Inhibition of leukocyte endothelial cell interactions and inflammation by peptides from a bacterial adhesin which mimic coagulation factor X

Inhibition of leukocyte-endothelial cell

interactions and inflammation by peptides from

a bacterial adhesin which mimic coagulation

factor X.

E Rozdzinski, … , S Putney, R Starzyk

J Clin Invest.

1995;

95(3)

:1078-1085.

https://doi.org/10.1172/JCI117754

.

Factor X (factor ten) of the coagulation cascade binds to the integrin CD11b/CD18 during

inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C., O.R.

Etingin, D.S. Fair, T.K. Brunk, J.E. Geltosky, D.P. Hajjar, and T. S. Edgington. 1991.

Science [Wash.DC]. 254:1200-1202). Filamentous hemagglutinin (FHA), an adhesin of

Bordetella pertussis also binds to the CD11b/CD18 integrin (Relman D., E. Tuomanen, S.

Falkow, D.T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Cell. 61:1375-1382). FHA

and the CD11b/CD18 binding loops of Factor X share amino acid sequence similarity. FHA

peptides similar to Factor X binding loops inhibited 125I-Factor X binding to human

neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue

prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and

blood brain barrier disruption in vivo. Interference with leukocyte migration by a

coagulation-based peptide suggests a novel strategy for antiinflammatory therapy.

Research Article

Inhibition of

Leukocyte-Endothelial Cell Interactions

and Inflammation by

Peptides

from

a

Bacterial Adhesin which Mimic Coagulation Factor

X

EvaRozdzinski, Jens Sandros, MichielvanderFlier, Alison Young, Barbara Spellerberg, Chandrabali Bhattacharyya, Julie Straub,* Gary Musso,*ScottPutney,* Ruth Starzyk,* and Elaine Tuomanen

Laboratory of Molecular Infectious Diseases, The Rockefeller University, New York 10021; and *Alkermes, Inc., Cambridge, Massachusetts 02139

Abstract

Factor X (factor ten) ofthe coagulation cascade binds to the integrin CD11b/CD18 during inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C.,0.R. Etingin, D.S. Fair, T. K. Brunk, J. E. Geltosky, D. P.

Hajjar,

and T. S. Edgington. 1991. Science [Wash. DC]. 254:1200-1202). Filamentoushemagglutinin (FHA), an adhesin ofBordetellapertussis also binds to the CDllb/ CD18 integrin (RelmanD., E. Tuomanen,S. Falkow,D. T. Golenbock, K. Saukkonen, and S. D. Wright. 1990. Cell.

61:1375-1382). FHA and the CD11b/CD18 binding loops of Factor X shareaminoacid sequencesimilarity. FHA pep-tides similar to Factor Xbinding loopsinhibited

"2I-Factor

X binding to human neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue

prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and blood brain barrierdisruption

in vivo.Interference with leukocytemigrationbya coagula-tion-based peptide suggests a novel strategy for

antiin-flammatory therapy. (J. Clin. Invest. 1995. 95:1078-1085.) Key words: pertussis-filamentoushemagglutinin *

meningi-tis *integrin * selectin

Introduction

The leukocyte integrin CDllb/CD18 (Mac-i, CR3) playsan

important role in leukocyte trafficking during inflammation.

Ac-tivatedCDI lb/CD 18 promotes the firm adhesion ofleukocytes

toendothelial cells(1-3).Eukaryotic ligandsfor CDIib/CD18

areknowntoincludecoagulationFactorX,C3bi,and intercellu-lar adhesion molecule-i

(ICAM-i

)' onendothelial cells, and fibrinogen (3-7). This combination ofbinding specificitieshas been suggestedtoprovide apointof convergence of the

path-AddresscorrespondencetoE.Tuomanen,TheRockefellerUniversity, Box 104, 1230 York Ave., New York, NY 10021. Phone: 212-327-8276;FAX: 212-327-7428.

Receivedfor publication9September1994 andinrevisedform3 November1994.

1. Abbreviations used in this paper: CSF, cerebrospinal fluid; FHA, filamentous hemagglutinin; HUVEC,humanumbilical veinendothelial cells; ICAM-1, intercellular adhesionmolecule-1;TEM, transendothe-lialmigration.

ways for leukocyte adhesion and coagulation which operate during inflammation (8).

Monocytic/myeloid cells stimulated withADPexpress acti-vatedCDI lb/CD 18 which binds Factor X with high affinity in

asaturable reaction (5).Inasecond step, CDI lb/CD18-bound Factor X is converted to the proteolytically active Factor Xa, thereby initiating the coagulation cascade on the surface of monocytes and neutrophils (9). Binding of Factor X to CDI lb/ CD18 has been demonstratedto bemediated by three surface loops: GYDTKQED (residues 366-373), IDRSMKTRG (422-430), and LYQAKRFKV (238-246) as evidenced by theability of peptides representing these three binding regions

to block radiolabeled Factor Xbinding to the monocytic cell line, THP-1 (8). These peptides also inhibit the generation of

Factor Xaprocoagulant activity.

Filamentoushemagglutinin (FHA) of Bordetella pertussis is

a220-kD nonfimbrial membraneprotein that binds to cilia and macrophages of the human respiratory tract during whooping cough (10, II). Of the several interactions FHA exhibits with macrophages, the ability to bind to the integrin CDllb/CD18 hasreceived the most attention. To enter phagocytes without an oxidative burst, many prokaryotes bind to carbohydrate

substitu-ents onthe integrin CDiIb/CD18 orbecome coatedwithC3bi (12).Incontrast,B.pertussis binds directly to the integrin in a protein-protein interaction mediated by FHA. This direct interac-tion has been demonstratedto occurwithpurified FHA and inte-grin (13). Previously, the region ofFHAsuggestedtoparticipate in this directbinding wasadomain containing an Arg-Gly-Asp (RGD) sequence (14). More recently, it has been recognized that,althoughtheRGD in FHAclearlypromotesbindingofFHA orbacteriatothe integrin presentedoncells,binding ofpurified

FHAtopurifiedintegrin isnotinfluencedby RGD peptides (13).

Non-RGD-mediated interactions may therefore explain the

di-rectbinding between FHA and

CDi

Ib/CD18.While delineation of themechanism of this interaction is of obviousimportanceto

understanding thepathogenesisofwhoopingcough, it is also of

potentialvalueas aunique probeinto thebiology oftheCDiIb/ CD18 adhesion molecule.

Here wepresent evidence thatregionsofFHAmimic motifs of Factor X of the coagulation cascade, a natural ligand of CDI lb/CD18. In additiontopreventingthegenerationof proco-agulant activity, this interaction also appears to interfere with

adherenceandtransendothelialmigrationofleukocytes. Usinga

rabbit model of meningitis, we also demonstrate that peptides

derived fromFactorX and FHAinhibitCD18-dependent leuko-cyte migration in vivo and protect against blood brain barrier

injury.

Methods

Peptides. PurifiedFHAofB.pertussiswasobtained from List Biologi-cals(Campbell, CA). Peptides derived from FHA andFactor Xwere

J.Clin. Invest.

C) The American Society for ClinicalInvestigation, Inc. 0021-9738/95/03/1078/08 $2.00

Table I. Inhibitionof TransendothelialMigration(TEM)

of Neutrophils by FHA and Factor X- and FHA-derived Peptides

Percentinhibition Peptide Amino acid sequence of TEM

Native FHA 82±1

Factor X I 238(G)LYQAKRFKV(G)246 10±6 FHA Ia 1979LGYQAK'98 23±5 FHA lb 2523GLIQGRSVKVD2533 7±10 Factor X II 366GYDTKQED(G)373 14±9 FHA II 2062ETKEVDG2068 21±10 Acetyl/amide FHA II 61±6 Scrambled FHA II DEETVK 4±5 Factor X III 422IDRSMKTRG430 5±7

FHA III 32GRTRG36 5±6

Peptides derivedfromFactor X weresynthesizedwithglycine added tothe termini in some cases(G)tomatch thepeptidesoriginally de-scribedinAltierietal.(8).Residues, identicaltoFactor Xpeptides,are highlightedin bold.Fluorescein-labeledneutrophilswereincubatedwith 10

_MM

ofpeptidesor0.1 Mof nativeFHAinM199 and, without washing,wereaddedtoHUVECmonolayers.TEMwasterminatedafter 1 h. In control wellscontaining M199alone,51±10% of neutrophils transmigrated. Percent inhibitionbyFHAorpeptideswascalculated in referencetocontrolwells setat 100% TEM. mAbW6/32 (25Htg/ml,

negative control)didnotinhibit TEMsignificantly (6±6%), whereas mAb IB4(25

_jtg/ml,

positive control)inhibited by73±14% (n =6). Values represent mean±standard deviation of at least 3 experiments with 6wells/peptide. Valuesof> 15% aresignificantlydifferent from controlatP< 0.05(t test).

synthesized byTheRockefeller University Protein Sequencing Facility orAlkermes, Inc. using FMOCchemistryand purifiedby HPLC (Ta-bleI).

Binding ofFactorXtoneutrophils. Human FactorX(Sigma Chemi-cal Co.orHematologic Technologies Inc.,St.Louis, MO)waslabeled with 1251Iusinglodogen (PierceChemicalCo., Rockford,IL) (5). Hu-man neutrophils were isolated from heparinized blood according to the manufacturer's specification using neutrophil isolation medium (CardinalAssociates, Santa Fe, NM)and washed three times with 5 mM EDTA-phosphate-buffered saline(PBS),pH 7.2, at 4°C. To eliminate plateletcontamination, neutrophilswere incubated withautologous se-rumcontaining 5 mM EDTAfor 30 minat37°C,washed threetimes withice-cold PBS, pH 7.2, and resuspendedin RPMI1640 tissue culture medium (Whittaker M. A. Bioproducts, Walkersville, MD) at 4°C. Bindingof125I-FactorX toneutrophilswasmeasured based on a method describedbyAltierietal.(8).Briefly,200 pl ofneutrophils (1.5x 107/ ml)wassupplemented with 17.5 y1 of 50 mMCaCl2,stimulated with 3.5

_Ml

of 100

l1.M

NH2-formyl-Met-Leu-Phe (Sigma Chemical Co.), and mixed with 30

_isl

of10.3

Mg/ml

"2I-Factor Xand100p1of1.75 mMpeptide. Buffer alone and apeptideof scrambled sequence served

as controls in eachof the three experiments. Afterincubation for 20 minat roomtemperature,neutrophil-bound '"I-FactorXwasseparated from unbound'251I-FactorXbycentrifugationof300-ki1aliquots through 50

_isl

ofamixture of Hiphenylsilicone oil DC 550 andmethylsilicone oilDC 200 5:1 (NyeInc.Specialty Lubricants, NewBedford,MA)at 12,000g for 2min. Aliquotsof the supernatant (cell free l'2I-Factor

X) and the cellpellet (cell bound "2I-Factor X) were counted in a gammacounter. Nonspecific binding (-50,000cpm) wasdetermined by addinga100-foldexcessofunlabeled Factor X, and subtracted from totalradioactivity (-500,000cpm forcontrols).

Generationof FactorXabymonocytes. FactorXagenerationwas determined by a modification of the method of Miletich et al. (14a). THP-1 cells 3 x 10'/ml;AmericanType Culture Collection, Rockville, MD) resuspendedin RPMI 1640 were incubated with 100

_jsM

ADP (SigmaChemicalCo.),2.5 mMCaCl2,5

tsg/ml

FactorX,and0.5mM peptides at room temperature. After 20 min, 100 MLl of the reaction mixture was transferred to37°Candsupplementedwith100 ,d of bovine Factor VII- and Factor X-deficient plasma (Sigma Chemical Co.). Coagulation was initiated by adding 100

_isl

of 25 mM CaCl2 and the clotting time was determined. Factor Xa procoagulantactivity (nano-grams per milliliter) was calculated by means of a standard curve con-structedbyserialconcentrations of Factor Xa(Hematologic Technolo-giesInc.) from 10 to 250 ng/ml.

Adherenceof neutrophilstocultured endothelial cells. Human um-bilical vein endothelial cells(HUVEC) (firstpassage; CloneticsCorp., San Diego,CA) were subcultured at confluence into Terasaki tissue culture wells. After 24 h, confluent monolayers were used for adherence assays (3). Monolayers were stimulated with 10 ng/ml TNF alpha (Boehringer Mannheim, Indianapolis, IN) at37°C for 4 h. 30

_ILI

of humanneutrophils (106/ml)waslabeled withfluorescein (3),and pre-incubated for15 min at37°C with 20

_ILI

of peptides (finalconcentration 0.5 mM in HAP) orHAP(PBS containing 0.5 mg/mlhuman serum albumin, 3 mMglucose, and0.3 U/ml aprotinin)as acontrol buffer. FHA was used at 0.1 gM(theupper limit ofsolubility).Neutrophils were allowed to adhere to the monolayer for 15 min at 37°C, and unboundcellswereremovedbysubmersion of the plate in PBSbuffer. The number of adherent neutrophils was counted in a 40x microscope field andexpressedas apercentage ofadherence in controlwells with neutrophilstreated with HAPbufferalone. ThemAbIB4against CD18 (MerckInc., Rahway, NJ)served as apositivecontrol.

Transendothelial migrationofneutrophils.Transendothelial migra-tionofneutrophilswasstudied based on a method described by Muller andWeiglin whichleukocytesthat have traversed and lie underneath themonolayerarecounted(15). Briefly, human neutrophils suspended in ice-coldHanks' balanced salt solution(HBSS) containing Ca and Mg(SigmaChemicalCo.)werefluoresceinated by adding 3.3

Il/ml

5-(and-6)-carboxyfluorescein diacetate, succinimidylester(CFSE; Mo-lecularProbes, Inc., Eugene, OR).Afterincubationfor 30 minonice, neutrophils were washed twice in cold HBSS with Ca and Mg and resuspendedin cold Medium 199 (MI99; SigmaChemical Co.) to a final concentrationof106/ml. HUVECweresubculturedatconfluence into 96-well tissue cultureplates(BectonDickinson Labware, Lincoln Park, NJ)thatwerecoated withhydratedcollagen gel (Vitrogen; Colla-genCorp.,PaloAlto,CA)andfibronectin.Endothelial cells were stimu-lated with 10 ng/ml TNF-alphafor 4 h and washed three times with warmM199. Neutrophils (106/ml) werepreincubated with 0.01 mM ofpeptides for5 min on icebefore theywereadded in

_100-ILI

aliquots tothe endothelial cells. Forpositive control, neutrophilswere preincu-bated with25,ug/ml anti-CD18mAbIB4.Fornegative control, neutro-phils weretreated with 25

_Ag/ml

mAb W6/32 against HLA class I antigen(Dako Corp., Carpinteria, CA).The cellswereincubated for1 hat370C,5%CO2. Toterminate transmigration, thesupernatant fluid wasaspiratedand thewellswerefilledwithwarm1 mMEGTA (Sigma ChemicalCo.)in HBSS without Ca andMg.Theplateswerecovered withplate sealer (Dynatech, Alexandria, VA) andcentrifugedin an invertedpositionat250 g for 3 minat roomtemperature. To remove nontransmigratedneutrophils, monolayerswerewashed twice with 200

,al

of warm Hanks' solution without Ca and Mg. Fluorescence was quantitated usingafluorescencecounter(Cytofluor' 2300; Millipore Corp.,Bedford, MA).The percentage oftransmigrated neutrophilswas

determinedby comparingthefluorescence oftestwells tocollagen-and fibronectin-coated wells without endothelial cells. Fluorescence counts

were corrected forcontamination of the neutrophil preparation with lymphocytes (<10%)whichdo nottransmigrate; contamination with monocytesasassessedby lightmicroscopywas<5%. Percent inhibition

compar-ing the number of transmigrated cells in wells containing neutrophils treated withpeptide to neutrophils treated withMl99alone.

Rabbit model for meningitis. The rabbit model was performed ac-cording to an established protocol (16. 17). Specific pathogen-free, New Zealand White rabbits (2 kg; Hare Marland, Nutley, NJ) were anesthetized with Valium (2.5 mg/kg, subcutaneously; Hoffman-LaRoche, Nutley, NJ), ketamine (35 mg/kg, intramuscularly; Aveco Co., FortDodge, IA), andxylazine(5 mg/kg, intramuscularly;Miles Laboratories,Shawnee, KS), and a helmet of dental acrylic was affixed to the calvarium. 24 h later, the rabbits were anesthetized with ethyl carbamate (1.75 g/kg; Aldrich Chemical Co., Milwaukee, WI) and pentobarbital (15 mg/kg; Abbott Laboratories, Abbott Park, IL) and placed in a stereotaxic frame.A spinal needlewasintroduced into the cisterna magna, and 300

MIl

ofcerebrospinal fluid (CSF) was withdrawn. The animals werechallengedintracistemallywith 108heat-killed, unen-capsulated pneumococcus strain R6 introduced in 200

_pi

ofsaline (time 0). 1 h later animals were treated with peptides dissolved in I ml of saline by intravenousinjection into theright marginalearvein.Samples of CSF were withdrawnathourlyintervals after pneumococcal inocula-tion, and leukocyte density was measured using a counter (Coulter Corp., Hialeah,FL). CSF samples werecentrifuged at 10,000 g for 5 min, and thesupernatantwasstored at -70°C until assayed for protein concentration using the bicinchoninic acid method (BCA kit; Pierce ChemicalCo.)andfor lactate concentration(lactatedetection kit; Sigma Chemical Co.). Differences between treatments were determined by one-way ANOVA.

Determinationof stability ofpeptidesinrabbitplasma. Rabbit blood

was heparinized and centrifuged to yield plasma. Peptides were dis-solved in PBS (120 mMNaCl, 2.7 mM KCI, 1.9 mM NaH2PO4,and 8.1 mMNa2HPO4) at aconcentration of 62.5

pM.

Thepeptides were

mixed with anequal volume ofplasma, and the sampleswereanalyzed by HPLC at0, 2, 4, 6, 8, 24, and 48 h. Analysis wasperformedon a

Hewlett-Packard (Palo Alto, CA) 1090HPLCusingaprotein and pep-tide column (C18; VYDAC,Hesperia, CA) (4.6 mm x 250 mm,

5-prm

particle size) equilibrated with 0.1 M sodium perchlorate/0.1% phosphoricacid (85%) in water, atpH2.5,and eluted with acetonitrile in agradient from 0to 40%over40 min (flow I ml/min; monitored

at210 nm).

Results

Sequencesimilaritybetween FHA and the CD] b/CD18

bind-ing sites ofFactor X. The amino acid sequence ofFHA was

compared with that of the three regions of humancoagulation

Factor X that are known to bind to the leukocyte integrin

CD1lb/CD18(8). Sequencesimilarity(40-70% identity)was

found between amino acid residues 238-246, 366-373, and 422-430ofFactor X(accordingtoFungetal. [18]) andFHA

residues 1979-1984,2062-2068, 32-36,and2523-2533 (ac-cording toRelman etal. [19]) (Table I).

FHApeptides blockFactor Xbindingtoleukocytes. In ac-cordance with previous observations (8), three peptides from Factor X inhibited 125I-FactorX bindingto leukocytes by 50-90% (Fig. 1 A, stippled bars). Native FHA and FHApeptide

II showed strong inhibition of'25I-Factor Xbindingto

neutro-philsof 70±4 and87±8%,respectively (Fig. 1A,shadedbars).

The other FHA peptides also demonstrated significant

inhibi-tion, but to a lesser extent than the corresponding Factor X

peptides. Native FHA, FHA peptide II, and the corresponding

Factor XpeptideII inhibited

1251-Factor

Xbindingin a

concen-tration-dependent manner (Fig. 1 B).

FHA peptides inhibit Factor Xa procoagulant activity on monocytes. Binding of Factor X to leukocytes is followed by conversion to Factor Xa and initiation of coagulation. Factor

A

C

.2

-B

concentration[log AM]

Figure 1. Inhibition of '25I-FactorXbinding to neutrophils by FHA, FHA-,orFactorX-peptides. Stimulatedneutrophils(3 x 106)were

mixed with 15 nM 'I51-FactorXandpeptides (asdesignated in Table I) ornative FHA. Neutrophil-bound '251-FactorX wasseparated from unlabeled FactorXby centrifugation throughsilicone oil. A, peptides 500,uM, native FHA 0.3

pM.

Values represent the mean±standard deviation oftriplicateexperiments. B, FHApeptideII (filledsquare), Factor Xpeptide II (opencircle),andFHA(opentriangle). Values

are representativeoftriplicateexperiments (standard deviations

< 15%).

Xagenerationonactivated THP-1cells in the absence ofpeptide

or in the presence ofa control peptide (a scrambled FHA II

analogue) was > 110 ng/ml (Table II). The concentration of Factor Xa correlatedinverselywith theclottingtime. Factor X

peptides I and II decreased Factor Xa production (<40 ng/ ml), thereby significantly prolonging clottingtime (TableII). FHA peptides Ia, Ib, and III also significantly reducedFactor Xaproduction by 50-70%. AlthoughFHApeptideII reduced thegenerationof Factor Xaprocoagulantactivity by only 25%,

theNH2-terminal acetylatedand COOH-terminal amidated form ofthis peptide significantlyenhanced this effect.

mi-Table IL Inhibition of Generation of Factor Xa by Peptides Derivedfrom Factor XorFHA

Peptide Clotting time FactorXa

s ng/ml

FactorX I 37±4 37.8*

FHAIa 34±1 57.4*

FHAlb 38±7 31.3*

FactorXII 55±4 <10*

FHAII 30±5 86.0

Acetyl/amideFHAII 40±3 28.5* FactorX III 26+2 107.5

FHAIII 36±3 43.4*

ScrambledFHAII 23±1 165.0 HAPbuffer 26±2 112.2 Leupeptin(25 jug/ml) 63±4 <10*

THP-l cells (3 x 107/ml) wereincubated with 100

_MM

ADP, 2.5 mM CaCl2,5

_jg/ml

FactorX, and 0.5 mMpeptidesat roomtemperature. After 20min,aliquots of the reaction mixtureweresupplementedwith FactorVII- and Factor X-deficientplasmaandrecalcified with 25 mM CaCl2. Factor Xacoagulantactivitywascalculatedby usingastandard

curveconstructed byserialdilutions of FactorXa. Values represent mean±standard deviation ofatleastthreeexperiments. *Statistically significantdifferenceascompared with scrambled FHApeptideIIat P <0.05 (t test).

grationofleukocytes. FHApeptidesweretestedfor theirability

to interfere with neutrophil adherence to stimulated HUVEC

(Fig. 2 A). The anti-CD18 mAb 1B4 (50

M.g/ml)

inhibited neutrophil adherence to HUVECs by 38+10% (not shown).

Native FHA(25 jug/ml = 0.1 ,pM)reduced neutrophil binding by 51+48%. The FHApeptideIawas significantly more active than the Factor Xpeptide I, whereas the FHA peptide II and

Factor X peptideIIdemonstrated similar activities. The

scram-bledFHA II peptidedid not interfere with neutrophil binding.

Asshown inFig.2B, nativeFHA,FactorX-, andFHA-peptides

were found to inhibit neutrophil adherence in a

concentration-dependent manner.

SinceCDl 8-dependentleukocyteadherencetoactivated

en-dothelia isaprerequisitetotransendothelialmigration,the FHA and Factor X peptides were tested for the ability to prevent

neutrophil transmigration invitro(TableI). mAbIB4 (25,ug/ ml) reduced transendothelial migration by

73±14%

(not

shown).Inconcordance with the results in theadherenceassay, FHApeptides Iaand II showedconsistently detectable activity, althoughthemagnitude of theeffectwaslow ( 20%). (Table I). This activity was greatly augmented by the acetyl/amide modification ofFHA II, leading to an inhibition of

61t+6%.

Fromthis and theneutrophiladherenceanalyses,FHApeptides

Ia and II were identified as candidates for more extensive

testing.

Several experiments were done to investigate the mecha-nismbywhich these peptidesinhibited adherence and transen-dothelialmigrationof neutrophils. First, we sought to determine whether they could enhance the ability of mAb IB4, a strong

inhibitorofneutrophiltransendothelial migration, to block

neu-trophil adherence to endothelial cells. Table III shows that the

A

a 0

-0 ._

< .X0 = =

a) _{5t J.. LL}0 _U.. L1

>

_t3

0 1

I _L . X;₀ 'O

C IL _{D lL}

E

1!m

ZU

h

B

60

-40

-o~

._

I

= 0D

U. .

-5 -2.5 0 2.5 5

[logp.MJ

Figure 2. Inhibition of neutrophil adherence toactivated endothelial cellsbyFHA, FactorX-,and FHA-derived peptides. Fluoresceinated human neutrophilswereincubated with peptides (designated as in Table I) and thenallowed to adheretoactivated HUVEC monolayers. After washing, thenumber of adherent neutrophils was countedina 40x microscope field and expressedas apercentage of adherence in control wells with neutrophils treated with buffer alone. A, peptides 500pM,

nativeFHA 0.1 pM; values represent themean±standarddeviation of

TableIII. Ability of Antibodies to Augment the Inhibition of Neutrophil Adherence Achieved by FHA Peptides

Antibody

Peptide None IB4

None 100* 39±8

FHAIa 63±12 20±71

FHAII 40±23 11±61

*Values arepercentages±standarddeviation of control binding in at least fourexperiments.In controlwells, 228±46 neutrophils were adher-entper40x field. Peptideswereadded at 5 ,ug/ml. mAb IB4 is directed against CD18 and was added to leukocytes atlg/ml for 1010 min before the assay. *_{Statisticallysignificant difference compared with}

antibody orpeptide alone.

combination of IB4 andFHA laand TI peptides showed greater inhibition of neutrophil adherence than either agent alone, indi-cating that theadhesion-inhibiting effects were additive. Maxi-mum inhibition with antibody orpeptide alone was -40-60% ofcontrol values. Preincubation ofneutrophils with FHA pep-tides Ta orIItogether with 10

_Mg/ml

anti-CD18 mAb IB4

re-sulted in an additive reduction of neutrophil adherence to

2 80%. In addition, IB4only modestly reduced '25I-Factor X

binding to leukocytes. The amount ofFactor X bound in the presence of 50

_Mg/ml

mAb IB4 was 81+6% of that boundin

the absence of antibody, yet 25 ,sg/ml of mAb IB4 strongly inhibited leukocyte transmigration (73% inhibition, Table I). Takentogether, these resultsindicate that the peptides are acting

atasiteindependent of that bound by IB4.

We alsoinvestigated thepossibility that theproteolytic

ac-tivity associated with the X to Xa conversion might promote leukocyte transmigration. Thishypothesis would predicta

cor-relation between inhibition of thegeneration of Factor Xa (i.e.,

ananticoagulanteffect) and inhibition of leukocyte transmigra-tion. Thediscordance of theanticoagulant effects of FHA pep-tidesTa, Tb,andIIIand the poorantimigration effects (compare Tables I and II) argues against thishypothesis. Similarly, the protease inhibitor leupeptin inhibited generation of Factor Xa (> 90% inhibition, Table II) but failedtoblockleukocyte

trans-migration (6±6% inhibitionasperformedaccordingtoTable I

at25

_itg/ml).

This result suggests that proteolytic activity is

not correlated with transmigration and that the peptides are

unlikelyto exert their effectonneutrophil migrationby inhib-iting proteolysis associated withcoagulationfactors.

Theantiinflammatory effectofFHApeptideswithFactor X

homologyin vivo. Todetermineif theabilitytoblockneutrophil

attachment andtransmigrationin vitropredicted antiinflamma-toryactivity in vivo, severalpeptidesweretested inananimal

model of pneumococcal meningitis for thecapacity toprevent leukocyte extravasation from the bloodstream into the CSF. Native FHA and FHA- and Factor X-derived peptides were

injected intravenously 1 h after intracistemal challenge with pneumococci. Fig. 3depicts the leukocyteresponse over 7 h. NativeFHA, FHApeptides I andII, and Factor Xpeptide II resultedinasignificantreductionofCSFleukocytosisof>60% (P < 0.05). The scrambledFHApeptideII wasineffective (P

> 0.5).

Todetermine thestabilityof theFHAIIpeptide,the

unmod-A

V)

I

Hours

B

8000

6000

U2

U)

'a

4000

2000

0

0 2 4 6

Hours

8

Figure 3.Antiinflammatory activityofFHA,andFHA-,and Factor X-derivedpeptidesinexperimentalbacterialmeningitis. Meningeal in-flammationwasinduced in rabbitsby intracisternalchallengewith 108 heat-killed pneumococci. 1hlater,animals receivedanintravenous

injec-tionof 10 nmol ofpeptidein 1 ml(n24),25_pgFHA(n= 5),or

PBS (n= 10).LeukocytedensityinCSFwasdeterminedat5,6, and 7 hafterpneumococcalchallenge.A, FHA o; PBSo.B, FHApeptideI

o;FHApeptideII*; Factor XpeptideII A; scrambledFHA peptide II *. *Statisticallysignificantdifference from control(P<0.01).

ified ortheacetylated-amidatedpeptidewas incubated in50% rabbit plasmaat37°C.Theconcentration ofunprotectedFHA

peptide IIwasdecreasedbyhalf in 4-6h,whereasthe

un-10000

-8

7500-

5000-2500

-acetyaide

FHA H saline

Figure4.Ability of acetyl/amide FHAIIpeptidetoinhibit accumulation of leukocytes in CSF. 2groupsof 10 animalswerechallenged with pneumococci asinFig. 3. 1 hlater, animals received anintravenous injection of 10 nmol of peptide (8.2

_Mg)

orPBS. Leukocyte density in CSFwasdeterminedat6 h after pneumococcal challenge. Mean values

asindicated by the bars arestatistically significantly differentatP

=0.0015 byANOVA.

changedover48 h. Rabbits (n = 10)weretreatedwith 10 nmol

ofthe acetyl-amide-FHAII.Control animals (n = 10) exhibited

a meanof6,480+684 leukocytes/,ul CSFat6 h after

pneumo-coccal challenge. In contrast, animals receiving the acetyl-amide-FHAIIdemonstratedasignificant reduction of leukocyte migration into the CSF to 3,011+684 cells/,ul (P = 0.0015,

ANOVA) (Fig. 4).

Protection against blood brain barrier permeability af-fordedbyFHApeptides.Lossofbarrierfunction of the cerebral capillary endothelium during inflammation is reflected by an

influx of serum proteins into the CSF, and impaired cerebral

metabolism has been shown tocorrelate with increased lactate concentrations in CSF (17). An effective antiinflammatory agentshould ameliorate both of theseparameters of injury. To examine if the ability of the peptides to decrease leukocyte transmigration correlated with improvements of thesemeasures

of cerebral injury, CSF protein and lactate concentrationswere

measured in peptide-treated and control animals (Table IV). FHA peptide II and its acetyl-amide derivative significantly

reduced protein influx into the CSF and generation of lactate (P<0.02). FHA peptide I and Factor X peptideIIdidnotcause a significant reduction in protein influx, afinding indicating a

greaterpotency of FHA peptide II.

Discussion

The binding of Factor X to the integrin CD1lb/CD 18 onthe

surface of ADP-activated monocytic cells initiates cleavage of the zymogen Factor X to Factor Xa and thereby activates the coagulation cascade in the microenvironment of an inflamed

tissue (9). The binding sitesonFactor X have been mappedto three spatially separate regions whichcontactthealpha subunit

Table IV. Effect of Peptides on Blood Brain Barrier Permeability andLactateMetabolism during MeningealInflammation

Peptide Lactate Protein

mg/I00ml mg/ml CSF

Saline control 38.7±4.8 2.1+0.4 FHA 25.0±5.3* 1.3+0.3* FHA I 36.0±6.7 1.8+0.4 FHA II 25.5+4.1* 1.4+0.6* Acetyl/amideFHA II 24.3+3.2* 1.2+0.6* Factor X II 37.6+6.9 1.6±0.7 Scrambled FHA II 40.7+5.8 1.9+0.3

Mean values for CSF lactate and protein at time 0 were 18.6+5.8 and 0.8±0.2, respectively. Animals received FHA (0.1 nmol) orpeptides (10nmol/animal in 1 ml saline) intravenously 1 hafter intrathecal challengewith pneumococci as inFig. 3. 10 control animals received PBS. Values represent mean±standard deviationof concentrations in CSF at 6 hafterpneumococcalchallenge(n 2 4 forpeptide-treated animals; n = 4 for FHA). *Statistically significant differenceas com-paredwith controlat P < 0.02.

of the integrin CDl1b/CD18 (8). The present studyprovides evidence that the binding of a prokaryotic adhesive protein, FHA, to CD1lb/CD18 mimics at least one binding loop of Factor X. FHA binds to cell-associated and purified CDllb/

CD18 (13, 14). Binding of FHA strongly inhibits subsequent

bindingofradiolabeled Factor X to the activatedintegrin.This property appears to reside, at least in part, in the sequence ETKEVDG since this FHA peptide inhibited'25I-FactorX

bind-ing to stimulated neutrophils by > 80%, while three other

re-gions of FHA were less effective. This peptide resembles DTKQED,the Factor Xpeptide which definesonebinding loop

forCD1 lb/CD18. The differences inthesequences ETKEVDG and DTKQED suggest that alteration of three residues in the Factor Xpeptidesequence does notinfluencethe activities mea-sured: aspartic acid to glutamic acid, glutamine to glutamic acid, and glutamic acidto valine. Thefactthat both FHA and Factor Xpeptidesdisplayedactivitysuggeststheimportance of

the conservedthreonine, lysine, and asparticacid orthe spatial

arrayof three acidic and onebasic amino acid side chains. Theinhibition of binding of 125IFactor X to leukocytes by

the FactorX-derived peptide DTKQEDGwasassociatedwith a stronganticoagulant effect asmeasured byclotting time and

generation ofFactorXa.TheFHApeptide ETKEVDG showed

amodest anticoagulant effect which was enhanced by acetyl/ amide modification of the peptide. Significant inhibition of

co-agulation was also characteristic of two other FHA peptides. Thus, three regions of FHA present sequence and functional

similarity to binding loops of Factor X. To our knowledge,

this is the first example of a bacterial peptide modulating the

triggeringof thecoagulationcascade on theleukocyte surface.

Itis of interest to note that anotherpathogen,Herpessimplex virus, encodes a Factor X receptor which recognizes the same three surface loops ofFactor X as does CD1lb/CD18 (8). It now appears that the conversesituation alsoexists, i.e.,a

bacte-riumB. pertussis,displaysFactor Xhomology regionsthat bind to nativeCDllb/CD 18 by sequences similarto native Factor X.Giventheimportance ofthe CD1 lb/CD18-dependententry

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ofthis pathogen into macrophages, this mimicry may provide an important mechanism to promote intracellular localization of the bacteria and thereby prolong the course of whooping cough. A predictable consequenceof this binding is the inhibi-tion of procoagulant activity. However, an additional effect of the FHA peptides, especially ETKEVDG, is to interfere with theability of CD1 lb/CD18 to mediatebinding and transmigra-tion ofleukocytes across endothelia. This unexpected effect and itsrelationshipto activities ofFactor X weredissected in more detail.

CDllb/CDI8 promotes the firm adherence ofneutrophils and monocytes to inflamed endothelia (1, 3, 6). The effect of thebindingof Factor Xtotheintegrinonitsabilitytoparticipate in neutrophil transmigration has not been reported previously. We examined whether the Factor X-peptides and FHA-pep-tides could block adherence and transendothelial migration of leukocytes. Native FHA and FHApeptides LGYQAK and ET-KEVDG inhibited neutrophil adherence to activated cultured endothelial cells. LGYQAK was clearly more effective than the corresponding Factor X peptide, GLYQAK (42 vs 18%, respectively); Factor X-derived DTKQEDG was similar in inhibitory activity to FHA-derived ETKEVDG (38 vs 32%, respectively). When peptides were tested for their ability to

preventmigration of neutrophils acrossmonolayers of activated endothelial cells, again LGYQAK and ETKEVDG were the

mosteffectivepeptides (23±5 and 21+10%inhibition, respec-tively) and showed greater inhibition than the relatedFactor X

peptides

(10±6

and 14±9% inhibition). Activity of the

ET-KEVDG peptide was strikingly increased from 21 to 61% by introducing acetyl/amide modifications. This effect may arise from greater stability of the modified peptide (static plasma half-life prolonged from4to>48h) (20), improved presenta-tion of the relevant sequencetotheintegrinreceptor, or seques-tration ofcharge effects.

Severalpossiblemechanismsunderlyingtheeffect of Factor X-like peptides on CD18-mediated adherence of leukocytes

wereexamined. TheproteolyticactivityofFactor Xaisunlikely

to be involved sinceleupeptin did notaffectleukocyte

migra-tion. In addition, strongly anticoagulant peptides (e.g.,

GLY-QAKRFKVG)failedtoblockneutrophiladherenceand

migra-tion. However, aformal elimination of thishypothesis will

re-quire development of Factor X with mutations ablating proteolytic action butnotbinding tocells.

Inhibition ofleukocyteadherencetoendothelium could also arise if the region ofCDllb/CD18 which binds endothelium

was identical with orlocated near the Factor X binding site. This doesnotappeartobethecasesinceAltieri has evidence that Factor X doesnotbindtothe

ICAM-l

binding domainof CD1 lb (Altieri,D.C.,personalcommunication).Our

observa-tions also appear to be independent of ICAM-1 in that the

peptides didnotalter theinhibition ofleukocyte adherence by

IB4 which interferes with CDllb/CD18 binding to ICAM-1;

rather, theywereadditive with IB4. Thus, it appears thatFactor Xinfluences adherence between leukocyteandendotheliumby

an interaction that is distinct from CDllb/CD18 binding to

ICAM-1.These experimentsdo not distinguish if Factor X or

itsderivative, FactorXa, actsdirectly orindirectlyon suchan

adhesion process.

TheCD1lb/CD18adhesionmolecule isanimportant target ofstrategies todevelopantiinflammatory agents oftherapeutic value. In the animal model ofpneumococcal meningitis used

here, leukocyte migration is strongly CD18 dependent (21). Intravenous injection of native FHA, the FHA peptides LGY-QAK, ETKEVDG, acetyl/amide ETKEVDG, or the corre-sponding Factor X peptide GYDTKQEDG resulted in a > 70% reduction ofleukocyte recruitment into the CSF at 7 h after intracisternal challenge with pneumococci. This effect is com-parable with that achieved in this system by treatment with steroids (21-23). To be of therapeutic significance,

antiin-flammatoryagents must also preventcompromise of the barrier function of the cerebral capillary endothelium. ETKEVDG and its acetyl-amide derivative, the peptides most effective at inhib-iting leukocyte migration, also effectively reduced influx of serum proteins into the CSF and accumulation of CSF lactate. Currently, antiinflammatory therapy is recommended as an

adjunct to antibiotics to decrease the morbidity and mortality ofmeningitis (22, 23). One such therapeutic candidate is an

anti-CD18 antibody(21). The data presented here indicate for the first time that peptides are also viable therapeutic alterna-tives.Peptides have theoretical advantages in that they are more easily produced and aremoreamenable to multiple dose therapy

astheyarelessantigenic. The precise mechanism of the in vitro and in vivo action of thepeptidesdescribed here remainstobe determined. However, the potential to deploy peptides which block bothleukocytetransmigrationaswell as the coagulation cascade at sites ofinflammation may affordadded therapeutic advantage. The use of peptides patterned after regions of Factor

X to reduce leukocyte accumulation in the CSF represents a novel antiinflammatory strategy.

Acknowledgments

We thank D. Altieri foradvice on methods of measuring Factor X-CDllb/CD18 bindingandfor discussionsas tothe mechanism of the effects of the FHA peptides, T. Edgington for continued stimulating advice,Parul Parmar for technicalassistance,and Tracy Olson for help-ful discussions. Peptide synthesis was performed by the Protein Se-quenceFacilityof TheRockefellerUniversity supported bya Biomedi-calResearchSupportGrant Shared InstrumentationGrantfrom the Na-tionalInstitutes ofHealth.

This work was supported by National Institutes ofHealth grant 23459 (E. Tuomanen), the Swedish Institute(J. Straub), and Alker-mes, Inc.

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