Evidence for an asociation betwen an enhanced reactivity of interleukin-6 levels and reduced glucocorticoid sensitivity in patients with fibromyalgia

Evidence

for

an

association

between

an

enhanced

reactivity

of

interleukin-6

levels

and

reduced

glucocorticoid

sensitivity

in

patients

with

fibromyalgia

Andrea

Geiss

*

,

Nicolas

Rohleder

,

Fernand

Anton

a

DepartmentofPsychobiology,UniversityofTrier,D-54290Trier,Germany

b_{UniversityofCologne,D-50931Ko¨ln,Germany}

c_{BrandeisUniversity,MS062,POBox549110,Waltham,MA02454,USA} d

LaboratoryofPsychobiologyandNeurophysiology,FLSHASE/INSIDE,UniversiteduLuxembourg, L-1511Luxembourg,Luxembourg

Received19November2010;receivedinrevisedform27July2011;accepted28July2011

KEYWORDS

Fibromyalgiasyndrome; Interleukin6;

Tumornecrosisfactor-a; Hypothalamus—pituitary— adrenalaxis; Cortisol; Glucocorticoid sensitivity; Fatigue; Pain; Chronicstress;

Pressurepainthresholds

Summary Painandfatiguehavebeenidentifiedascoresymptomsoffibromyalgiasyndrome (FMS). Sinceboth symptomsare alsocharacteristic ofhypocortisolemic disorders, reduced cortisollevelshavebeenthoughttopromoteanexacerbationoftheseFMScoresymptomsbyan enhanced reactivity of interleukin-6 (IL-6) levels. The aim of the current study was to investigate the pathophysiologicrelevance ofreduced cortisol levels for manifestation of FMS core symptoms. Twelve female FMS patientswith 15 female controls were compared regardingthefunctionofhypothalamus—pituitary—adrenal(HPA)axisandbehavioral, endo-crineandIL-6responsesaftermeasuringthepressurepainthresholds(PPTs)attenderpoints. FunctionofHPAaxiswasassessedbydeterminingthecortisolawakeningresponse,daytime profileofcortisolsecretion, lowdoseovernightdexamethasonesuppression test(DST)and glucocorticoidsensitivity(GC)ofinflammatorycytokineproduction.WhileendocrineandIL-6 responsesweredeterminedbycollectingbloodandsalivasamplesbehavioralresponseswere assessedbypainandfatiguerecordingsofparticipantsbeforeandafterPPTmeasurementusing visualanaloguescale(VAS).WhereasFMSpatientswerefoundnottodifferfromcontrolsin cortisolawakeningresponse,daytimeprofileofcortisolsecretionandcortisolsuppressionafter overnightDST,theydidexhibitareducedGCsensitivityofinflammatorycytokineproduction. PPTmeasurementdidinducethreetimeshighercortisolandfourtimeshigherIL-6levelsin FMS patients,but no changeintheir ACTHlevels. The enhanced IL-6 reactivityafter PPT

* Correspondingauthorat:UniversityofCologne,DepartmentofPsychology,Gronewaldstraße2,D-50931Ko¨ln, Germany. Tel.:+4902214704723;fax:+4902214705105.

E-mailaddress:geissa@uni-koeln.de(A.Geiss).

Availableonlineatwww.sciencedirect.com

jo u rn al ho m e p age : w w w. el s ev i er. co m / lo c at e /p s y n eu en

1.

Introduction

Fibromyalgiasyndrome(FMS)isachronicdisorder character-ized by core symptoms widespread musculoskeletal pain, fatigue and sleep disturbances for a duration of at least threemonths(Wolfe,1989).FMSisapproximatelyninetimes morecommonamongwomenthanmenandtheprevalence increaseswith age, affectingnearly 7%of womenover 60 years(Wolfeetal.,1995).

Onset of FMShasbeen found to bepreceded byeither priorlife traumaor high workload andworkplace bullying (Imbierowicz andEgle, 2003; Kivima¨ki etal., 2004). Both typesofchronicstressorshavebeenreportedtobe accom-paniedbyalterationsofthe hypothalamus—pituitary—adre-nal (HPA) axis as this neuroendocrine system is mainly involvedinthemediationofthestressresponse(forreview Tanriverdietal.,2007).Stress-inducedglucocorticoid(GC) secretioniscurrentlybelievedtoprotecttheorganismfrom harmful effects of overshooting defense mechanisms, for exampleby suppressing the synthesis of pro-inflammatory cytokines,suchasinterleukin-6(IL-6)inimmunecells(e.g. Sapolskyetal.,2000).Twodifferentpatternsofanaltered neuroendocrineHPAaxisfunctionhavebeencharacterized, termed hypocortisolism and hypercortisolemia. Prior life traumaandcumulativeadversityhavebeenassociatedwith both patterns of dysfunctional HPA axis function as this chronicstressorwasfoundtoincreasetheriskfordeveloping posttraumaticstressdisorder(PTSD)followingalatertrauma andmajor depressivedisorder(MDD)inlaterlife(Edwards etal.,2003;Keaneetal.,2006).Ahypocortisolemicpattern ismainlycharacterizedbyreducedcortisollevels,enhanced feedbacksensitivity andincreasedsensitivityofperipheral blood mononuclear cells (PBMCs) to glucocorticoids and hence enhanced glucocorticoid (GC) sensitivity (Yehuda etal.,1991,2004).Mainneuroendocrinefeaturesof hyper-cortisolemiaareenhancedcortisollevels,adiminished feed-back sensitivity and reduced sensitivity of PBMCs to glucocorticoids(GillespieandNemeroff, 2005).Whilepain andfatiguehavebeenidentifiedascoresymptomsof hypo-cortisolemicdisordershypercortisolemiahasbeenreported tobemanifestedmainlyinfatigueandcognitivedysfunction (Geissetal.,2005;Friesetal.,2005;Milleretal.,2009).Both neuroendocrinepatternshavebeenfoundtobeaccompanied byelevatedcytokinelevels.Whileareducedcortisol avail-abilityisthoughttoaccountforelevatedcytokinelevelsin hypocortisolismanattenuatedsensitivityofPBMCsto gluco-corticoidshasbeen relatedto increased cytokinelevelsin hypercortisolemia (Geiss et al., 2005; Fries et al., 2005; Milleretal.,2009;Rohlederetal.,2010).

Thereisevidenceforbothahypercortisolemicand hypo-cortisolemicstateinFMS.Inmoredetail,FMSpatientshave beenreportedtoexhibitanelevatedcortisolprofile(Catley et al., 2000), but also to display an attenuated cortisol responseduringthefirsthourafterawakening(Weissbecker

et al., 2006; Klingmann et al., 2008). Furthermore, FMS patientshavebeenidentifiedtoexhibitalower glucocorti-coidreceptor(GR)bindingoftheirPBMCstoglucocorticoids andhencereduced GCsensitivity(Lentjesetal.,1997).In contrast,neitherWingenfeldetal.(2008)norMacedoetal. (2008) did find any difference between FMS patients and controls in their GC sensitivity of circulating monocytes. Inconsistentfindingshavebeenreportedalsoforthestatus of the feedback sensitivity in FMS patients as enhanced feedbacksensitivityhasbeen detectedbyonestudy( Win-genfeldetal.,2007),but severalotherstudiesdidshowa reducedfeedbacksensitivity(Ferracciolietal.,1990;Griep etal.,1998).

Likewise,increasedIL-6levels havebeendetected pre-viouslyinsupernatantsofstimulatedFMSpatients’PBMCsby Wallace et al. (2001), but recent studiesdid not find any differencebetweenFMSpatientsandcontrolsin theirIL-6 levelsaftercontrollingfordepressivesymptoms(Gu¨retal., 2002;Bazzichietal.,2006;Wangetal.,2008).

Sincepainandfatiguehavebeenidentifiedascore symp-tomsofFMSweassumedthatFMSpatients’hypocortisolemic patternmightpromoteanexacerbationofpainandfatigue through increased inflammatory signaling. The aim of the presentstudywastotestwhetherlowcortisolresponsesto themeasurementofpressurepainthresholds(PPTs)attender pointswereassociatedwithenhancedreactivityofIL-6levels inFMSpatientsascomparedwithcontrols.

2.

Methods

TwelvefemalepatientswithFMSwererecruitedfromalocal supportgroupinTrier,Germany.Allpatientswerediagnosed with FMS by their rheumatologists according to the 1990 American College of Rheumatology classification criteria (Wolfeetal.,1990).Allpatientsfulfilledtheinclusion cri-teriaof(1)being<60yearsold;(2)havingabodymassindex (BMI)<35kg/m2_{;(3)nothavingahistoryofmajorphysical}

(includingrheumatic conditions),or psychiatricillness;(4) hadnotreceivedepiduralcorticosteroidinjectionsand anti-depressantmedicationattheclinicalandlowdoseduringone yearprecedingthestudy;and(5)wereabletodiscontinue eitheranti-inflammatorymedicationand/orhormone repla-cementtherapyforatleast4weekspriortothestudy.Asa control group, fifteen female, pain-free participants matchedforageandeducationwererecruitedfrom adver-tisementsplacedonbulletinboardsandinlocalnewspapers. Donors havingelevated Westergen erythrocyte sedimenta-tionrates(WESR)andincreasedtotalneutrophilcounts,for instance,twoweeksprecedingthestudywereexcludedfrom participation. Thestudy was approved bythe Ethics Com-mittee of theUniversity of Trierandall participants gave theirinformedconsenttoparticipation.

measurementwasaccompaniedbyanincreaseintheseverityofFMSpatients’painandfatigue ratings.Thefindingsofthepresentstudyprovideevidenceforthepathophysiologicrelevance ofadisturbedglucocorticoidreceptor(GR)function,ratherthanreducedcortisollevelsfor themaintenanceofFMScoresymptoms.

2.2. Experimentalprotocol

AsdepictedinTable1,theexperimentalexaminationswere conductedonfourconsecutivedays.Onthefirstday,cortisol awakening response (CAR) and daytime profile of cortisol secretionwereassessedbyinstructingparticipantstocollect onesalivasampleimmediatelyafterawakeningat0700h,as well as four saliva samples in intervals of 15min after awakening.Further sevensalivasampleswereobtainedby the participants at intervals of 2h, beginning at 1000h. Participantswereaskedto refrainfromeating forat least 30min before saliva collection. On the second day, the experimentalsessionwasstartedbyinsertingacatheterinto theantecubitalvein(Braun,Melsungen,Germany)within1h afterarrivalofparticipantsinthelaboratoryoftheCenter forPsychobiologicalandPsychosomaticResearch,University ofTrier(Germany)at1300h.Inordertoavoidastimulatory effectofphysicalactivityorpsychologicalstressonIL-6and cortisol levels participants wereinstructed to take a rest between1400hand1630h(Ostrowskietal.,1998;VonKa¨nel etal.,2006).

Baseline measurement of differential white blood cell (WBC) count, freecortisol, ACTH, catecholamine andIL-6 levelswereperformedbytakingbloodandsalivasamples30, 20, 10 and 1min before PPT measurement. Participants underwent the algesimetric procedure for a duration of 45min,startingat1700h.Post-algesimetricmeasurements offreecortisol,ACTH,catecholaminesandIL-6levelswere obtainedbycollectingsalivaandbloodsamples1,10,20,30, 90,150,195,240,285,330and375minafterPPT measure-ment,endingat2400h.

Additionally,atallpre-andpostalgesimetrictimepoints, participantsratedtheirpainandfatigueintensitiesbyusinga 100mm visual analogue scale (VAS) for pain (VAS-P) and fatigue (VAS-F), respectively. The subjects also measured their core body temperature (CBT) at four time points, beginningat3p.m.andthereafterintimeintervalsof3h. FourhoursafterconclusionofPPTmeasurement,all parti-cipants received a standard meal. On day 3, two blood samples werecollected at 1100h to determine the gluco-corticoid(GC)sensitivityofinflammatory cytokine produc-tionbycirculatingmonocytes.Inthenightofday3at2200h,

participants ingested 0.5mg dexamethasone (DEX; Jena-pharm,Jena,Germany)andcollectedonthedaythereafter fivesalivasamplesduringthefirsthourafterawakeningat 0700handadditionalsevensalivasamplesinintervalsof2h (Yehudaetal.,1993).

2.3. Psychological measurements

Forassessment oftheseverity offatiguesymptoms a vali-datedGermanversionoftheMultidimensionalFatigue Inven-tory(MFI)(Gaabetal.,2002a)hasbeenemployed.Asinthe Englishversion,theMFIconsistsoffivescales,eachwithfour items, which address different manifestations of fatigue, includingGeneral,PhysicalandMentalFatigue.The partici-pantswereaskedtoassessonaseven-pointratingscalehow frequentlytheyexperiencedspecificfatiguesymptoms dur-ingpreviousdays.

Forassessmentofchronicstresslevelsthevalidated Ger-man version of the Trier Inventory for the Assessment of Chronic Stress (TICS) (Schulz and Schlotz, 1999) has been used.The39-itemTICSconsistsofsixscales whichaddress differentoriginsof subjectivestresslevels,includingwork overload,worries, social stress, lack of recognition, work discontent,andintrusivememories. Theparticipants were askedtojudgeon afive-point rating scalehow frequently they experienced specificstresssituations during theyear precedingtheonsetoftheirdisease.

Presence of major depressive disorder (MDD) was screened by using the German version of the Center for Epidemiological Studies Depression Scale (CES-D) and the GermanversionofthePatientHealth Questionnaire (PHQ-D)intheirvalidatedforms(HautzingerandBailer,1993;Lo¨we etal.,2002).TheCES-Dassessesthefrequencyofdepressive symptomsduringthepreviousweek.Sincesomaticsymptoms comprising depression are thought to be confounded with symptomsofchronicpainpatientsacutoffscoreof19has beenrecommendedtoidentifyMDDinchronicpainpatients (Magni et al., 1994; Turk and Okifuji, 1994). The PHQ-D assessesthefrequencyofninedepressivesymptomsduring theprevioustwoweeksand13physicalsymptomsduringthe previousmonthaccordingtocriteriaofDiagnosticand Statis-ticalManualofMentalDisorders(DSM)(Spitzeretal.,1999).

Table1 Overviewoftheexperimentalexaminationsthesubjectsundergoingeachday.

Day1 Day2 Day3 Day4

Procedure Collectionofsaliva samplestodetermine thecortisolawakening response(CAR) immediatelyafter awakeningat0700h

Collectionofsalivaand bloodsamplesbefore andaftermeasurement ofpressurepainthresholds (PPTs)attenderpoints, startingat1630hand endingat2400h

Asumscoregreaterthan15providesathresholddiagnosisfor MDD and subthreshold diagnosis for somatoform disorder (Kroenkeetal.,2001,2002).

Subjective pain experience was assessed with the vali-datedGermanversionoftheMcGillPainQuestionnaire(SES) (Geissner, 1995). This questionnaire allows the separate assessment of the sensory (MPQ-S) and affective (MPQ-A) qualities of pain, as well as providing a total pain score. Writteninstructionswere slightlymodified to ask patients specificallytodescribetheirmusculoskeletalpain.

2.4. Measurementofpressurepainthresholds (PPTs)attenderpoints

Pressurepainthresholds(PPTs)weredeterminedbilaterally atfourdifferenttenderpointsincludingsuboccipitalmuscle insertions(occiput),themidpointoftheupperborder (tra-pezius) origins, above the scapuala spine near the medial border(supraspinous)andinupperouterquadrantsof but-tocksinanteriorfoldofmuscle(gluteal)byusingapressure algometer(SomediaSalesAB,Munich,Germany).Ascending series of pressure in a rate of 20kilopascal (kPa)/s were appliedtoeachmuscleuntilthepressurewasfirstperceived aspainful.PPTsweredeterminedtwiceateachtenderpoint ontherightandleftsideofthebodyinarandomizedorder. Applicationofeachpressureserieswasseparatedbyatleast 20s.TheparticipantsunderwentthemeasurementofPPTsin aproneposition.ForeachtenderpointPPTswereaggregated fortherightandleftside.

Participantswerefamiliarizedwiththepressure algome-trypriortoexaminationbybilaterallymeasuringPPTsonthe jointcapsulesofbothhandsandankles.Thispre-trial assess-ment was conducted with participants being in a sitting position.

2.5. Biochemicalmeasures

SeparateEDTAbloodsamplesweredrawnfordetermination of IL-6, norepinephrine (NE), epinephrine (EPI) and ACTH levels(Sarstedt,Nu¨mbrecht,Germany).Inordertoprevent IL-6 from being metabolized, blood was drawn into pre-chilledtubesandcentrifugedat1000gfor10min.Blood samplesfordeterminationofcatecholaminesandACTHwere centrifugedat3000rpmfor10min.Plasmafromallsamples wasaliquotedandstoredat 808Cuntillateranalysis.

Catecholamine levels were determined by high-perfor-manceliquidchromatography with electrochemical detec-tion(Chromsystems,Germany;Smedesetal.,1982),anda commercialchemiluminescenceassay(NicholsInstitute,San Juan Capistrano, USA) was used to detect ACTH levels. Plasma IL-6 levels were determined by a high sensitivity quantitative sandwich enzyme immunoassay (ELISA, R&D Systems,Minneapolis,MN,USA).Thelowestlimitof detec-tionwas0.156pg/ml.Theintra-andinterassaycoefficientof variation(CVs)ofaconcentrationat1.99pg/mlwas0.15% and5%, respectively. Saliva samples for cortisol measure-mentwerecollectedusingsalivacollectiondevice(Salivette, Sarstedt,Nu¨rnbrecht,Germany)andstoredat 208Cuntil analysis. Free cortisol in saliva was measured by a time resolved fluorescence immunoassay as described in detail previously(Dressendo¨rfer etal.,1992).

Glucocorticoidsensitivity ofinflammatory cytokine pro-ductionbycirculatingmonocyteswasmeasuredasdescribed earlier(DeRijketal.,1996).Inbrief,5mlwholebloodwas diluted10:1with0.9%saline,400mlofdilutedwholeblood was coincubatedwith 50mloflipopolysaccharide(LPS) (E. coli, DifcoSigma, Augsburg, Germany) and 50ml of dexa-methasone(DEX)indifferentconcentrations(Sigma, Deisen-hofen, Munich, Germany) on a 24-well-plate (Greiner, Nuertingen,Germany)for6hat378Cinahumidified incu-bator containing 5% CO2. The final concentrations were

30ng/ml LPS, and 0, 10 10, 10 9, 10 8, 510 8, 10 7 and 10 6M DEX. After centrifugation for 10min at 48C and 2000g the supernatants were aliquotedand stored at 808Cuntilanalysis.Supernatantconcentrationsoftumor necrosis factor-a (TNF-a) and IL-6 were determined by employingacommercialELISA(BDPharmingen,SanDiego, CA,USA).The detectionlimits oftheELISAs were9.4and 15.5pg/mlforIL-6andTNF-a,respectively.Theintra-and interassayCVswerebelow10%.

2.6. Statisticalanalysis

Cortisolawakeningresponses(CAR)wereassessedby com-putingtwoindices: Thetotalcortisolsecretionwithin1hr postawakeningwasquantifiedusingtheareaunderthecurve (AUC)with respectto ground(AUCG1),andtheincreaseof

cortisolafterawakeningwasquantified usingtheAUCwith respecttothebaseline(AUCI)(Pruessneretal.,2003).

Day-timecortisolsecretionwasestimatedbycomputingtheAUC withrespecttogroundforcortisollevelsbetween1000hand 2200h (AUCG2;Wu¨stetal.,2000).Reactivity ofIL-6levels

andchangesinpainandfatigueratingsafterPPT measure-mentwerequantifiedbycomputingtheAUCwithrespectto groundbetweenbaselineand375minpost-testing(Pruessner etal.,2003).

Basedonpreviousfindings,feedbacksensitivityoftheHPA axiswasassessedbyquantifyingtheextentanddurationof cortisolsuppressionafterovernightdexamethasone suppres-siontest(DST)(Yehudaetal.,1991,1993).

Anenhanced HPA axisfeedbacksensitivityis character-izedbypost-dexamethasone (post-DEX)freecortisollevels whicharesignificantlyreducedincomparisonwithcontrols andbelow2nmol/Lduringthefirsthourafterawakeningand time interval ranging from 1000h to 2200h (Gaab et al., 2002b;Geissetal.,2005).Areducedfeedbacksensitivityhas beencharacterizedbypost-DEXlevelswhicharesignificantly elevated in comparison with controls and above 5nmol/L during the first hour after awakening and time interval rangingfrom1000hto2200h(Liebetal.,2004).Toquantify theextentanddurationofcortisolsuppressionareasunder post-DEXcortisolvalueswithrespecttogroundwere calcu-latedforthefirsthourafterawakening(AUCD1),andbetween

1000hand2200h(AUCD2).

Glucocorticoidsensitivity (GC) ofcirculating monocytes wasassessedbydeterminingthedosageofDEXrequiredto inducea50%inhibitionofLPS-inducedTNF-aandIL-6 pro-duction(IC50;Geissetal.,2005).

variable ‘‘time’’. For analyzing DEX inhibition of LPS-induced TNF-a and IL-6production, two-way analysesof variance (ANOVA) for repeated measurements with the variables ‘‘group’’ and ‘‘dose’’ were computed. Degrees of freedom were adjusted where appropriate employing theGreenhouse-Geisserapproachtocorrectforviolationof the sphericity assumption (Hays, 1981). Subsequent uni-variate analyses (ANOVAs) were employed to ascertain which subgroup, dose or time point accounts for the revealed significant main effect. Comparisons between groups in demographic characteristics and psychometric variableswereconductedbyperformingmultipleStudent’s t-tests. A Bonferroni correction of the error probability wasperformed taking into account the multiple compar-isons(Hays,1981).Sincetheaimofthepresentstudywas totestwhethermeasurementofPPTsattenderpointsisa suitable method to induce enhanced IL-6 levels in FMS patients and not to analyze the change process of IL-6 levelsafter PPTmeasurementwe didnottakeadvantage of more potent analytical approaches such as multilevel modeling. Statistical significance was determined at an alphalevelof0.05.Forrelatingpsychologicalparameters toIC50 andtotal-time integrated in vivoIL-6response to

PPT measurement, Pearson correlation coefficients and partialcorrelationcoefficientswerecomputed.Effectsizes

(ES)were additionally computed. While product moment (r)wascomputedforbetween-subjectcomparisonspartial etasquared(h2)wascalculatedforwithin-subject compar-isons(Cohen,1987).

3.

Results

3.1. Demographicand psychometricvariables

AsreportedinTable2,FMSpatientsdidnotdifferfromcontrols onmostdemographiccharacteristics,withtheexceptionthat themajorityofFMSpatientsdidtakeanti-inflammatory med-icationincludingDevil’sclawextractandnonsteroidal anti-inflammatorydrugssuchasdiclofenacuntilfourweeksbefore examinations.FMSpatients scoredhigher on theTICS sub-scales‘‘lackofrecognition’’[F(1,24)=6.512,p<0.018]and ‘‘work discontent’’ [F(1,24)=4.826, p<0.038]. Further-more,FMSpatientsdidscoresignificantly higheronallMFI subscales(allPs<0.05),andtheGerman versionofMcGill PainQuestionnaire(SES)indicatingthattheyexperiencedthe sensory [F(1,23)=14.125, p<0.001] and affective [F(1, 23)=92.576, p<0.000] qualities of pain more intensely. FMS patients reported a significantly higher frequency of depressive symptoms than controls [F(1, 22)=5.938,

Table2 Demographicandpsychometriccharacteristicsofpatientswithfibromyalgiasyndrome(FMS)andcontrols. PatientswithFMS Controls Demographiccharacteristics

Age(years) 502.07 412.98 Timesincediagnosisoffibromyalgia(inmonths) 143.7033.925

Bodymassindex(meansSE) 26.300.363 26.471.01 Anti-inflammatorymedicationand/orhormonereplacementtherapy(%,n) 75(9) 7(1) Nomedication(%,n) 25(3) 93(14) Menopause(%,n) 25(3) 13(2) Psychometriccharacteristics

TrierInventoryfortheAssessmentofChronicStress(TICS)

Workoverload 24.2501.919 18.7852.425 Worries 16.75001.349 14.8570.740 Socialstress 15.00000.8528 15.5000.521 Lackofrecognition 20.5001.0624** _17.0710.848 Workdiscontent 14.2500.970* _11.9280.518 Intrusivememories 16.0001.193 14.5710.992 GermanversionoftheMcGillPainQuestionnaire(SES)

Sensoryqualitiesofpain 26.903.128** 16.600.86 Affectivequalitiesofpain 42.203.268** _15.470.70

GermanversionofMultidimensionalFatigueInventory(MFI)

Generalfatigue 14.8001.331** _6.5000.817

Physicalfatigue 13.8001.459** 7.3570.9978 Mentalfatigue 13.6001.641** 5.7140.8012 Reducedactivation 13.5001.4624** _5.6420.874

Reducedmotivation 12.1001.754** 7.1420.776 GermanversionofCenterforEpidemiologicStudiesDepressionScale(CES) 24.103.99* _14.571.695

GermanversionofPatientHealthQuestionnaire(PHQ)

Thresholddiagnosisofmajordepressivedisorder(%,n) 17(2) Subthresholddiagnosisofsomatoformdisorder(%,n) 17(2)

DataarerepresentedasmeanSEM.

p<0.023]andtwoFMSpatientsdidconformwithcriteriafora thresholddiagnosisofamoderatelysevereMDD.

3.2. Baselinemeasurements

3.2.1. Pressurepainthresholds(PPTs)attenderpoints FMS patients hadsignificantly lower PPTs at all examined tenderpoints,includingsuboccipitalmuscleinsertions (occi-put) [FMS patients: 219.56kPa47.08; Controls: 433.19kPa15.63],themidpointoftheupperborder (tra-pezius)origins[FMSpatients:249.25kPa55.26;Controls: 431.72kPa7.11],abovethescapualaspinenearthe med-ialborder(supraspinous)[FMSpatients:255.75kPa49.38; Controls:446.28kPa10.32]andinupperouterquadrants ofbuttocksinanteriorfoldofmuscle(gluteal)[FMSpatients: 258.87kPa46.96; Controls: 437.35kPa11.33] (all Ps<0.001).

3.2.2. Differentialwhitebloodcell(WBC)count

FMSpatientsdiddifferfromcontrolsonsubsetsofwhiteblood cells, showing significantly higher leukocyte [FMSpatients: 7.18106_{cells/ml0.75; Controls: 5.5410}6_cells/ml

0.37; F(1, 21)=4.79, p<0.04], neutrophil [FMS patients: 4.29106_{cells/ml0.65; Controls: 2.8110}6_cells/ml

0.36;F(1,21)=4.67,p<0.04] and monocytecounts [FMS patients: 0.55106cells/ml0.07; Controls: 0.37106 cells/ml0.01;F(1,21)=10.47,p<0.004].

3.3. Assessment ofHPAaxisfunction

3.3.1. Cortisollevelsinresponsetoawakeningand duringtheday

Fig.1displaysfreecortisollevelsduringthefirsthourafter awakeningandtheinterval rangingfrom1000hto 2200h. ANOVAfor repeated measures revealed a significant main effectoftime[F(2.04,51.13)=9.66,p<0.001,h2=0.28], but no significant group by time interaction [F(2.05, 51.14)=0.85, p<0.44, h2=0.03],thereby indicatingthat FMS patients and controls did exhibit a similar cortisol responseduringthefirsthourafterawakening.

InfurthersupportofthisnotionFMSpatientsdidnotdiffer significantlyfromcontrolsintheircortisolsecretionduringthe first hour after awakening (AUCG1), and adrenocortical

response to awakening (AUCI) [F(1, 25)=2.72, p<0.11,

r=0.07;F(1,25)=1.331,p<0.26,r=0.04,respectively]. ComparisonbetweenFMSpatientsandcontrolsregarding theircortisollevelsduringthedayrevealedneithera signifi-cant group by time interaction [F(4.14, 91.05)=1.53, p<0.20, h2=0.07] nor main effect of group [F(1, 22)=0.06,p<0.81,h2=0.003]indicatingsimilarityof FMS patientsandcontrolsintheircortisollevelsthroughouttheday. Furthermore,therewasnosignificantdifferencebetweenFMS patientsandcontrolsintheirAUCofcortisollevelsbetween 1000hand2200h(AUCG2)[F(1,22)=0.12,p<0.72].

3.3.2. Feedbacksensitivityofthepituitary—adrenal axis

Fig.2displaysthepost-DEX cortisolvalues duringthefirst hourafterawakeningandtheintervalrangingfrom1000hto 2200h. Statistical analysis of thepost-DEX cortisol values duringthefirsthourafterawakeningrevealedasignificant

group by time interaction [F(1.76, 44.00)=3.60, p<.04, h2=0.13]. Post hoc analysis ascertained FMS patients as displaying significantly higher post DEX cortisol values 45min [F(1,25)=6.59, p<0.01] and 60min [F(1, 25)=3.94,p<0.05]postawakening.Therewasastatistical trend for a higher post-DEX cortisol secretion within 1 hr postawakening (AUCD1) in FMS patients as compared with

controls[F(1,25)=3.21,p<0.09,r=0.10].

Again,comparingpost-DEXcortisolvaluesbetween1000h and2200hrevealedasignificantgroupbytimeinteraction [F(1.45,33.42)=4.63,p<0.03,h2=0.17].Posthoc univari-ateANOVAsconfirmedthatFMSpatientshadsimilarpost-DEX values as controls between1000h and2000h, but signifi-cantly higher post-DEX values at 2200h [F(1,24)=7.41, p<0.01]. FMS patients did display similar AUCD2 [F(1,

23)=0.001,p<0.98,r=0.00]ascontrols.

3.3.3. Glucocorticoid(GC)sensitivityofinflammatory cytokineproductionbycirculatingmonocytes

AsdepictedinFig.3A,significantlyhigherconcentrationof DEXwasnecessarytoinducea50%inhibitionofFMSpatients’ LPS-induced IL-6 production in comparison with controls

[F(1,20)=4.38,p<0.05,r=0.18].FortheDEXdosebeing requiredfora50%inhibitionoftheLPS-inducedTNFa pro-ductionthedifferencebetweenFMSpatientsandcontrolsin their IC50 failed to reach significance [F(1,20)=2.13,

p<0.16,r=0.09].

3.3.4. Bivariateassociationsofpsychologicalvariables withglucocorticoidsensitivity

Inallparticipants,dosageofDEXwhichisrequiredtoinduce a50% inhibitionof LPS-inducedTNF-a andIL-6 production wassignificantlycorrelatedwithsubscalesoftheMFI, includ-ing ‘‘General Fatigue [r=0.475, p<0.01 and r=0.533, p<0.008, respectively], ‘‘Mental Fatigue’’ [r=0.375, p<0.05andr=0.531,p<0.008,respectively], ‘‘Reduced motivation’’ [r=0.407, p<0.03 and r=0.57, p<0.004, respectively] and ‘‘Reduced Activation’’ [r=0.405, p<0.03 and r=0.375, p<0.05, respectively]. Whereas MFIsubscale‘‘PhysicalFatigue’’wassignificantlycorrelated with IC50 for LPS-induced TNFa production [r=0.361,

p<0.05]thecorrelationwiththeDEXdosebeingrequired toinducea50%inhibitionoftheLPS-inducedIL-6production failed to reach significance [r=0.283,p<0.11].Withthe exceptionofthecorrelationbetweenMFIsubscale‘‘General Fatigue’’andrequiredDEXdosetoinducea50%inhibitionof LPS-inducedIL-6 production the correlations between MFI subscalesandrequiredDEXdosetoinducea50%inhibitionof LPS-inducedTNFa andIL-6 production diddisappear after controllingfortheeffectoftotal-timeintegratedinvivoIL-6 responsetoPPTmeasurement[allPs>0.07].

3.4. Behavioral,endocrineand interleukin-6 responsestopressurepainthreshold(PPT) measurement

3.4.1. FatigueandpainresponsetoPPTmeasurement Thetimecoursesoffatigueandpainratingsbeforeandafter PPTmeasurementaredepictedinFig.4.SinceFMSpatients

Figure3 Differences betweenFMS patients and controls in dosages of dexamethasone being required to induce a 50% inhibition of the LPS-induced (A) IL-6 production and (B) TNFa-production in peripheral blood monocytes (IC50). The

IC50isinverselyrelatedtothesensitivityofmonocytestothe

immunosuppressiveeffectsofcortisol.AhigherIC50indicatesa

lowersensitivityofmonocytestotheimmunosuppressiveeffects of cortisol. * Indicates significant difference between FMS patients and controls (p<0.05). Data are expressed as meansSEM.

reported significantly higher fatigue scores 30min [F(1, 23)=779.64, p<0.0001] and 20min [F(1, 23)=781.23, p<0.0001]beforePPTmeasurement theaveragedfatigue scores were considered as a covariate in the subsequent analysis.ANOVAforrepeatedmeasuresrevealedasignificant interaction between group and time [F(3, 69.07)=89.96, p<0.0001, h2=0.80] (Fig. 4A). Post hoc analysis showed that pressure application on tender points did aggravate sleepiness in FMS patientsas their VAS-Fscores increased significantly 60min [F(1,23)=1726,74, p<0.0001] and 90minpost-testing[F(1,23)=22417.72,p<0.001].

ForchangesinpainratingsafterPPTmeasurement,there wasastatisticaltrendforgroupbytimeinteraction[F(2.19, 43.8)=2.7, p<0.08, h2=0.12] after controlling for FMS patients’significantlyhigherpainscoresunderpre-algesimetric

conditions[F(1,23)=39.01,p<0.001].Univariateanalyses revealed thatVAS-Pscores of FMSpatients did increaseby 10 units on average within 10minafter pain measurement [F(1, 20)=43.05, p<0.0001], thereby revealing that PPT measurement exacerbated sleepiness and pain intensity in FMSpatients.

3.4.2. CatecholamineresponsetoPPTmeasurement Fig.5showsepinephrine(EPI)andnorepinephrine(NE)levels beforeandafterPPTmeasurement.Forepinephrinelevels, ANOVAforrepeatedmeasuresindicatedastatisticaltrendfor group by time interaction [F(2,44)=2.89, p<0.07, h2=0.12].Univariateanalysesconfirmedastatisticaltrend for higher epinephrine levels 30min pre-testing [F(1, 22)=3.69, p<0.07] in controls and identified significant lowerepinephrinelevels60minpost-testinginthissubgroup [F(2,44)=5.55, p<0.007] (Fig. 5A). For norepinephrine levels, ANOVA for repeated measuresshowed a significant maineffect ofgroup[F(1,22)=11.3,p<0.003,h2=0.34], but group by time interaction failed to reach significance

Figure4 Changesintheseverityof(A)fatigueand(B)pain ratingsafterconclusionofpressure painthreshold(PPT) mea-surement.Subjectsscoredtheirfatigueandpainintensityona visual analoguescale for fatigue (VAS-F) and visual analogue scaleforpain(VAS-P),respectively.**Indicatessignificant dif-ferencesbetweenFMSpatientsandhealthycontrols(p<0.01). DataareexpressedasmeansS.E.M.

[F(2,44)=2.22,p<0.12,h2=0.09](Fig.5B).Posthoc ana-lysesrevealedthatthesignificantmaineffectisduetoFMS patients’significantlyelevatednorepinephrinelevels10min before[F(1,22)=6.04,p<0.02]and10minafterPPT mea-surement [F(1,22)=13.08,p<0.002],thereby pointingto theirhighersympathoadrenalactivity.

3.4.3. ACTHresponsetoPPTmeasurement

Comparedwithcontrols,FMSpatientsexhibitedsignificantly lower ACTHlevels30and10minbefore PPTmeasurement (F(1,21)=7.24,p<0.02;F(1,21)=5.69,p<0.03, respec-tively)(Fig.6A).Inordertocontrolforthisgroupdifference, theaverage ofthebaseline ACTHlevels wasaddedto the

repeatedmeasuresANOVAasacovariate.Asignificant inter-action between group and time emerged for ACTH levels (F(1.78,37.37)=7.4,p<0.003,h2=0.26)aftercontrolling for baseline ACTH levels. While controls’ ACTH levels declined from pre- to 10min post-testing [F(1,21)=3.71, p<0.07],FMSpatients’ACTHlevelsdidnotchangeduring theobservationperiod.

3.4.4. CortisolresponsetoPPTmeasurement

SimilartoACTH,FMSpatientsexhibitedsignificantly lower cortisollevels30min(F(1,23)=4.50,p<0.04)before PPT measurement (Fig. 6B). ANOVA for repeated measures revealed a significant group by time interaction after

controllingfordifferencesinpre-algesimetriccortisolvalues [F(2.09,46.02)=4.9,p<0.011,h2=0.18].Posthocanalyses revealed that FMS patients had significantly higher cortisol levels after PPT measurement for 10min [F(1,23)=11.04, p<0.003], 90min [F(1,23)=7.01, p<0.01] and 375min [F(1,23)=27.22, p<0.0001]. Frombaseline levels, cortisol increased approximately 300% at 10-min post-testing, then returnedbelow baseline levels and increased again at 240-minpost-testing, thereby indicating that PPT measurement inducedconsiderableadrenocorticalactivationinFMSpatients. 3.4.5. Interleukin-6responsetoPPTmeasurement Statisticalcomparisonof pre-algesimetricwith post-algesi-metricIL-6levelsrevealedasignificantgroupbytime inter-action[F(3.23,61.35)=4.77,p<0.004,h2=0.20](Fig.6C). WhileFMSpatientsdidnotdifferfromcontrolsinIL-6levels before PPT measurement [F(1, 21)=2.71, p<0.11] they exhibitedsignificantlyenhancedIL-6levelsduringthewhole post-testing session including 30min [F(1,21)=10.5, p<0.004], 90min [F(1,20)=22.22, p<0.0001], 150min [F(1, 21)=22.53, p<0.0001], 195min [F(1,21)=28.57, p<0.0001], 240min [F(1,21)=15.62,p<0.001], 285min [F(1,21)=18.51, p<0.001], 330min [F(1, 21)=15.03, p<0.001]and375min[F(1,20)=7.81,p<0.011] post-test-ing.IntermsofFMSpatients’IL-6increase,theirIL-6levels peaked at approximately 300% above baseline at 240-min post-testinganddidnotreturntobaselinelevelsattheendof post-testingsession, therebyrevealingtheirenhanced IL-6 reactivityinresponsetoPPTmeasurement.Infurther sup-port of FMS patients’ enhanced IL-6 reactivity after PPT measurementtheyexhibitedasignificantlyhighertotal-time integrated IL-6 response as compared with controls [F(1, 19)=16.26,p<0.001,r=0.42].IL-6AUC wassignificantly associatedwithfatigue(r=0.77,p<0.0001)andpain rat-ings(r=0.60,p<0.001)AUC.Therewasastatisticaltrend forapositivecorrelationbetweeninvitroGCsensitivityof LPS-inducedIL-6,but notTNFa productionwithtotal-time integrated in vivo IL-6 response to PPT measurement [(r=0.34,p<0.09)and(r=0.21,p<0.21),respectively].

4.

Discussion

ThisstudywasconductedtoexaminewhetherFMSpatients’ reducedcortisollevelsareassociatedwithanexacerbationof pain and fatigue ratings, which may be mediated by an increasedIL-6reactivity afterPPT measurement.This isto thebestofourknowledgethefirststudytotestforendocrine andimmunecharacteristicsinFMSpatientsthatarein agree-mentwiththehypothesizedpathophysiologicalrelevanceofa hypocortisolemicpatternformanifestationofFMScore symp-toms.Insummary,FMSpatients’awakeningresponse,daytime profile of cortisol secretion and cortisol suppression after ingestion of a low dose DEX were found to be normal. In contrast, IL-6 production in circulating monocytes of FMS patientswasfoundtobelesssensitivetothe immunosuppres-siveactionsofglucocorticoids.WhilePPTmeasurementdidnot changeACTHlevelsinFMSpatients,wefoundelevated nor-epinephrinelevels,togetherwiththreetimeshighercortisol levelsandfourtimeshigherIL-6levelsinFMSpatients com-paredwithcontrols.TheenhancedIL-6reactivitywas accom-paniedby areducedsensitivityof circulatingmonocytesto

glucocorticoidinhibitionofIL-6productionandbyan aggrava-tionofpainandfatigueratingsafterPPTmeasurement.

Overall,thefindingsofthepresentstudyareinagreement with thehypothesisofa pathophysiologicalrelevanceof a disturbedGRsignalingpathway,ratherthanreducedcortisol levelsforthemaintenanceofFMScoresymptomsfatigueand pain.Thereducedsensitivityofcirculatingmonocytestothe immunosuppressive effects of glucocorticoids may permit the maintenance of FMS core symptoms fatigue and pain byaninsufficientinhibitionofcytokineproduction.

TherevealedsimilaritybetweenFMSpatientsandcontrols intheir cortisolawakening responsedoesconflictwith pre-viousfindingsasFMSpatientswerefoundtoexhibitan atte-nuatedcortisolawakeningresponse(Weissbeckeretal.,2006; Klingmann et al., 2008). Lack of control for confounding variablesofcortisolawakeningresponsesuchastrauma his-toryorgestationallengthmayaccountfortheinconsistency. DuringthefirsthourafterawakeningFMSpatients’post-DEX cortisol valueswere foundto bebelowthecut-offvalueof 2nmol/L,butsignificantlyelevatedincomparisonwith con-trols. Duringtheremainderof thepost-DEXstudy day,FMS patientsdidexhibitsimilarpost-DEXcortisolvaluesascontrols. Onaverage,FMSpatients’post-DEXcortisolvalueswerebelow 5nmol/L during the time interval ranging from 1000h to 2200h.SinceFMSpatients’post-DEXcortisolvaluesdidnot fulfillallcriteriaofeitheranenhancedorareducedHPAaxis feedbacksensitivitythepresentfindingsdopointtoanormal HPAaxis feedbacksensitivityinthispopulation.Differences betweencurrentandpreviousstudiesinDEXdosage,amountof post-DEXcortisolmeasurementsandmeasuredcortisol frac-tionmayaccountfortheinconsistentfindings.While Ferrac-cioli etal.(1990) did use 1mgDEX for assessing feedback sensitivitysubjectsinthepresentstudydidingest0.5mgDEX. Bothdosageshavebeenreportedtodifferintheirsensitivityto discriminate normal from alteredfeedback sensitivity as a dosage of 1mgDEX causes a total and a dosage of0.5mg DEXinducesapartialsuppressionoftheadrenalcortisol secre-tion(Yehuda,1997).Limitationtoonepost-DEXtotalcortisol measurementat0800hinWingenfeld’sstudymayinfluence DSTresultsaspost-DEXcortisolvalueshavebeenshowntovary markedly9and16hfollowingDEXingestion(LowyandMeltzer, 1987).DSTresultsmaybealsoinfluencedbylimitationofthe measuredcortisolfractiontototalcortisollevelsbecausetotal cortisollevelsaremodulatedbycorticosteroid-binding globu-lin(CBG)levels(Rosner,1990).SinceFMSpatientshavebeen shown to exhibit reduced CBG levels, exaggeratedcortisol suppression inWingenfeld’s studymay reflect,inpart, FMS patient’slowerCBGlevels(Lentjesetal.,1997).

Inthecurrentstudyasignificantly higherDEXdose was foundtoberequiredtoinducea50%inhibitionofthe LPS-induced IL-6 production in FMS patients. This finding is indicativeofa reducedGC sensitivityof circulating mono-cytesinthesepatients.Thereducedsensitivityofcirculating monocytestotheimmunosuppressiveeffectsof glucocorti-coidsisaccompaniedbyahigherfrequencyofexperienced fatiguesymptoms,thereby suggestingthepathophysiologic relevanceofreducedGCsensitivityofcirculatingmonocytes forFMScoresymptoms.

signalingpathwayby meansof an increased productionof glucocorticoidinducibleleucinezipper(GILZ)inmonocytes hasbeenidentifiedasanimportantstepwithinthis transduc-tionprocess(Clark,2007;Eddlestonetal.,2007;DeBosscher andHaegeman,2009).PBMCsofFMSpatientshaverecently been foundto contain significantly lower levels of GILZin comparisonwithcontrols(Macedoetal.,2008).Thisraises thepossibilitythatFMSpatients’higherIC50forLPS-induced

IL-6productiondoespartlyreflectareducedexpressionof GILZandhenceabnormalNF-kBactivationintheircirculating monocytes(Eddlestonetal.,2007).

Exclusionofsubjectshavingtakenlong-term antidepres-sant medication from participation and examination of mainly premenopausal women in the current study may provideanexplanation fortheinconsistency between pre-sent and findings by Wingenfeld etal. (2008)and Macedo etal.(2008)asparticipantsinMacedo’sstudywereallowed toparticipateafterdiscontinuingmedicationfortwoweeks, andWingenfeldetal.didexamine mainlypostmenopausal women.Thereisinvitroevidencesuggestingthat transduc-tionofcortisolsignalintocytokineinhibitionmaybe decel-eratedbyreducedestradiolavailabilityduringmenopauseas fewerestrogenreceptors(ER)areoccupiedbythishormone (Liuetal.,2005).Long-termantidepressantmedicationmay accelerate thetransductionofcortisolsignalintocytokine inhibitionbecauseincubationofwholebloodsamplesfor24h with amitriptyline has been demonstrated to induce an upregulationofGRexpression(Vedderetal.,1999).

TherevealedreducedGCsensitivityofcirculating mono-cytes in FMS patients is indicative of a hypercortisolemic patterninthispopulation.Inconflictingwitha hypercortiso-lemic pattern FMS patients did exhibit a normal daytime cortisolprofileandHPAaxisfeedbacksensitivity.FMSpatients were,onaverage,abovethethresholdforadiagnosisofMDDon theCES-D.ThePHQ-DidentifiedtwoFMSpatientsas conform-ingwithcriteriaforathresholddiagnosisofMDDindicativeof MDDpresenceinasubgroupofFMSpatients.Inlinewiththis finding, subgroups and not the general population of FMS patientshavebeenreportedpreviouslytoexperience signifi-cantdepressivesymptomatology(Ahlesetal.,1987; Ferrac-ciolietal.,1990).AlthoughthresholddiagnosisofMDDinFMS patients needs to beconfirmed bystructured interviews it seemsunlikelythatadepressive-likehypercortisolemiamay accountforalteredHPAaxisfunctioninFMSpatients.

ItmightbearguedthatexclusionofFMSpatientshaving takenantidepressant medicationfor along time increases the risk of having a sub-group of patients which are not representativeofFMSpatientsingeneral.ExclusionofFMS patientshavingtakenantidepressantmedicationforalong time was necessaryas long-termamitriptyline medication has been identified as a confounder of GC sensitivity of circulatingmonocytesininvitroandourpilotstudies( Oku-gawaetal.,1999;Vedderetal.,1999).Thecurrently exam-ined FMS patients are representative of FMS patients in generalastheyare comparabletopreviouspopulationson psychosocial variables. For instance, the meanage of the currentlystudiedFMSpatientsisrepresentativeoftheage grouphavingahighprevalenceforthisdisorder(Wolfeetal., 1995). Consistent with previous findings a minority of the currentlyexaminedFMSpatientsdidexperiencesignificant depressivesymptomatology(Ahlesetal.,1987;Ferraccioli etal.,1990).

WhereasFMSpatientsdidnotdifferfromcontrolsintheir IL-6 levels before PPT measurement, their IL-6 levels increased approximately 300% above baseline after PPT measurement.TheenhancedIL-6reactivityinFMSpatients afterpressureapplicationontheirtenderpointsprovidesthe first lineof evidence for elevated IL-6 levelsafter painful stimulationinthispopulation.

MusclecellsmayrepresentacellularoriginofenhancedIL-6 reactivityafterpressureapplication ontenderpointsinFMS patientsas painful stimulation ofmuscles has been demon-stratedtoincreasecirculatingIL-6levelsinhumansandanimals (Loram et al., 2007; Edwards et al., 2008).Inmore detail, muscle-derivedIL-6mayelevatecirculatingIL-6levelsbythe demonstrated potency of this cytokine to sensitize muscle nociceptorsthroughinductionofprostaglandin(PG)E2synthesis

(Lahirietal.,2001).Sensitizedmusclenociceptorsreleasethe monocyteattractantneuropeptidesubstanceP(SP)whichhas beenshowninvitrotobeabletoincreasecirculatingIL-6levels bybindingtoanon-neurokininSPreceptoronmonocytes( Kave-laarsetal.,1994;Schratzbergeretal.,1997;Mense,2003).

Although FMS patient’s epinephrine concentrations remainedat lowlevels atall timepoints beforeand after PPTmeasurement theirnorepinephrine concentrations did increasesignificantlyfrom10minpre-testingto10min post-testing.Inlinewithpresentfindingslowepinephrinelevels have been detected consistently in FMS patients after exposuretodifferenttypesofstressors(Giskeetal.,2008; KadetoffandKosek,2010).

Highnorepinephrinealongwithlowepinephrinelevelsare indicativeofareducedcortisolproductionintheadrenalcortex asdecreasedcortisolproductiongivesrisetoalower norepi-nephrine to epinephrine conversion in theadrenal medulla (WurtmanandAxelrod,1966).Astressorinflammation-related dissociationbetweenthehypothalamic—pituitary-systemand theadrenalcortexhasbeenrelatedtoanadrenalinsufficiency (Nussdorfer,1996;Ehrhardt-Bornsteinetal.,1998).

responseincontrols.Increasedcortisollevelshavebeenshown tobedependentonprecedingelevationofACTHlevels( Kirsch-baumet al., 1993). Decline in control’s ACTH levels from 30minto10minpre-testingmayaccountforthelackintheir pre-algesimetriccortisolresponse.

Severallimitationsofourstudyshouldbenoted.First,the studyislimitedbyasmallsamplesize.Second,wedidnot measuretotalcortisol levelsandDEXlevelsfollowing late-night administration of DEX. As FMS patients’ circulating monocyteswerefoundto belesssensitiveto the immuno-suppressiveactionsofDEXitisconceivablethattheir post-DEXcortisolvaluesfollowingDEXadministrationhavebeen modulatedbytheirreducedGCsensitivity(LowyandMeltzer, 1987).

To summarize, the present findings donot support the hypothesis that FMS core symptoms pain and fatigue are promotedbyreducedcortisollevels.Instead,theyindicate thatdeficiencies intheGRsignalingpathwayincirculating monocytesmaycontributeto themaintenanceofFMScore symptomspainandfatiguebyfacilitatinganenhancedIL-6 reactivity.

Conflict

of

interest

Allauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgements

WeespeciallythankProf.Dr.AlexanderL.Gerlach,Institute for Clinical Psychology and Psychotherapy, University of Cologne,for his adviceinscreeningfor presenceofmajor depressivedisorder(MDD)inpatientswithfibromyalgia syn-drome(FMS).WeextendourgratitudetoRenateEngeland NicoleReinertfortheperformanceofthecytokine measure-ments.Thisresearchwassupportedinpartbygrantsfromthe Center for Psychobiological and Psychosomatic Research, University of Trier and Centre de Recherche Public de la Sante,Luxembourg,Luxembourg.

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