The TNF Receptors p55 and p75 Mediate Chemotaxis of PMN Induced by TNFα and a TNFα 36–62 Peptide

Research Paper

Mediatorsof Inflammation 3, 347-352 (1994)

Tt present studywas performed to examine whether residues36-62ofTNFcxcontainthe chemotactic domain ofTNF,and whether the p55 and p75TNFreceptors are involvedinTNFczinduced chemotaxis.The chemotactic effect ofTNFczonPMNwas inhibitedbythemAbsHrt-Tb and Utr-1, against thepandpT TNFreceptors,respec- tively. Bothreceptors maytherefore berequiredforme- diating the chemotactic effect ofTNFcz. The synthetic TNFcx36-62,similartoTNFcx,hadchemotacticeffectson bothPMNand monocytes. The chemotactic activity of the TNFcz 36-62 peptide on PMN, was inhibitedby Htr-Tb, Utr-I and soluble p5 receptor, which shows that the peptide possessed the ability to induce chemotaxis through the TNF receptors. In contrast to TNFcx, the peptidedid notshow a cytotoxic activity against WEHI 164flbrosarcoma cells. Itissuggestedthatdifferentdo- mains of theTNFcx molecule induce distinct biological effects.

Key words: Chemotaxis, Molecular modelling, Synthetic peptide,TNF, TNFreceptors

The TNF receptors p55 and p75 mediate chemotaxis of PMN

induced by TNFo and a TNFcx 36-62 peptide

13t. Rekdal,^,c* Z. Konopski,

J.

S. Svendsen, J.-O. Winberg, T. Espevik⁴and B. Esterud

lnstitute of Medical Biology, ^lnstitute of Mathematical and Physical Sciences and Polar Institute of Medical Genetics, University of Troms, 9037 Troms, and "Institute of

Cancer

Research, University ofTrondheim, N-7006Trondheim,

Norway

CA Corresponding Author

Introduction

Circulating polymorphonuclear cells (PMN) and monocytes ^{are activated} by chemotactic factors for recruitment to sitesofinflammation. Thepleiotropic cytokine,tumour necrosisfactor-(TNF),has been reported^tobe chemotactic, as itinduces directional locomotion of PMN and monocytes in vitro.^’-5 Fur- thermore, in vivo studies show that TNF0t plays ^a crucial role in the recruitment ofneutrophils at an earlystage, and monocytes^atalater stage ofimmune

complex-induced inflammatory reactions.^6,7 Many biological effects induced by TNF:^-11 have been shown to involve binding to the 55kDa (p55) and the75^kDa(p75) TNFreceptors,^{which are}expressed onalmost all cell types,^mHowever, theinvolvement ofthese receptors in theTNFx induced chemotaxis has not been studied.

An interesting approach for the study of distinct TNF{x activities istheuseofTNFpeptides.Different TNF peptides have recently been reported ^{to in-} duce distinctTNF effects,^2-14andinhibitbindingof TNF to the TNF receptors. The authorshave per- formed molecular dynamic calculations¹⁵ combined with studies on the three-dimensional structure of TNF0t¹⁶ in order to design TNF0t peptides which couldinteractwithTNF receptors, andinduce TNF0t effects.Itwasfound thatapeptideincluding residues

36-62

^hadconformational properties whichcould be related to the corresponding parent molecule. ^This

sequence is also one of the most homologous do- mainsbetweenTNFand

TNF,

^{whichbothbindto}

theTNFreceptors.^eIn the presentstudy,thispeptide was investigated for two crucial TNF0t effects, chemotaxisand cytotoxicity. The involvement ofp55 anp75 TNFreceptorsinthechemotacticresponseof TNF and TNF{x 36-62, was also studied.

Materials and Methods

Molecular modelling: Themolecular modelling stud- ies of TNF peptides were performed using the MolecularSimulationInc.Quanta3.2/CHARMm21.2 program package,on aSiliconGraphics Personal IRIS 4D/30EG(USA). Thepeptideatomcoordinates were obtained from the TNF0tX-ray structure (pdbl tnf), andpeptide candidates wereminimizedbymolecu- lar mechanics using 2000 steps ofadopted^basis set

Newton-Raphson minimization, before the mini- mized structures were subjected ^to molecular dy-

namic calculations with a total simulation of250 ps

at 300^K.

Synthesis

of

TNF peptides: TNF: peptides^weresyn- thesized asdescribedpreviously,⁷usingFmoc chem- istry on a semi-automatic peptide synthesizer (Milligen, Model 9020). The peptides were purified andanalysedusingreverse-phase HPLC,and FIB-MS on aVGTribidMS instrument(VGAnalytical, ^Man- chester, ^UK).

Mediators of Inflammation.Vol 3. 1994 347

.

^{Rekdalet al.}

Materials: Human recombinant TNF0t (Hr TNF00, with a specific activityof1.0 x 108 U/mg, was pur- chased from _Boeringer (Mannheim, Germany). The generation of the mAbs Utr-1 an Htr-7b, specific for p75 ^and p55 respectively, ^{is described} elsewhere,^TM and soluble p55 ^was kindly provided by ^Dr Hansruedi Loetscher, Hoffman-La Roche (Basel, Switzerland). Anti-IL-8 and anti-MCP-1 were pur- chased from British Biotechnology (UK).

Formylmethionyl-leucyl phenylalanine (FMLP) was

purchasedfrom Sigma Chemical Co. (StLouis, MO).

Endospecy from Seikagaku Co. (Tokyo, Japan) was used to check endotoxin contamination.

PMNisolation: Polymorphonuclearcells (PMN)were isolated as follows: 2 ml of freshly drawn heparinized blood (10 U/ml) from healthy adults was applied ^on top of a bilayer consisting of3 ^ml polymorphoprep and 3 ml lymphoprep ⁱⁿ polycarbonate tubes (Nycomed Pharma AS, Nor- way). Aftercentrifugation^at 530 x g^for 20 min, the PMN band in the polymorphoprep layer was iso- lated. The cells were washed once with ice cold sterile 0.15 M NaCl, and centrifuged ^at 185xg for 10min.ContaminatingerythrocytesⁱⁿthePMN band werelysedwithicecold0.2%NaCl for 90^s.The cells were resuspended ^at 106/ml in ice cold RPMI-1640 and used immediately. The PMN preparation con- tained at least 95% neutrophils.

Monocyte^isolation:The monocyte bandwasisolated using themethod previously ^describedby Byum.¹⁹ In brief, mononuclear cells (PMBC) from healthy adults, fromeither freshlydrawn heparinized blood or buffy ^coats (10U/ml), were centrifuged on lymphoprep, ^isolatedand washedwith 0.15M NaCl.

The PMBC, resuspended ^at 106/ml in RPMI-1640 were used directly for chemotaxis studies.

Assay

for

chemotaxis:TNFandTNFpeptideswere tested for chemotactic activity on PMN and PBMC.

Chemotactic activity wasassayedⁱⁿa48-wellmicro- chemotaxis chamber (Neuro Probe Inc. CabinJohn, MD, USA), as described previously,ⁱ In brief, the upper wellswerefilled with 50

l-tl

^{ofcells, and 25}

l.tl

of the compounds tested forchemotaxis were filled in the bottom wells. For checkerboard analysis, the stimulants werealsoplacedⁱⁿthe upper wells. In the pretreatmentstudies, the anti-TNFreceptor antibod- ies, or other antibodies were added to the cells for 10min at 4C, before they ^were placed ⁱⁿ the upper wells. The soluble p55 ^{was mixed with} 0.5

l.tM

^TNF0t

36-62

^{in a 1:1} molar ratio at 20C for 10min, before addition to the lower wells. A polycarbonate-polyvinyl pyrrolidone (PVP) filter with

51.tm

^{pore size was used in the PBMC}

chemotaxis assay, while a PVP-free polycarbonate filter,withthesameporesize, wasused for the PMN chemotaxis assay. Chemotaxis chamber assemblies

wereincubatedat37Cinhumidified95%^airand 5%

CO2

for 3 hⁱⁿassayswithPBMC and40min inassays with PMN. Then the filters were removed, fixed in 2.5% glutaraldehyde (Merck, Damstad, Germany), andstained withGiemsa(Sigma,Cleveland,USA) for 30^min. Cells that had migrated through to the bot- tom of the filter were counted in 6-10 high-power fields (HPF) (x60 ^or 100 objective). Chemotactic bioactivity was expressed as the mean number of cells per HPF. Variations in response to the tested agents, ^were dependent ^onthe blood donor.

Assay

for

cytotoxicity: Cytotoxicity of TNF

36-62

wastestedusingthefibrosarcomacelllineWEHI 164 clone13,asdescribedbyEspeviketal.²¹Cell viability inthe assay^wasmeasured colorimetrically,byusing tetrazolium salt(MTT), ^{as described}byMosmann et al.²²

Results

Molecular modelling

of

^TNFe

^36-62

^{peptide: Starting}

fromaminimizedX-raystructure, theconformational propertiesofTNF

36-62

^{in a vacuum}environment were calculated _using molecular dynamics. The re- suitsfrom thecalculationssuggestedthat the peptide would possessapartially conservedtertiarystructure similar to the conformation inthe minimizedcrystal

structure ofTNF (Fig. 1). Parts of

[-strands

^from

each of the two

[-sheets

^inthe TNF0t monomer are included in TNF0t

36-62

(Figs. 1A and 1B). The strandsinTNF0t

36-62

^{were stabilized} by hydrogen bonds, but some of these were different from the corresponding hydrogen bonds observed in the

TNF{x structure. A

-strand

interruption in TNF0t could also be recognized ⁱⁿ the peptide. The ^two loops (38-41and 50-54) locatedatthe baseof TNF{x, and important residues surrounding ^a shallow de- pression which ^are suggested ^to be involved in receptor binding,⁶ were all exposed ⁱⁿ a similar manner in the peptide as in TNFx. The distance between the carbon in the C-terminal carboxyl and thenitrogenintheN-terminalaminogroupinTNF0t

36-62

wasonly 2.89

A

compared^to18.19

A

ⁱⁿTNF, indicating an attraction between the oppositely charged C- and N-terminals. The effect ofthis elec- trostatic attraction is, however, expected to be of much less importance in aqueous environment.

Chemotactic

effects of

^TNFoandTNF 36-62peptide on PMN: TNF and the TNF0t

36-62

peptide have been tested for the abilityto attractPMN. Theexperi- mentswererepeatedatleast threetimes, andsimilar resultswereobtaineddespite donorvariations.Both TNF0t and TNF

36-62

showed a dose dependent chemotactic effect on PMN. Migration of approxi- mately120cellswasachieved with either 2nMTNF or10

l.tM

^TNF0t

36-62

(Fig. 2). Thatahigherconcen-

p55 andp75 mediatechemotaxis inducedby TNF36-62

A C

FIG.1. Drawing of theTNFamonomerand theTNFa36-62 peptide.(A)Drawing of the a-carbon backbone of the minimizedcrystal structureofTNFa.

Thesequenceconsisting of residues 36-62areblackened. (B)Drawing of the a-carbon backbone of theTNFasequence36-62. (C)Drawingofthe minimizedstructure oftheTNFa36-62a-carbonbackbone based on molecularmodelling, usingComputerGraphics.

200

*’’o

_E= ¹⁰⁰

z

0 RPMI20nM2nM lnM

501M 101M IIM

FIG.2.Dosedependenteffect ofTNFaandTNFa36-62onPMNmigra- tion. Indicated concentrations ofTNFa (,), andTNFa36-62 (=1)were tested for chemotactic effectonPMNwithRPMI(F"I)as acontrol. Migrated cellswerecounted in high-power fields(HPF) (x60 objective). Resultsare presentedas means + S.E.M.(n 9).

Table^1,Effect of varyingconcentrationsofT.NF36-62 peptide onPMNmigration.

TNF36-62 concentration inlower compartment

TNF(36-62concentration inupper compartment

0

M

¹⁰

IM

⁵⁰

M

IM

^{91+11 63+ 13" 54+13 61+ 11}

10

M

^{129 + 10 89+7 64+ 8" 57+14}

50M ^{197+9 161 +16 111 +8 51 +9"}

Theindicated concentrations ofTNF36-62 peptideormedium alone were added to the upper compartmentsof thechemotaxis chamber,toneutralizethe chemotactic effectof^TNF(36-62 used inthelowercompartments. MigratedPMNwerecountedin high- power fields (HPF) (x⁶⁰ objective). The data represent the mean + S.E.M. (n= 9). *The number of migrating PMN with the peptidepresentatthesameconcentrationinbothcompartments.

trationofTNF0t

36-62

was neededto induce migra- tionofthesamenumber of cellsasinducedbyTNF,

is probably due to lack of domains necessary for optimalbinding of T.NF0t

36--62.12

^{FMLPwasusedas}

a positive control at 10^-7 M, and gave an l1-fold migration ofPMNcompared^tothe negative control RPMI. As forTNF,aZigmond-Hirsch checkerboard analysis confirmed that TNF0t

36-62

^displayed

chemotactic, and not chemokinetic effects on PMN (Table 1). A different TNFot peptide including residues 78-96, used at the same concentrations as TNF 36-62, ^{did notexhibit} any chemotactic effect on PMN. We also tested whether ^TNF{x and TNF

36-62

^had chemotactic effects on PBMC, and ob- served that both stimulants induced chemotatic ac- tivitiesonPBMCinadosedependent^mannerandat similarconcentrationsasforPMN (data notshown).

Both stimulants with their buffers were tested and confirmedfree ofLPS using the Endospecy assay.

Inhibition

of

^{TNF and TNF{x} 36-62-induced

chemotaxis onPMN: Both mAb, Utr-1 and Htr-7b, specific for p75 and p55 respectively, significantly inhibitedthechemotacticresponseofTNFonPMN (Fig. 3A), when used separately orin combination.

Likewise, the chemotactic effect of TNF0t

362

peptide ^was also significantly inhibited by Utr-1 or Htr-7b (Fig. 3B) or a combination of both mAb.

These mAb alone, or incombination, didnotinduce chemotaxis on PMN. FMLP induced migration was notinhibitedbythese mAb aloneor incombination.

The experimentswererepeatedfourtimes, andsimi- lar results were obtained despite donor variations.

Antibodies against IL-8 or MCP-1 didnot show any inhibitory effectoneithertheTNF, TNF

36-62

or

.

^Rekdaletal.

z

A

20

10

0

30

20

10

0

B

RPMI TNF

HtrTb Utrl HtrTb

RPMI TNF

HtrTb Utrl HtrTb

+Utr ^{3 6 6} +Utr

FIG.3. Inhibitoryeffectof anti-p55 and anti-p75 antibodiesonthe chemotacticeffectonPMN,inducedbyTNFandTNF36-62. Utr- 1(10mg/ml)andHtr-7b(10 mg/ml)(anti-p75 and anti-p55, respectively)werepreincubatedwithcells for 10 minat 4C.thePMN werethen tested for chemotaxis toward(A)0.6nMTNFand(B)10IM^{TNF36-62 with()orwithout()antibodies}(I-l:control).Migrated cells werecounted in high-power fields(HPF)(x100objective).^{The data}representthemean

_

^S.E.M.(n 8). "p<0.001compared toTNFor TNF36-62 induced chemotacticeffectwithout antibodies.

100

z

NoS.

RPMI FMLP FMLP TNF- TNF-

+ p55 36-62 36-62 + p55 FIG.4. Inhibitoryeffect ofthe solublep55receptor on PMNchemotaxis inducedbyTNF36-62.TNFe36-62orFMLPweretested for their ability toinduce migration ofPMNwith()orwithout()solublep55([]:control).

Solublep55 waspreincubated withTNF36-62orFMLPfor 10 minat 20C.Migrated cellswerecounted in high-power fields(HPF)(xl00objec- tive). Theresultsare expressed as mean+S.E.M.(n 6)."p< 0.001 com- paredwithTNF36-62 alone.N.S. notsignificant.

FMLPinduced chemotaxis onPMN when usedinthe same concentrations as Utr-1 and Htr-7b.

TNFot36-62interacted withsolublep55:Thesoluble p55 was ableto significantly inhibit thechemotactic responseoftheTNF0t

36-62

peptideonPMN (Fig. 4).

Soluble p55 didnotinhibitthe chemotacticeffect of FMLP (Fig. 4), indicating aspecific binding ofTNF0t

36-62.

TNF{x

36-62

^hadno cytotoxic

effect:

^TNF0

^36-62

^was

tested for cytotoxic activity. In contrast to TNF0t,

TNF

36-62

^didnotshow anycytotoxiceffectonthe WEHI 164 clone 13 fibrosarcoma cell line, when tested up ^{to a} 104-fold higher concentration than TNF.

Discussion

In the present study it is shown that both TNF0t

36-62

^andTNFisachemoattractanttoPMNin vitro.

It is also shown that the chemotaxis induced by TNF0t and TNF0t

36-62

^{is mediated} through both p55 ^and p75 TNF receptors. The finding regarding TNF0t as a chemoattractant is in line with previous reports showing that antibodies against TNF inhibitTNFc induced chemotaxis in vitro on PMN and monocytes, respectively. It is noticeable that there have been conflicting reports about the chemotactic property of ^{TNF(x.2-5,23,24} In the authors’_opinionthediscrepancymight beexplained by ^variations in experimental conditions, donor variations andsensitivitiesofthetest systems.TNF has also been shown to stimulate production of the chemoattractant IL-8 by granulocytes and monocytes.²⁵ Since interleukin-8 (IL-8) shows chemotactic activityforPMN,^2,26TNFcmightinduce release of IL-8 by PMN, which in turn could be responsible for the chemotactic effect observed in our experiment. However, the present work shows that anti-IL-8 and anti-MCP-1 antibodies did not in- hibit the chemotactic response of TNF0t. This ex- cludes IL-8 and MCP-1 as responsible for the chemotactic activity observed.

Itis shown that the chemotactic activityTNF on PMNinvolvesbothp55andp75receptors. Evennon- redundancyin thefunction of thetwo receptors for

p55andp75mediate chemotaxis inducedbyTNF 36-62 some effects has been observed,²⁷ a simultaneous

involvementofbothp55andp75receptors has been reported for a series of TNF0t activities, such as induction of differentiation of ML-1 cells, NF-cB activation, cytotoxicity ^on U937 cells and IL-6 pro- ductionbyendothelialcells.^27-29Tartagliaetal.³have suggested that the high affinity p75 receptor may regulate the rate ofTNF0t association with the p55 receptor, by increasing the local concentration of TNF0tthrough rapid ligandassociationanddissocia- tion. In contrast to this, Brouckaert et al.¹ have proposed ^an alternative cooperation between the two receptors where p75 interferes with the p55 signalling pathway. This hypothesis is basedonthe fact that p75 triggering is not sufficient to initiate the redundant signals and that p75 triggering can diminish p55 ^mediated c-fos induction. However, ^it

remains to be explored how the two receptors cooperate ⁱⁿ TNF0t induced chemotaxis.

Similar to TNF0t, the TNF0t

36-62

peptide also induced chemotaxis onPMN and PBMC. Thefinding that soluble p55 inhibited this effect (Fig. 3), ^indi-

cates that the peptide possesses the conformation needed for interaction with the receptor. ^This sup- ports the results of the molecular dynamic calcula- tions(Fig. 1). That^TNF{x

36-62

^isableto interactwith soluble p55, ^is also in line with the recentwork by Ratjen etal.¹whofound that severalbioactiveTNFx peptideswere ableto inhibitbinding ofTNFtothe TNFreceptors.Antibodies againstp55andp75^inhib- itedTNF0t

36-62

induced chemotaxis onPMN,which indicates that this peptide, like TNF, ^induces the chemotactic activitythroughbothTNFreceptors. It^is noticeable that Postlewaite et al.

’

^{have observed}

chemotaxis on fibroblasts, by using another TNF peptide, which also included the sequence

36-62.

Although these authors did not show that the chemotaxis inducedbythe peptidewasTNFreceptor mediated, desensitisation studiessuggested ^involve-

ment ofTNF receptors. The presentwork supports and extends this hypothesis.

TNF0

36-62

was not cytotoxic, suggesting the existence ofdistinct TNFc regions for the cytotoxic and chemotactic effects. It is noteworthy that two other TNF peptides, whichoverlap ^with onlyfour residues (54-58),wereshownto becytotoxic,¹sug- gesting a critical domain forTNF cytotoxicity. This sequenceisincludedinourTNF

36-62

peptide and the molecular calculations of the peptide suggested that this specific domain encompassing residues

54-58 did not possess the conformation needed for optimalinteractionwiththeTNFreceptors. ^Thiswas duetotheattractionbetween the oppositelycharged C- and N-terminals (Fig. 1).

Crystallographicstudies onthe

TNF[3/TNF

receptor complex have shown that three

TNF]3

monomers bind threeTNFreceptors ⁱⁿ asymmetricalfashion.² It has been suggested that a crosslinking of TNF

receptor ^is also necessary for signal transduction leadingtoTNFo effects,^3-5 and is based,onstudies with antibodies and TNFot mutants against TNF receptors.^3-6Ourand otherinvestigations withsyn- theticTNFot peptide fragments,^1’-14however,suggest thatatleastsomeof theTNFceffectsarenotdepend-

ent on crossreaction of TNF receptors ^with three TNFotmonomers.

In conclusion, ^differentTNFot peptidesmayinduce distinct activities, indicating that TNF possess dis- tinct domains critical for different TNF activities.

This property opens the possibility of designing TNFx fragments with specific TNFot effects. We are currently investigating this hypothesis by studying TNFot

36-62

and several otherTNF peptidesfortheir bioactivities and specificities ^to target cells.

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ACKNOWLEDGEMENTS. We thank EliBerg and LivTone Elliassen for excellent technicalassistance.The contribution of ArnfinnKvarsnesinperforming the FABoMS analysisof the syntheticpeptidesisgratefullyacknowledged. Thecostof the MS instrument ispartiallypaid bythe Norwegian Council forScienceand theHumanities (NAVF),andthe Norwegian Council for Agriculture Research (NLVF).

Received 29 March 1994;

accepted in revised form 25 May 1994