Serum soluble interleukin 6 (IL 6) receptor and IL 6/soluble IL 6 receptor complex in systemic juvenile rheumatoid arthritis

Serum soluble interleukin 6 (IL-6) receptor and

IL-6/soluble IL-6 receptor complex in systemic

juvenile rheumatoid arthritis.

F De Benedetti, … , D Novick, A Martini

J Clin Invest.

1994;

93(5)

:2114-2119.

https://doi.org/10.1172/JCI117206

.

By using a sandwich ELISA, soluble human IL-6 receptor (sIL-6 R) levels were measured in

the sera of 20 healthy children and of 25 patients with systemic juvenile rheumatoid arthritis

(JRA). In patients with systemic JRA, serum sIL-6 R levels (114.6 +/- 37.7 ng/ml) were

significantly lower (P < 0.01) than those of healthy children (161.2 +/- 45.5 ng/ml). Serum

sIL-6 R levels were negatively correlated (r = -0.610, P < 0.001) with serum IL-6 levels

measured with the B9 cells. The serum IL-6/sIL-6 R complex was detected using an ELISA

based on a monoclonal antibody to IL-6 for capture and on a monoclonal antibody to human

sIL-6 R for detection. Healthy controls had little, if any, detectable serum IL-6/sIL-6 R

complex (OD 0.024 +/- 0.027), while the majority of patients with systemic JRA presented

measurable serum IL-6/sIL-6 R complex (OD 0.492 +/- 0.546). IL-6 levels estimated in the

circulating IL-6/sIL-6 R complexes were in the range of nanograms per milliliter and

approximately 20-fold higher than those measured by the B9 cells. Since serum C-reactive

protein concentrations were much more correlated with serum levels of IL-6/sIL-6 R

complexes (r = 0.713, r2 = 0.51, P < 0.0001) than with the serum IL-6 levels measured with

the B9 cells (r = 0.435, r2 = 0.19, P = 0.05), the large quantities […]

Research Article

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Serum

Soluble Interleukin 6

(IL-6) Receptor

and IL-6/Soluble IL-6

Receptor Complex in Systemic Juvenile Rheumatoid Arthritis

FabrizioDe Benedetti, Margherita Massa, Patrizia Pignatti, SalvatoreAlbani, DanielaNovick,*and AlbertoMartini

Clinica Pediatrica, Universita degliStudi diPavia,Instituto di RicoveroeCuraaCarattereScientificoPoliclinico San Matteo,

27100Pavia,Italy; and*DepartmentofMolecular Genetics andVirology, WeizmannInstitute, 76100Rehovot, Israel

Abstract

By using a sandwich ELISA, soluble human IL-6 receptor

(sIL-6R) levelsweremeasuredintheseraof20

healthy

chil-dren andof 25patientswithsystemicjuvenilerheumatoid

ar-thritis

(JRA).

Inpatients with

systemic

JRA,

serumsIL-6R levels(114.6±37.7ng/ml)weresignificantly lower (P<0.01) than those ofhealthy children (161.2±45.5 ng/ml). Serum

sIL-6 R levels were negatively correlated (r = -0.610, P

<0.001) withserumIL-6levels measuredwith the B9 cells. The serum IL-6/sIL-6 R complex was detected using an ELISA basedon amonoclonalantibodytoIL-6 for capture and on a monoclonal antibody to human sIL-6 R for detection. Healthycontrols hadlittle, ifany,detectableserum

IL-6/sIL-6Rcomplex(OD

0.024±0.027),

while themajorityofpatients

withsystemic JRA presented measurableserumIL-6

/sIL-6

R

complex(OD0.492±0.546).IL-6 levels estimated in the

cir-culating

IL-6/sIL-6

R

complexes

wereinthe range of

nano-grams per milliliter and 20-fold

higher

than thosemeasured

by the B9 cells. SinceserumC-reactive

protein

concentrations were much morecorrelated withserumlevelsofIL-6

/

sIL-6R

complexes (r

=

0.713,

r2 = 0.51,P<

0.0001)

than with the serum IL-6 levels measuredwith the B9 cells

(r

=

0.435,

r2

=0.19,P=

0.05),

thelarge

quantities

ofserumIL-6 present in

IL-6/sIL-6Rcomplexesappear to bebiologically relevantin

vivo, at least as far as the induction by IL-6 ofacute phase protein production.(J. Clin.Invest.

1994.93:2114-2119.)

Key words: interleukin 6*interleukin 6receptor *C-reactive protein

*juvenile rheumatoid arthritis

Introduction

IL-6 hasmultiple

biological

effectsonimmune and

inflamma-tory responses(for reviewseereference 1). Ithasbeenshown

to induce fever andhepatocyteacute phase response, to en-hanceproliferation and maturation ofBcellsaswell as immu-noglobulin production, andtoactivateTlymphocytefunction.

Elevatedlevelsof IL-6havebeenfoundinperipheralbloodor

otherbiological

fluids

inavariety of diseases, including bacte-rialandviral infections, neoplasia,trauma,andchronic

inflam-matorydiseases(2).

The receptormediatingthebiological activities of IL-6 has been identified as twodifferent membrane glycoproteins, an

Address correspondence to Alberto Martini, M.D., Clinica Pediatrica, IRCCS San Matteo, P. le Golgi 2, 27100 Pavia, Italy.

Receivedfor publication16September1993.

80-kD protein, referred to as the ligand-binding protein (IL-6 receptor[IL-6

R]),'

and a 130-kD protein, referred to as the signal transducing protein (3, 4). cDNA encoding both mole-cules has been cloned and expressed and its biochemical proper-ties were analyzed (3-5). In particular, a genetically

engi-neered truncated form of the 80-kD IL-6

R,

lacking the trans-membrane and the intracytoplasmatic domains, has been expressed successfully and has been shown to be able to asso-ciate with the 130-kD protein in the presence of IL-6 and to mediate IL-6 functions (6). Moreover, a soluble form ofIL-6 R (sIL-6 R) has been purified as a 50-kD protein from human urine(7) and has been shown to be present in human serum and to bind recombinant human IL-6 with a binding affinity similar tothatof theentirerecombinant IL-6Rmolecule (8). Juvenile rheumatoid arthritis (JRA) is a clinically

heteroge-nousconditioncurrently divided into differentclinkalforms based on symptoms at onset (9). The systemic form is charac-terizedby chronic arthritis associated with highspikingfever,

other systemic features, includingevanescent cutaneous rash,

hepatosplenomegaly, lymphoadenomegaly, and serositis,and

withprominentlaboratory evidence ofinflammation (9).The other JRAonset forms arecharacterized mainly by chronic joint inflammation and are subdivided according to the num-ber ofjoints involved duringthefirst 6 moof diseasein pau-ciarticular(fourorlessjointsinvolved) orpolyarticularJRA

(fiveormorejoints involved)(9). We have shown previously that serum andsynovial fluid IL-6 levels are elevated in pa-tients with JRA during active disease and normalize during remission(10, 11).Inpatients withsystemicJRA,serum IL-6

levelsaremuchhigherthan those presentin patients with

poly-articular or pauciarticular JRA (10, 11) or in patients with adult rheumatoid arthritis (12, 13). In addition, in patients

withsystemicJRA, synovialfluid IL-6 levels are significantly

higher thanthose presentin patientswith polyarticular or pau-ciarticular JRA or inpatients with adult rheumatoidarthritis

(DeBenedetti,F., V.Gerloni,M.Massa, P. Pignatti, R.

Capo-rali, C. M. Montecucco, K. Matsushima, F. Fantini, and A.

Martini,

manuscript in preparation). Moreover, in systemic

JRA,serumIL-6 levels are correlated with the extent and

sever-ity ofjoint involvementand withplatelet counts(10).Taken

together, these data suggestthat IL-6playsanimportantrole in the pathogenesis of systemic JRA. To better understand the

biologyof IL-6 anditsrelationto thesIL-6 R in systemic JRA, inthis studywemeasured serum sIL-6 R levels, evaluated their

relation with IL-6levels, and developed an assay system for the

detection of IL-6/sIL-6 Rcomplexesinsera ofpatients with

systemicJRA.

1. Abbreviations used in thispaper:AP,alkalinephosphatase; CRP,

C-reactiveprotein; HGF,hybridoma growth factor;IL-6R,IL-6

recep-tor;JRA, juvenilerheumatoidarthritis;sIL-6R,soluble IL-6 R.

2114 DeBenedetti,Massa,Pignatti, Albani, Novick, andMartini J.Clin.Invest.

©TheAmericanSocietyforClinicalInvestigation,Inc.

Methods

Patients. 60 children(meanage7.2yr, range2-17 yr)whofulfilledthe AmericanCollegeofRheumatology's (formerlytheAmerican Rheu-matismAssociation) proposed criteria for the diagnosis ofJRA(9)

wereincluded in thestudy. 25 had systemic, 15polyarticular, and 20

pauciarticularJRA. HLA B27orrheumatoid factor-positive(by Latex

agglutination test) patientswereexcludedfrom the study. All patients

werestudiedduring active diseaseasdefinedby thepresenceof

synovi-tison examination. Six patients werereceiving no treatment. The othersweretreated with nonsteroidal antiinflammatory drugs,

asso-ciatedinapproximatelyhalfofthemwithmethotrexate (singleweekly

dose) and/or prednisone (onanalternate-day regimen inthe majority

ofcases). Duringthecourseof thisstudy,somepatientsweretested morethanonceduringdifferentphasesofthediseaseorduring

differ-enttreatments.Sera obtained from 20healthychildren(meanage8.1

yr, range3-18yr), hospitalizedfor bonemarrowdonationorfor minor

surgical procedures,wereusedascontrols. Permission fordrawingof extrabloodduringroutinevenipuncturewasobtained fromparentsof all children.

Bloodwasallowedtoclot for2 h at roomtemperature and

centri-fugedat1,500gfor 10min.Serawerestored at-70'Cuntil used. 12

synovialfluidsamples,3 frompatientswithsystemicJRA and 9 from

patientswithpauciarticular JRA,obtainedbefore intraarticular steroid

injectionwerealso studied.Synovialfluidswerecentrifugedatl0,OOOg

for 10minand stored at-70'Cuntiluse.

AssayprocedureforsIL-6R.sIL-6Rlevelsweremeasuredusinga

sandwichELISA,basedontheuseof monoclonal antibodies 34.14 and alkaline phosphatase (AP)-conjugated 22.3. These two monoclonal antibodies were selected for the ELISA because previous studies showed thattheybindtodifferentepitopesonthe sIL-6 R molecule

( 14). Polystyrene plates (Costar Corp., Cambridge, MA)werecoated

overnightat4°Cwith monoclonalantibody34.14(20,g/mlinPBS),

andnonspecific bindingsiteswereblockedbyfurther incubation with

PBS/2%BSA for1h at37°C.Serumandsynovialfluidsampleswere addedata1:100dilution inPBS/1%BSA,and theplateswere incu-bated for 3 h at37°C.Natural sIL-6R,purifiedtohomogeneityfrom human urine asdescribedpreviously ( 15), was usedas astandard.

Afterwashing, plateswereincubated for 2 h atroomtemperaturewith

AP-conjugated monoclonal antibody 22.3 (0.5 ug/ml in PBS/1% BSA).Thesubstratep-nitrophenyl phosphatewasadded,andthe

opti-caldensity read at 405 nM. The detection limit of the ELISA is 40

pg/mlofpurifiedhuman sIL-6 R.

DetectionofIL-6/sIL-6Rcomplex.To detect thepresenceof IL-6/

sIL-6 Rcomplex, weconstructedan ELISA based ontheuse ofa monoclonalantibodytohuman IL-6(34.1 ) (kindly provided by

Inter-pharm Laboratories, Nes-Ziona, Israel) ( 16)forcaptureanda mono-clonalantibodyto the sIL-6 R(AP-conjugated 22.3)for detection. Monoclonalantibody22.3 waschosen because itwasshown not to inhibit thebindingof radiolabeled humanrecombinant IL-6 tohuman

natural sIL-6 R( 14). Polystyrene plateswerecoatedovernightatroom

temperaturewithmonoclonalantibody34.1 to IL-6ataconcentration of 50_tg/mlinPBSorwith PBS alone. Afterblockingthenonspecific bindingsites witha2-hincubationat37°CwithPBS/2% BSA,each testserum,diluted 1:1 inPBS,wasadded to wells coatedwith monoclo-nalantibody34.1 to IL-6and to wells coated with PBSalone,and the

plateswereincubated for 2 h at37°C.Plateswerenotwashed,and the

AP-conjugatedmonoclonalantibody22.3tothesIL-6 Rwasaddedto

thewellscontainingtestsamplesatafinalconcentrationof 1.3,g/ml.

Theplateswereincubated for 1hat 37°Cwithgentle swirling.This

approachwaschosen becausepreliminary experimentswithpatients'

seraand withnormalserainthepresence of exogenousIL-6 showed thatwashingtheplatesfollowedbya 1-hincubationwith AP-conju-gated22.3 inPBS/1%BSA resulted inamarkeddecrease in theoptical density, conceivablybecause of dissociation of theIL-6/sIL-6R

com-plex.Afterwashingtheplates,theassaywasdevelopedasdescribed for the sIL-6 R ELISA. Thebackground optical density usingPBS/2%

BSAasatestsamplewasalwaysbelow 0.190. The resultsareexpressed

asspecific optical density obtainedby subtractingthenonspecific opti-cal density found in wells coated with PBS alone to the optiopti-cal density observed in wells coated with the monoclonal antibody 34.1 to human IL-6. To comparethe levelsof IL-6 present in the IL-6/sIL-6 R com-plexwith the amount ofIL-6 measured by thehybridoma growth factor (HGF) assay (see below), the amountof IL-6wasextrapolated from a standardcurveobtained by addingincreasingconcentrations ofhuman recombinant IL-6 derived from Chinese hamster ovary cells (Genzyme Corp., Cambridge, MA)to a referenceserum fromahealthyadult control. Thisserumhad 100 ng/ml of sIL-6 R and contained neither detectable IL-6 in the HGF assaynordetectableIL-6/sIL-6Rcomplex

(specificOD0.000).Toobtainacceptableresolutionover awide range

ofIL-6concentrations, opticaldensityat405 nMwasread after

differ-enttimes ofincubationwith the substratep-nitrophenyl phosphate (see Results andFig. 5).

AssayforIL-6. IL-6 levels weremeasured using the hybridoma cell line B9 (kindly provided by Dr. L. Aarden, Netherland Red Cross, Amsterdam), asdescribed previously (10). Chinese hamster ovary cell-derived recombinant human IL-6 (Genzyme Corp.)wasusedto

culture B9 cells and as a standard in the assay.L-6 levels were ex-pressed, unless otherwise indicated,in HGFunits-(HGFU), where 1 HGF Uis the amount ofIL-6 required to achieve half-maximal prolifer-ation of B9 cells. The conversion from HGF U/ml to pg/ml was per-formed usingthe amount ofhumanrecombinant IL-6 required to achieve half-maximal proliferation of B9 cells in each assay, that is,

- 4.5 pg/mlof human recombinant IL-6.

Statistical analysis.Data wereanalyzedusingtheMann-Whitney

Utestforunpairedsamples and theSpearman correlationcoefficient.

Results

sIL-6Rlevelsinpatients with JRA.Patients with systemicJRA presented serum sIL-6 R levels (114.6±37.7 ng/ml)

signifi-cantlylower(P < 0.01 ) thanthose found in healthy children

( 161.2±45.5ng/ml),whilepatients with polyarticularor pau-ciarticular JRA had serum IL-6 R levels (176.7±66.9 and

165.9±51.5 ng/ml, respectively) comparable with those of healthychildren (Fig. 1). To rule out thepossibility that the

lowserumsIL-6 R levelsinpatients with systemicJRA could be due to thebindingofendogenousIL-6present in patients' sera tothe sIL-6R,thusmaskingthe epitope recognized by the monoclonalantibodiesusedinthe ELISA, we tested theeffect

of preincubation of normal sera

_:with

exogenous IL-6. As

li

E

300-CM

250-n -i

LUJ

J200 --J

r

(D 150

--J

100-w 50

0n

St.

CTRL SYST POLY PAUCI

JRA

Figure1.Serum sIL-6Rlevels inhealthycontrols

(CTRL)

and in patientswithsystemic (SYST),

polyarticular

(POLY),

or

pauciarti-cular(PAUCI)JRA.Thehorizontal continuous line_representsthe meanofeach group.

-4:"

4,,0

I

0

t 0

shown in Fig. 2 for two representative sera, the addition of 20 ng/mlofhumanrecombinant IL-6did not have an effect on the measurement of the sIL-6 R in the assay system.

Whenpatients with JRA weredivided accordingtotheir

treatment, nodifferences in serum sIL-6 R levels were observed

(data not shown), thus suggesting that nonsteroidal antiin-flammatory drugs, prednisoneormethotrexate, donot have a

directeffect on the release of sIL-6 R in vivo. No significant

correlationof serum sIL-6 R levelswith erythrocyte

sedimenta-tionrate values or with C-reactive protein (CRP)

concentra-tionswas found in the three JRA onset types (data not shown). Inpatients with systemic JRA,serum sIL-6Rlevelswere

negatively correlated (r=-0.610,P <0.001 ) withserumIL-6 levelsmeasured with the B9 cells (Fig. 3).Tofurther evaluate the relationship between serum IL-6 andsIL-6 Rlevels, we measuredIL-6 and sIL-6Rlevels inserumsamplesobtained during the'febrile peakfrom twopatientswithsystemicJRA. AsshowninFig.4foronerepresentative patient, the increase inserumIL-6 levelswasassociated withadecreasein sIL-6

R,

whose levelswereinverselyrelated to body temperature.

sIL-6 Rwasalso found in synovial fluid samplesat nano-gram permilliliter concentrations withnoevident difference

amongpatients with systemic or pauciarticular JRA. As

re-portedpreviously ( 10,11), while IL-6 levelswere muchhigher insynovialfluidsthaninthecorrespondingserum,thiswas not truefor sIL-6 R,

suggesting

that, unlike IL-6, sIL-6Risnot

preferentially producedat,and thus releasedfrom, sites of in-flammation (Table I).

Presence of circulating IL-6/sIL-6Rcomplexinpatients

with

systemicJRA. To identify the circulating IL-6/sIL-6 R

complex,weconstructedanELISA basedonthe use ofa

mono-clonal

antibody

tohuman IL-6(34.1 ) forcaptureanda

mono-clonal

antibody

tothesIL-6R

(AP-conjugated

22.3) for detec-tion of theIL-6/sIL-6Rcomplex.Tocontrolforpossible

non-specific reactivity

secondarytothebinding of theserumsIL-6

Rtoplastic,all seratestedwereincubated in wells coated with PBS alone and in wellscoated with the monoclonal

antibody

34.1toIL-6.Alow backgroundwasfound withno

significant

differences in thebackground

optical

density obtained in wells coated with PBS alone between sera from patients with

sys-temicJRA(n =_{25) and}serafrom healthy controls (n = 12) (0.030±0.031 and 0.021±0.020,

respectively;

P=0.79). The

0

I.--J Li

-J (0

-j

Li (/)

100

50

* .0 0 * 0

* 0

0 * * *

. 0

80 110 140 170 200

SERUM sIL-6 R LEVELS (ng/ml)

Figure 3.Relationship between serum IL-6 and serum

sIL-6

Rlevels in patients with systemic JRA. SerumIL-6levels were measured using anHGF assayasdescribedin Methods. The dottedlinerepresents thedetectionlimit of the HGF assay.r = -0.618;P<0.001.

addition

ofincreasing concentrations of human recombinant IL-6to normal seraresulted ina dose-dependent increasein

specific optical density, demonstratingthat this assay system is

abletodetect theIL-6/sIL-6R

complex

formed

by

the

binding

ofexogenous IL-6 to its soluble receptor in human serum.

Dose-response curvesobtainedatdifferent times ofincubation with the substrate for the referenceserum areshown inFig. 5.

Asshown in

Fig.

6, healthy children had little ifany detect-able IL-6/sIL-6 R complex in their serum

(specific

OD

0.024±0.027),

while the

majority

ofserafrom

patients

with

systemic

JRAhad detectable levels ofIL-6/sIL-6 Rcomplex

(specific

OD

0.492±0.546)

withahighly significantdifference

withserafrom healthy controls (P<0.0001).

Comparison

of IL-6 levelsmeasuredby the HGFassayand thoseestimated in the

IL-6/sIL-6

Rcomplex,asmeasured by the

ELISA,

in the same 24 serum

samples,

lead us to

unexpected

results. As

shown in TableII,in

patients

with

systemic

JRA,serumIL-6 levelsestimated in the IL-6/sIL-6Rcomplexwerein therange

ofnanograms permilliliter andweremuch

higher

thanthose measuredby the HGFassay(mean ratio IL-6 in

IL-6/sIL-6

R

complex/IL-6 measured by the HGFassay:22.4). Of

interest,

when thesamecomparisonwasmadefor IL-6 levelspresentin

synovial

fluid

samples,

suchadifferencewas notobserved

(Ta-ble

II).

Moreover,

comparison

of the correlation between IL-6 levelsestimated by thetwoassaysystemsinsera orin

synovial

fluids showedthatresultsby thetwoassay systems arestrictly correlatedinsynovial

fluid

samples (r2 =0.78) butare much

SERUM 1

SERUM 2

0.0 0.5 t.o

O.D-Figure 2. Effect of preincubation of normal sera with recombinant human IL-6(20 ng/ml) on the detection of sIL-6 R. Normal sera wereincubated at 37°C for 45 min in the absence or in the presence of 20 ng/ml of recombinant human IL-6 and then tested in the ELISA for sIL-6R asdescribed in Methods. Results from two representative

sera areshown. Background, white box; no IL-6, dotted box; IL-6 (20 ng/ml), striped box.

E

Co

c

a:

ni

100-

90- 80- 70- 60- 50-40

400-

t-\

300-C, I

*s

200-J

D

100-0

- 18 20 22 24

HOURS

-39 C.)_{_} -38 O

0.-0

0

ui

-37

I-a o

136

2 _

Figure 4. Serum IL-6 andsIL-6Rlevels during the febrile peak in

representative patientswith systemic JRA. Body temperature, closed squares;serumIL-6, closedtriangles; serum IL-6 R, closed circles.

2116 DeBenedetti,Massa,Pignatti,Albani,Novick, andMartini

-TableI.IL-6andsIL-6 RLevelsinSynovial Fluid and

intheCorresponding Serum Samples ofRepresentativePatients

withSystemic orPauciarticularJRA

sIL-6 R IL-6

Synovial Synovial

fluid Serum fluid Serum

ng/ml HGFU/ml Systemic

1 64.8 99.4 18500 100

2 142.6 148.9 13000 39

3 138.3 168.9 5600 19

Pauciarticular

1 175.6 185.1 5800 18

2 80.2 148.1 6200 <4

3 126.7 167.1 7600 <4

4 60.0 134.9 600 <4

lesscorrelatedin serum (r2= 0.44)(Fig. 7). Taken together,

the datainTableIIand Fig.7 suggestthatquantities of IL-6

presentinserum aremuch

higher

than thoseestimated by the

HGF assay andthatthefactor(s) interfering with the detection of IL-6 by the HGFassayispresentinserum,butnotin syno-vial fluid.

To evaluate the in vivo

biological

relevance ofthe large quantities of IL-6 estimated in

circulating

IL-6/sIL-6 R com-plexes, we compared thecorrelation betweenserumCRP

con-centrations and the levels ofthe IL-6/sIL-6Rcomplex with the correlation betweenserumCRP concentrations andserum

IL-6 levels measuredwith theHGFassay. Asshown in

Fig.

8, CRP concentrationsweremuch morecorrelated withthelevelsof

thecirculating IL-6/sIL-6 Rcomplex (n = 24,r = 0.713, r2

=0.51, P<0.0001 ) than with theserumIL-6levelsmeasured

with the HGFassay (n= _24,r= _{0.435, r2} =0.19, P=0.05).

Discussion

InthisstudywehavemeasuredserumsIL-6 R levels in

patients

with JRA and

developed

an ELISA for the detection of the

Ut) 1.0

z

0.011

0~

UJ 0A. 00

0.002

0.04 0.1 1 10 100

EXOGENOUS HUMAN REC. IL-6 (ng/ml)

Figure 5. Dose-responsecurvesof the ELISA for theIL-6/sIL-6 R

complex. Results fromonerepresentativeserumfromahealthyadult controlareshown. Thetestsamplewasincubatedat37°Cfor 45 min in the absenceorin the presence ofincreasingconcentrations of humanrecombinant IL-6 (range 0.062-128 ng/ml) and then tested in theELISAfor the IL-6/sIL-6 Rcomplexasdescribed in Methods. Theoptical densityat405nMwasreadaftera30-min(closed square),a1-h(closed triangles),anda4-h(closed circles)incubation withthesubstratep-nitrophenylphosphate.

U)

z

wL

0 -J

0

C-)

CLA

0-C/)

2.0

1.5

1.0

0.5-0.0

Figure 6.Serum IL-6/sIL-6 R complex in healthy controls

(CTRL)and inpatientswith sys-* temic JRA (SYSTJRA). The re-: sultsareexpressedasspecific

opti-caldensity calculatedasdescribed . s...----.k--- in Methods. The horizontal dotted

CTRL SYST line represents the upper limit of

JRA controls (mean +2 SD).

IL-6/sIL-6 R complex in biological fluids, demonstrating that ahighamountof IL-6 circulatesas anIL-6/sIL-6Rcomplex. The serumsIL-6 R levelsfound in our age-matched con-trols areapproximately twofold higher than thosereported

re-cently byHonda et al.(8) and by Gaillardetal.(17)in healthy adults. Thesedifferences could be because ofdifferencesin the

antibodies usedorpossibly differences inthe ageofthe popula-tiontested.Indeed,wefoundthat serumsIL-6Rwashigherin ourage-matchedcontrolpopulation(mean level 161.2ng/ml) thanin a group of healthyadults (n = 10; mean level 104.5 ng/ml).

Inour study, among patients with JRA, only those with

systemicJRA werefoundto have serum sIL-6 R levels

signifi-cantly lowerthanthose ofhealthy controls comparable with age. As far as weknow,only twostudiesareavailable on serum

sIL-6Rlevels in humandiseases. Patientswith HIVinfection and patients with multiple myeloma have been reported to

have serum sIL-6 R levels significantly higher than healthy

controls(8, 17). These datasuggest thatdifferentmodulation ofthe invivo levels of sIL-6Roccursin differentdisease condi-tions. This maypossibly be because of quantitative differences inIL-6production in vivo.Indeed, serum IL-6 levels foundin patients withHIVinfectionorwith multiplemyeloma (18-20) are muchlowerthanthose presentin patientswith systemic JRA(10).Apossibleroleofthe increased production ofIL-6 indownregulating the levels of sIL-6 R in patients with

sys-temicJRAis suggested by the finding ofanegative correlation

TableII.Comparisonofthe Levels ofIL-6Measured by theHGF Assayand Those EstimatedintheCirculating IL-6/sIL-6R ComplexinSerafromPatients with SystemicJRA andin

SynovialFluidsfromPatientswith Systemic orPauciarticularJRA

HGF assay IL-6/sIL-6RELISA

ng/ml ng/ml

Serum

(n=24) 0.335±0.334 6.850±8.971

(0.022-1.44) (0.26-32.0)

Synovial fluid

(n= 12) 45.2±43.2 39.8±35.4

(1.4-150.0) (1.6-118.0)

24 serafrompatientswithsystemicJRA and 12synovialfluid

sam-ples, 3 frompatientswithsystemicand 9frompatientswith pauciar-ticular JRA,weretested in both assay systems. Resultsareshownas

A

r=0.665

R2=0.44

0.1

***

.0

0 * * *

3 10 100

SERUM IL-6 (HGF U/ml)

B

r=0.881

R2=0.78

0.1

0.0 10

0

0

300 1000

0

10000

SYNOVIAL FLUID IL-6 (HGF U/mi)

Figure7.Comparisonof the correlation between IL-6 levels measured

bythe HGFassaywiththe levels ofIL-6/sIL-6Rcomplexinserum

(A)and insynovialfluid(B).

ever, preincubation of control sera withincreasing concentra-tions of exogenous human recombinant IL-6 resulted in a

dose-dependent

increase in

optical density, demonstrating

that thisassay systemis abletodetect the

IL-6/sIL-6

R

complex

formed

by

the

binding

ofIL-6toits solublereceptorpresentin human serum. This also confirms thefindingsof Honda et al.

(8)

and ofGaillardetal.( 17)thatcirculatingsoluble IL-6 R is

abletobind IL-6. Almost allseraof

patients

with

systemic

JRA

displayed high optical density

inthisassaysystem,

demonstrat-ingthepresenceof elevated serumIL-6/sIL-6Rcomplex lev-els. These resultssuggest

strongly

that

IL-6,

oratleastpartof

it,

circulates bound toits soluble receptorin patients with

sys-temicJRA. To the best ofourknowledge,this isthe first demon-stration of thepresenceofcirculating cytokine/soluble

cyto-kinereceptorcomplexesin human diseases. Our data are con-sistent with the following hypothesison thebiology ofIL-6: IL-6 produced at sites ofinflammation (e.g., joints) or

pro-duced in theperipheralblood inresponsetostimuli releasedby inflammatorysites(e.g., IL-lorTNF-a)binds the serum sIL-6 R and it is vehiculated in the blood as anIL-6/sIL-6 R

com-plex.Thisbinding may protectIL-6 fromprotease

degradation,

in a manner similar to what has been described for IL-4 and the

soluble IL-4receptor(24), and/or stabilize IL-6 bioactivity,as has been demonstrated for TNF-a and its soluble receptors (25). TheIL-6/sIL-6 R complex will thus associate withthe 130-kDsignal transducing proteinonthe surface oftarget cells and mediate IL-6bioactivities.

Comparison ofserum IL-6 levels measured by the HGF

assay andthose estimated in circulating IL-6/sIL-6 R

com-betweenserumsIL-6 R levels withserumIL-6 levels, measured

with the HGF assay, and by the decrease in serum sIL-6 R

levels thatwas_{associated with the increase in}serumIL-6 levels

during the febrile peak. The mechanisms of the production of sIL-6Rhavenotyetbeen clarified (21, 22). Nevertheless,our

datasuggestthattwopossible mechanisms,notnecessarily

mu-tually exclusive, may account for the decrease in theserum

sIL-6 R levels in patients with systemic JRA: (a) since IL-6 has

beenreportedtoinhibittheexpressionof the IL-6 Rgeneboth

in vivo and in vitro (23), adirect effect of increased in vivo

IL-6 productionontheexpression ofthe IL-6Rgeneis

conceiv-able; and (b) the decrease inserum sIL-6 R levelsmaybea

consequenceofaconsumption ofcirculating sIL-6Rduetothe

formation of IL-6/sIL-6 R complexes in thepresenceofIL-6

andof the subsequent binding andinternalizationofthe

com-plex bytarget cellsexpressing the 130-kD signaltransducing

protein.

This second hypothesis is based on the assumption that

circulating IL-6/sIL-6Rcomplexesarepresentinpatients with

systemic JRA. Since theserumsolubleformof IL-6Rand the

genetically engineered truncated form ofIL-6 R have been

showntobind IL-6 withabinding affinitysimilartotheentire

80-kD molecule (6, 8)andgiventheserumconcentrationsof

the sIL-6 R found in thisstudy,it is conceivabletohypothesize

that IL-6 circulates boundtoits solublereceptor.Toverify this hypothesis, we constructed an ELISA based on the use ofa

monoclonal antibody toIL-6 forcaptureand amonoclonal

antibodytosIL-6 Rfor revealingthepresenceof the

IL-6/sIL-6 Rcomplex. Using this approach we found noevidence of

circulatingIL-6/sIL-6 R complex in healthy controls.

How-r=0.435 R2=0. 19

p=0.05

E

D

l

en

D,

100

10

A

0

0 * .

0

* 0 0

.0 _*

0

0.6 10

SERUM CRP (mg%)

a

C:

co

-J

en

r=0.713 R2=0.51

p<0.0001

1.0

0.1

0.0

0.6 1

B

.

0

-@00

0000

* *. *

*

10

SERUM CRP (mgX)

Figure8.Correlation ofserumCRPconcentrations withserumIL-6

levels measuredby the HGFassay(A)orwith circulatingIL-6/sIL-6 Rcomplexlevels(B)inpatients with systemicJRA.

2118 De Benedetti, Massa, Pignatti, Albani, Novick, and Martini

a

0

Ix

-J

W

(D

-J

D en

Co o

J

(O

N CD

-J

0

-J

La.

-J

0 z

(n

.

plexes led us totheunexpected finding that, in patientswith

systemic JRA, IL-6 was present in nanogram per milliliter amountsinthecirculating IL-6/sIL-6 R complexes compared withthefewhundreds of picogramspermilliliter measured by

the HGF assay. Therefore, ourdata suggestthat in systemic

JRA, butalsopossibly in other disease conditions, IL-6

circu-lates in much higher amounts than those measured by the

HGFassay.Thepossibility that the HGFassaydetects onlya

portion of IL-6presentin humanserumis alsosupported by

theconsistent findingthatIL-6 levels measured with the

hepa-tocyte-stimulating

factor assayhave been found tobemuch

higher than those measured by the HGFassayina

variety

of disease states(26-29). More recently, May et al. (30) have demonstratedthat the bulk of IL-6presentin

peripheral

blood circulates intwohigh molecular weight complexes (100-150

and400-500 kD) and that

this

IL-6isnotdetectedby the HGF

assay (30). Ofnote, amongtheproteinspresentinthe 100-150-kD complex, a 50-kD material was present, consistent with the molecular weight of the human natural sIL-6 R.

Taken together, the

discrepancies

betweenserum IL-6 levels measuredby the HGFassayandby thehepatocyte-stimulating factorassay(26-29), ourresults, and those

of

Mayetal.(30) raisetheissue of the biological role ofthelargequantities of IL-6 present in the peripheral blood. In ourstudy, thestrict correlation

of

serumIL-6 levels measured in the IL-6/sIL-6 R complex ELISA withCRP concentrations in patients with

sys-temic JRAsuggests thatthe

high

amounts

of

IL-6presentin the

circulating IL-6/sIL-6

R

complex

are

biologically

relevant in vivo,at least as farasthe induction by IL-6

of

acutephase proteinproduction.

Inconclusion, in this studywereportedadecrease in sIL-6

Rlevels inpatients with

systemic

JRA,adisease characterized byamarked increase in serumIL-6 levels. We

developed

an assay systemfor detectionand measurementof

circulating

IL-6/sIL-6Rcomplexes and demonstrated that IL-6

circulates

as anIL-6/sIL-6Rcomplex. We have also

found

thatlarge

quan-tities of IL-6,notdetectedby the HGFassay, are presentin the

peripheral

blood of

patients

with

systemic

JRAas

IL-6/

sIL-6R

complexes, whose in vivo

biological

relevanceis suggested by their strict correlation withserumCRP concentrations.

Acknowledgments

This workwassupportedby theIstituto di Ricovero e Cura a Carattere

ScientificoPoliclinico San Matteo, Pavia, Italy and by the Istituto di

RicercaCesare Serono, Ardea, Italy.

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