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On the binding of tumor necrosis factor (TNF) to heparin and the release in vivo of the TNF binding protein I by heparin

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On the binding of tumor necrosis factor (TNF) to

heparin and the release in vivo of the

TNF-binding protein I by heparin.

M Lantz, … , E Nilsson, I Olsson

J Clin Invest.

1991;

88(6)

:2026-2031.

https://doi.org/10.1172/JCI115530

.

Tumor necrosis factor (TNF), a protein released by activated macrophages, is a central

mediator of the host response to infection and inflammation. The TNF-binding protein I

(TNF-BP-I) is a soluble fragment of the p60 transmembrane TNF receptor and an antagonist

to TNF. The level of serum TNF-BP-I was found to be increased in patients with renal

insufficiency as a result of a decrease in the glomerular filtration rate. During hemodialysis

of patients with renal failure there was a rapid but transient increase in serum TNF-BP-I.

This increase was found to be caused by heparin given before dialysis and a similar

dose-dependent response to heparin was observed also in healthy individuals. The finding of a

repeated release of TNF-BP-I into the circulation with intermittent injections of heparin

indicates that TNF-BP-I is present both in a storage pool and in a circulating pool. The

mechanism for the heparin-mediated release of TNF-BP-I was not explained; TNF-BP did

not show affinity for heparin. On the other hand, TNF was found to have affinity for heparin

and it could also be dissociated from heparin by TNF-BP-I. It is suggested that heparin-like

molecules of the extracellular matrix can retain TNF in physical proximity with target cells

and restrict the actions of TNF and protect against systemic harmful manifestations.

Research Article

(2)

On

the Binding of Tumor

Necrosis Factor (TNF)

to

Heparin

and the Release In Vivo

of the TNF-binding Protein I by

Heparin

Mikael Lantz,* Hans Thysell,t Eva Nilsson,* and Inge Olsson*

*Division ofHematology, DepartmentofMedicine, andtDepartmentofNephrology, University Hospital, S-22185 Lund,Sweden

Abstract

Tumor necrosis factor (TNF), a protein released by activated macrophages, is a central mediator of the host response to

in-fection and inflammation. TheTNF-binding protein I (TNF-BP-I) is a soluble fragment of the p60 transmembrane TNF receptor and an antagonist to TNF. The level of serum TNF-BP-I wasfound to be increased in patients with renal insuffi-ciency as a result of a decrease in the glomerular filtration rate. During hemodialysis of patients with renal failure there was a rapid but transient increase in serum TNF-BP-I. This increase wasfound to be caused by heparin given before dialysis and a

similardose-dependent response to heparin was observed also in healthy individuals. The finding of a repeated release of

TNF-BP-I into the circulation with intermittent injections of heparin indicates thatTNF-BP-I is present both in a storage

pooland inacirculating pool.Themechanism forthe

heparin-mediatedrelease of TNF-BP-I was notexplained;TNF-BP did not show affinity for heparin. On the other hand, TNF was

found to haveaffinity for heparin and it could also be

disso-ciatedfromheparinby TNF-BP-I. It is suggested that heparin-like molecules of the extracellular matrix can retain TNF in physicalproximitywith target cells and restrict the actions of TNF and protect againstsystemic harmful manifestations.(J.

Clin.Invest. 1991.88:2026-2031.)Key words: inflammation-endotoxin*glomerular filtration*lymphotoxin*growthfactors

Introduction

Tumornecrosis factor

(TNF)'

wasdiscovered inanattempt to

explain hemorrhagic necrosis oftumors in relation toacute

bacterial infections (1, 2).TNFisproduced byactivated

macro-phages asaresponsetolipopolysaccharides of Gram-negative bacteria (1, 2)andplaysamajorroleinendotoxic shock(3, 4),

which can lead tocardiovascularcollapse, organ failure,and death. It is also knownas apleiotropicagent which

participates

in a numberofways in the defense ofthe organism

against

infectious agents (5). Obviously some ofthe effects can be

harmfulif they are notproperly controlled, leadingtocascade

reactions, suchasinendotoxic shock. Mechanisms of regula-tion are therefore important and mechanisms by which the

AddresscorrespondencetoMikael Lantz,M.D.,ResearchDepartment

2, E-block, LundHospital,S-22185Lund,Sweden.

Receivedfor publication19 June1990 andinrevisedform17July

1991.

1. Abbreviations usedinthis paper: TNF,tumornecrosisfactor;

TNF-BP-I, TNF-binding proteinI.

destructivepower of TNF is suppressed have potential clinical

applications. The action ofTNFis likely to be regulated at

differentlevels, e.g., at the levelof production ofTNF, at the levelof interactionwith TNF anditsreceptor, and at the post-receptor level. In addition, the newly discoveredTNF-binding protein I (TNF-BP-I) may play a protective role against the

adverseactivities ofTNF(6-10).TNF-BP-Ihas beenpurified from urine and consists ofa 30-kDcysteine rich glycoprotein

withouthomologiestoknownproteinsequences (8-10). It in-hibits thebiological activitiesof TNF bybindingto TNF with

high affinityand thus prevents TNF frombindingtoits trans-membrane cellular receptor (6-10). Immunological

cross-reac-tivity of antibodiesto TNF-BP-IwithcellsurfaceTNF

recep-torssuggests that TNF-BP-Iconstitutesasolublefragment of

the p60 transmembrane TNF-receptor (11, 12). This finding

wasconfirmedby molecularcloning ofthep60TNF receptor

(13). Recentlyasecond TNF-binding protein,TNF-BP-II,has

been

purified

fromhumanurineandmolecularly cloned (12,

14).TNF-BP-IIexhibits identity with the extracellular ligand-binding domain ofthe p80 TNF receptor(14).Thep60 and p80 TNF receptors show homology, but not identity, in the extracellular

ligand-binding

domain butpossessdifferent intra-cellular domains, suggesting alternative modes in signalling

anddifferent function (13-16).

In our initial work on TNF-BP-I (7, 8) excessively high

levels ofTNF-BP-I were observed in serumofpatients with

renalfailure.This findingled us toinvestigatetherelationship

between serum TNF-BP-I and renal function as well as the

effectonTNF-BP-I of

hemodialysis

treatment.The resultsof

these

investigations

arepresentedinthisreport. Wedetecteda

transient effect of

heparin

ontheserumlevelofTNF-BP-Iand

ourresultssuggest thatapool ofTNF-BP-Iispresent invivo from whichTNF-BP-Icanberapidlyreleased. Theaffinity for

heparin

wasinvestigated forboth TNF and TNF-BP-I andwe

foundthat TNF representsa

heparin-binding cytokine

while TNF-BP-I showed no affinity for

heparin.

Our observations

may suggest the existence of mechanisms by which TNF is

restricted

to alocalfocus

leading

to

protection against

its

sys-temic effects. Methods

Materials. Heparin,

Fragmin®,

and protamine chloride were from KabiVitrum,Sweden.Heparin-SepharosewasfromPharmacia,

Upp-sala, Sweden. Phorbol12-myristate 13-acetate(PMA)wasfrom

Sigma

Chemical Co.,St.Louis,MO. Endothelial cellgrowthfactorwasfrom

BoehringerMannheim, Bromma, Sweden. RecombinantTNF

(pro-ducedbyGenentechInc.,SanFrancisco, CA)andamonoclonal anti-body to TNF were kindly provided by Dr. Gunter

Adolf,

Ernst

Boehringer Institute, Vienna,Austria. Native TNF-BP-Iwas

purified

asdescribed before and containedone30-kD chain under both reduc-ing andnonreducingconditions(8).Amonoclonalantibody

(TBP-1)

wasobtained by immunizing mice with purifiedTNF-BP-I

(Adolf,

G.R., andI. Apfler.Submitted for

publication.) Polyclonal

antibodies

toTNF and TNF-BP-Iwereobtainedbyimmunizingrabbits with

re-2026 M.Lantz, H. Thysell,E.Nilsson, andI.Olsson J.Clin. Invest.

© The American Society for Clinical Investigation, Inc. 0021-9738/91/12/2026/06 $2.00

(3)

combinant TNF and native TNF-BP-I, respectively.The antibodies againstTNF werespecific and did not cross-reactwith TNF-BP-Ior

lymphotoxin; neither did the anti-TNF-BP-I antibodies cross-react withTNF orlymphotoxin (1 1).

Collectionofserumsamples.Informedconsent wasobtained from allindividualsparticipatinginthisstudybefore collection of the sam-ples. Serumwaspreparedfromvenousblood drawn frompatientson regularhemodialysis,continuousambulatoryperitoneal dialysis,with different renal disordersnot ondialysis,andhealthyindividuals. The glomerular filtration ratewasdeterminedby useof 51Cr-EDTA (17) andserumcreatinineasdescribed(18). Sampleswerecollected from 19

patientsbeforehemodialysis,at30 min andatthe end ofdialysis.The

following membranes were used for dialysis: cuprophane, polysul-phone, polycarbonate,cellulosa acetate, polyacrylonitrile, and

poly-amide. Forcorrection of changes inserumTNF-BP-I duetoreduction of extracellular volumeduringdialysis,determination ofcystatinC of serum wasperformedasdescribed(19).Allpatientsonhemodialysis

received5,000-15,000Uof heparinatthebeginningofdialysis; sam-plesweredrawnbefore and after theinjectionofheparin.Inaddition, sampleswerecollectedatfrequentintervals fromhealthyindividuals

receivingintravenousinjectionsof500, 2,000,5,000,and10,000Uof heparin, 5,000 U ofFragmin",or20 mgprotaminechloride. Serum sampleswerealso collectedduringcirculation in vitro ofheparinized

blood. Fresh heparinized donor blood (500 ml) was recirculated

throughacuprophanedialyzerfor 2 h. The blood volumewaskept

constantbyinfusion ofaRingersolution.

Statistical parameterswerecalculatedbyuseofStatViewSE, Mac-intosh.

TNF-BP-I- andTNF-freesera wereprepared byapplyingpooled serumfrom healthy individualsto acolumnof

anti-TNF-BP-I-Sepha-rose equilibratedwith column buffer(0.15 mol/literNaCl, 5 mmol/

liter Hepes, pH 7.4)at aflowrateof 10 ml/h. Fractions of 10 mlwere collected and tested forthe presence ofTNF, TNF-BP-I, and anti-TNF-BP-Iwith ELISA technique.

Aninhibition ELISAfordeterminationofTNF-BP-I.Aninhibition ELISA, where TNF-BP-I competes for bindingto aconstantamountof

polyclonalanti-TNF-BP-I,wasdevelopedfor detection ofserum TNF-BP-I.Samples of 50 Mlwereloaded in 96-well round-bottom incuba-tionplates(2-62170; Nunc, Roskilde, Denmark).A 50-Mlaliquot of

polyclonal anti-TNF-BP-I diluted 1:4,000in incubation buffer(0.1 mol/liter NaCl,0.05mol/liter NaH2PO4,0.05% Tween-20,adjustedto pH 7.5 withNaOH)wasaddedtoeach wellfollowed by incubation

overnight at 4°C. In parallel, 96-well flat-bottom immunoplates

(4-39445;Nunc)werecoatedwith 100 Ml per well of TNF-BP-I dilutedto 7.5ng/ml in 0.1 mol/literNaHCO3, and incubatedovernight at room temperature followedby three washes inwashingsolution(0.15 mol/

literNaCl,0.05% Tween-20). Thecontentof the incubation plate was transferredtothe plate coated with TNF-BP-I whichwasincubated for 3 h at4°C and washed three times.Analiquot of 100Ml secondary

antibody,goat anti-rabbitantibody conjugatedtobiotin (Vectastain ABCkit,AK5001; Vector Laboratories Inc., Burlingame, CA), diluted 1:200 inincubation buffer supplemented with 1 mg/ml BSA was added toeach well. The plateswereincubated for 1 h atroom temperature followedbythree washes. To enhance thesignal, 100Mlofasolution

containingavidin-biotincomplexeswithalkaline phosphatase

conju-gated tobiotin(VectastainABCkit, AK5001; Vector Laboratories) wasadded toeach well. The plates were incubated for 1 h at room temperature, washed three timesfollowed by addition of phosphatase substrate(Sigma 104) dissolvedin 1 mol/liter diethanolamine buffer pH 9.8supplemented with 0.5

Mmol/liter

MgCl2, 100

Ml/well.

The ab-sorbancewasmeasured at405nminanautomatic Titertek multiscan ELISA plate reader. Valueswere calculated from a standard curve basedonfreshlyprepareddilutions ofTNF-BP-I(0.3-30

Mg/liter)

in TNF-BP-I-free poolserum.Thelowest detectable amount was 0.3

jg/

liter.

Sandwich ELISA technique for determinationofTNFand TNF-BP-I.Asandwich ELISA was developed for detection ofTNF, in incu-bation buffer and serum, and for detection of TNF-BP-I in cellular

supernatants containing 10% FBS. Nunc 96-well immunoplates (4-39445) were coated with monoclonal antibody to TNF or TNF-BP-I dilutedto2.5 ug/ml (40Ml/well) in 0.1 mol/liter NaHCO3 and incu-bated foratleast 3 horovernightatroomtemperature, followedby

three washes inwashingsolution(0.15 mol/liter NaCl,0.05% Tween-20). Samples of 100,lwereloaded in triplicate and incubated over-nightat40C followed by three washes in washing solution. Analiquot

of 100,lofapolyclonal antibodytoTNForTNF-BP-I, diluted1:3,600

in incubation buffer (0.1 mol/liter NaCl, 0.05 mol/liter NaH2PO4, 0.05% Tween-20, adjustedtopH 7.5 withNaOH) supplemented with 1 mg/ml BSA was addedtoeach well. Plates were incubated for 3 hat room temperature followed by three washes in washing solution. A peroxidase-conjugated goat anti-rabbit antibody (170-6513; Bio-Rad Laboratories, Richmond, CA) diluted 1:2,500 in incubation buffer sup-plemented with 1 mg/ml BSAwasprepared andanaliquot of 100M1

was added to each well. Plates were incubated for 1 h at room tempera-ture, washed three times inwashing solution followed by addition of

100,gl

oftetramethyl benzidine peroxidase (172-1066; Bio-Rad Labora-tories)toeach well. The absorbancewasmeasured at 660 nm inan automaticTitertekmultiscan ELISA plate reader. Valueswere calcu-latedfromastandardcurvebased onfreshlyprepared dilutions ofTNF (0.02-2Mg/liter) in incubation buffer or TNF-free serum. The standard for TNF-BP-I (0.03-3 Mg/liter) was diluted in RPMI 10% fetal bovine serum(FBS). The lowest detectableamountofTNFandTNF-BP-Iwas 0.02alg/literand 0.03 Mg/liter, respectively, and the detection of neither TNFnorTNF-BP-Iwasaffected byheparin.

Investigation of heparin affinity. Heparin-Sepharose (0.5 g) was swollen and washed in 100 ml of distilledwaterfor 15min. Freshly preparedheparin-Sepharose (1-2 ml)waspacked in acolumn, equili-brated with column buffer (0.15 mol/liter NaCl, 5 mmol/liter Hepes, and 0.1% BSA, pH 7.4). TNF (15-150 ng) or TNF-BP-I (100-1,000 ng) wasapplied,allowed tobind for 30min,andelutedwith column buffer containing 0.2-1.5 mol/literNaClorheparin 1-100 U/ml, withaflow rateof 20 ml/h. Fractions of 1-3ml werecollected anddialyzed against 0.1mol/liter NaCl, 0.05 mol/L NaH2PO4, pH 7.5, followed by determi-nation of TNF or TNF-BP-I with ELISA technique.

Preparationofsupernatants from cells treated with heparin. Cells wereincubated at 1 x106/ml for 3, 8, and 24 h at370CinRPMI 1640 with 10% FBS in the presence or absence ofheparin, 50 U/ml, or PMA, 10ng/ml. Themedium of the human umbilical cord endothelial cells was supplemented with endothelial cell growth factor (200Mg/ml).The following cellswereobtained from American Type Culture Collection, Rockville, MD: HL-60, human promyelocytic leukemia; U-937, hu-manhistiocytic lymphoma; Hep G2, human hepatoma; HeLa, human epitheloid cervix carcinoma;A549, humanlung carcinoma and HU-VEC, human umbilical cord endothelial cell. PMN and mononuclear cells (mono)wereisolated from human heparinized whole blood by centrifugation inLymphoprep'according to the manufacturers

de-scription;NyegaardCo., Oslo, Norway. Samples were stored frozen at -20'C until analyzed with a highly sensitive and specific sandwich ELISAforTNF-BP-I.

Results

Serum

levels

ofTNF-BP-I inrenal

insufficiency.

TNF-BP-I

de-termined in serum from 10 healthy individuals was foundto varybetween5and10,ug/liter.Serumfrompatients on regular

hemodialysis or continuous ambulatory peritoneal dialysis contained 117±9.2

jig/liter

(X±SD), n=24, and 114±7.8

,g/

liter(X±SD), n=14,TNF-BP-I, respectively. We investigated

ifthe increased levels of serum-TNF-BP-I in patients with

chronic renal failure couldbe explained by a decrease of

glo-merular filtration rate. Clearance of Cr-EDTA correlated nega-tively and S-creatininepositivelywith serum-TNF-BP-I (Fig. 1).However,thelevels ofserum-TNF-BP-I in patients on

(4)

A

0

* .0

00

0

VSO@

40 60 80 100 120 140 Cr-EDTA-clearance ml/min-1 (1.73m2)1

B

200 00 00 80100

S-Creatinine (jimol /L)

Figure1.The relationshipbetweenserumTNF-BP-I and the values of

Cr-EDTAclearance,r=0.506,P=0.0003, (A) andserumcreatinine, r=0.774,P=0.000 1,(B)in patientswith varyingglomerular

filtra-tionrate.

levels expected solelyonthe basis of glomerular filtrationrate.

Thus, factors in addition to kidney function may affect the level ofserumTNF-BP-I.

Effects ofhemodialysison serumlevelsofTNF-BP-L Dur-inghemodialysis the level of serum-TNF-BP-I increased

rap-idlyfrom 117±9.2,ug/liter (X±SD)toamaximum of240±8.2

,gg/liter

(X±SD) and decreased slowly to 160±11.3 Ag/liter

(X±SD)attermination ofdialysis.Fig.2showsatypical time course for serum TNF-BP-I during hemodialysis. The

in-creasedlevelsofserumTNF-BP-I returnedtothe basal initial

levels after termination of dialysis (datanotshown). The maxi-malincrease ofserumTNF-BP-Iwasreached after5-15minin

allpatients investigated. Thesametimecourseforserum

TNF-BP-Iwasobserved independent onthetypeofdialysis mem-brane used. We furtherinvestigated the role of the dialysis membrane by circulating heparinizedblood in vitro and found

-i

~1-Im

a20

0-LL

z

2 cn,.

1001

120

180

Tmne

(nu*tes)

Figure2. Results from determinationsofserumTNF-BP-Iduring hemodialysiswithacuprophane dialyserinapatientwithchronic

renal failure.

that the levels of serum TNF-BP-I were unaffected (data not shown). Thus the increase in serum TNF-BP during dialysis is not causedby the dialysis membrane itself.

Theeffect ofheparinonreleaseofTNF-BP-Iintothe

circu-lation. Allpatients undergoing hemodialysis receivedabolus

injection of 5,000-15,000 U of heparin before dialysis. Actu-ally, the heparin injection by itself was found to double the level of serum TNF-BP followed by a slow decrease with time exactly as in patientsundergoing hemodialysis (Fig. 2).

In more than three independent control experiments the standard for TNF-BP-I was diluted in serum supplemented with increasing amounts ofheparin to rule out that the increase ofTNF-BP-I was caused by heparin itself. Results from one controlexperiment are given as absorbances for the 10 ng/ml value of the standard in the presence of 0, 10, 100, and 1,000

U/mlof heparin and the values were as follows: 0.585±0.080 (X±SD), 0.567±0.071 (X±SD), 0.546±0.039 (X±SD), and 0.597±0.051 (X±SD). No significant difference could be dem-onstrated in the values obtained in the presence of various amounts of heparin.

To rule out that the heparin-induced release of TNF-BP-I was restricted to patientswith renal failure, heparin was

in-jected intravenously into healthy individuals. A twofold in-crease in serum TNF-BP-I was observed within 5 min after

injection of2,000 U or more ofheparin(Fig. 3). Lowmolecular weight heparin, FragminO, also induced release of TNF-BP-I

intothecirculationbut theeffectwas less pronounced as

com-paredtothatofheparin (Fig.3). In oneexperimentheparinwas

injected repeatedly four times with 1-h intervals (Fig. 4). An

identical increasein serumTNF-BP-Iwas observed eachtime.

Theseresults suggest that TNF-BP-I is equilibratedbetweena

circulating and a storage pool. An alternative explanation is

30

Imt 20 30.

z

10°

-I~~~~~~~C

20J

0.

0 60 12018

iniiuaa

fe adinstato oftvaiou doesofhearn:50

laiosi bewenhpri n

Smnvausof

eu N-PIi

U-z E 2

CO) 10

0

o

~~

~~do

1~o 18o0

Time(minutes)

Figure3.Results from determinationofserumTNF-BP-1 inahealthy

individual after administrationof various dosesofheparin:500U

(.

.),

2,000U(o

~~o),

5,000U

(.

in), 10,000U

(0

o),or5,000Uof

Fragmin* (A~-t)

Adose-dependent

re-lationshipbetweenheparinand5-mmnvaluesofserumTNF-BP-I is

2028 M.Lantz,H. Thysell,E.Nilsson,andI Olsson

-i

,30-0C

m

20-z

10.

cn n.

Im60

a)O40

E

22

(5)

1.

120

180

Time (minutes)

Figure 4.Results fromdetermination ofserumTNF-BP-I ofahealthy

individualwhoreceivedhourlyintravenousinjectionsof2,000U of heparinfour times(indicatedbyarrows).

thatheparin inducesproteolytic cleavageof cell surfaceTNF

receptors.However, treatmentofvarious cell lines with hepa-rin did not increase the release of TNF-BP-I into thecellular

supernatants (Table I). When protaminechloride (20 mg), a

competitive antagonisttoheparin, wasinjected 5 minbefore

administration ofheparin, the release of TNF-BP-I intothe

circulationwaspartiallyblocked(Fig. 5). However, injectionof anadditional dose ofheparin60 min later gaveafull response

onthe release of TNF-BP-I. Injection ofprotaminechloride

alone did not affect the serum level of TNF-BP-I (data not

shown).

Investigation of

heparin-binding

ofTNFand TNF-BP-L TNF-BP-Idid not show anyaffinityforheparin (Fig.6A).On

theother hand TNF boundto aheparincolumn in 0. 15mol/ liter NaCland waselutedby0.5mol/literNaCl

(Fig.

6B).In a

separate experiment increasing concentrations ofNaCl were

used for elution and it wasfound that noelution took place

Table I. Production of TNF-BP-I In Vitro in the Presence orAbsenceofHeparin

Cells Control Hepann PMA

HL-60 0.22 0.18 0.60

U-937 0.24 0.25 2.9

HeLa 2.1 2.0 6.0

A549 0.65 0.60 3.0

Hep G2 0.06 0.06 0.20

HUVEC 0.16 0.15 0.29

Mono 0.17 0.15 0.25

PMN 0.25 0.27 0.50

Supernatantswerecollectedfrom HL-60, U-937, HeLa, A 549, Hep

G2,human umbilical cord endothelial cells (HUVEC), mononuclear cells(mono), andPMNafter24 hin the presence or absence of

heparin(50 U/ml)orphorbolester, PMA, (10 ng/ml) as describedin Methods.PMAserved asapositive control. The amount ofTNF-BP-I

wasdetermined with a sensitive sandwich ELISA as described in Methods.Values are given in

gg/liter.

-J

m 20

U-.

I-E

10-2

U)

Cl)

II

II

__'4ll

120

180

Time

(minutes)

Figure5. Effect of protamine chlorideonheparin-inducedrelease of TNF-BP-I into the circulation. Protamine chloride, 20 mg (arrowI),

wasinjected 5min before administration of 2,000 U ofheparin

(arrow II).Anadditional dose of 2,000 U ofheparinwasgiven 1 h later(arrow III).

untilthe salt concentrationwasincreasedto0.5 mol/liter(data

notshown). TNF could also be eluted from the heparin column by heparin itself (Fig. 6 C). No binding of TNFtoSepharose

alone was observed (data notshown). These results

demon-stratethat TNF binds specificallytoheparin in vitro.

Complexes of TNF and TNF-BP-I prepared by mixing

TNF and TNF-BP-I inaratio of1: 10wereappliedto a

heparin-Sepharose column. No TNF-BP-Iandonlyasmall fractionof the applied TNF was found to bind under these conditions (Fig. 7 A). Obviously complexes betweenTNF andTNF-BP-I lack affinity for heparin sincenoTNF-BP-I wasfound inthe TNF containing fractions eluted from the column. Actually it

waspossibletoelute TNF from the heparin-Sepharose column withasolution of TNF-BP-I (Fig. 7 B).

Discussion

The major findings of this work are that TNF representsa

heparin-binding cytokine and that TNF-BP-I, although lacking heparin-binding capacity by itself, is released into the circula-tion by injeccircula-tion of heparin. These observacircula-tions may be of importance for the understanding of how the biological effects ofTNFareexpressed and regulated.

Like other low molecular weight serum proteins, serum

TNF-BP-I was found to increase with decreasing glomerular filtrationrate.Patients with terminal renal failure had 10 to 20 times higher levels ofserum TNF-BP-I than healthy individ-uals. In addition,weobserved that serum TNF-BP-I increased rapidly but transiently during hemodialysis. The transient

in-crease of serumTNF-BP-I during hemodialysis turned outto

be caused by heparin which is routinely injected before dialysis. Intravenous administration of heparin to healthy individuals

alsoresultedin anincrease of serum-TNF-BP-I.

(6)

7E

6I--0)

>400

z

200

20

1 1 0

0.5 M NaCI

5 10 15

0.5 M NaCI

I'

A

0.2M OAM 0.6M A

NaCI NaCI NaCI

E 3- A -40

0~~~~~~~~~4

U-o10 20

04

0 10 20

20

B

E

z

5 1b 15 20

b___~~T ii

0 5 10

Fraction no.

C

15 20

Figure 6. Investigation of the binding of TNF and TNF-BP-I to hep-arin.TNF(B and C) or TNF-BP-I (A) was applied to a 2-ml column of heparin-Sepharose which was eluted with 0.5 mol/liter NaCI (indi-catedbyarrowsinAandB) or 10(I),100(11),and 1,000 (III)U/ml

ofheparin (indicated by broken arrows inC).

heparin suggests ananalogy with other responses to heparin.

Lipoprotein lipase anddiamine oxidase are both rapidly re-leased into the circulation by heparin (20, 21). These sub-stancescanbindtoendothelial cells,abinding which is sensi-tivetoglycosaminoglycan degradingenzymes(22). However,

unlike lipoprotein lipase and diamine oxidase, TNF-BP-I lackedaffinity for heparin.Itis thereforeunlikelythat the re-lease ofTNF-BP-I is due to dissociation ofTNF-BP-I from heparan sulphateoftheendothelialcells as has been suggested

for

lipoprotein

lipase and diamine oxidase. The heparin-me-diated release ofTNF-BP-I ismorelikely explainedby an indi-recteffect ofheparinand not bydissociation ofa hypothetical

complex between TNF-BP-I and the extracellular matrix.

Any-how, ourdata suggestthat TNF-BP-Iispresentin vivo in a storage pool from which it canbe released by heparin. This

may alsoexplainthedifference in peaklevelsofserum TNF-BP-Ifoundforhealthy humansandpatients with chronicrenal

failure. Thus the basal levelsofserum TNF-BP-I mayreflect

thesize ofthesuggestedpool from whichTNF-BP-Iis released

andtherebyexplain theabovedescribeddifferences in the basal

levels ofserumTNF-BP-I. Thefinding ofrepeated releaseof

TNF-BP-I intothecirculation withintermittent injections of heparinalsostrongly supports thetheory ofastoragepool for

TNF-BP-I.Thispoolisinequilibrium with circulating

TNF-BP-Iand theequilibriumbetweenbound andcirculating TNF-BP-Iis affectedbyheparin.Theroleof heparin inthe releaseof TNF-BP-Iisstrengthenedby thefinding ofapartial inhibition

z1

I-B

Figure7. Prevention of binding of TNF to heparin byTNF-BP-I. TNF-BP-I(o o) and TNF ( )were mixed in a ratio of

10:1,applied to a 2-ml column of heparin-Sepharose, and eluted with

NaCiasindicated in A. In another experiment TNF alone was applied to acolumnofheparin-Sepharose, eluted with 200(I),400(II), and 600ng/ml(III)of TNF-BP-I (as indicated by broken arrows in B) followed by washing with 0.6 MNaCI(as indicated by a broken arrow,IV, in B).

of releaseofTNF-BP-Ibyacompetitive antagonisttoheparin, protamine chloride.

Whatmechanismsareresponsiblefortheheparin-induced

releaseofTNF-BP-I? Recentobservationsbased on immuno-logical cross-reactivity ofTNF-BP-Iwithp60transmembrane TNFreceptorsindicatethatTNF-BP-I isasoluble fragment of

the

p60

TNF receptor,which canbegenerated by cleavage of

theextracellular

ligand-binding

domain (1 1, 12). Inaddition

resultsfrom sequencing ofcomplementaryDNAclones encod-ingthep60TNF receptorchain have shownthatthe

extracellu-larligand binding domain encodesthe soluble TNF-BP-I (13-16).There was noindicationthatalternative

splicing

is

respon-sible forthe

generation

ofTNF-BP-I.Ourresults have shown that the

production

ofTNF-BP-Iis

rapidly

increased whenthe TNF receptors aredownregulated incellsby activation of

pro-tein kinase C with

phorbol

esters

(1 1). Heparin

maytherefore

actby activation of

protein

kinaseCorby direct activation ofa

proteaseleadingtocleavage ofthe

p60

TNF receptor

resulting

in release ofTNF-BP-I.This

explanation

is

unlikely

becausewe

couldnotdemonstrateanydirect effectonthe releaseof TNF-BP-Ifrom cells treated with

heparin

invitro

(Table

I).

Thefinding of

heparin affinity

forTNFhas

implications

for

the

understanding

of

regulation

oftheeffects ofTNF.

Interac-tion of cellswith

heparin-like

componentsoftheextracellular matrix seems to be

important

for

regulation

of

growth

and

differentiation.Thusseveral

growth

factorssuchasacidicand

basic fibroblast

growth factor, granulocyte macrophage

colony

stimulating factor, interleukin-3,

andinsulin-like

growth

factor

have

heparin-binding

abilities

(23-25). Obviously

abound

fac-torcanbeastimulus for

growth.

Growth factorsnecessary for

hematopoiesis

are

being synthesized by

thestromal cells ofthe bonemarrowand immobilizedoncomponents of the surface

(7)

of these cells, such asheparan sulphate, before being presented tohematopoieticstemcells(24). Bindingtoheparan sulphate may protectagainstenzymatic degradation.It is also possible thatsequestration by molecules in the extracellular matrix can be a way of protection against systemic effects of cytokines.

Therefore,the presentfinding ofaheparin affinityfor TNF is ofbiological importance.It suggests that TNF may be retained by extracellular matrix,e.g., by heparan sulphatein physical

proximity withtarget cells such asfibroblasts,endothelial cells, andhematopoieticcells.However,injection of heparin did not result inincreasedserumTNF,which may beexplained bya local releasewithoutresultingin increasedserum TNF(data notshown).Atthe same time the extracellularmatrixcould act as abuffer and retain TNF to protect the host against its

sys-temiceffects, whichmay be harmfulasinendotoxic shock (3, 4). Itwillbeinterestingtoinvestigateifaffinityforheparinand extracellular matrix is a feature of othercytokinestoo.

The finding that heparin-bound TNF can be dissociated

withTNF-BP-Iisalsoofinterestbecause TNF-BP-I is a soluble

fragment ofthep60transmembrane TNF receptor(1 1). Thusa

highaffinity binding betweenTNF and TNF-BP-Iis expected.

Alow affinity bindingbetween TNF and aheparin-like

sub-stance wouldallowpresentationof bound TNF to a TNF re-ceptor on the same(autocrineregulation) or anadjacent cell (paracrine regulation) with concurrent dissociation and

spe-cificbindingtothe receptorwithtriggering ofabiological re-sponse.

Weconclude that TNF, butnotTNF-BP-I, has

affinity

for heparinand suggest thatbinding ofTNF tothe extracellular

matrix of cellsmayrestrict its actions. Thefinding that TNF-BP-Icoulddissociate TNFfrom heparin, suggeststhat TNF-BP-I may alsofunctionas acarrierforTNFinvivo. The

mecha-nismbywhich heparin inducesthe observedreleaseof TNF-BP-Iinto the circulationremainsto beelucidated.

Acknowledgments

This work was supported by the Swedish Cancer Society, John and Augusta PerssonFoundation,AlfredOsterlundFoundation, the Swed-ish Associationfor Physicians,and theMedical Faculty of Lund.

References

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References

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