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Tissue Factor-Dependent Chemokine Production Aggravates Experimental Colitis

Queiroz, K.C.S.; van 't Veer, C.; van den Berg, Y.; Duitman, J.; Versteeg, H.H.; Aberson, H.L.;Groot, A.P.; Verstege, M.I.; Roelofs, J.J.T.H.; te Velde, A.A.; Spek, C.A.Published in:Molecular Medicine

DOI:10.2119/molmed.2011.00138

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Citation for published version (APA):Queiroz, K. C. S., van 't Veer, C., van den Berg, Y., Duitman, J., Versteeg, H. H., Aberson, H. L., Groot, A. P.,Verstege, M. I., Roelofs, J. J. T. H., te Velde, A. A., & Spek, C. A. (2011). Tissue Factor-Dependent ChemokineProduction Aggravates Experimental Colitis. Molecular Medicine, 17(9-10), 1119-1126.https://doi.org/10.2119/molmed.2011.00138

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https://doi.org/10.2119/molmed.2011.00138

INTRODUCTIONInflammatory bowel diseases are

chronic and recurrent disorders of thegastrointestinal tract, of which both etiol-ogy and pathogenesis remain only partlyunderstood (1–3). Crohn’s disease andulcerative colitis, the two major forms ofinflammatory bowel disease, are charac-terized by intestinal inflammation as aresult of transmural infiltration of granu-locytes, lymphocytes, monocytes ormacrophages and plasma cells, accompa-nied by the excessive production of freeoxygen radicals, ultimately leading tomucosal disruption and ulceration (4).

Inflammatory bowel disease patientshave an approximately three-fold in-creased risk of venous thrombosis com-pared with age, sex and geographicallymatched controls (5), and these throm-botic complications significantly con-tribute to morbidity and mortality (6,7).The frequency of venous thrombosis inpatients suffering from inflammatorybowel disease lies between 1 and 8%,whereas postmortem studies showthrombotic events in up to 40% of pa-tients (8). Thrombotic risk increases withactive disease, although >30% of patientshave inactive disease at the time throm-

bosis is diagnosed (6). Overall, it is thusapparent that a “hypercoagulable state”is frequently observed during inflamma-tory bowel disease and that coagulationabnormalities are an intimate part of theclinical picture of inflammatory boweldisease (9). However, whether exacer-bated coagulation participates in the eti-ology of inflammatory bowel disease re-mains controversial. It is interesting tonote that the risk of inflammatory boweldisease is lower than expected in patientswith inherited bleeding disorders (10)and that several observational studiesshow a beneficial effect of anticoagulanttreatment (11–14). However, a random-ized clinical trial did not confirm thisbeneficial effect of heparin treatment(15). In line with a limited effect of bloodcoagulation in the etiology of inflamma-tory bowel disease, prothrombotic factorV Leiden mice were indistinguishablefrom wild-type animals in dextran sul-

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Tissue Factor–Dependent Chemokine Production AggravatesExperimental Colitis

Karla C S Queiroz,1 Cornelis van ‘t Veer,1 Yascha van den Berg,2 JanWillem Duitman,1 Henri H Versteeg,2

Hella L Aberson,1 Angelique P Groot,1 Marleen I Verstege,3 Joris J T H Roelofs,4 Anje A te Velde,3 and C Arnold Spek1

1Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, the Netherlands; 2Einthoven Laboratoryfor Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; 3Tytgat Institute for Liver andIntestinal Research, Academic Medical Center, University of Amsterdam, the Netherlands; and 4Department of Pathology, AcademicMedical Center, University of Amsterdam, the Netherlands

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatoryprocesses. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation ofTF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium(DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histo-logical parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice comparedwith controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlowmice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels toa similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studiesrevealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocytemigration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Takentogether, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction–dependent KC produc-tion in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2011.00138

Address correspondence and reprint requests to C Arnold Spek, Center for Experimental

and Molecular Medicine, Academic Medical Center, Meibergdreef 9, NL-1105 AZ Am-

sterdam, the Netherlands. Phone: +31-20-556-8750; Fax: +31-20-6977192; E-mail:

[email protected].

Submitted April 18, 2011; Accepted for publication June 21, 2011; Epub

(www.molmed.org) ahead of print June 22, 2011.

fate sodium (DSS)-induced experimentalcolitis (16).

Tissue factor (TF), a 47-kDa transmem-brane glycoprotein, initiates blood coag-ulation via formation of an enzymaticcomplex with factor (F)VIIa, eventuallyleading to the activation of thrombin andthe formation of fibrin (17,18). Beyond itswell-established role in coagulation, TFhas been suggested to play a paramountrole in inflammatory conditions. Indeed,TF may activate protease activated recep-tor (PAR) signaling leading to the pro-duction of (among others) inflammatorycytokines (19,20). The increase of both in-terleukin (IL)-6 and IL-8 levels in coagu-lating blood (21) is indicative of TF- induced cytokine production eitherthrough direct cellular effects or viadownstream coagulation factors. Becauselipopolysaccharide-induced IL-6 and ker-atinocyte-derived chemokine (KC) (amouse analog of IL-8) production in cul-tured macrophages and in anticoagu-lated whole blood is lower in heterozy-gous TF-deficient cells than in wild-typecells, one might argue that TF influencescytokine production without interven-tion of other coagulation factors (22). Thein vivo significance of TF for host defenseis most evident because TF blockingagents reduce the risk of lipopolysaccha-ride-induced mortality. For instance, pre-treatment with a monoclonal TF anti-body attenuates coagulopathy andmortality in a lethal Escherichia coli sepsismodel in baboons (23). Moreover, immu-nization of mice with a polyclonal TF an-tibody protects against death upon ad-ministration of lethal amounts oflipopolysaccharide (24), whereas treat-ment with site-inactivated FVIIa delaysor prevents death upon LD100 E. coli ad-ministration in baboons (25). In line withan important role of TF in inflammation,it was recently shown that a murine TF-blocking antibody reduced DSS-inducedexperimental colitis (26). Most promi-nently, blocking TF reduced blood cell re-cruitment and tissue injury in mice sub-jected to DSS-induced colitis. Asexpected, the TF-blocking antibody alsoreduced thrombin–anti-thrombin levels

(TAT; a marker of ongoing coagulation),and it blunted thrombus formation inDSS-treated mice. This latter study thusshows that TF mediates inflammatorycell recruitment and tissue injury in thecolon during experimental colitis. Al-though the authors suggest that the anti-coagulant properties of TF may be im-portant, the underlying mechanism bywhich TF aggravates experimental colitisremains elusive.

The current report aims to create betterunderstanding of the role of TF in colitisand directly compares experimental coli-tis in mice with a genetic ablation of TFwith wild-type mice treated with the an-ticoagulant dalteparin. Our data confirmthe importance of TF in experimental co-litis and extend these findings by provid-ing evidence that TF aggravates colitis ina coagulation-independent manner. Wesuggest that TF-dependent signal trans-duction induces KC production in colonepithelial cells, thereby provoking granu-locyte influx with subsequent inflamma-tion and organ damage.

MATERIAL AND METHODS

AnimalsTFlow mice, previously generated (27)

and provided by Dr. Mackman (Univer-sity of North Carolina, Chapel Hill, NC,USA), were bred at the animal care facil-ity of the Academic Medical Center. Ho-mozygous TFlow mice are mice deficientfor murine TF that are rescued from em-bryonic lethality by introducing a humanTF minigene containing the human TFpromoter and human TF cDNA leadingto TF activity of around 1% (relative tomouse TF). Heterozygous TFlow mice ex-press 50% murine TF and also contain thehuman TF minigene. C57BL/6J wild-typemice were purchased from Charles River(Someren, the Netherlands). All micewere bred and maintained at the animalcare facility at the Academic MedicalCenter according to institutional guide-lines, with free access to food and water.Animal procedures were carried out incompliance with the Institutional Stan-dards for Humane Care and Use of Labo-

ratory Animals. All mice were housed inthe same temperature-controlled roomwith alternating 12-h light/dark cycles.Mice at an age of 8–10 wks were used inthe colitis model as described below.

Induction of Colitis and Assessment ofDisease Progression

Mice received 1.5% DSS (molecularweight of 40 kDa; TdB Consultancy, Up-psala, Sweden) in filter-purified drinkingwater for 7 d ad libitum as described be-fore (16). Control mice received filteredwater alone. For the anticoagulant exper-iments, mice received 80 IU/kg/d dal-teparin (based on [28]). Body weight loss(calculated as the percentage differencebetween the original weight and theweight at the time of sacrifice) and stoolconsistency were used for clinical assess-ment of disease severity.

Histological AnalysisHistological analysis was performed

essentially as described before (29,30). Indetail, the longitudinally divided colonswere rolled up, fixed in 4% formalin andembedded in paraffin for routine histol-ogy. The following parameters werescored in a blinded fashion by a patholo-gist: (i) percentage of area involved, (ii)number of follicle aggregates, (iii) edema,(iv) fibrosis, (v) erosion/ulceration and(vi) crypt loss and infiltration of (vii)mono- and (viii) polymorphonuclearcells. Percentage of area involved andcrypt loss were scored on a scale rangingfrom 0 to 3 as follows: 0, normal; 1, <10%;2, 10–50%; and 3, >50%. Erosions weredefined as 0 if the epithelium was intact,as 1 for the involvement of the laminapropria, as 2 for ulcerations involving thesubmucosa and as 3 when ulcerationswere transmural. The severity of theother parameters was scored on a scale of0–3 as follows: 0, absent; 1, weak; 2, mod-erate; and 3, severe. The score rangesfrom 0 to a maximum of 24 points.

Cytokines and ChemokinesMeasurement

IL-6, monocyte chemoattractant pro-tein (MCP)-1 and tumor necrosis factor

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T I S S U E F A C T O R A N D E X P E R I M E N T A L C O L I T I S

(TNF)-α were measured in colon ho-mogenates using the BD CytometricBead Array Mouse Inflammation Kit(Becton Dickinson, Lincoln Park, NJ,USA) as described before (31). Detectionlimits were 10 pg/mL. KC was measuredusing an enzyme-linked immunosorbentassay (ELISA) (R&D Systems, Minneapo-lis, MN, USA) according to the manufac-turer’s instructions with a detection limitof 31.25 pg/mL.

Colon Tissue CultureColons, collected from wild-type and

TFlow mice, were washed in Hanks’ bal-anced salt solution (HBSS) medium con-taining penicillin and streptomycin, afterwhich colon sections of approximately2 mm were placed in RPMI 1640 me-dium containing 1% fetal calf serum,penicillin and streptomycin. Colon sec-tions were stimulated with FVIIa (10 and100 nmol/L) for 24 h, after which the su-pernatant was collected for analysis ofKC levels.

PatientsRest material of ulcerative colitis pa-

tients was used for immunohistochemistryaccording to the guidelines of the MedicalEthical Committee of the Academic Med-ical Center (AMC), University of Amster-dam, Amsterdam, the Netherlands.

(Immuno)histochemistryColon sections were harvested, fixed,

paraffin embedded and stained withhematoxylin and eosin according to rou-tine procedures. For immunohistochem-istry, 4-μm sections were deparaffinized,endogenous peroxidase activity wasquenched with 0.3% hydrogen peroxidefor 15 min at room temperature and anti-gen retrieval was performed for 10 minat 100°C in 10 mmol/L sodium citratebuffer, pH 7.4 (PAR-2 and TF stainings),for 15 min at 37°C with 0.025% pepsin in0.1 mol/L HCl (granulocyte staining) orfor 30 min at 37°C with 0.1% trypsin.After subsequent washing at room tem-perature and rehydration in phosphate-buffered saline, sections were blockedwith TENG-T (100 mmol/L Tris-HCl, pH

8.0, 50 mmol/L EDTA, 1.5 mmol/LNaCl, 2.5% gelatin, 0.5% Tween) for 10min (lymphocyte antigen 6 complex,locus G [LY-6G]) or with 10% normalgoat serum in phosphate-buffered salinefor 30 min (PAR-2) or with normal rabbitserum for 10 min (fibrin) and subse-quently incubated with antibodiesagainst LY-6G (fluorescein isothiocyanatelabeled) (1:1,000; Pharmingen, San Diego,CA, USA), TF (1:50; American Diagnos-tica product number 4509; Stamford, CT,USA), PAR-2 (1:80; SAM11; Santa Cruz,CA, USA) or fibrin(ogen) (1:1,000; bi-otinylated goat anti–mouse fibrinogenantibody; Accurate Chemical and Scien-tific, Westbury, NY, USA [32]) overnightat 4°C. For LY-6G staining, the slideswere first incubated for 15 min with arabbit–anti-FITC antibody (1:1,000), afterwhich the slides were incubated with theappropriate horseradish peroxidase– conjugated secondary antibodies. For fib-rin stainings, slides were incubated for30 min with avidin-biotin-peroxidasecomplex (DAKO K0355). For all stain-ings, 3,3′-diaminobenzidine (DAB) stain-ing was used to visualize peroxidase ac-tivity. Slides were photographed with amicroscope equipped with a digital cam-era (Leica CTR500; Leica Microsystems,Wetzlar, Germany). For granulocytestainings, we scored the number of posi-tive cells in 10 different fields (200× mag-nification).

Granulocyte MigrationGranulocytes were isolated from

EDTA-anticoagulated blood by polymor-phprep density gradient centrifugation(Axis-Shield, Oslo, Norway). Granulo-cytes were collected, diluted and labeledwith CellTracker Green as described be-fore (33,34). Briefly, the fluorescent dyewas incubated with the cells for 30 minat 37°C. Cells were washed once in me-dium to remove excessive soluble dyeand then incubated for 1 h at 37°C in me-dium without dye. Subsequently, cells(1 × 105) were transferred to 3 μmol/Lpore size HTS FluoroBlok Cell Cultureinserts, which were inserted in fitting 24-well plates containing IL-8 as the

chemoattractant in the presence or ab-sence of FVIIa. Promptly, fluorescencevalues representing the number of cellson the bottom side of the insert wereread at 37°C on a Series 4000 CytoFluorMulti-Well Plate Reader (PerSeptiveBiosystems, Framingham, MA, USA).The raw fluorescence data were cor-rected for background fluorescence andfading of the fluorophore; the data werethen plotted with GraphPad Prism 4.

Data AnalysisStatistical analyses were performed

using one-way analysis of variance, fol-lowed by the Mann-Whitney U test. Allvalues are reported as means ± SE. Statis-tical significance was set at P < 0.05.

RESULTS

TFlow Mice Are Protected againstDSS-Induced Colitis

The development of DSS-induced coli-tis was determined in wild-type, het-erozygous and homozygous TFlow mice.Importantly, wild-type mice developedcolitis, as evident from weight loss, re-duced length of the colon and increasedweight of the colon (Figure 1). Althoughthe TFlow mice did develop colitis aswell, it was clearly not as severe as in thewild-type mice. In detail, TFlow miceshowed a clear reduction in weight losscompared with wild-type mice. More-over, the colon length of wild-type micethat received DSS decreased by 23%,whereas the colon length decreased byabout 10% for both homozygous and het-erozygous TFlow mice. Finally, the colonweight increased by around 57% in wild-type animals, while in the TFlow animals,the colon weight increased by approxi-mately 18% in homozygous TFlow ani-mals and 27% in heterozygotes.

Reduced Inflammation andCoagulation in TFlow Mice

To confirm the macroscopic data show-ing that TFlow mice are protected againstDSS-induced colitis, we next scorededema, crypt loss, granulocytes, mononu-clear cells, fibrosis and ulceration on HE-

R E S E A R C H A R T I C L E


stained colon slides (Figure 2A). This dis-ease severity score increased from 1 incontrol animals to 12.8 ± 1.1 in DSS-treated wild-type mice and 10.2 ± 2.1 or7.8 ± 3.4 in DSS-treated heterozygous andhomozygous TFlow mice, respectively(Figure 2B). Most importantly, the num-ber of granulocytes and edema were sig-nificantly reduced in TFlow comparedwith wild-type mice. As shown in Fig-ure 2C, Ly-6G stainings confirm the re-duced number of granulocytes in theTFlow mice. In line with these reducedgranulocyte numbers, DSS-induced in-flammatory cytokine production was dra-matically diminished in wild-type micecompared with both heterozygous andhomozygous TFlow mice (Figure 2D). Toassess local coagulant activity, we scoredthe levels of fibrin(ogen) in the colon ofDSS-treated or untreated control animals.As shown in Figure 2E, DSS induces thelevel of fibrin(ogen) in both wild-typeand TFlow animals. Interestingly, how-ever, fibrin(ogen) levels were significantlyreduced in a dose-dependent manner inthe TFlow mice.

Anticoagulant Treatment Does NotLimit DSS-Induced Colitis

TF may exert its aggravating effect incolitis by either its procoagulant activityor by its cellular signaling properties. Todiscriminate between these two potentialmechanisms by which TF is detrimental,we evaluated the effect of anticoagulanttreatment in DSS-induced colitis. Asshown in Figure 3A, dalteparin treat-

ment did significantly reduce DSS-in-duced colonic levels of fibrin(ogen) tosimilar levels as observed in DSS-treatedheterozygous TFlow mice. Interestingly,however, dalteparin treatment did notprotect mice against DSS-induced colitisas opposed to TFlow mice. As shown inFigure 3B, clinical parameters of experi-mental colitis tested (body weight loss,colon length and colon weight) were notsignificantly different in dalteparin-treated versus -untreated control mice.

As shown in Figure 3C, dalteparintreatment did limit DSS-induced proin-flammatory cytokine production, al-though not as dramatically as in TFlowmice (compare Figures 2D and 3C).However, the reduced cytokine levels donot diminish tissue damage, as evidentfrom similar scores for edema, crypt loss,granulocytes, mononuclear cells, fibrosisand ulceration (Figure 3D). Ly-6G stain-ings do not show any difference in thenumber of granulocytes in dalteparin-versus control-treated mice (Figure 3E),although KC levels were reduced in thedalteparin-treated mice.

TF-Dependent Signaling in ColonEpithelial Cells Induces KC Levels

It is well accepted that granulocytesplay a detrimental role during the devel-opment of inflammatory bowel disease.Interestingly, a major difference betweenwild-type animals and TFlow mice in re-sponse to DSS-induced colitis is the in-flux of granulocytes into the inflamedcolon. This is particularly interesting be-

cause TF is known to influence cell mi-gration (mainly in the setting of tumorcells) (35,36). Consequently, we deter-mined whether TFlow granulocytes (ob-tained from TFlow mice) are affected intheir migratory response compared withwild-type granulocytes. However, FVIIa-stimulated wild-type and TFlow granu-locytes did migrate to IL-8 to a similarextent (data not shown). Thus, TF seemsnot to alter the intrinsic capacity of gran-ulocytes to migrate into inflamed tissues.

An alternative explanation for the in-creased influx of granulocytes in wild-type versus TFlow colon might be thelocal expression of granulocyte chemoat-tractants (IL-8, or its murine homolog KC).Indeed, KC levels are high in colon ho-mogenates of wild-type mice treated withDSS but not in both heterozygous and ho-mozygous TFlow animals (Figure 4A). Tosupport the hypothesis that low numbersof granulocytes in the colon of TFlowmice are due to impaired TF- dependentKC production, we first analyzed expres-sion levels of TF and its cognate receptorPAR-2 in ulcerative colitis (Figure 4B) andCrohn’s disease patients (data notshown). As shown in Figure 4B, TF andPAR-2 expression is most prominent oncolonic epithelial and goblet cells, al-though TF is also expressed on the in-flammatory infiltrate. Next, we deter-mined whether the colon would produceKC in response to FVIIa. Indeed, ex vivostimulation of small colon sections ob-tained from wild-type animals with FVIIasignificantly induced KC production in adose-dependent manner (Figure 4C). Sim-ilar experiments using TFlow colon sec-tions did not show increased KC produc-tion after FVIIa stimulation (Figure 4C).Finally, we stimulated colon sections witha PAR-2 agonist peptide and observedthat PAR-2 activation also led to KC pro-duction, thereby suggesting an importantrole for TF/FVIIa/PAR-2 signaling inlocal KC production (data not shown).

DISCUSSIONWe studied the role of TF in DSS-

induced colitis, and we have shown thatTF deficiency protects mice from DSS-



Figure 1. TF deficiency reduces the severity of clinical indices of DSS colitis in mice. (A)TFlow mice fed 1.5% (w/v) DSS in their drinking water for 7 d were protected against bodyweight loss compared with wild-type mice. The TFlow mice presented smaller changes incolon length (B) and colon weight (C) (shown in mg/cm) than wild-type mice. Data aremeans ± SEM (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001.

induced experimental colitis. Both ho-mozygous as well as heterozygous, TFlowmice have significantly reduced scores forboth clinical and histological parameterscompared with wild-type mice. The re-duced severity of experimental colitis cor-related with reduced levels of proinflam-matory cytokines in the TFlow mice,whereas the number of granulocytes atthe site of inflammation was also signifi-cantly reduced in TFlow mice. These dataare in perfect agreement with previousstudies showing that a monoclonal TF-blocking antibody limits experimental co-litis in wild-type animals (26), and ourdata thus underscore the importance ofTF in inflammatory bowel disease.

To determine whether TF would aggra-vate experimental colitis by activatingblood coagulation, wild-type mice weretreated with the low–molecular weightheparin dalteparin. The selected dose ofdalteparin is within the dose range usedin patients and has previously beenshown to effectively block coagulant ac-tivity and to limit murine diabeticnephropathy (28). However, dalteparintreatment did not ameliorate DSS-in-duced colitis, as evident from similar in-creases in colon weight, decreases incolon length and DSS-induced disease ac-tivity scores between treated and un-treated animals. Although dalteparintreatment reduced fibrin levels and lim-ited inflammatory cytokine production asefficient as heterozygous TF deficiency,anticoagulant treatment also did not limitgranulocyte influx into the inflamed area.Thus, it seems that the procoagulant ac-tivity of TF does not significantly con-tribute to the development of experimen-tal colitis. This result is particularlyinteresting considering the ongoing con-troversy on the importance of activatedblood coagulation in inflammatory boweldisease. Indeed, a vascular component tothe pathogenesis of inflammatory boweldisease was already proposed in 1934(37), and small observational studies didshow dramatic responses in patients withulcerative colitis (11–14). Nine out of tenpatients on combined heparin and sul-fasalazine therapy became asymptomatic,



Figure 2. TFlow mice present reduced inflammation and coagulation during experimentalcolitis. (A) Hematoxylin and eosin staining of colon sections of wild-type and TFlow miceshow that inflammation is less prominent in TFlow than in wild-type mice. (B) The totalhistopathological score is reduced in TFlow mice in comparison to wild-type mice. (C)Granulocyte infiltration is specifically decreased in colon sections of TFlow mice com-pared with wild-type mice, as shown by LY-6G staining. (D) Decreased levels of inflamma-tory cytokines in colon homogenates of TFlow mice. (E) Reduced levels of fibrin(ogen) inTFlow mice compared with control animals. Data are means ± SEM (n = 7). *P < 0.05, **P <0.01, ***P < 0.001.

whereas patient number 10 had a partialresponse (12). Nadroparine treatmentalone significantly improved endoscopicand histological signs of inflammation in20 out of 25 patients (11), whereas dal-teparin treatment resulted in complete re-mission in 6 out of 12 patients (13). Al-though a small clinical randomized trialcould not confirm these beneficial effects(15), the inclusion criteria of mainlymildly affected patients and the short du-ration of LMWH treatment did not allowconfirmative conclusions.

Probably the most important findingof our study is the large difference in in-

flux of granulocytes into the inflamedcolon between wild-type and TFlowmice in response to DSS treatment. Thisis particularly interesting, since granulo-cytes are of pivotal relevance in the clini-cal setting of inflammatory bowel dis-ease, as evident from, for instance, thepromising effect of Natalizumab andMLN02 for the treatment of Crohn’s dis-ease and ulcerative colitis (38,39). More-over, it is well know that TF induces mi-gration of a large variety of cells (35,36).Although the presence of TF on granulo-cytes is under debate (40), we deter-mined whether granulocytes derived

from TFlow mice did have a hamperedmigratory capacity compared with gran-ulocytes from wild-type mice. However,FVIIa-stimulated wild-type granulocytesmigrated toward IL-8 as efficient as wild-type granulocytes, suggesting the ob-served differences in granulocyte influxare not due to the intrinsic migratory ca-pacity of the granulocytes. Instead, weshow that the low numbers of granulo-cytes in the colon of TFlow mice are mostlikely due to impaired TF-dependent KCproduction. Indeed, KC levels are signifi-cantly induced in wild-type mice treatedwith DSS but not in both homo- and het-



Figure 3. Anticoagulant treatment does not ameliorate DSS-induced colitis. (A) Dalteparin treatment does reduce the levels offibrin(ogen) in DSS-treated animals. (B) Dalteparin does not limit DSS-induced changes in body weight and colon length or weight. (C)Cytokine levels in colon homogenates of dalteparin- and control-treated mice after 7 d DSS treatment. Histopathological score (D), num-ber of LY-6G–positive cells and KC production (E) in dalteparin- and control-treated mice. Data are means ± SEM (n = 10). *P < 0.05. A.U.,arbitrary units.

erozygous TFlow mice. In an apparentdiscrepancy, the absence of KC was pre-viously shown to increase the suscepti-bility to DSS-induced colitis, mainly bypreventing granulocyte infiltration intothe inflamed colon (41). However, granu-locyte numbers are tightly regulated dur-ing inflammatory conditions and bothlow as well as high numbers may bedetrimental. Dalteparin treatment re-duces KC levels in wild-type mice, butthese levels are still significantly in-creased compared with TFlow animals.The decreased KC levels after anticoagu-lant treatment are most likely due to re-duced thrombin levels. Indeed, it is wellknown that thrombin induces IL-8/KClevels in different cell types (42,43).

Interestingly, IL-8 (the human counter-part of KC) expression is significantly in-duced in the colonic mucosa of inflam-matory bowel disease patients, and itsexpression is in direct proportion withthe degree of inflammation (44,45), sug-gesting an essential role of IL-8 in thepathogenesis of inflammatory bowel dis-ease. Moreover, colon epithelial cells area known source of IL-8 in inflammatoryconditions (46), and the FVIIa/TF com-plex was previously shown to induce IL-8 levels in several different cell lines(47–49). In agreement, we show that exvivo FVIIa-dependent KC production isdramatically reduced in TFlow colon sec-tions compared with colon sections ofwild-type mice. These data suggest an

important role for the TF/FVIIa complexin KC production and subsequent granu-locyte influx during colitis.

Our data suggest that the TF/FVIIacomplex aggravates experimental colitisdepending on the signaling properties ofTF. In line with this notion, activation ofPAR-2 by either the TF/FVIIa complex oragonist peptides leads to IL-8 productionin several cell lines, including epithelialcells of the gastrointestinal tract (47,50,51),but also in the whole colon section (thisreport). Moreover, promoter methylationof PAR-2 seems to correlate with theseverity of ulcerative colitis (52), andPAR-2 deficiency was protective in DSS-,2,4,6-trinitrobenzenesulfonic acid (TNBS)-and oxazolone-induced colitis (53). Mostimportantly, endogenous PAR-2 seemedto aggravate experimental colitis by in-ducing leukocyte recruitment toward thecolon. In DSS-induced colitis, the lack ofPAR-2 in hematopoietic cells did not pro-tect the animals, suggesting an importantrole for the epithelial cells (54).

In conclusion, our data show that TFplays a detrimental role in experimentalcolitis. We suggest that TF-dependentPAR-2 activation in colon epithelial cellsleads to local KC production, therebyprovoking granulocyte influx with subse-quent inflammation and organ damage.

ACKNOWLEDGMENTSWe would like to thank Joost Daal-

huisen, Marieke ten Brink and DanielleKruijswijk for expert technical assistance.This work is supported by grant WO 06-02of the Dutch Digestive Diseases Founda-tion (Maag Lever Darm Stichting [MLDS]).

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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Figure 4. TF-dependent signaling in colon epithelial cells induces KC levels. (A) KC produc-tion in colon homogenates of wild-type and TFlow mice after 7 d DSS treatment. (B) TFand PAR-2 expression in colon sections of ulcerative colitis patients. (C) KC productionafter ex vivo stimulation of wild-type and TFlow colon section with FVIIa for 24 h. Data aremeans ± SEM of three independent experiments. *P < 0.05.

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