Inhibition of leukocyte-endothelial cell
interactions and inflammation by peptides from
a bacterial adhesin which mimic coagulation
E Rozdzinski, … , S Putney, R Starzyk
J Clin Invest.
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.
Leukocyte-Endothelial Cell Interactions
and Inflammation by
Bacterial Adhesin which Mimic Coagulation Factor
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
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
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
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
CDiIb/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
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
Peptides. PurifiedFHAofB.pertussiswasobtained from List Biologi-cals(Campbell, CA). Peptides derived from FHA andFactor Xwere
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
MMofpeptidesor0.1 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
l1.MNH2-formyl-Met-Leu-Phe (Sigma Chemical Co.), and mixed with 30
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
islofamixture 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
jsMADP (SigmaChemicalCo.),2.5 mMCaCl2,5
tsg/mlFactorX,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
islof 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
ILIof humanneutrophils (106/ml)waslabeled withfluorescein (3),and pre-incubated for15 min at37°C with 20
ILIof 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/ml5-(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-ILIaliquots tothe endothelial cells. Forpositive control, neutrophilswere preincu-bated with25,ug/ml anti-CD18mAbIB4.Fornegative control, neutro-phils weretreated with 25
Ag/mlmAb 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
,alof 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
MIlofcerebrospinal fluid (CSF) was withdrawn. The animals werechallengedintracistemallywith 108heat-killed, unen-capsulated pneumococcus strain R6 introduced in 200
piofsaline (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
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-prmparticle 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
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. ) andFHA
residues 1979-1984,2062-2068, 32-36,and2523-2533 (ac-cording toRelman etal. ) (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
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
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
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
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
MMADP, 2.5 mM CaCl2,5
jg/mlFactorX, 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
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
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
Fromthis and theneutrophiladherenceanalyses,FHApeptides
Ia and II were identified as candidates for more extensive
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
< .X0 = =
a) 5t J.. LL0 U.. L1
I L . X;0 'O
C IL D lL
-5 -2.5 0 2.5 5
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
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/mlanti-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/mlmAb 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
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
Todetermine thestabilityof theFHAIIpeptide,the
0 2 4 6
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),25pgFHA(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
-acetyaideFHA 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
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.
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
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
(10±6and 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.,
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-lbinding 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
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.
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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