Yassera.Korani , Alaa T.Hassan , Effata.E.Tony , Madleenadela.Abdou

Theimpactofsmokingoninflammatorybiomarkersinpatientswithchronicobstructivepulmonarydisease.

YasserA.Korani¹, Alaa T.Hassan ¹, EffatA.E.Tony², MadleenAdelA.Abdou³

¹Departmentsofchest,²Internalmedicineand³ClinicalPathology,FacultyofMedicine,AssiutUniversity,Assiut,Egypt.

ABSTRACT

Background:Chronicobstructivepulmonarydisease(COPD)isachronicprogressiveinflammatorydiseasecharacterizedbylimitationsairflowthatisnotfullyreversible(42).ThepathophysiologyofCOPDisnotcompletelyunderstood. Cigarette smoking is amajorriskfactorforchronicobstructivepulmonarydisease(COPD).ElevatedCRPhasbeenincreasinglyusedasasurrogatemarkerofsystemicinflammationindiverseconditions.TNF-α,apowerfulpro-inflammatorycytokineprimarilyproducedbyactivatedmacrophages,isthoughttoacriticalroleinthepathogenesisofCOPD[13,14].

Theaimofthework:toevaluatetheimpactofsmokingoninflamatorybiomarkersandrelationsbetweenthesebiomarkersandthedeclineoflungfunctioninCOPDpatients.

Methods:Thiscase–controlobservationalprospectivestudywasconductedonfifty-eightclinicallystableCOPDpatients(26non-smokersand32current smokers; at differentstagesrangedfrommildtoverysevere),theirmeanage53.1±14.25and53.9±5.95yearsrespectively),recruitedfromChestDepartment,AssiutUniversityHospitals.AllpatientsmettheGlobalInitiativeforObstructiveLungDisease(GOLD)[4].Allparticipantsweresubjectedtothoroughhistorytaking,fullclinicalexamination,anthropometricmeasurementswithspirometryandchest X-ray. Peripheral hemogram,liverfunctiontests,kidneyfunctiontests,highsensitivityC-reactiveprotein(hsCRP)andserumlevelofTNF-αweremeasuredforbothpatientsandcontrols.

Results:Theconcentrationsofcirculatinghs-CRPandTNF-α,werehighlysignificantlyelevatedinpatientswithCOPDincomparisontothecontrolgroup(3.74±0.2vs.1.30±0.14forhs-CRP;33.88±5.97vs.8.79±0.57forTNF-αwithp0.0001foreach)andthelevelsofmeasuredTNF-αweresignificantlyincreasedwiththeincreaseddegreeandseverityofCOPDandincreasedseverityofsmokingstatus.RegardingthesmokingstatusofCOPDpatients,therewasahighlysignificantlydifferencefor the measuredTNF-α(53.74±9.52versus12.73±1.20withp0.0001)withnosignificantdifferenceforthemeasuredhs-CRP(3.87±0.29versus 3.58±0.27withp˃0.05).Interestingly,therewere significant negativecorrelationsbetweenthelevelsof TNF-α and hs- CRP, andFEV1instagesII,III,IVofCOPD.

Conclusions:Thecirculatinglevelsoftheinflammatorymarkershs-CRPandTNF-alphaaresignificantlyelevatedinpatientswithstableCOPDandthesebiomarkerscouldbeusedaspredictorfactorsforseverityofinflammationinCOPDpatients..Longitudinalstudiesevaluatingtheeffectsofsmokingcessationonbronchialandsystemicinflammationareneededtoallowbetterunderstandingoftheserelationshipsandtheirconsequences.

Keywords:COPD,FEV1,inflammatorymarkers;hs-CRPandTNF-alpha.

Introduction

Chronicobstructivepulmonarydisease(COPD)isasyndromecharacterizedanddefinedbyasinglephysiologicalparameter:limitationofexpiratoryair-flowwhich,mostoften,isslowlyprogressiveovertheyearsAccordingtothewidelyaccepteddefinitionfromGlobalInitiativeonObstructiveLungDisease(GOLD)COPDis"adiseasestatecharacterizedbyairflowlimitationthatisnotfullyreversible.Theairflowlimitationisusuallyprogressiveandassociatedwithanabnormalinflammatoryresponseofthelungstonoxiousparticlesandgases"[AgarwalRetal2013].COPDasaconditioncharacterizedbyanabnormalinflammatoryresponsebeyondthelungswithevidenceoflow-gradesystemicinflammationwhichcausessystemicmanifestationssuchasweightloss,skeletalmuscledysfunction,anincreasedriskofcardiovasculardisease,osteoporosisanddepression,amongothers[GarrodRetal2007andSamyNetal2010].SeveralstudieshaveshownsystemicinflammationinCOPDpatientswithincreasedneutrophil,macrophage,andT-lymphocytenumbersandhighconcentrationsofinflammatorymediators in peripheralblood(C-reactiveprotein(CRP)andTNF-α)[Pinto-PlataVMetal2006andQuintJKandWedzichaJA2007].Airwayinflammatorymarkersarehigherin more severedisease andincreaseduringCOPDexacerbations.However,noexhaledbiomarkerhasbeenwidelyusedinclinicaltrialsinCOPD(Smithetal,2005).Kostikasetal.,2008statedthatsystemicinflammationispresentinstableCOPDandtheintensityoftheinflammatoryprocessrelatestotheseverityoftheunderlyingdisease.IntheinterestofimprovingthediagnosisofCOPD,severaltypesofbiomarkerhavebeenmeasuredthatarerelatedtodiseasepathophysiologyandtheinflammatoryanddestructiveprocessinthelung.However,thereislittleinformationaboutbiomarkersreproducibilityandtherelationshiptodiseasedevelopment,severity,orprogression(Franciosietal., 2006).SeveralinflammatorymarkerssuchasC-reactiveprotein(CRP),fibrinogenandIL-6,areincreasedinpatientswithCOPDinbothstablediseaseandexacerbations,withCRPbeingthemoststudiedbiomarker(Dahletal.,2007).OneoftheinflammatorymarkerswhichisincreasinglyevaluatedinCOPDpatientsisCRP.CRPisanacutephaseproteinsynthesizedpredominantlybythehepatocytesinresponsetotissuedamageorinflammation.IthasbeenacceptedthatlevelsofCRPrelatetothepresenceofairflowobstruction[AgarwalRetal2013].Elevated CRP has beenknownto beusedas asurrogatemarkerofsystemicinflammationindiverseconditions.TNF-α(tumornecrosisfactor-α)isaglycoprotein,astronginflammatorycytokinewhichhasakeyroleininflammationprimarilyproducedbyactivatedmacrophages.TNF-αisthoughttoplayacriticalroleinthepathogenesisofCOPDbypromotingandmaintainingtheexpressionandreleaseofvariousproinflammatorymediatorswhichleadtotissuedamageandremodelling.[MarevicS.etal2008].VerylittleisknownaboutthemechanismofincreasedTNF-αconcentrationintheplasmaofCOPDpatientsanditsrelationshipwithdiseaseseverityandactivesmokinghasnotbeenestablished[FranciosiLGetal2006andMukhopadhyaySetal2006].Tobaccosmokingisthemainriskfactorofchronicobstructive pulmonary disease (COPD) but not all smokers develop the disease. An

abnormalpulmonaryandsystemicinflammatoryresponsetosmokingisthoughttoplayamajorpathogenicroleinCOPD,butthishasneverbeentesteddirectly.Yet,onlyaproportionofsmokers,socalled‘‘susceptiblesmokers’’,developthedisease[VestboJ,etal.2013andRosaFaneretal2014].Thegeneticandepigeneticbackgroundofeachsmokerislikelytoregulatethetypeandintensityofhis/herinflammatoryresponsetosmoking[DeMeoDLetal2005].In‘‘susceptiblesmokers’’,thisresponseisthoughttobe‘‘enhanced’’,bothinthelungsandinthesystemiccirculation,andisbelievedtodrivediseaseprogression[AgustiA,etal2012].However,despitethewideacceptanceofthisnotion,nopreviousstudyhasactuallystudiedthe‘‘response’’to smoking (i.e., thespecificinflammatorychangesthatoccurbeforeandaftersmoking)insusceptiblesmokers(i.e.,patientswithCOPD)andresistantsmokers(i.e.,smokerswithnormalspirometry)[RosaFaneretal2014].Long-termsmokingcausesairwayinflammationcharacterizedbyneutrophil,macrophage,andactivatedTlymphocyteinfiltrationandbyincreasedcytokineconcentrationssuchastumournecrosisfactor-alpha(TNF-α),interleukins(IL)-6andIL-8[BattagliaSetal2007andGambleEetal2007].Althoughnearlyallsmokersshowsomeevidenceoflungandsystemiccellularand/orhumoralinflammation,onlyafewwillsufferanamplifiedresponseanddevelopCOPD.TheaimofthisstudywastoidentifywhethertheinflammatoryprocessinstableCOPDpatientsispowerfulenoughtoproducesignificantchangesintheselectedinflammatorymarkers(TNF-αandhsCRP).TheroleandlevelsofTNF-αandhsCRPintheperipheralcirculationofCOPDpatientsandtherelationshipbetweenthesebiomarkerswithprognosticfactorsinCOPDwillbeinvestigatedandaswehypothesizedthatsmokingexposurewillinduceadifferentinflammatorysignature,theimpactofsmokingoninflammatorybiomarkersinpatientswithchronicobstructivepulmonarydiseasewillbeevaluated.WiththedevelopmentofmanynewdrugsthattargetinflammationinCOPD,thereisapressingneedtoidentifyreliablebiomarkersthatmayindicatewhetheranantiinflammatorytherapyislikelytohaveclinicalbenefit.AmajorproblemisthelackofanygoldstandardantiinflammatorytherapythatiseffectiveinCOPD,asayardsticktocomparepotentialtherapies.

5.95yearsrespectively),recruitedfromChestDepartment,AssiutUniversityHospitals.AllpatientsmettheGlobalInitiativeforObstructiveLungDisease(GOLD)[4]recommendedspirometriccriteriaforCOPD(ratioofpost-bronchodilator forcedexpiratoryvolumeinonesecond[FEV1]toforcedvitalcapacity[FVC]lessthan70%)[17].TheCOPDpatientsweresubdividedaccordingtotheirtreatmentinto: thirteenpatientsuseonlyshortactingbronchodilatorasneeded,theremainingfortyfivepatientswereusinglong-termbronchodilatorsfromthose45patients,twentyseven(13COPDcurrentsmokersand14non-smokers)patientswereregularlyusinginhaledcorticosteroid(800mcgof budesonide), sixteenpatientsof them hadbeenonstable oxygenflow

therapy for the last six months. No patients were medicated with theophylline orleukotrienemodifiers.

Patientsonoralsteroiduseorexacerbationsinthelastthreemonthsbeforeenrolmentinthestudy,ordiagnosedwithotherrespiratorydiseaselikeasthma,bronchiectasis,bronchiolitis ortuberculosis,orpresenceofotherchronicdiseases like diabetes, renalfailure,cancerwereexcluded.Inaddition,age,sexandBMImatchedapparentlyhealthysubjectswithnoevidenceofCOPDorusingregularmedicationsforotherdiseasesfrommedicalstuffandfamilieswhounderwenthealthexaminationatAssuituniversityhospitalswereenrolledinthestudyascontrolgroup..thecontrolsweresubdividedintofifteencurrentsmokercontrols(atleast10pack-years)andfifteennever-smokercontrols.Allparticipantsweresubjectedtothoroughhistorytaking,fullclinicalexamination,anthropometricmeasurementswithchestX-rayandspirometry.Forcedexpiratoryvolumeinthefirstsecond(FEV1)andforcedvitalcapacity(FVC)wereobtainedfromtheflow-volumecurveusingaspirometer[Conventionalspirometrywasa10-LclosedcircuitSpirographs(Zan300,SensorMedicsMGAUSB,Germany)]tostudypatientsandcontrolgroups.Staticlungvolumesweremeasuredbyclosed-circuitheliumdilutionmethod.ThereferencevaluesusedwerethoseoftheAmericanThoracicSocietystandards)beforeand20minutesafterβ-agonist(fenoterol400mcg)inhalation.Thehighestvalueofatleastthreemeasurementswasselectedandexpressedasapercentageofreferencevalues[18].Venousbloodsampleswerecollected fromparticipantsunderstandardizedconditions.samples werecentrifuged(3,000 g for 10min.)andserumsamplesweredividedandstoredinaliquotsat –20ºCuntilanalysis.Peripheralhemogram,liverfunctiontests,kidneyfunctiontests,HighsensitivityC-reactiveprotein(hs-CRP)measurementwasdonebyAccu-BindELISAMicrowells(MonobindInc.,USAcode3125-300AlotEIA-31KIC2).AssayofserumlevelsofTNF-αwasperformedusingAviBionHumanTNF-αELISAKit(Orgenium,Finland,Rev02.10).Smokinghistorywascalculatedinpack-yearsastheproductoftobaccouse(icurrent smokernyears)andtheaveragenumber ofcigarettessmokedperday/20(yearsxcig.perday/20).ThestudywasapprovedbytheInstitutionalEthicsCommitteeofFacultyofMedicine,AssiutUniversityandwritteninformedconsentwasobtainedfromeachparticipant.

Statisticalanalysis:DatawereanalyzedwithStatisticalpackageforsocialsciences(SPSS)version17Chicago.USA.Datawereexpressedasmeans±SDormeans±SEandfrequenciesaccordingiftheyarequantitativeorqualitativerespectively.Non-parametrictestswereusedinthecurrentstudy.

1.Mann-WhitneyTest(equivalenttoindependentstudentttestinparametrictests)wasusedforcomparisonofquantitativevariablesamongtwoindependentgroups

2.WilcoxonSignedRanksTest(equivalenttopairedttestinparametrictests)wasusedforcomparisonofquantitativevariablesamongtwodependentgroups.

3.Chi-SquareTestwasusedforcomparisonofdistributionofqualitativevariablesamongdifferentgroups.

4.ComparisonsbetweengroupswereachievedbyKruskal-WallisTest(equivalenttoonewayANOVAtest).

5.CorrelationbetweencontinuousvariableswasperformedusingSpearman'scorrelationcoefficient.

Pvalue<0.05isconsideredstatisticallysignificant.Multipleregressionanalysiswasusedtodete-rmineindependentfactorsaffectingdependentvariables.

Results:Demographics,baselinelungfunction,andbodymassindexofCOPDandcontrolsaccordingtotheirsmokingstatussmokerswerepresentedinTable1whereitshowedCOPDpatientstendedtohaveanearlysameagetocontrolsregardlesstheirsmokingstatuswithsimilarsmokinghistory(pack-years)incurrentsmokerCOPDpatientsandcontrols.Asexpected,COPDpatientshadsignificantsevereairflowlimitationanddecreasedSpO2withhighlysignificantdeclineinFEV1comparedtocontrols.

Table(1):Demographicdata,PulmonaryfunctionsandbloodgasesamongCOPD

patientscomparedtocontrolsaccordingtotheirsmokingstatus.

Variables / ControlNon-smoker
(N=15) / Control-smoker
(N=15) / COPD
Non-smoker(N=26) / COPD
Smoker(N=32) / P-valueSignificance
FVC%pred / 94.6±8.47 / 91.8±7.5 / 73.2±3.6 / 71.5±6.3 / 0.009*
RV%pred / 92.3±4.2 / 88.3±4.2 / 129.33±8.4 / 133.5±12.4 / 0.004*
TLC%pred / 91.7±6.8 / 85.2±3.4 / 127.8±5.1 / 131.1±10.6 / 0.003*
FEV1%pred / 83.2±3.1 / 81.5±2.0 / 54.23±23.4 / 51.6±22.2 / 0.007*
FEV/FVC% / 79.56±9.1 / 76.4±6.42 / 63.9±5.46 / 59.2±10.7 / 0.011*
PaO2mmHg / 88.3±6.3 / 82.6±5.4 / 67.3±4.9 / 61.5±8.7 / 0.041*
PaCO2mmHg / 39.1±3.6 / 40.5±3.8 / 58.5±8.4 / 61.3±11.9 / 0.037*
Age(years) / 49.37±14.37 / 52.56±8 / 53.1±14.25 / 53.9±5.95 / 0.746
BMI(kg/m2 / 24.1±5.0 / 24.2±4.51 / 22.8±4.9 / 21.4±5.7 / 0.149

Kruskal-WallisTest*Statisticalsignificantdifference

Tables2(a,b),Tables3(a,b)andFigures(1and2):showedhighlysignificantelevatedlevelsofcirculatingTNF-αandhs-CRPinCOPDpatientsincomparisontothecontrolgroupregardlesstheirsmokingstatuswithsignificanthighestlevelsincurrentsmokerspatientsandcontrols.

Table(2a):TNF-alpha(pg/ml)

Parameters / Patients(n=58) / Controls(n=30) / P-value
Mean±SE / 35.88±5.97 / 8.79±0.57 / 0.000*
Range / 3.7–265.5 / 3.7–14

Mann-WhitneyTest*Statisticalsignificantdifference(P0.05)

Table(2b):TNF-alpha(pg/ml)

Parameters / Smoker / Non-smoker / P-value
Mean±SE / Mean±SE
Patients / 53.74±9.52 / 12.73±1.20 / 0.000*
Controls / 10.70±0.60 / 6.88±0.69 / 0.001*

Mann-WhitneyTest*Statisticalsignificantdifference(P0.05)

Figure(1)levelsofTNF-alphainCOPDpatientsandcontrols(pg/ml)accordingtotheirsmokingstatus.

Table(3a):hs-CRP(ug/ml)

Parameters / Patients(n=58) / Controls(n=30) / P-value
Mean±SE / 3.74±0.20 / 1.30±0.14 / 0.000*
Range / 1.2–8.0 / 0.2–2.5

Mann-WhitneyTest*Statisticalsignificantdifference(P0.05)

Table(3b):hs-CRP(ug/ml)

Parameters / Smokers / Non-smokers / P-value
Mean±SE / Mean±SE
Patients / 3.87±0.29 / 3.58±0.27 / 0.764
Controls / 1.96±0.14 / 0.64±0.06 / 0.000*

Mann-WhitneyTest*Statisticalsignificantdifference(P0.05)

Figure(2):Levelsofhs-CRPinCOPDpatientsandcontrols(ug/ml)accordingtotheirsmokingstatus.

Table(4):ThemeanlevelsofcirculatingTNF-alphaaccordingtoincreasedthedegreeofCOPD,theseverityofdyspneaandthesteroidinhalationtherapyinCOPDpatients.

Parameters / Mean±SE / P-value
DegreeofCOPD: / 0.000*
Mild / 10.16±0.92
Moderate / 21.86±0.92
Severe / 46.63±4.39
Verysevere / 149.90±33.04
Dyspnea: / 0.000*
GradeII / 17.06±1.49
GradeIII / 38.76±5.09
GradeIV / 137.26±30.66
Steroidinhalationtherapy: / 0.077
Yes / 29.61±12.33
No / 46.16±9.29

Kruskal-WallisTest*Statisticalsignificantdifference(P0.05)

Table4:showedthesignificantincreasedlevelsoftheTNF-αwiththeincreasedseverityofCOPDandincreasedgradeofdyspnea.Moreover,nonsignificantdifferencesforthemeasuredTNF-αwerefoundbetweenthepatientsaccordingtotheuseoftheinhaledcorticosteroidstherapy.

Table(5):RegressionmodelswithrobuststandarderrorsforTNF-αandCRPinCOPDPatients.

Parameters / TNF-alpha(pg/ml) / HsCRP(ug/ml)
r-value / P-value / r-value / P-value
Smokingindex / 0.850 / 0.000* / 0.614 / 0.000*
DegreeofCOPD / 0.936 / 0.000* / 0.198 / 0.122
Gradesofdyspnea / 0.708 / 0.000* / 0.179 / 0.163

Spearman correlation*Statisticalsignificantdifference(P0.05)

TheregressionresultsshowedahighsignificantpositiveassociationamongthemeasuredTNF-αwithsmokingindex,increaseddegreeofdyspneaanddegreeofCOPD,whileanonsignificantpositiveassociationamongthemeasuredhs-CRPwithincreaseddegreeofdyspneaanddegreeofCOPD with a high significantpositiveassociationamongthemeasuredhs-CRPwithsmokingindexwerefoundinTable5

Table(6):ThecorrelationbetweenthemeasuredTNF-alphaandhs-CRPversussmokingindexinCOPDpatientsandcontrolgroups.

Variables / SmokingIndex
r-value / P-value
Patientsmokers / TNF-alpha / 0.788 / 0.000*
HsCRP / 0.614 / 0.000*
Controlsmokers / TNF-alpha / 0.889 / 0.000*
HsCRP / 0.858 / 0.000*

Spearman correlation*Statisticalsignificantdifference(P0.05)

Table6showedhighsignificantpositiveassociationsamongthemeasuredTNF-αandhs-CRPwithsmokingindexinCOPDpatientsandcurrentsmokercontrols.

Table(7): ThecorrelationbetweenthemeasuredTNF-alphaandhs-CRPversusFEV1inCOPDpatientsandcontrolsregardingtotheirsmokingstatus.

Variables / FEV1
r-value / P-value
COPDsmokers / TNF-alpha / -0.843 / 0.000*
hsCRP / -0.501 / 0.002*
COPDnon-smokers / TNF-alpha / -0.464 / 0.015*
hsCRP / -0.067 / 0.741
Controlsmokers / TNF-alpha / -0.881 / 0.000*
hsCRP / -0.848 / 0.000*
Control non-smokers / TNF-alpha / -0.879 / 0.000*
hsCRP / -0.857 / 0.000*

Spearman correlation*Statisticalsignificantdifference(P0.05)

Table7showedhighsignificantpositiveassociationsamongthemeasuredTNF-αlevelswithFEV1inCOPDpatientsandcontrolsregardlesstotheirsmokingstatus.However,RegardingtosmokingstatusinCOPDpatients,therewasahighsignificantpositiveassociationamongcurrentsmokerswithnosignificantdifferenceamongthenonsmokersbetweenFEV1withthemeasuredhs-CRP.Incontrolsregardlesstotheirsmokingstatushighsignificantpositiveassociations among the measured hs-CRPwithFEV1weredetected.

Discussion:

Chronicobstructivepulmonarydisease(COPD)isaslowlyprogressivediseaseinducedprimarilybysmokingtobacco.OtheraetiologicalfactorsofCOPD,likeairpollution,poornutrition,professionalriskatworkandgeneticfactorsmaybeprobablyparticipated.ThepathophysiologyofCOPDisnotcompletelyunderstood.(SamyNetal,2010).AccordingtoGlobalInitiativeonObstructiveLungDisease(GOLD)definition,TheairflowlimitationinCOPDisnotfullyreversibleandisusuallyprogressiveandassociatedwithanabnormalinflammatoryresponseofthelungstonoxiousparticlesandgases"(AgarwalRetal2013).IntheinterestofimprovingthediagnosisofCOPD,severaltypesofbiomarkerhavebeenmeasuredthatarerelatedtodiseasepathophysiologyandtheinflammatoryanddestructiveprocessinthelung.TheaimofthisstudywastotoidentifywhethertheinflammatoryprocessinstableCOPDpatientsispowerfulenoughtoproducesignificantchangesintheselectedinflammatorymarkers(TNF-αandhsCRP)andtoevaluatetherelationshipbetweensystemicinflammationandsmokingstatusinCOPDpatients.

CRPisanacutephaseproteinsynthesizedpredominantlybythehepatocytesinresponsetotissuedamageorinflammation.IthasbeenacceptedthatlevelsofCRPrelatetothepresenceofairflowobstruction.ElevatedCRPhasbeenknowntobeusedasasurrogatemarkerofsystemicinflammationindiverseconditions(AgarwalRetal2013).Interestingly,Ourstudyinfact,showedthattheserumlevelofhsCRPwashighlysignificantlyelevatedinCOPDpatientscomparedwithcontrolsregardlesstotheirsmokingstatus.ThisresultconfirmsthatthehigherlevelsofcirculatinghsCRPmaybethusregardedasavalidbiomarkeroflow-gradesystemicinflammationintheCOPD. OurresultswereinagreementwithNillawarANetal2013whoreportedthatCOPDpatientshadhigherlevelsofhs-CRPduetothefactoftheearlierresponseofhs-CRP,asitissecretedasacutephasereactantsbytheliverinresponsetoIL-6.Moreover,GanWQetal2004,Pinto-PlataVMetal2006andQuintJKandWedzichaJA2007statedthatsystemicinflammationinCOPDpatientsisassociatedwithincreasedneutrophil,macrophage,andT-lymphocytenumbersandhighconcentrationsofinflammatorymediatorsinperipheralblood(C-reactiveprotein(CRP)andTNF-α).Theirstudyshowed asignificantlyhigherlevels ofcirculatinghsCRPinCOPDpatientsthaninsmokingandnonsmokingcontrols.However,thelevelofhsCRPinthesmokingCOPDpopulationremainednonsignificantlyhigherthaninthenon-smokingpatients.Wesuggestthat,althoughcigarettesmokinghasaroleinpromotinginflammatoryprocessinCOPDpatients,itisnottheleadingcauseofincreasedinflammatorymarkers.itshouldbenoticedthatonlysomecasesdevelopinflammatoryreactionfollowingcigarettesmoking,andthiscanbeduetogeneticdifferences.Incontrasttotheabovefindings,AgarwalRetal2013,showedthelevelofhsCRPinthesmokingCOPDpopulation remainedsignificantlyhigherthaninthenon-smokingpatients.

TNF-α,apowerfulpro-inflammatorycytokineprimarilyproducedbyactivatedmacrophages,isthoughttoacriticalroleinthepathogenesisofCOPDbypromotingandmaintainingtheexpressionandreleaseofvarious proinflammatory mediatorswhichleadtotissuedamageandremodelling(HigashimotoYetal2008).VerylittleisknownaboutthemechanismofincreasedTNF-αconcentrationintheplasmaofCOPDpatientsanditsrelationshipwithdiseaseseverityandactivesmokinghasnotbeenestablished(FranciosiLGetal2006andMukhopadhyaySetal2006).However,MarevicS.etal2008reportedthatttobaccosmokinginducesthereleaseofTNF-αandhasanimportantroleinthepathophysiologyofCOPD.TNF-αinfluencesthegeneexpression ofinterleukin-8and endothelin-1(ET-1)inhumanendothelialcells,andparticipatesinbronchoconstrictionthroughsecondaryreleaseofET-1.

Ourstudyshowedthatthelevelofseruminflammatorymarker;TNF-αwashighlysignificantlyelevatedinCOPDpatientsregardlesstotheirsmokingstatusindicatingapersistentsystemicinflammationinsubjectswithCOPDwith significant highestlevelsofTNF-αincurrentCOPDsmokers.TheseresultssuggestthatsmokingmaybeassociatedwithhigherTNF-αmediatedsystemicinflammationin COPD patients.ThesefindingswereinagreementwithHigashimotoYetal.2008whoreportedtheincreasedlevelsofserumTNF-α,IL-6andtissueinhibitorsofmetalloproteinase-1inasthmaandCOPDpatientswithhighestlevelsincurrentsmokerCOPDpatientsandcontrols.Moreover,ourresultswereinconsistentwithPinto-PlataVMetal2006,QuintJKandWedzichaJA2007,MarevicS.etal2008andSamyNetal2010andwhoreportedsignificantincreaseinseruminflammatorymarkersTNF-α,hsCRPandinterlukin-6indifferentstagesofCOPDpatientsandthisincrementwassignificantlyhigherthanthoseofthecontrols.WemaythusspeculatetheassociationofsmokingwithhighersystemicTNF-αlevelsobservedinthisstudymaypartiallyexplainsomeofbenefitsassociatedwithsmokingcessation.Incontrasttoourfindings,VernooyJHetal2002reportedincrease intheconcentrationofsolubleTNF-αreceptors,whiletheconcentrationofTNF-αwaswithinthereferencerangeforhealthyindividuals.possibleexplanationforunchangedconcentrationofTNF-αisitslocalandshorttermeffect,itsdegradationanditshalflifeofapproximately6-7minutes,aswellasitsbindingtoreceptorsandrenalclearance.

FEV1iseasilymeasuredandisaspirometricpredictorofmortalityinpatientswithCOPD.FactorsthataffectthedeclineofFEV1are,therefore,ofprognosticimportanceinCOPD.(SamyNetal,2010).OurstudyrevealedtheincreasedTNF-αlevelsinCOPDpatientswithincreasedairwayobstructionseverity ,increasedanddegreeofdyspnoeawithasignificantnegativecorrelation between TNF- α withFEV1.TheseresultsclearlyconfirmthefactofthattheintensityoftheinflammatoryprocessinCOPDcouldberelatedtotheseverityoftheunderlyingdisease.OurfindingswereinagreementwithWagnerPD,2008whostatedthehigherserumTNF-αlevelsinCOPDpatientswasassociatedwithFEV130%incomparisonwith

mild-moderateCOPDpatients.Furthermore,Kostikasetal.,2008andSamyNetal,2010whoreportedTNF-αlevelsincreasedaccordingtothestageoftheCOPD.Interestingly,nonsignificanthigherhs-CRP concentrations have been associatedwithincreasedseverityofairwayobstructionandincreaseddegreeofdyspnoeawereobservedinourstudy.ThesefindingswereinagreementwithGodoyIetal2003andMukhopadhyaySetal2006whoreportedthathigherhs-CRPconcentrationhasbeenassociatedwithairwayobstructionseverity,increasedrestingenergyexpenditure,andimpairedexercisecapacityandqualityoflifeinCOPDpatientsAgarwalRetal2013concluded thatthecirculatinglevelsof the inflammatorymarkerhs-CRParesignificantlyelevatedinpatientswithCOPD,supportingtheviewthatCOPDisinpartaninflammatorydisorder.Hs-CRPlevelsinstable COPDpatientsarebestcorrelatedwithFEV1and6-minutewalkdistance(6MWD).Clearly

,intheourstudyhsCRPlevelswereinverselycorrelatedwithFEV1instableCOPDpatients.Thestrongestnegativeassociationbetween FEV1 and CRP andhigherCRPlevelsovertimewereassociatedwithafasterFEV1decline.theseresultswereinconsistentwithManSFetal2006andAgarwalRetal2013whostatedCRPlevelsassociatedwithaccelerateddeclineinFEV1andmortalityinpatientswithmildtomoderateCOPD,indicatingthatCRPmeasurements might enableidentificationofpatientsathighriskofdiseaseprogressionandmortality.

Tobaccosmokingisthemainriskfactorofchronicobstructivepulmonarydisease(COPD)butnotallsmokersdevelopthedisease.AnabnormalpulmonaryandsystemicinflammatoryresponsetosmokingisthoughttoplayamajorpathogenicroleinCOPD,butthishasneverbeentesteddirectly.Long-termsmokingcausesairwayinflammationcharacterizedbyneutrophil,macrophage,andactivatedTlymphocyteinfiltrationandbyincreasedcytokineconcentrationssuchastumournecrosisfactor-alpha(TNF-α),interleukins(IL)-6andIL-8(BattagliaSetal2007andGambleEetal2007).Althoughnearlyallsmokersshowsomeevidenceoflungandsystemiccellularand/orhumeralinflammation,onlyafewwillsufferanamplifiedresponseanddevelopCOPD.

Interestingly,highlysignificanthigherconcentrationsofTNF-αandhs-CRPinCOPDpatientshavebeenassociatedwithincreasedseverityofsmokingstatuswereobservedinourstudy.ThecorrelationsofTNF-αandhsCRPconcentrationwithsmokinghabit(pack/years)supportstheassumptionthattobaccosmokehasaninfluenceonsystemicinflammation.Hs-CRPandTNF-αindicatethatCOPDisasystemicinflammatorydisease,andtheycouldserveaspredictorsforexacerbations.However,itisunclearwhetherestablishedinflammationinsmokersreturnstonormalaftersmokingcessation.SmokingcessationistheonlymanagementmeasureassociatedwithreducedFEV1declineinCOPDpatients;however,todate,noreductioninairwayinflammationhasbeendemonstratedwhenex-smokersarecomparedtocurrentsmokersbyGambleEetal2007whodidnotfindanydifferencesincellcountsorinflammatorymarkers(CD8+,CD4+,CD68+,orTNF-α)

inbronchialbiopsiesofCOPDcurrentsmokersandCOPDex-smokers.Nevertheless,BarnesPJetal2006reportedthatneutrophilandlymphocytenumbers,andIL-8concentrationinthesputumofCOPDpatientswereincreasedoneyearaftersmokingcessation.

Unfortunately,inspiteofourCOPDpatientswereclassifiedaccordingtoGOLDclassesanduseofLTOTorinhaledcorticosteroids,wecouldnotrevealedtheinfluenceofinhaledcorticosteroidsonsystemicinflammationinCOPD patientsowingtosmallersizedsampleused,poorpowertodetectpotentialdifferencesandlongertimeobservationwasneededtoconfirmtheseresults.

Conclusions:

Thecirculatinglevelsoftheinflammatorymarkershs- CRP and TNF-alpha aresignificantlyelevatedinpatientswithCOPD,supportingtheviewthatCOPDisinpartaninflammatorydisorder.ThesecirculatingbiomarkersinstableCOPDpatientsarebestcorrelatedwithFEV1suggestingthatthesecirculatingbiomarkersaregoodcandidatesaspredictorsforrapiddeclineofFEV1inCOPD,althoughalargersizestudywithlongertermobservationisneededtoconfirmthesefindings.Itislikelythatinthefuturebiomarkerswillbecomeincreasinglyrequiredtoaidindeterminingthosepatientswhowillbenefitfromagivendrugtherapytoimproveriskand/orcostbenefit,especiallysinceitisbecomingmorewidelyconsideredinCOPD.SmokingisassociatedwithhigherlevelsofTNF-alphaandhs-CRPmediatedsystemicinflammationinpatientswithorwithoutCOPD,thereforeactivesmokingmaybeassociatedwithevenhigherdegreeofthesystemicinflammation.Longitudinalstudiesevaluatingtheeffectsofsmokingcessationonbronchialandsystemicinflammationareneededtoallowbetterunderstandingoftheserelationshipsandtheirconsequences.

Abbreviations:

BMI:BodyMassIndex;COPD:ChronicObstructivePulmonaryDisease;Hs-CRP:HighlysensitiveC-reactiveprotein;FEV1:ForcedExpiratoryVolumeinonesecond;FEV1/FVC:ForcedExpiratoryVolumeinonesecond/ForcedVitalCapacity;BMI:BodyMassIndex;FVC:ForcedVitalCapacity;GOLD:GlobalInitiativeforChronicObstructiveLungDisease;IL:Interleukin;SpO2:PeripheralOxygenSaturation;TNF-α:Tumor NecrosisFactoralpha.

Competinginterests

Theauthorsdeclarethattheyhavenocompetinginterests.

References

1.QuintJK,WedzichaJA:Theneutrophilinchronicobstructivepulmonarydisease.JAllergyClinImmunol2007,119:1065-1071.

2.GambleE,GrootendorstDC,HattotuwaK,O'ShaughnessyT,RamFSF,QiuY,etal.:AirwaymucosalinflammationinCOPDissimilarinsmokersandex-smokers:apooledanalysis.EurRespirJ2007,30:467-471.

3.BaraldoS,TuratoG,BadinC,BazzanE,BegheB,ZuinR,etal.:NeutrophilicinfiltrationwithintheairwaysmoothmuscleinpatientswithCOPD.Thorax2004,59:308-312.

4.BattagliaS,MauadT,vanSchadewijkAM,VignolaAM,RabeKF,BelliaV,etal.:Differentialdistributionofinflammatorycellsinlargeandsmallairwaysinsmokers.JClinPathol2007,60:907-911.

5.FletcherC,PetoR:Natural-HistoryofChronicAir-FlowObstruction.BrMedJ1977,1:1645-1648.

6.LøkkeA,LangeP,ScharlingH,FabriciusP,VestboJ:DevelopingCOPD:a25yearfollowupstudyofthegeneralpopulation.Thorax2006,61:935-939.

7.AgustíAG,NogueraA,SauledaJ,SalaE,PonsJXB:Systemiceffectsofchronicobstructivepulmonarydisease.EurRespirJ2003,21:347-360.

8.GanWQ,ManSFP,SenthilselvanA,SinDD:Associationbetweenchronicobstructivepulmonarydiseaseandsystemicinflammation:asystematicreviewandameta-analysis.Thorax2004,59:574-580.

9.FranciosiLG,PageCP,CelliBR,CazzolaM,WalkerMJ,DanhotM,etal.:Markersofdiseaseseverityinchronicobstructivepulmonarydisease.PulmPharmacolTher2006,19:189-199.

10.TakabatakeN,NakamuraH,AbeS,HinoT,SaitoH,YukiH,etal.:Circulatingleptininpatientswithchronicobstructivepulmonarydisease.AmJRespirCritCareMed1999,159:1215-1219.

11.Pinto-PlataVM,MullerovaH,TosoJF,Feudjo-TepieM,SorianoJB,VesseyRS,etal.:C-reactiveproteininpatientswithCOPD,controlsmokersandnon-smokers.Thorax2006,61:23-28.

12.KonyS,ZureikM,DrissF,NeukirchC,LeynaertB,NeukirchF.AssociationofbronchialhyperresponsivenessandlungfunctionwithC-reactiveprotein(CRP):apopulationbasedstudy.Thorax2004;59:892–896.

13.HigashimotoY,YamagataY,TayaS,IwataT,OkadaM,IshiguchiT,etal.:Systemicinflammationinchronicobstructivepulmonarydiseaseandasthma:Similaritiesanddifferences.Respirology2008,13:128-133.

14.ChurgA,DaiJ,TaiH,XieCS,WrightJL:Tumornecrosisfactor-alphaiscentraltoacutecigarettesmoke-inducedinflammationandconnectivetissuebreakdown.AmJRespirCritCareMed2002,166:849-854.

15.MukhopadhyayS,HoidalJR,MukherjeeTK:RoleofTNF-alphainpulmonarypathophysiology.RespirRes2006,7:125.

16.BroekhuizenR,WoutersEF,CreutzbergECAS:RaisedCRPlevelsmarkmetabolicandfunctionalimpairmentinadvancedCOPD.Thorax2006,61:17-22.

17.VernooyJH,KucukaycanM,JacobsJA,ChavannesNH,BuurmanWA,DentenerMA,etal.:Localandsystemicinflammationinpatientswithchronicobstructivepulmonarydisease.AmJRespirCritCareMed2002,166:1218-1224.

18.GOLD.GlobalStrategyfortheDiagnosis,Management,andPreventionofChronicObstructivePulmonaryDisease:GOLDExecutiveSummaryupdated2013[ webcite

19.KnudsonRJ,LebowitzMD,HolbergCJ,BurrowsB:Changesinthenormalmaximalexpiratoryflow-volumecurvewithgrowthandaging.AmRevRespirDis1983,127:725-734.

20.KyleUG,BosaeusI,DeLorenzoAD,DeurenbergP,EliaM,GómezJM,etal.:CompositionoftheESPENWorkingGroup.Bioelectricalimpedanceanalysis--partI:reviewofprinciplesandmethods.ClinNutr2004,23:1226-1243.

21.KyleUG,PichardC,RochatT,SlosmanDO,FittingJW,ThiebaudD:Newbioelectricalimpedanceformulaforpatientswithrespiratoryinsufficiency:comparisontodual-energyX-rayabsorptiometry.EurRespirJ1998,12:960-966.

22.KyleUG,GentonL,KarsegardL,SlosmanDO,PichardC:Singlepredictionequationforbioelectricalimpedanceanalysisinadultsaged20-94years.Nutrition2001,17:248-253.

23.ScholsA,SoetersPB,DingemansAMC,MostertR,FrantzenPJ,WoutersEFM:PrevalenceandcharacteristicsofnutritionaldepletioninpatientswithstableCOPDeligibleforpulmonaryrehabilitation.AmRevRespirDis1993,147:1151-1156.

24.SigmaStat:ManualofSigmaStat3.5forWindows.2006,Chapter8:308-326.

25.CroftM:TheroleofTNFsuperfamilymembersinT-cellfunctionanddisease.NatRevImmunol2009,9:271-85.

26.BalkwillF:Tumournecrosisfactorandcancer.NatRevCancer2009,9:361-71.

27.O'ShaughnessyTC,AnsariTW,BarnesNC,JefferyPK:Inflammationinbronchialbiopsiesofsubjectswithchronicbronchitis:inverserelationshipofCD8+TlymphocyteswithFEV1.AmJRespirCritCareMed1997,155:852-7.

28.SaettaM,DiStefanoA,TuratoG,FacchiniFM,CorbinoL,MappCE,etal.:CD8+T-lymphocytesinperipheralairwaysofsmokerswithchronicobstructivepulmonarydisease.AmJRespirCritCareMed1998,157:822-826.

29.SaettaM,TuratoG,MaestrelliP,MappCE,FabbriLM:Cellularandstructuralbasesofchronicobstructivepulmonarydisease.AmJRespirCritCareMed2001,163:1304-1309.

30.KeatingsVM,CollinsPD,ScottDM,BarnesPJ:Differencesininterleukin-8andtumornecrosisfactor-alphaininducedsputumfrompatientswithchronicobstructivepulmonarydiseaseorasthma.AmJRespirCritCareMed1996,153:530-534.

31.TakanashiS,HasegawaY,KanehiraY,YamamotoK,FujimotoK,SatohK,etal.:Interleukin-10levelinsputumisreducedinbronchialasthma,COPDandinsmokers.EurRespirJ1999,14:309-314.

32.BarcelóB,PonsJ,FerrerJM,SauledaJ,FusterA,AgustíAG:PhenotypiccharacterisationofT-lymphocytesinCOPD:abnormalCD4+CD25+regulatoryT-lymphocyteresponsetotobaccosmoking.EurRespirJ2008,31:555-562.

33.BarnesPJ,ChowdhuryB,KharitonovSA,MagnussenH,PageCP,PostmaD,etal.:Pulmonarybiomarkersinchronicobstructivepulmonarydisease.AmJRespirCritCareMed2006,174:6-14

34.WillemseBWM,tenHackenNHT,RutgersB,Lesman-LeegteI,PostmaDS,TimensW:Effectof1-yearsmokingcessationonairwayinflammationinCOPDandasymptomaticsmokers.EurRespirJ2005,26:835-845.

35.TeoKK,OunpuuS,HawkenS,PandeyMR,ValentinV,HuntD,etal.:INTERHEARTStudyInvestigators.Tobaccouseandriskofmyocardialinfarctionin52countriesintheINTERHEARTstudy:acase-controlstudy.Lancet2006,368:647-658.

36.TønnesenP,CarrozziL,FagerströmKO,GratziouC,Jimenez-RuizC,NardiniS,etal.:Smokingcessationinpatientswithrespiratorydiseases:ahighpriority,integralcomponentoftherapy.EurRespirJ2007,29:390-417.

37.GodoyI,CampanaAO,GeraldoRR,PadovaniCR,PaivaSA:Cytokinesanddietaryenergyrestrictioninstablechronicobstructivepulmonarydiseasepatients.EurRespirJ2003,22:920-925.

38.AronsonD,RotermanI,YiglaM,KernerA,AvizoharO,SellaR,etal.:InverseassociationbetweenpulmonaryfunctionandC-reactiveproteininapparentlyhealthysubjects.AmJRespirCritCareMed2006,174:626-632.

39.DebigareR,MarquisK,CoteCH,TremblayRR,MichaudA,LeBlancP,etal.:Catabolic/anabolicbalanceandmusclewastinginpatientswithCOPD.Chest2003,124:83-89.

40.WagnerPD:PossiblemechanismsunderlyingthedevelopmentofcachexiainCOPD.

EurRespirJ2008,31:492-501.

41.vonHaehlingS,HopkinsonNS,PolkeyMI,NiethammerM,AnkerSD,Genth-ZotzS:ElevatedTNFαproductioninwholebloodinpatientswithseverCOPD:thepotentiallinktodiseaseseverity.WienKlinWochesnschr2009,121:303-308.

42.NillawarAN,JoshiKB,PatilSB,BardapurkarJS,BardapurkarSJJ.EvaluationofHS-CRPandLipidProfileinCOPD.ClinDiagnRes.2013;7(5):801-3

43.AgarwalR,ZaheerMS,AhmadZ,AkhtarJ.TherelationshipbetweenC-reactiveproteinandprognosticfactorsinchronicobstructivepulmonarydisease.LungIndia.2012Jan;29(1):24-9.

44.SamyN,AbdEl-MaksoudM.D,ElKhayyalAandImamA:Clinicalutilityofbiomarkersaspredictorsoflungfunctioninchronicobstructivelungdisease.NewYorkScienceJournal2010;6(3):25-32.

45-ManSandSinD:Effectsofcorticosteroidsonsystemicinflammationinobstructivepulmonarydisease.PROCAmThoracSOC2005:2:78-82