of March 26, 2014.This information is current as

without Affecting Protective ImmunityDevelopment of Murine Lyme Arthritis

Antagonist Prevents1β4αA Small Molecule

Röllinghoff and André GessnerMould, Martin J. Humphries, Rupert Hallmann, Martin

PaulUlrich Stilz, Jonathan D. Humphries, G. Paul Curley, A. Joachim Gläsner, Horst Blum, Volkmar Wehner, Hans

http://www.jimmunol.org/content/175/7/47242005; 175:4724-4734; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/175/7/4724.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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A Small Molecule �4�1 Antagonist Prevents Development ofMurine Lyme Arthritis without Affecting Protective Immunity1

Joachim Glasner,* Horst Blum,† Volkmar Wehner,† Hans Ulrich Stilz,†

Jonathan D. Humphries,‡ G. Paul Curley,‡ A. Paul Mould,‡ Martin J. Humphries,‡

Rupert Hallmann,§ Martin Rollinghoff,* and Andre Gessner*2

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammationis dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesionmolecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyzethe functional importance of �4�1-dependent cell adhesion for the development of arthritis and for the antibacterial immuneresponse. S16197 is shown to interfere specifically with the binding of �4�1 integrin to its ligands VCAM-1 and fibronectin in vitro.Treatment of B. burgdorferi-infected C3H/HeJ mice with the �4�1 antagonist significantly ameliorated the outcome of clinicalarthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflam-matory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor thedevelopment of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere withAb-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protectiveimmune response, in Lyme arthritis is dependent on the �4�1-mediated influx of inflammatory cells. The onset of inflammationcan be successfully targeted by treatment with S18407. The Journal of Immunology, 2005, 175: 4724–4734.

L yme disease caused by three genospecies of the spirocheteBorrelia burgdorferi sensu lato is the most common tick-borne disease in humans in the Northern Hemisphere. In-

fection with B. burgdorferi provokes a multiorgan inflammatoryailment, but the precise underlying mechanisms are not well un-derstood (1). The disease is characterized by some or all of thefollowing clinical manifestations: an initial erythematous rash,lymphocytoma cutis, acrodermatitis chronica atrophicans, neuro-logical disorders, carditis, and, especially, arthritis. Intermittent orchronic oligoarticular arthritis primarily affecting large joints suchas the knees is a debilitating late complication. Although mostpatients with acute Lyme arthritis can be treated effectively withantibiotics, �10% of patients have persistent knee swelling formonths to years after �2 mo of medication. This condition hasbeen termed antibiotic treatment-resistant Lyme arthritis (2).

In a murine model of Lyme borreliosis, used to delineate mech-anisms of inflammation and protective immunity, genetically sus-ceptible mouse strains like C3H/He develop severe carditis andarthritis when infected with B. burgdorferi. The disease pattern

arising in joints of mice resembles human Lyme arthritis, withedema, hyperproliferation of synovial cells, as well as massiveinfiltration of neutrophils and monocytes, but markedly few lym-phocytes into tissue and joint space (3). The severity of inflam-mation peaks at 2–3 wk after infection and regresses spontane-ously in the presence of B. burgdorferi-specific Abs.

Leukocytes are recruited from circulating blood to the inflamedtissue by sequential adhesive interactions with the endothelium inpostcapillary venules. This process involves capture on, rollingalong, and firm adhesion to the microvascular endothelium, fol-lowed by transmigration through the vessel wall and further mi-gration across basal lamina/extravascular tissue (4). All of thesesteps, along with a costimulation of cellular activation, are orches-trated by cell adhesion molecules (CAMs)3 expressed on both leu-kocytes and endothelial cells (EC). CAMs are often divided intofour major subsets responsible for the different steps in extravasa-tion, each revealing a partial functional redundancy of its mem-bers. The selectins and heavily glycosylated selectin ligands me-diate the initial phases (tethering and rolling, i.e., slowing thepassage of leukocytes), whereas leukocyte integrins and their en-dothelial counter-receptors of the Ig superfamily are mainly in-volved in firm attachment of leukocytes to endothelial surfacesfollowing activation by chemokines or other stimuli (5). Membersof the Ig superfamily (PECAM-1, junctional adhesion molecules)are engaged furthermore in diapedesis, when leukocytes respond-ing to a chemoattractant concentration gradient polarize andsqueeze through the endothelial junctions (6). Blockade of any ofthe hierarchically proceeding steps leads to an interruption of leu-kocyte extravasation.

*Institute for Clinical Microbiology, Immunology, and Hygiene, University of Er-langen-Nurnberg, Erlangen, Germany; †Aventis Pharma Deutschland GmbH, a com-pany of the Sanofi-Aventis Group, Frankfurt am Main, Germany; ‡Wellcome TrustCentre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester,Manchester, United Kingdom; and §Department of Experimental Pathology, Univer-sity Hospital, Lund, Sweden

Received for publication December 29, 2004. Accepted for publication June 30, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Federal Ministry of Education and Research(Bundesministerium fur Bildung und Forschung) and the Interdisciplinary Center forClinical Research (Interdisziplinares Zentrum fur Klinische Forschung), SubprojectA2, at the University Hospital of the University of Erlangen-Nuremberg (Erlangen,Germany).2 Address correspondence and reprint requests to Dr. Andre Gessner, Institut furKlinische Mikrobiologie, Immunologie und Hygiene, Wasserturmstrasse 3, D-91054Erlangen, Germany. E-mail address: gessner@mikrobio.med.uni-erlangen.de

3 Abbreviations used in this paper: CAM, cell adhesion molecule; EC, endothelialcell; ECM, extracellular matrix; MS, multiple sclerosis; MAdCAM-1, mucosal ad-dressin CAM-1; RT, room temperature; HBS, HEPES-buffered saline; HEC, hy-droxyethylcellulose; p.i., postinfection; COX-2, cyclooxygenase-2; HPRT, hypoxan-thine phosphoribosyltransferase; PMN, polymorphonuclear leukocyte.

The Journal of Immunology

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The expression level and relevance of CAMs during B. burg-dorferi infection has been studied in several settings. In patientswith antibiotic treatment-resistant Lyme arthritis-enhanced expres-sion of P-selectin, vascular adhesion protein-1, ICAM-1, LFA-1,VCAM-1, and �4�1 was detected in affected joints (2). In the mu-rine model of acute Lyme arthritis, B. burgdorferi was shown toup-regulate CAMs such as E-selectin, P-selectin, ICAM-1, andVCAM-1 in synovia (7). However, E- and P-selectin double-de-ficient mice displayed no significant differences to their wild-typelittermates during infection with B. burgdorferi in terms of arthritisdevelopment and tissue spirochete levels (8).

In addition, some reports described in vitro the enhanced ex-pression of functional P-selectin, E-selectin, ICAM-1, andVCAM-1 on murine as well as human ECs upon contact withintact B. burgdorferi, spirochetal lysates, and OspA or OspB, re-spectively (9–13). Inhibition experiments conducted by Burns andFurie (14) and by Sellati et al. (11) using mAbs to �4, CD18, andE-selectin demonstrated that monocytes and CD4� lymphocytesmainly engage �4 and CD11/CD18 integrins for migration acrossB. burgdorferi-activated endothelium, whereas extravasation ofneutrophils involves E-selectin- and CD11/CD18-dependentpathways.

The integrin �4�1 (very late Ag-4, VLA-4, CD49d/CD29) is akey cell surface receptor that is expressed constitutively on mostleukocytes such as lymphocytes, monocytes/macrophages, eosin-ophils (15, 16), mast cells (17), basophils (18), and NK/NKT cells(19). On neutrophils, a minor baseline expression is enhancedupon activation/emigration of these cells (20–24). �4�1 is a non-covalent heterodimer composed of an �4 (155 kDa) and a �1 (150kDa) chain. It is known that the subunits, like in most integrins, areconformationally mobile, moving between high-affinity, active andlow-affinity, inactive states in response to stimuli (25). �4�1 bindsto vascular CAM-1 (VCAM-1, CD106), an inducible EC surfaceprotein, to the CS1 region in the alternatively spliced type III con-necting segment of the extracellular matrix (ECM) protein fi-bronectin (26, 27), and to a number of other ligands (28–33),thereby mediating cellular adhesion and activation through a va-riety of cell-cell and cell-matrix interactions (16).

Because the association of �4�1/VCAM-1 in numerous animalmodels of chronic inflammatory, autoimmune, and allergic humandiseases (e.g., multiple sclerosis (MS), asthma, inflammatory ar-thritis, atherosclerosis, and inflammatory bowel disease) is welldocumented (reviewed in Ref. 34), the efficacy of blocking �4�1

with specific agents has been evaluated in many studies. The useof Abs directed against the �4 subunit of �4�1 for intervention inseveral animal models was successful (35–39) and has led to thedevelopment of the humanized mAb Natalizumab (approved inNovember 2004) that was shown to be effective in both MS andinflammatory bowel disease (40, 41).

Natural ligands of �4�1 (fibronectin, VCAM-1) and anti-�4

mAbs have served as the basis for the design of highly selective,peptide-based small molecule �4�1 antagonists with low or evensubnanomolar IC50 values (42). To date, targeting of �4�1 withsynthetic antagonists has been examined in mouse models of lunginflammation/asthma (43, 44) and experimental autoimmune en-cephalomyelitis/MS (45, 46) with predominantly beneficial results.However, to our knowledge, no small molecule antagonists spe-cific to �4�1 have been tested so far for treatment in an animalmodel of an infectious disease.

Therefore, in this study, we attempted to determine the effects ofthe newly developed �4�1 inhibitor S18407 on the onset and res-olution of B. burgdorferi-induced Lyme arthritis, the antibacterialimmune response, and the elimination of spirochetes in systemi-cally treated C3H/HeJ mice. S18407 is a small molecule �4�1

antagonist, which belongs to the class of hydantoin-based inhibi-tors. The compound represents a prodrug that is physiologicallymetabolized at a rate of �25% to the active carboxylic acidS16197 inherently exhibiting poorer oral bioavailability. S16204 isthe sodium salt of S16197 with an improved solubility in water(Fig. 1).

Materials and MethodsMice

Female C3H/HeJ mice were obtained from Harlan Winkelmann. All of themice were housed under specific pathogen-free conditions and were 4–5wk old at the time of infection. Animals destined for treatment were se-lected randomly and divided into three groups consisting of 6–12 miceeach. All of the experiments were conducted according to the institutionalguidelines for animal welfare of the Institute for Clinical Microbiology,Immunology, and Hygiene at the University of Erlangen-Nuremberg andapproved by the District Government of Middle Franconia.

Cell lines

The following cell lines were obtained: mlEnd.1, murine mesenteric lymphnode-derived EC line, created in the same fashion as the line bEnd.3 (47,48); U-937, human promonocytic leukemia cell line (49) (American TypeCulture Collection (ATCC)); JY, EBV-transformed human B lymphoblas-toid cell line (50); KL/4, LFA-1-transfected K562 cell line (highly undif-ferentiated human chronic myelogenous leukemia cell line) (51); MOLT-4,human thymic T lymphoblastoid leukemia cell line (52) (ATCC); and HT-1080, human fibrosarcoma cell line (53) (ATCC).

In vitro binding assays

Cell attachment assays as well as cell-free binding assays were performedto test the inhibitory efficacy of the drug S16197 to the interaction ofselected pairs of adhesion molecules in vitro.

Cell attachment assays (54)

Assays with JY and KL/4 cells. Ninety-six-well tissue-culture-treated mi-crotiter plates (round-bottom; Corning/Costar) were coated with purifiedfusion proteins consisting of the Fc portion of human IgG and the extra-cellular domain of mucosal addressin CAM-1 (MAdCAM-1) (2 �g/ml) orICAM-1 (10 �g/ml), or with human vitronectin (2.5 �g/ml) for 60 min atroom temperature (RT) (all dilutions in HEPES-buffered saline (HBS) with20 mM HEPES and 150 mM NaCl; pH 7.4). Wells were aspirated andblocked with heat-denatured BSA (10 mg/ml) for 30 min at RT. Serialdilutions of the compound S16197 were prepared in HBS containing 0.4mM Mn2�. For the KL/4-ICAM-1 assay, an activating anti-�2 mAb(KIM185) was added at 50 �g/ml to the HBS/Mn2� buffer. JY and KL/4cells were cultured in RPMI 1640 and resuspended at 1 � 106 cells/ml inDMEM/25 mM HEPES.

Fifty microliters of cells and 50 �l of the diluted compound were addedto the wells for 20 min at 37°C with 5% (v/v) CO2. To determine thereference value for 100% attachment enabling quantification of the per-centage of cells specifically bound, cells were added directly to uncoated/unblocked control wells. The control wells were fixed without washingwith 20 �l of 50% (w/v) glutaraldehyde. Unbound and loosely attachedcells in the rest of wells were aspirated, and specifically bound cells werefixed with 100 �l of 5% (w/v) glutaraldehyde for 30 min. After aspiratingthe fixative, the wells were washed gently three times with dH2O, andattached cells were stained for 60 min with 0.1% (w/v) Crystal Violet in100 mM MES (pH 6.0). Finally, the wells were washed four times withdH2O before adding 100 �l of 10% (v/v) acetic acid to solubilize dye.Results were assessed by spectrophotometric measurement of absorbanceat 570 nm using a plate reader.

FIGURE 1. Chemical structures of the orally administered prodrugS18407 and its corresponding active drugs S16197 and S16204, used forinhibition studies in vitro.

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Assays with U-937 cells (All steps of the assays were performed at RT).Ninety-six-well microtiter plates with round-bottom (MaxiSorp; Nunc)were coated with 10 �g/ml goat anti-human Ig (MP Biomedicals) in 50mM Tris buffer (pH 9.5) and incubated for 60 min. Plates were washedwith PBS and blocked for 30 min with 1% BSA in PBS. After washingwith PBS, the huVCAM-1-IgG fusion protein (VCAM-1 (I–VII)-IgG; Bio-tech Australia) was added at 0.37 �g/ml in blocking buffer and incubatedfor 1.5 h. Plates were washed again with PBS, and a Fc receptor blockingbuffer (1 mg/ml �-globulin; Sigma-Aldrich) in binding buffer (100 mMNaCl, 100 �M MgCl2, 100 �M MnCl2, 1 mg/ml BSA in 50 mM HEPESbuffer; pH 7.5) was added for 30 min.

The plates were washed with PBS and preincubated for 20 min withserial dilutions of compound S16197 in binding buffer before adding U-937cell suspension (1 � 106 cells/ml, preblocked in Fc receptor blockingbuffer). After 20 min liquid was poured off gently, and plates were drainedin a tank with stop buffer (100 mM NaCl, 100 �M MgCl2, 100 �M MnCl2in 25 mM Tris; pH 7.5). Exposure to stop buffer was repeated once beforestaining cells with the DNA-specific fluorochrome Hoechst 33258 (Sigma-Aldrich; 16.7 �g/ml in PBS with 4% (w/v) formaldehyde and 0.5% (v/v)Triton X-100) for 15 min. Liquid was poured off, and plates were drainedtwice in stop buffer as described above. Number of cells was assessed bymeasurement of fluorescence in a microtiter plate cytofluorometer (Milli-pore; single measure, filter A/A (360/40/460/40)).

Cell-free binding assays

Integrins (�4�1, MOLT-4 cell extract; �2�1, HT-1080 cell extract; �5�1,placental extract) were diluted in PBS without NaHCO3 and immobilizedon 96-well microtiter plates (Immulon 3; Dynex) overnight at RT. Dilu-tions applied were 1/100 for �4�1, 1/200 for �2�1, and 1/400 in the case of�5�1. Plates were blocked for 3 h at RT with 5% BSA, 0.05% Na-azide inTBS (150 mM NaCl, 25 mM Tris-HCl; pH 7.4). After washing three timeswith TBS containing 0.1% BSA and 2 mM Mn2�, the appropriate coun-terligands of the integrins were added with or without compound S16197and incubated for 3 h under the following conditions: VCAM-1-IgG, 0.5�g/ml at 37°C; biotinylated fibronectin fragments 50 kDa and H/120, each0.1 �g/ml at RT; and biotinylated collagen I, 1 �g/ml (rough estimate) atRT. Plates were washed three times as before and incubated for 20 minwith peroxidase-conjugated ExtrAvidin (Sigma-Aldrich; diluted 1/500 inTBS) or in the case of VCAM-1-IgG with a peroxidase-conjugated anti-Ig-Fc Ab (diluted 1/1000 in TBS). After washing four times as before, theperoxidase substrate ABTS (11 mg of ABTS dissolved in 0.5 ml of dH2O� 10 ml of 0.1 M NaOAc/0.05 M NaH2PO4 (pH 5.0) � 100 �l of 0.3%H2O2) was added. Enzymatic reaction was stopped after 15 min with asolution of 2% SDS in TBS, and absorbance was measured spectrophoto-metrically at 405 nm in a plate reader.

Cell attachment to B. burgdorferi-activated endothelium

To assess the inhibitory efficacy of S16204 to the adhesion of specificleukocytes to B. burgdorferi-activated ECs, 3 � 104 mlEnd.1 cells wereseeded onto Lab-Tek chamber slides (Nunc) and grown to confluency.Cells were activated by coincubation with 1 � 107 spirochetes in 100 �l ofDMEM for 50 h at 37°C. Murine thioglycollate-elicited peritoneal cellscontaining �65% neutrophilic granulocytes, or U-937 cells (each 1 � 106

in 100 �l of medium) were preincubated with the drug at a final concen-tration of 3 nM for 30 min at RT and added to ECs that were washed threetimes with medium before. Leukocytes were permitted to adhere for 20 minunder shear (85 rpm on a horizontal shaker; Buhler) at RT. Cells were thenwashed cautiously three times in DMEM for removal of nonadherent cellsand fixed with 2.5% glutaraldehyde in DMEM for at least 2 h. Measure-ments were done in quadruplicate. The assays were evaluated by directcounting of four random visual fields.

Infection with B. burgdorferi

The N40 isolate of B. burgdorferi was grown at 32°C in BSK-H mediumcontaining 6% rabbit serum (Sigma-Aldrich) (55) and underwent fewerthan five in vitro passages before inoculation. Spirochetes were enumeratedwith a blood cell counting chamber by dark-field microscopy and dilutedwith sterile medium. Mice were infected by s.c. injection of 5 � 105 bac-teria in 50 �l of BSK-H into the right hind footpad.

Because inflammation predominantly emerges unilaterally dependent oninoculation site, only joints derived from the affected side of the body wereused for examination in all respective experiments.

In vivo treatment with the �4�1 antagonist

Before application, S18407 was ground up in an agate mortar followed bysuspending in a solution of 5 mg/ml hydroxyethylcellulose (HEC) in tap

water. Groups of 6–12 mice were treated once daily starting on the day ofinoculation for a period of 21 days. At this time the extent of ankle swellingin untreated control mice was, as expected, spontaneously decreasing.Doses of 3 mg or 30 mg S18407 per kilogram body weight or the vehicleHEC alone were administered in a total volume of 200 �l per mouse by theoral route using a bulb-headed cannula (1.0 � 55 mm, straight; Acufirm).

Measurement of joint swelling

The development of joint swelling, grossly correlating with the severity ofarthritis, was monitored by measuring the thickness of the infected and, asreference, of the noninfected contralateral tibiotarsal joint by means of ametric caliper (Kroeplin). Values obtained were used to calculate the foldincrease in swelling of the infected joint over the contralateral joint. Mea-surements were taken in the anterior to posterior orientation with extendedankles through the thickest portion.

Histology of ankle joints

Rear limbs were excised above and below the tibiotarsal joint and embed-ded in Tissue-Tek O.C.T. compound into vinyl cryomolds (Sakura Fi-netek), snap-frozen on liquid nitrogen, and stored at �70°C until analysisby histology. To preserve morphology, ankle joints were dissected in totowithout preceding decalcification at �25°C on a HM 500 OM cryostat(Microm) using a tungsten carbide-tipped microtome knife (16 cm, D-profile; Microm). Sagittal sections (10 �m) were mounted on slides pre-viously coated with 50 �g/ml poly-L-lysine in 10 mM Tris-HCl (pH 8.0),fixed with ice-cold acetone for 20 min, and stained with hemalaun (Dr. K.Hollborn & Sohne) and eosin Y (Sigma-Aldrich) for 4 min each. Thesections were dehydrated in an ascending sequence of ethanol and twice inRotihistol (Roth) before embedding in DePeX (Serva). Pictures were takenwith a digital video camera (Spot RT Color; Diagnostic Instruments)adapted to an Axiophot microscope (Zeiss) using the MetaVue 4.6 software(Universal Imaging).

Determination of spirochete burden in ankle joints byquantitative PCR

DNA was extracted from rear ankle joints of infected mice using theQIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instruc-tions. Elution volume was 200 �l of AE buffer (10 mM Tris-HCl, 0.5 mMEDTA; pH 9.0).

Simultaneous detection and quantification of B. burgdorferi was con-ducted with the LightCycler PCR system (Roche) using the followingprimers: forward, 5�-TCTTTTCTCTGGTGAGGGAGCT-3� and reverse,5�-TCCTTCCTGTTGAACACCCTCT-3�, which amplify a 70-bp frag-ment of the Borrelia flagellin B gene ( flaB, chromosomal, single copy);and forward, 5�-CCAGCCACAGAATACCATCC-3� and reverse, 5�-GGACATACTCTGCTGCCATC-3�, which flank a product 154 bp inlength corresponding to the murine nidogen-1 gene that was used as ref-erence for normalization. HPLC-purified primers were provided by Meta-bion. PCRs for flagellin B and nidogen-1 were performed in separate runs.The 20-�l reaction volume contained 1 U Platinum Taq polymerase, 2 �lof 10� reaction buffer, 4 mM MgCl2 (all obtained from Invitrogen LifeTechnologies), 0.4 �M each primer, 0.25 mM deoxynucleosid triphosphatemix (Amersham Biosciences), 0.5 � SYBR Green I dye (Roche), 0.5mg/ml BSA (New England Biolabs), 5% DMSO (Sigma-Aldrich), and 2 �lof extracted DNA in a dilution of 1/10 in H2O or 2 �l of external standardtemplate. DNA preparations extracted from the reference strain of B. burg-dorferi sensu stricto were used to establish a standard curve for flagellin BPCR. Serial dilutions of a pGEM-T Easy plasmid (Promega) harboring amouse nidogen-1 fragment were included as standards in each normaliza-tion PCR for nidogen-1. Applying the LightCycler 5.32 software (Roche),the number of spirochetes was calculated on the basis of the standards andnormalized to 105 copies of nidogen-1.

The amplification program consisted of the initial denaturation step at95°C for 10 min and 50 cycles of denaturation at 95°C for 15 s, annealingat 61°C (flagellin B)/60°C (nidogen-1) for 10 s and extension at 72°C for15 s. The temperature transition rate was 20°C/s. Fluorescence was mea-sured at the end of each extension step. After each amplification, meltingcurves were acquired to determine the specificity of PCR products.

Restimulation of lymphocytes and cytokine assays

Thirty-five days postinfection (p.i.), spleens of mice were removed, andsingle-cell suspensions were prepared. Cells (4 � 106/ml) were cultured invitro for 48 h in the presence of B. burgdorferi Ag (10 bacterial equivalentsper one spleen cell) or Con A (7.5 �g/ml final; Sigma-Aldrich) in Click-RPMI (Biochrom), supplemented with 2 mM L-glutamine, 10 mM HEPESbuffer, 7.5 mM NaHCO3, 0.05 mM 2-ME, 165 IU/ml penicillin G, 80

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�g/ml streptomycin, and 10% selected FCS with a total LPS content �100pg/ml.

The concentrations of IL-4 and IFN-� in supernatants of stimulatedspleen cells were measured by specific two-site ELISAs, with referencestandard curves obtained from known amounts of the respective murinerecombinant cytokine. Matched Ab pairs for the detection of cytokineswere purchased from BD Pharmingen (anti-mouse IL-4, clone 11B11; anti-mouse IL-4-biotin, clone BVD6-24G2; anti-mouse IFN-�, clone R4-6A2;anti-mouse IFN-�-biotin, clone XMG1.2). They were used according to thesupplier’s recommendations with a streptavidin/biotin amplification(StreptABComplex/HRP; DakoCytomation) and tetramethylbenzidine(Sigma-Aldrich) as a substrate for HRP.

Results were assessed by spectrophotometric measurement of absor-bance at 405 nm wavelength.

Cell isolation from joints and flow cytometry

For the isolation of single-cell suspensions, rear hind limbs were excisedjust above and below the tibiotarsal joint. After removal of skin and thor-ough dissection with scissors, the tissue particles were digested for 1 h at37°C in 0.1% collagenase D (Roche) in HBSS. The resulting cell suspen-sion was passed through a 70-�m cell strainer (BD Falcon) to removeinsoluble material and washed with Click-RPMI containing FCS. Totalyield was determined by counting of isolated cells in a hemacytometerchamber with trypan blue exclusion and ignoring erythrocytes. Cells wereanalyzed by flow cytometry in the presence of 1 �g/ml propidium iodideon a FACSCalibur cytometer using CellQuest Pro software (BD Bio-sciences). Low fluorescence detritus and propidium iodide-positive cellsconsidered as nonviable were gated out before analysis. Abs used wereanti-mouse Fc�RIII/II (CD16/CD32) (clone 2.4G2), anti-mouse CD45-al-lophycocyanin (clone 30-F11), and anti-mouse Gr-1(Ly-6G and Ly-6C)-PE (clone RB6-8C5) (all obtained from BD Pharmingen).

Detection of B. burgdorferi-specific IgG1and IgG2a and totalIgE

On days 12 and 35 p.i., serum was collected by bleeding the retro-orbitalplexus of B. burgdorferi-infected mice at sacrifice and analyzed for B.burgdorferi-specific Ig isotypes using Ab capture ELISAs. Microtiterplates were coated with sonicated B. burgdorferi at a concentration of 5�g/ml. Serum samples diluted 1/100 (IgG1 and IgG2a) or 1/5 (IgE) inPBS/10% FCS were added to plates and incubated overnight at 4°C. Boundmurine Ig was detected by addition of alkaline phosphatase-conjugatedAbs to murine IgG1 (clone G1-6.5) or IgG2a (clone R19-15) (both 1/1000diluted; BD Pharmingen) for 1 h. Total IgE was measured by a standardsandwich ELISA applying anti-mouse IgE (clone R35-72) and anti-mouseIgE-biotin (clone R35-118) (both 2 �g/ml) as matched Ab pair and purifiedmouse IgE as standard, all obtained from BD Pharmingen. Plates weredeveloped by incubation with 1 mg/ml p-nitrophenyl phosphate (Sigma-Aldrich) followed by measuring of absorbance in a spectrophotometer at405 nm.

RNA preparation and cDNA synthesis

At various time points after infection indicated in the figure legend, the rearankle joints were excised, snap-frozen in liquid nitrogen, and stored at�70°C until use. After mechanical processing with scissors, total RNAwas isolated from ankles using the RNAqueous kit (Ambion). DNase treat-ment was performed with the DNA-free kit (Ambion) applying 4 U ofDNase I for 1 h at 37°C. Absence of detectable DNA contamination wasverified by a subsequent PCR with primers matching a murine genomicsequence.

For cDNA synthesis 8 �l of total RNA were mixed with 500 ng ofoligo(dT) primer, 0.5 mM deoxynucleosid triphosphate mix (both obtainedfrom Amersham Biosciences), 4 �l of 5� first-strand buffer, 10 mM DTT,and 50 U of SuperScript II RT polymerase (all obtained from InvitrogenLife Technologies). Samples were incubated for 50 min at 42°C followedby heat inactivation at 70°C for 15 min.

Quantitative RT-PCR

Quantitation of mRNA expression profiles of Vcam-1, Il-6, Il-1�, and cy-clooxygenase (COX)-2 in ankle joints was conducted by RT-PCR with theLightCycler PCR system (Roche). In each quantitative PCR run, an exter-nal standard curve was generated using a 4 log spanning serial dilution ofthe vector plasmid pGEM-T Easy (Promega) containing one copy of therespective target sequence per plasmid. The standard curves were createdby the LightCycler 5.32 software (Roche) and applied for calculation ofamplification efficiency and mRNA expression levels. The content of thehousekeeping gene hypoxanthine phosphoribosyltransferase (Hprt) was

determined in a separate run on the basis of an external standard as welland used for normalization of the acquired values. In addition, porphobi-linogen deaminase (synonym: hydroxymethylbilane synthase) levels in thesamples were assessed to validate the results of Hprt normalization (datanot shown).

PCR was performed in a final volume of 20 �l containing 1 U PlatinumTaq polymerase, 2 �l of 10� reaction buffer, 4 mM MgCl2 (all obtainedfrom Invitrogen Life Technologies), 0.4 �M each primer (for primer char-acteristics, see Table I), 0.25 mM deoxynucleosid triphosphate mix (Am-ersham Biosciences), 0.5� SYBR Green I dye (Roche), 0.5 mg/ml BSA(New England Biolabs), 5% DMSO (Sigma-Aldrich), and 2 �l of cDNA ina dilution of 1/10 in H2O or external standard plasmid template. PCRparameters were set as described above, adapting the annealing tempera-tures according to Table I.

Statistical analysis

Statistical analysis of data was performed by two-tailed Student’s t testsusing GraphPad PRISM (GraphPad). Comparison of values obtained frommore than two groups of animals was also made by one-way ANOVA.Differences were considered significant at p � 0.05.

ResultsCompound S16197 specifically inhibits binding of �4�1 toVCAM-1 and CS1 fibronectin

The potency of the newly developed compound was initially an-alyzed in vitro in cell-free binding assays and cell-based attach-ment assays. As listed in Table II, this compound inhibited the�4�1-VCAM-1 interaction in the low nanomolar concentrationrange (IC50, 1.23 nM for cell-free �4�1 and 0.29 nM for U-937-linked �4�1, respectively). An even higher inhibitory efficacy ofS16197 was observed toward the interaction of cell-free �4�1 withthe CS1-containing fibronectin fragment H/120 (IC50, 0.22 nM).The binding of JY cells that also express �4, but in combinationwith the �7 subunit, to the �4�7 ligand MAdCAM-1 was onlyblocked at a much higher concentration (IC50, 6.23 �M). No effectof S16197 was detected on the binding properties of other �1-containig integrins (�2�1, �5�1, and �6�1) or on integrins thatcontain subunits other than �4 and �1 (IC50 � 50 �M). Thus, thecompound S16197 targets selectively the heterodimeric molecule�4�1.

The �4�1 antagonist inhibits leukocyte binding toB. burgdorferi-activated ECs

Previous studies have shown that coincubation of B. burgdorferiwith mouse ECs (bEnd.3) resulted in enhanced expression ofVCAM-1. As a consequence of this up-regulation, �4�1-express-ing murine L1.2 B lymphoma cells were found to bind much moreefficiently to the bEnd.3 cells (9).

Expanding these results, we examined in vitro the efficacy ofS16204 to antagonize binding of primary leukocytes to mlEnd.1ECs that were specifically activated by B. burgdorferi in a celladhesion assay. As depicted in Fig. 2, both a mixture of inflam-matory cells derived from murine peritoneal cavities, consistingmainly of neutrophils, macrophages, and lymphocytes, and the�4�1-expressing monocytic U-937 line bound to a marginal extentto resting endothelium. Prestimulation of mlEnd.1 cells with B.burgdorferi for 50 h, however, led to an �6-fold increase in thenumber of cells adhering to the EC monolayer. This adhesion ofperitoneal cells and U-937 cells to B. burgdorferi-activated ECwas efficiently inhibited by �75% and by nearly 100%, respec-tively, following addition of 3 nM S16204.

The severity of Lyme arthritis in ankle joints is significantlyreduced by the �4�1 antagonist

We next investigated whether �4�1-mediated processes might beof relevance for the pathogenicity of B. burgdorferi in the mousemodel of Lyme arthritis. For this purpose, C3H/HeJ mice, which

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are known to develop severe arthritis in the course of Borreliainfection, were systemically treated with S18407 at two differentdoses starting from the day of inoculation and continuing for aperiod of 21 days.

Ankle swelling, which grossly correlates with arthritis progres-sion, increased rapidly after inoculation in the control groups ofanimals (vehicle-treated and untreated, respectively) and peaked2–3 wk p.i., as expected (Fig. 3). In contrast, the clinical course ofdisease in mice that received the small molecule compound ateither dose was markedly alleviated, although a dose-response ef-

fect was not evident. The maximum inhibition was achieved byapplication of 30 mg S18407/kg body weight daily, resulting insignificantly lower levels of swelling by days 15 and 19 of infec-tion compared with controls. Of special interest was the findingthat the cessation of the treatment did not obviously lead to anexacerbation of arthritis.

FIGURE 2. Leukocyte binding to B. burgdorferi-activated ECs. Bind-ing of murine thioglycollate-elicited peritoneal cells and human monocyticU-937 cells to mlEnd.1 cells, activated by coincubation with 1 � 107

spirochetes for 50 h, was measured in a standard adhesion assay. Theinhibitor (3 nmol/l) was applied to the leukocytes (1 � 106 each) 30 minbefore they were added to activated ECs. Measurements were done in qua-druplicate. The assays were evaluated by direct counting of four random visualfields. Mean adhesion � SD from one representative experiment of two isshown. (control, ECs without stimulus, B. b., B. burgdorferi.)

Table I. Primers used for quantitative RT-PCR in this studya

Target Gene Primer Sequence (5� to 3�) Tann. (°C)

Amplicon

bp Tm (°C)

Il-6 ForwardAAC CAC GGC CTT CCC TAC TTC

54 154 83.0ReverseGCC ATT GCA CAA CTC TTT TCT CAT

Vcam-1 ForwardTGC CGG CAT ATA CGA GTG TGA ATC

58 350 83.5ReverseGAG GGG GCG GGG CTG TAA TA

Il-1� ForwardTCC CAA GCA ATA CCC AAA GAA GAA

58 236 85.5ReverseTGG GGA AGG CAT TAG AAA CAG TC

COX-2 ForwardCCC TGA CCC CCA AGG CTC AAA TA

61 228 85.0ReverseGGG GGA TAC ACC TCT CCA ATG

Hprt ForwardGTT GAA TAC AGG CCA GAC TTT GTT G

63 163 81.0ReverseGAT TCA ACT TGC GCT CAT CTT AGG C

PBGD ForwardATG TCC GGT AAC GGC GGC

59 135 89.5ReverseCAA GGC TTT CAG CAT CGC CAC CA

a All primers were HPLC-purified and provided by Thermo Hybaid. Tann., Annealing temperature applied in quantitativePCR; Tm, melting point of PCR product as determined by melting curve analysis.

Table II. Inhibitory efficacy of S16197 to the interaction of selectedpairs of adhesion molecules in vitroa

Interaction Partners IC50 (S16197)

�4�1-VCAM-1 1.23 � 0.32 nM�4�1-H/120b 0.22 � 0.03 nM�5�1-50 Kc �100 �M�2�1-Collagen I �100 �M

U-937 (�4�1)-VCAM-1 0.29 � 0.09 nMJY (�4�7)-MAdCAM-1 6.23 � 1.59 �MKL/4 (�L�2)-ICAM-1 �50 �MJY (�v�3)-Vitronectin �100 �MU-937 (�6�1)-Matrigeld �100 �M

a Values are the means of data � SEM (where indicated) from three independentexperiments. Assays were conducted as described in Materials and Methods.

b H/120, Fragment of fibronectin, comprising modules III12–III15 including IIICSregion, which binds �4�1.

c 50 K, Fragment of fibronectin, comprising modules III6–III10 including centralcell binding domain, which binds �5�1.

d Matrigel, Solubilized basement membrane preparation, major components arelaminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen.

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Because we have observed essentially no differences betweenuntreated and HEC-treated mice regarding the parameters ana-lyzed in the in vivo experiments described below, for reasons ofclarity only data obtained from the untreated mouse group as rep-resentative control group are included in Figs. 4-9.

The antagonist suppresses characteristic histological features ofmurine Lyme arthritis

To evaluate the arthritis-inhibiting effects of �4�1 blockade moreclosely, we analyzed the rear ankle joints of B. burgdorferi-in-fected mice histopathologically at the peak level of arthritic swell-ing on day 16 of infection. In joints of untreated animals (Fig. 4,A and C) the typical histological changes arising in the course of

Borrelia infection were identified. Massive and dense cell accu-mulations were evident in the joint space as well as in tendonsheaths and other areas of connective tissue. In addition, the sy-novial membrane was thickened due to hyperproliferation, withfragments breaking off from the synovial surface into the joint.Moreover, numerous fibrin clots were discernable in the jointspace. Examination at a higher magnification (Fig. 4C) disclosedneutrophilic granulocytes as the major infiltrating cell population.

In antagonist-treated animals, the overall lesion was less severecompared with their untreated counterparts (Fig. 4B), although dis-tinct foci of inflammatory cell infiltrates were present especially inareas close to tendons and tendon sheaths, in agreement with theankle swelling measurements. Besides a reduced total influx ofcells, these mice revealed only moderate proliferation of the sy-novial lining cells, a marginal exfoliation of cells into the jointcavity and fibrin exudates to a much lesser extent (Fig. 4D). Thus,

FIGURE 3. Development of Lyme arthritis in ankle joints. Mice wereinfected s.c. into the right hind footpad, and the �4�1 antagonist S18407was administered daily to two groups of animals in different doses specifiedin the figure. Another group of mice received the vehicle HEC alone.Treatment was terminated at day 21 p.i. when signs of spontaneous diseaseregression in untreated controls were evident. The level of arthritis, indi-cated as relative ankle swelling, was monitored by measuring the diameterof the rear ankle joints and by calculating the ratio of right to left side. Datapoints depict the averages and error bars the SEM of values obtained from6 to 12 infected animals. One representative experiment of three is dem-onstrated. ���, p � 0.001; ��, p � 0.005, comparing the animals treatedwith 30 mg/kg/day and with 3 mg/kg/day to the untreated group.

FIGURE 4. Histopathology of ankle joints. His-topathological evaluation of rear ankle joints and asso-ciated tissues comparing treated and untreated mice wasperformed on day 16 of infection with B. burgdorferi.The drug (30 mg/kg) was administered once a day untilexamination. Cryosections (10 �m) were fixed in ace-tone and stained with H&E. A and C, sections from un-treated control mice with cell accumulations throughoutthe ankle joint. B and D, sections from treated mice,demonstrating moderate overall lesion and nearly un-touched joint cavities. In B, the arrow is placed into anarea with a dense inflammatory cell infiltrate, character-ized by abundant nuclei. Small arrows in C indicate rep-resentative neutrophils, and the thick arrow in the samepicture points to a fibrin clot. Original magnifications: Aand B, �100; C and D, �400. JC, Joint cavity; B, bone;C, cartilage; T, tendon; M, muscle; SL, synovial celllining.

FIGURE 5. Leukocyte recruitment to inflammatory sites. Total numberof cells isolated by digestion of tissue with collagenase D was determinedby direct counting, and the fractions of CD45- and Gr-1(high)-positivecells were identified in a FACS analysis. As inflammation predominantlyemerges unilaterally dependent on inoculation site, only joints derivedfrom the affected side of the body were used for examination. Treatment ofanimals with S18407 (30 mg/kg/day) was performed from day 0 to the dayof analysis. Values at day 0 represent cell composition in ankles of unin-fected mice. Data were acquired from two experiments each with threemice per group and are depicted as means � SD. ��, p � 0.01.

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blockade of �4�1 integrin prevented the key hallmarks of B. burg-dorferi-induced murine arthritis.

Quantitation of total cells directly isolated out of rear anklejoints and determination of leukocyte (CD45-expressing) and neu-trophil (Gr-1high-expressing) fractions were performed on the dayof inoculation as well as 2 days and 1 wk thereafter to gain insightin the process of initial cell migration and the influence of �4�1

antagonism in the phase before clinical disease onset. In the pre-symptomatic stage (day 2), no differences were found betweentreated mice and controls in terms of total articular cell numbers orcell composition: both remained at physiological levels compara-ble to that in uninfected C3H/HeJ mice (Fig. 5). At 1 wk p.i.,however, when signs of arthritic swelling were becoming visible incontrol mice, the recruitment of cells was markedly attenuated byS18407. The initial infiltration of neutrophils and other leukocytesinto joints of treated mice was effectively inhibited, supportinghistological findings at the acute stage of disease.

S18407 treatment attenuates transcription of inflammatorymediators in rear ankle joints

To determine whether the treatment with S18407 affects the ex-pression of proinflammatory mediators that may contribute to theinhibition of cell infiltration and arthritis development, we ana-lyzed the mRNA levels of the key cytokines IL-1� and IL-6 aswell as of COX-2 and VCAM-1 in joint tissue by quantitativeRT-PCR over a period of 25 days postinoculation.

In untreated, infected mice, the mRNA expression of IL-1�,IL-6, and COX-2, all of which are known to be crucially involvedin the pathogenesis of Lyme disease (56–59), was strongly in-creased concomitant with arthritis development, reaching maxi-mum values on day 8 p.i. (12-fold, 40-fold, and even 45-foldhigher levels of IL-1�, COX-2, and IL-6, respectively, comparedwith treated mice) and remaining elevated through the time ofacute clinical disease (Fig. 6).

In striking contrast, the expression level of these genes in jointsof antagonist-treated animals was only slightly enhanced over un-infected control (IL-1�, 2-fold and IL-6, 6-fold, respectively, onday 6 p.i.) or persisted approximately at a constitutive level

FIGURE 7. Spirochetal load in rear ankle joints. Total DNA was ex-tracted from the right joints of treated (day 0 to day 21 p.i.) and untreatedmice at indicated time points p.i. and assessed for B. burgdorferi DNAlevels by quantitative LightCycler PCR. Values reflect the number of B.burgdorferi flagellin B (flaB) gene copies normalized to 105 copies ofmurine nidogen-1. Calculation of copy numbers was based on purifiedborrelial DNA and a cloned nidogen-1 fragment as external standards usingthe LightCycler software. Levels at time point 0 are derived from inoculumquantity. Error bars indicate the SD of values obtained from three mice pertime point.

FIGURE 8. Ab levels in serum. Sera from mice were collected on day12 and day 35 p.i. and diluted 100-fold (B. burgdorferi-specific IgG1,IgG2a) and 5-fold (total IgE), respectively, for measurement of Ab titers byELISA. Absorbance data at 405 nm (A405, background subtracted) arepresented as means � SD of 6–8 mice per group from two independentexperiments. (n.s., Not significant.)

FIGURE 6. Expression of inflam-matory mediators in ankle joints. The�4�1 antagonist was applied from theday of inoculation to day 21 p.i. Attime points indicated, total RNA wasextracted from whole joints and re-versely transcribed with oligo(dT)primers. Measurement of Il-1�, Il-6,COX-2, and Vcam-1 expression levelswas performed by quantitative Light-Cycler PCR using the content of Hprtas housekeeping gene for normaliza-tion. The value of 1 was assigned to thenormalized mRNA amount of eachgene in uninfected and untreated con-trol mice (day 0). The normalized levelof mRNA in the rest of samples isrepresented as fold increase/decreaseover control. The assays were repeatedonce with similar results.

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(COX-2) during the medication period. Interestingly, after cessa-tion of treatment, transcript numbers of IL-6 and COX-2 escalated,even exceeding those of controls, although without any clinicalimpact.

Induction of VCAM-1, expressed exclusively on resident cells,was not affected by the antagonist and occurred roughly to thesame extent (3- to 6-fold increases) in all mice independent oftreatment, but, as opposed to controls, with a decreasing tendencyfrom day 2 to day 15 in joints of S18407-treated mice.

Treatment with the �4�1 antagonist has no impact on bacterialclearance in rear ankle joints

Given the profound inhibitory effect of S18407 on recruitment ofimmune cells to the site of infection and on the induction of pivotalinflammatory modulators, we investigated the effect of �4�1

blockade on the capability of B. burgdorferi-infected mice to elim-inate bacteria over the course of 48 days postinoculation. By theend of this time, Lyme arthritis in control mice was completelyresolved, and bacterial load in the host was usually diminished toa persisting low level of intact organisms (60). Using quantitativePCR, we found no differences between treated and untreated ani-mals in spirochetal DNA levels in ankles at any of the time pointsexamined (Fig. 7). A drop in bacterial burden to a number of sev-eral thousands per 105 mouse nidogen-1 copies was observed intreated as well as control groups as soon as 12 days p.i. and wasnot further reduced over the whole period analyzed. The persistentspirochetemia in infected host tissues for extended periods despiteappropriate activation of phagocytes and induction of adaptive im-munity is well known, yet there is at present no explanation for thisphenomenon (61). Nevertheless, global immunological mecha-nisms controlling the replication of B. burgdorferi were apparentlynot affected by suppression of �4�1 function. Likewise, long-last-ing protection against the disease was established despite �4�1

inhibition during the initial immune response, because we ob-served no arthritis development in mice reinfected several weeksafter primary infection (data not shown).

S18407 does not modify the regular development of the adaptiveantibacterial immune response

The differentiation phenotype of CD4� T cells during Lyme bor-reliosis is widely considered to affect the outcome of infection. Theexistence of highly polarized Th1 cell cytokine patterns and Th1-associated Ig subtypes has been previously described in arthriticmice infected with B. burgdorferi (62, 63). Other studies, however,have proven that immune responses yielding Th2 cells or IL-4 are

not sufficient to protect from Lyme arthritis (64, 65). Therefore, toevaluate possible effects of S18407 treatment on the generation ofa defined Th response phenotype, the production of Borrelia-spe-cific Igs (IgG1 and IgG2a) and of the Th cell-derived cytokinesIFN-� and IL-4 was assessed.

Serum titers of Th1-related IgG2a in untreated mice were 6.2-and 3.5-fold higher on day 12 and day 35, respectively, than thoseof Th2-related IgG1 (Fig. 8). Similarly, mice that received theantagonist exhibited 5.2- and 2.2-fold higher levels of IgG2a at thesame time. Production of total IgE largely remained at a low level,supporting the bias at IgG generation. In treated animals, slightlyelevated titers of IgG1 and IgE relative to control mice were ev-ident by day 35 p.i., but these differences were not statisticallysignificant.

Consistent with the Th1-driven IgG isotype switching, the quan-tification of Borrelia-induced cytokine secretion by splenic T cellsrevealed abundant amounts of Th1-derived IFN-� and barely de-tectable IL-4. Depletion of CD4� cells as well as the quantificationof IFN-�-expressing spleen cells using a recently establishedIFN-� reporter mouse (66) revealed that the majority (�80%) ofIFN-� was produced by B. burgdorferi-specific CD4� T cells (datanot shown). This cytokine pattern was essentially not affected by�4�1 blockade (Fig. 9).

These findings provide clear evidence that untreated as well astreated mice had developed a predominant Th1 immune responseto infection with B. burgdorferi.

DiscussionIn an attempt to clarify �4�1-dependent mechanisms of inflamma-tion and bacterial clearance in an experimental mouse model ofhuman Lyme borreliosis, we used the highly selective small mol-ecule �4�1 antagonist S18407 for treatment of mice after infectionwith B. burgdorferi. The compound inhibited production of in-flammatory mediators and leukocyte influx into joints, yet withoutany consequences on the host’s ability to control spirochetal re-jection. Thus, by inhibition of �4�1 overwhelming inflammatoryprocesses, known to be detrimental and most likely responsible forarthritis development in this model, were successfully suppressedwhile beneficial parts of the immune response to B. burgdorferiremained functional.

Innate immune cells, especially neutrophils, and their productsare thought to play a pivotal role in the development of Lymearthritis, and the level of polymorphonuclear leukocyte (PMN) in-filtration into joints of infected mice has been associated with theseverity of disease (67). Preventing or attenuating the recruitmentof these cells should therefore be a prerequisite to ameliorate thecourse of infection. As a consequence of �4�1 blockade, we ob-served histologically as well as by FACS analysis a reduced, butstill notable recruitment of neutrophils to infected ankle joints.This suggests that adhesion molecule pathways not involving �4�1

are still operative to govern PMN migration under this condition.Indeed, it has been proven that murine neutrophils generally en-gage �4�1 together with the two main �2 integrins LFA-1 andMac-1 to enter inflammatory sites and that there is a substantialfunctional overlap among these integrins (21). Moreover, an ad-ditional �4�1- and �2-independent pathway of PMN migration toarthritic rat joints has been proposed (68). Thus, LFA-1, Mac-1,and possibly other receptors not yet identified are likely to be in-volved in the residual neutrophil migration found in S18407-treated animals. In addition to an effect on decreased invasion, theaccelerated clearance of PMN from inflamed tissue as a further

FIGURE 9. Cytokine pattern of B. burgdorferi-specific splenic T cells.Five weeks after infection, cultures of spleen cells (4 � 106/ml) fromtreated (day 0 to day 21 p.i.) and untreated mice were restimulated with B.burgdorferi (multiplicity of infection 10) and as a control with Con A (7.5�g/ml) for 48 h. Levels of cytokines in the supernatants were analyzed bysandwich ELISA. Data are the mean � SD of quadruplicates, obtainedfrom two mice per group in one experiment of two with similar results.

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consequence of �4�1 antagonism may also contribute to the con-trol of leukocyte burden in joints, according to recent results ob-tained by �2 integrin inhibition (69). Quite intriguing, the spread-ing of infiltrated PMN within various compartments of thearticular cavity of treated animals showed a pattern uncharacter-istic for murine Lyme arthritis. Whereas cells seemed to accumu-late in tendon sheaths and synovial membrane, the joint fluid wasalmost free of pathologic signs. Fibronectin, in joints derived fromplasma and synthesized in situ by fibroblast-like synoviocytes, is amajor ECM component in normal and pathological synovium. Fol-lowing VCAM-1-mediated extravasation, CS1 fibronectin wasshown to be the most important ligand for �4�1-bearing cells toaccomplish (possibly in concert with other components of theECM) migration along a chemokine gradient to the site of activity(70). Under the experimental conditions of B. burgdorferi infec-tion, this interaction of leukocytes to CS1 fibronectin may beblocked more efficaciously by S18407 than binding to VCAM-1,as indicated in vitro in the cell-free binding assays. This is sup-ported by the finding that �4�1 binds with lower affinity to CS1fibronectin than to VCAM-1 (33, 71). �1 integrins appear to be thedominant class of integrins that mediate PMN interactions withfibronectin, whereas the contribution of �2 integrins to adhesion tofibronectin is minimal (72). Therefore, impaired �1-dependent mi-gration of neutrophils through ECM, unlike extravasation, canhardly be compensated by action of �2 integrins.

Interestingly, despite the apparent, although reduced, recruit-ment of leukocytes to joint tissue of treated mice, the local mRNAlevels of proinflammatory mediators known to be strongly up-reg-ulated by innate immune cells during B. burgdorferi infection wereessentially not elevated over baseline. Consistently, formation ofedema in ankles, induced particularly by products of COX-2 (J.Glasner, M. Rollinghoff, and A. Gessner, manuscript in prepara-tion), was largely abolished. Based on these findings, we proposethat �4�1 blockade in murine Lyme arthritis predominantly acts byimpeding (full) activation of �4�1-bearing innate immune cells,whereas the interference with cellular trafficking appears to play aminor role.

Numerous studies have disclosed the substantial contribution ofoutside-in signaling by integrins to the activation of various neu-trophil functions upon adhesion to matrix ligands (e.g., respiratoryburst, expression of inflammatory mediators, degranulation, andapoptosis inhibition) (69, 73–76). As shown in some previous invivo models, blockade of �4 integrin by mAbs has led to a partialor complete failure of cell activation, and, as in this study, thedisease-ameliorating effects could not fully be attributed to an im-paired cell recruitment (77–80). In studies using infectious agentssuch as Yersinia enterocolitica or Trichinella spiralis, the defect incell activation was directly correlated with an increase in the tissueburden of the respective pathogen (77, 78). This finding appears toconflict with our results, although the discrepancy may be ex-plained by the nonspecific targeting of �4�7 integrin in addition to�4�1 by the anti-�4 mAbs used. Binding of �4�7 to MAdCAM-1plays a critical role in lymphocyte homing to gut-associated lym-phoid organs (81, 82), and clearance of intestinal Y. enterocoliticaand T. spiralis, unlike B. burgdorferi, is known to be mediated byT cell-dependent mechanisms (83, 84).

�4�1-independent host control of spirochetal growth, despite re-stricted numbers of neutrophils at sites of infection, demonstratesthat these phagocytic cells are not essential for elimination of bac-teria. This is in line with previous work suggesting that in theLyme arthritis model, killing spirochetes is secondary to the role ofimmune regulation by neutrophils (67).

Numerous publications have pointed to the importance of thehumoral response in preventing infection by B. burgdorferi, in

controlling spirochete levels in tissues, and in clearing arthriticlesions in the mouse (85–87). Because Borrelia-specific activationof lymphocytes, i.e., priming of T cells to produce large amountsof IFN-� and secretion of Abs by plasma cells, are not impairedunder treatment with S18407, it is likely that, independent of �4�1

inhibition, the acquired host defense plays a dominant role forelimination of bacteria from tissues. It further implies that the con-tribution of �4�1 signaling to T cell activation, highlighted byprevious reports (88, 89), is functionally compensated by othercostimulatory pathways.

In conclusion, this study has shown that �4�1-mediated pro-cesses are involved in the PMN-dominated development of murineLyme arthritis, but dispensable for the induction of protective im-munity against B. burgdorferi, and that the novel �4�1 antagonistS18407 offers a safe approach to efficiently disturb �4�1-depen-dent cell adhesion associated with this infectious disease.

AcknowledgmentsWe thank Mrs. Claudia Giessler for her excellent technical assistance.

DisclosuresThe authors have no financial conflict of interest.

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