinophilic andnoneosinophilicNPsweredistinguishedaccordingtoimmunochemicalresultsofthe speci-men.Thehistological,mRNAandproteinexpressionfeatureswerecomparedbetweenthetwo groups.

Results:IneosinophilicNPs,weobservedasignificantlyhigherGATA-3,IL-5,IL-4,IL-13mRNA andproteinexpression.InnoneosinophilicNPs,IL-17,IL-23andRORcmRNAandprotein expres-sionwereincreased.Immunohistochemistrytestsshowed,moremastcellsandlessneutrophils ineosinophilicNPscomparedwithnoneosinophilicNPs.EosinophilicNPspatientpresentedmore severesymptomscoreswhencomparedtononeosinophilicNPs.

Conclusion:We demonstrate for the first time that Th2 is the predominant reaction in eosinophilicNPswhileTh17isthepredominantreactioninnoneosinophilicNPs.Ourstudymay providenewtreatmentstrategyforNPs.

© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE

Póliposnasais;

CélulasTh2;

CélulasTh17

Diferenc¸asnascaracterísticasdepóliposnasaiscomesemeosinofilia

Resumo

Introduc¸ão:Póliposnasais(PNs)eosinofílicosenãoeosinofílicossãodiferentessubtiposdePNs erequeremdiferentesmétodosdetratamento.

夽 _{Pleasecitethisarticle as:SunC,Ouyang H, LuoR. Distinct characteristicsofnasal polyps withand withouteosinophilia. Braz J}

Otorhinolaryngol.2017;83:66---72.

∗_{Correspondingauthor.}

E-mail:meishanoo@163.com(R.Luo).

♦ _{Bothauthorscontributedequallytothearticle.}

PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. http://dx.doi.org/10.1016/j.bjorl.2016.01.012

Objetivo: CompararascaracterísticashistológicaseaexpressãodemRNAseproteínasentre PNscomesemeosinofilia.

Método: Amostrasde PNs foramobtidosde86 pacientesduranteacirurgia. PNs eosinofíli-cosenãoeosinofílicosforamdiferenciadossegundoosresultadosimunoistoquímicosdecada amostra.AscaracterísticashistológicasedeexpressãodemRNAsedeproteínasforam com-paradasentreosdoisgrupos.

Resultados: Em PNs eosinofílicos, observamos uma expressão significativamente maior dos mRNAseproteínasGATA-3,IL-5,IL-4eIL-13.NosPNsnãoeosinofílicos,aumentouaexpressão dosmRNAseproteínasIL-17,IL-23eRORc.Nostestesimunoistoquímicos,observamosmaior númerodemastócitosemenornúmerodeneutrófilosnosPNseosinofílicos,em comparac¸ão comPNsnãoeosinofílicos.OspacientescomPNseosinofílicosobtiveramescoresdesintomas maisgravesvs.PNsnãoeosinofílicos.

Conclusão:Demonstramos,pelaprimeiravez,umareac¸ãoTh2predominanteemPNs eosinofíli-coseumareac¸ãoTh17predominanteemPNsnãoeosinofílicos.Nossoestudopodeproporcionar novasestratégiasterapêuticasparaarinossinusitecrônica.

© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Chronic rhinosinusitis(CRS) is characterizedby persistent

inflammationofnasalandparanasalmucosa,andisdivided

intotwotypesaccordingtotheabsenceorpresenceofnasal

polyps(NPs):CRSwithoutNPsandCRSwithNPs(CRSwNP).1

HistologicalfeaturesofNPincludeinflammationofTh2cells accompaniedbyinfiltrationwitheosinophils,thickeningof basementmembrane,andhyperplasiaoftheepithelium.2---5

In Western populations, eosinophil infiltration is found in most NPs and considered as a major pathological marker of NPs.6,7 _{However, more and more studies on Chinese}

NPs showed that many NPs patients in China presented as non-eosinophilic inflammation.8 _{For example, several}

studies have shown that a considerable proportion of Chinese NPs were neutrophil dominate, but the detailed difference between eosinophilic and non-eosinophilic NPs isstillunknown.9

Similar studies on asthma have demonstrated that eosinophilicasthmaispharmacologicallyresponsiveto glu-cocorticoid, butnon-eosinophilic asthma mayberesistant toglucocorticoid.10,11_{Inarecentstudyontheresponseof}

NPs patients to oral corticosteroid therapy conducted by Wen,theyfoundeosinophilicNPspatientsweremore sen-sitivetocorticosteroidcomparedwithnon-eosinophilic NP patients.12_{Besides,eosinophilicNPshadahighertendency}

ofrecurrenceaftersurgery.Allthesestudiesdemonstrated thateosinophilicandnon-eosinophilicNPsmaybedifferent subtypesofNPsandneededdifferenttreatmentmethods.

This study aimed to investigate the expression of key transcriptionfactorsandcytokinesforTh1/Th2/Th17cells betweeneosinophilicandnon-eosinophilicNPsandprovide newinformationonCRSwNP.

Methods

Patients

Eighty-sixpatientswithNPswereenrolledconsecutivelyin thisstudy.Thediagnosis wasmainlybasedonpathological

examination.Accordingtopreviousmethods,13_eosinophilic

and non-eosinophilic NPs were categorized based on immunochemical results by the presence of either <5 or ≥5eosinophils/highpoweredfields(HPF),respectively.The baselinedatawerecollectedandLund-Kennedyand Lund-MackeyscorewereobtainedtoevaluatetheseverityofNPs. Noneofthesubjectsusedoralornasalcorticosteroidsduring fourweeksbeforesurgery.Detailsofallsubjectsare sum-marized in Table1. This study wasapproved by thelocal ethicscommittee(No.20130106)andinformedconsentwas obtained.

Everyspecimenwascut intotwoportions. Oneportion wasstored at −80◦

C for mRNA andprotein analysis. The otherportionwasusedforIHCstaining.

Symptomscores

Attheclinicalvisit,thepatientsgaveanoverallassessment oftheirrhinitissymptoms.Thesymptomsofnasalblockage, nasalitching,sneezing,andrhinorrheawererated ona 4-pointscale,where0=nosymptoms,1=mild,2=moderate, and3=severe. Totalsymptom scores ranged from0to12 andrepresentedthesumofthescoresfornasalblockage, nasalitching,sneezing,andrhinorrhea.

Immunohistochemicalstaining

Forimmunohistochemistry,thesectionsunderwent dewax-ing,dehydration,andthenwereplacedin0.3%H2O2for20

minutesatroom temperaturetoreduce nonspecific back-ground staining. After antigen retrieval by 10mM citrate bufferfor15minutes,antihumanmonoclonalantibodiesfor MBP(eosinophils,1:100,SantaCruz),anti-HNE(neutrophils, 1:200, Dako) and anti-tryptase (mast cells, 1:100, Santa Cruz) were incubated overnight at 4◦_{C for}

Table1 Demographicandclinicalcharacteristicsofeosinophilicandnon-eosinophilicNPpatients.

EosinophilicNP Non-eosinophilicNP p-value

Sex(male/female) 22/24 21/19 0.834

Age,years 41.3 39.4 0.235

Durationofsymptoms,years 7.5 4.1 0.046

Absolutebloodeosinophilcount,109_{/L 0.28 0.11 0.013}

Proportionofeosinophils(%) 2.6 7.8 0.003

BloodIgElevel,kU/L 112 19 0.001

Patientswithallergy(%) 65 22 0.002

Lund-Kennedyscore 16.5 10.4 0.012

Lund-Mackeyscore 17.8 9.8 0.005

Timeofsurgery 2.3 1.1 0.042

NP,nasalpolyp.

Afterwashing,DAB(GeneTech,Shanghai,China)

stain-ingwasperformedundermicroscopeandthesectionswere

counterstained with Mayer’s hematoxylin (Dako) for 40s,

dehydratedwithseriesethanol,clearedwithxylene(three

times),and mounted withneutralbalsam (Dako). Control

for nonspecificstaining wasroutinely performed withPBS

insteadofprimaryantibodies,andallprovednegative.

The sections were visualized with an Olympus CX40

Microscope (Olympus Europa, GmbH --- Germany) and the

numberofpositivecellswascountedunderhigh-powerfields

(400×).Tenfieldswerecountedineachspecimenandthe

medianwascalculatedforeachantibody.

Real-timepolymerasechainreaction(PCR)analysis

Real-time PCR were performed as previously described.

Total mRNA was extracted from SIP or mucosa tissues

using TRIzol reagent (Life Technologies---Carlsbad, CA,

United States) according to the manufacturer’s

instruc-tions. Reverse Transcription (RT) was performed, and

cDNA was synthesized from 2␮g of total RNA using an

oligo (dT) 18 primer and M-MLV reverse transcriptase

(Takara---Syuzou,Shiga, Japan). The mRNA expression was

determined using an ABI PRISM 7300 Detection System

(Applied Biosystems---Foster City, CA, United States) and

SYBRPremixTaqTM_{(Takara).Thesequencesoftheprimers}

are listed in Table 2. PRISM samples contained 1× SYBR

GreenMasterMix,1.5␮Lof5␮Mprimers,and25ngof syn-thesizedcDNAina25␮Lvolume.Reactionswereheatedto 95◦

C for10min,followed by40 cyclesof denaturationat 95◦_{Cfor10s,andannealingextensionat60}◦_Cfor60s.All

PCRreactions wereperformed induplicate. Meltingcurve analysiswasusedtocontrolforamplificationspecificity.The meanvalueofthereplicatesforeachsamplewascalculated andexpressedasacyclethreshold(Ct)value.Therelative expressionofeachtargetgenewasdeterminedasthe differ-ence(Ct)betweentheCtvalueofthetargetgeneandthe Ctvalueof␤-actin.FoldchangesinthetargetgenemRNA weredeterminedas2−Ct_.

Enzyme-linkedimmunosorbentassay(ELISA)

Freshly obtained tissue specimen was weighed, and pro-tease inhibitor cocktail(Keygentec --- Nanjing,China) was

Table 2 Primers used for quantitative PCR analysis of transcriptionalfactorsandcytokinegeneexpression.

Primer Sequence

T-bet (s)59-GTCAATTCCTTGGGGGAGAT-39 (a)59-TCATGCTGACTGCTCGAAAC-39

GATA-3 (s)59-CTGGCCACAGTTGTTTCATG-39 (a)59-GCAACTGGTGAACGGTAACA-39

RORC (s)59-GCTGTGATCTTGCCCAGAACC-39 (a)59-CTGCCCATCATTGCTGTTAATCC-39

IL-4 (s)59-CCACGGACACAAGTGCGATA-39 (a)59-CCCTGCAGAAGGTTTCCTTCT-39

IL-5 (s)59-CCCACAAGTGCATTGGTGAA-39 (a)59-CCTCAGAGTCTCATTGGCTATCAG-39

IL-17 (s)59-CAAGACTGAACACCGACTAAG-39 (a)59-TCTCCAAAGGAAGCCTGA-39

IL-13 (s)59-TGAGGAGCTGGTCAACATCA-39 (a)59-CAGGTTGATGCTCCATACCAT-39

IL-23 (s)59-GAGCAGCAACCCTGAGTCCCTA-39 (a)59-CAAATTTCCCTTCCCATCTAATAA-39

GAPDH (s)59-ACCCAGAAGACTGTGGATGG-39 (a)59-TTCTAGACGGCAGGTCAGGT-39

PCR,polymerasechainreaction.

added per 100mg of tissue. The tissue was then

homog-enized using homogenizer (Kinematica --- Switzerland) for

1minonice.Afterhomogenization,thesuspensionwas

cen-trifugedat4000rpmfor20minat4◦_{C,andthesupernatants}

werestoredat−80◦_{Cuntilanalyzed.}

Enzyme-linked immunosorbent assay (ELISA) kits were

used for measuring tissue levels of IL-5, IL-4, IL-13,

IL-17, IL-23, IL-8, and MPO (R&D systems ---

Minneapo-lis, MN, United States) according to the manufacturer’s

protocols. Each sample was tested in duplicate. The

detection limits of the assays were as follows: IL-5,

3.9pg/mL, IL-4, 31.2pg/mL, IL-13, 62.5pg/mL, IL-17,

31.2pg/mL, IL-23, 39pg/mL, MPO, 1.56mg/mL; andIL-8,

Statisticalanalysis

All data were expressed as mean±SD. Statistical

signif-icance between two groups was determined using the

Mann---WhitneyUtest.Significantdifferencewasconsidered whenp<0.05.

Results

Subjects

Basedonthehistologicalcriterion,46NPpatients (53.5%)

were classified into the eosinophilic subgroup and 40

NP patients (46.5%) were classified into non-eosinophilic

subgroup. The demographic and clinical characteristics

of patients are displayed in Table 1. Compared with

non-eosinophilic NP patients, eosinophilic NP patients demonstratedalongerdurationofsymptoms,higherblood absoluteeosinophilcount,higherbloodIgElevel,higher per-centageofallergichistory,higherproportionofeosinophils,

higher Lund-Kennedy and Lund-Mackey score, more inci-dence of surgery, and higher symptom scores. In term of ageand sex, therewasno significant differencebetween eosinophilicandnon-eosinophilicNPpatients.

Comparisonofhistologybetweeneosinophilicand non-eosinophilicNPs

Itwas found that the number of total inflammatory cells in eosinophilic NPs increased significantly compared with non-eosinophilicNPs(datanotshown).Regardingcelltypes, eosinophilic NPs have increased mast cells except for eosinophils, whereas the non-eosinophilic NP presented more neutrophil infiltration (Table 3). The results also showed that 85% of eosinophilic NPs had more than six mastcells perHPF,whileallnon-eosinophilic NPshadless thanthreemastcellsperHPF.Meanwhile,non-eosinophilic NPspresentedassevereneutrophilinfiltration.90%of non-eosinophilicNPshadmorethanfivemastcellsperHPF,while alleosinophilicNPshadless thantwoneutrophil perHPF.

0.15

0.10

0.05

0.00

Relativ

e e

xpression

of GA

T

A3 mRNA

Relativ

e e

xpression

of IL-5 mRNA

Relativ

e e

xpression

of IL-13 mRNA

Relativ

e e

xpression

of IL-17 mRNA

0.15

0.10

0.05

0.00

0.08

0.06

0.04

0.02

0.00

0.3

0.2

0.1

0.0

Relativ

e e

xpression

of IL-4 mRNA

Relativ

e e

xpression

of R

O

RC mRNA

Relativ

e e

xpression

of IL-23 mRNA

NENP ENP NENP ENP

NENP ENP

NENP ENP

NENP ENP

NENP ENP

NENP ENP

*

*

*

*

*

* * 0.25

0.20

0.15

0.10

0.05

0.00

2.0

1.5

1.0

0.5

0.0

0.015

0.010

0.005

0.000

Table 3 Median score and 95% reference range (mean±SEM) of cell counts in eosinophilic and non-eosinophilicNPpatients(perhigh-poweredfield).

EosinophilicNP Non-eosinophilicNP p-Value

Eosinophil 9.3(8.8±2.1) 2.8(2.6±0.3) 0.02 Neutrophil 1.5(1.7±0.5) 6.3(5.4±1.5) 0.04 Mastcell 7.6(6.8±2.3) 2.9(2.8±0.1) 0.01

NP,nasalpolyps.

Whenthe relationshipbetween thenumberof eosinophils

andothercellswasalsoanalyzed,itwasfoundthatthe

num-berofeosinophilscorrelatedpositivelywiththenumberof

mastcells (r=0.68;p<0.001)andtotalinflammatorycells (r=0.46;p<0.05),butnorelationshipwasfoundbetween eosinophilsandneutrophils.

ComparisonofmRNAexpressionbetween eosinophilicandnon-eosinophilicNPs

IneosinophilicNPs,significantlyhigherTh2(GATA-3,IL-5, IL-4,IL-13)transcriptionfactorandcytokinesexpressionwere

observed, while in non-eosinophilic NPs, Th17 (RORC,

IL-17A,andIL-23)transcription factor andcytokinesshowed

increased expression (Fig. 1). For Th1 (T-bet and IFN-␥)

transcriptionfactorandcytokines,nodifferencewasfound betweentwogroups(datanotshown).

Comparisonofproteinexpressionbetween eosinophilicandnon-eosinophilicNPs

SimilarwiththemRNAexpression,Th2cytokines(IL-5and IL-13)proteinweresignificantlyhigherin eosinophilicNPs andTh17cytokines(IL-17A andIL-23)proteinwere signifi-cantlyhigherinnon-eosinophilicNPs(Fig.2).However,IL-4 proteinexpression was undetectablein both groups (data notshown).Asfor Th1cytokines(IL-12andIFN-␥) expres-sion,nodifferencewasfoundbetweentwogroups(datanot shown).Itwasalsofoundthatexpressionofmarkersof neu-trophil(IL-8,MPO)innon-eosinophilicNPswashigherthan thatofeosinophilicNPs.Therelationshipbetweencytokine expressionwasalsoanalyzed;itwasfoundthatIL-17Awas positivelyrelatedtobothIL-8andMPOexpression(r=0.53, p<0.01;r=0.57,p<0.05).

Discussion

In Western populations,NPsare considered tobe orches-trated by Th2 cells, and tissue eosinophilia is a very important feature.14---16 _{The infiltration and activation}

of eosinophils in nasal mucosa can promote secre-tion of specific granule proteins, synthesis, and release of lipid mediators, inflammatory cytokines, chemokines, and growth factors. Through these chemical mediators, eosinophilscontributetothedevelopmentofNPs.However, severalstudieshavedemonstratedthatlessthan50%ofNP patientsinChinaorotherAsiancountrieshadeosinophilic inflammation.17,18 _{These findings indicate that NPs are}

150 400

300

200

100

0

50

40

30

20

10

0

150

100

50

0 100

50

0

IL-5 protein e

xpression

(pg/mL)

IL-13 protein e

xpression

(pg/mL)

IL-17 protein e

xpression

(pg/mL)

IL-23 protein e

xpression

(pg/mL)

IL-8 protein e

xpression

(pg/mL)

MPO protein e

xpression

(ng/mL)

NENP ENP

NENP

* *

* *

* _*

ENP

NENP ENP NENP ENP

NENP NENP

ENP ENP

200

150

100

50

0

6000

4000

2000

0

heterogeneous and can be broadly divided into two sub-types: eosinophilic and non-eosinophilic NPs. Eosinophilic NPscanbewell-controlledbycorticosteroidtherapy,while non-eosinophilic NPs are responsive to a combination of endoscopicsinussurgeryandmacrolidetherapy.19---21

In thepresentstudy,theeosinophilicNPgroup demon-strated a higher prevalence of allergy and IgE levels. Since blood eosinophil count is significantly correlated witheosinophil infiltration in the nasal polyps, the blood eosinophilcountcouldbeagoodmarkerfortheeosinophilic inflammation of NPs. All these data suggested that the occurrenceofeosinophilicNPswasclosedrelatedtoallergy. However,theroleof allergyin thepathogenesis ofNPsis stillcontroversial.Afewstudieshavequestionedtheroleof allergyinthepathogenesisofNPs.Caplinetal.evaluated 3000 atopicpatientsandfound that only0.5% ofpatients hadNPs.Otherreportswerealsounabletosupporteithera higherincidenceofatopyinpatientswithNPsorapatternof allergicinflammationinthepathogenesisofNPs.22---24_Thus,

thepresentstudyrequiresfurthervalidationbystudieswith largersample size.It wasalsofound thateosinophil infil-trationwasdirectly correlatedwithdiseaseseverity,since bothtotalandeachitem’sscorewerehigherineosinophilic NPs.Besides,higherLund-KennedyandLund-Mackeyscores ineosinophilicNPswerealsofound. Takentogether,these results suggest that eosinophilic NPs predict long disease durationandpoorprognosis.

In the histologicaltest, it wasfound that moresevere inflammatory reaction in eosinophilic NPs presented as moremastcellsandeosinophilsinfiltration,andmore neu-trophil infiltration in non-eosinophilic NPs. Mast cells are themajoreffectorcellofIgE-mediatedallergicreactionsby releasinghistamineandotherchemicalsinvolvedinallergic inflammation; their infiltration into eosinophilic NPs sug-gestedthat a T-cell-mediated immune response mayplay animportantroleineosinophilicNPs.Consistentwiththese results, the data showed that the number of eosinophils was positively correlated with the number of mast cells andtotalinflammatorycells.Neutrophilinfiltrationisoften believed to be related with bacterial infection, but the present results demonstrated that neutrophil infiltration wasalso involved in chronic inflammation. Nagakura sug-gestedthathigh-molecular-weightneutrophil chemotactic activity wasrelated tonasalhypersensitivity. In addition, elastase released by neutrophils after degranulation may alsoplayanimportantroleintissuedamage.25,26

Previous studies had confirmed the role of Th cell in thepathogenesisofCRS,thus,thepresentstudycompared the expression of mRNA and protein expression between twogroups.TheresultsrevealedTh2predominantreaction in eosinophilicNPand Th17predominant reactionin non-eosinophilicNP.However,Th1(T-betandIFN-␥)transcription factorsand cytokinesexpression werefound between the two groups. GATA-3 is necessary for commitment toward Th2 cells and controls the expression of IL-5.27---30 _The

upregulation of GATA-3 in eosinophilic NP was reflected by the subsequent increase of the IL-5 mRNA and pro-tein. As a marker for Th17 cells, the transcription factor RORCwasanalyzedandsignificantlyhigherexpressionwas foundinnon-eosinophilicNPs.31,32_{Similarly,Th17cytokines}

(IL-17AandIL-23)proteinweresignificantlyhigherin non-eosinophilic NPs. Consistent with these results, previous

studiedhaveshownthattheIL-17-IL-23axisplaysimportant rolesinthe pathogenesisofNPs. As expected,markers of neutrophils(IL-8,MPO)innon-eosinophilicNPswerehigher thanthatofeosinophilicNPs.ItwasalsofoundthatIL-17A waspositivelyrelatedtobothIL-8andMPOexpression, sug-gestingthatIL-17mayberelatedtoneutrophilmigration; itsmechanismrequiresfurtherexploration.

Conclusion

Insummary,thedatademonstrate, forthefirsttime,Th2 predominant reaction in eosinophilic NPs and Th17 pre-dominantreactioninnon-eosinophilicNPs. Thisstudymay providenewtreatmentstrategyforNPs.

Ethical

standards

Theauthorsassertthatallprocedurescontributingtothis work comply with the ethical standards of the relevant nationalandinstitutionalguidelinesonhuman experimen-tationandwiththeHelsinkiDeclarationof1975,asrevised in2008.

Funding

Supported by Medical Scientific Research Foundation of GuangdongProvince,China,A2013518.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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