International Immunopharmacology 13 (2012) 424–431

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International Immunopharmacology

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Citrullinated mouse collagen administered to DBA/1J mice in the absence of adjuvantinitiates arthritis

Geoffrey M. Thiele a,b,d, Michael J. Duryee a,b,⁎, Anand Dusad a,b,c, Carlos D. Hunter a,b, Jordan P. Lacy a,b,Daniel R. Anderson e, Dong Wang c, James R. O'Dell a,b, Ted R. Mikuls a,b, Lynell W. Klassen a,b

a Experimental Immunology Laboratory, Omaha Veterans Administration Medical Center, NE 68105, United Statesb Experimental Immunology Laboratory, University of Nebraska Medical Center, Department of Internal Medicine, Division of Rheumatology, Omaha, NE 68198, United Statesc Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, United Statesd University of Nebraska Medical Center, Department of Pathology and Microbiology, Omaha, NE 68198‐6495, United Statese Experimental Immunology Laboratory, University of Nebraska Medical Center, Department of Internal Medicine, Division of Cardiology, Omaha, NE 68198‐3025, United States

⁎ Corresponding author at: Experimental Immunologbraska Medical Center, Wittson Hall Room 3005, OmStates. Tel.: +1 402 559 7030; fax: +1 402 559 7910.

E-mail addresses: gthiele@unmc.edu (G.M. Thiele), m(M.J. Duryee), anand.dusad@unmc.edu (A. Dusad), cdhujplacy@unmc.edu (J.P. Lacy), danderso@unmc.edu (D.R.(D. Wang), jrodell@unmc.edu (J.R. O'Dell), tmikuls@unmlklassen@unmc.edu (L.W. Klassen).

1567-5769/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.intimp.2012.05.007

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 22 March 2012Received in revised form 1 May 2012Accepted 7 May 2012Available online 21 May 2012

Keywords:Collagen‐induced arthritisCitrullination of proteinsAutoimmunity to self‐proteinsInflammationMicro-CTT-cell proliferation

Introduction: Citrullinated self‐proteins are thought to be involved in the onset/progression of rheumatoidarthritis (RA). Numerous studies have been performed to look for the self‐antigen that becomes citrullinatedand induces RA. Importantly, these studies have been performed using citrullinated self‐antigens injectedinto an animal model in the presence of a strong adjuvant in order to derive the response. However, todate no studies have been performed to determine if these phenotypes can be induced in the absence ofan adjuvant.Methods: To investigate this possibility, mice were immunized with citrullinated or non-citrullinated mouseType II collagen (Cit-Col or Col) in the presence or absence of Freund's Complete Adjuvant (FCA).Results: An autoimmune-like RA response was observed in mice immunized with Cit-Col in the absence ofFCA; by the increase in caliper score, visual observation, and micro-CT analysis of bone erosions. Antibodyand T-cell responses were increased in the Cit-Col injected mice to Cit-Col as well as antibody to Anti-Citrullinated Peptide Antigens (ACPA) as determined by a commercially available test kit.

Conclusions: Therefore, the use of citrullinated mouse collagen induces an autoimmune‐like RA in the absenceof an adjuvant. These data also suggest that citrullinate self‐proteins may be potential molecular adjuvantsthat assist in driving an inflammatory response, that increases the production of PAD in joint tissue, resultingin the citrullination of other self‐proteins to exacerbate the disease.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Rheumatoid arthritis (RA) is a severe and potentially debilitatingdisease characterized by inflammation and the progressive destruc-tion of joint tissues. While numerous studies have implicated the in-volvement of various antigens in the disease, the pathogenesis andcause of RA remain uncertain. In the last decade autoantibodies tocitrullinated protein antigens (ACPA) and/or peptides have been usedas diagnostic tools and have been implicated in the disease process[1]. A number of citrullinated proteins have been implicated as playinga central role in disease pathogenesis; including, filaggrin, fibrin, fi-brinogen, vimentin, alpha enolase, collagen, and even peptidylagrinine

y Laboratory, University of Ne-aha, NE 68198‐6350, United

duryee@unmc.edunter@unmc.edu (C.D. Hunter),Anderson), dwang@unmc.educ.edu (T.R. Mikuls),

rights reserved.

deiminase (PAD), the enzyme responsible for the post-translationalmodification of arginine into citrulline [2–9].

Animal models in RA have traditionally used type II or IV collagenfrom a variety of animal sources in the presence of Freund's completeadjuvant (FCA) to initiate the disease [10–12].With an increased under-standing of their potential role in disease pathogenesis, citrullinatedproteins have been evaluated as immunogens in the presence of FCA[4–6,13]. These studies showed that there was a slight increase in im-mune responses towards the injected immunogen, and a slight increasein joint damage reflected in the ankle. For example, citrullinated humanand rat fibrinogen were injected into rats and mice with FCA, resultingin increased joint damage compared to that observed in animals immu-nized with FCA plus unmodified collagen [4,6,14]. When citrullinatedrat fibrinogen was injected into rats in the presence of FCA, tolerancewas broken and an autoimmune response was initiated and character-ized by the production of IgG antibodies to both citrullinated fibrinogenand native fibrinogen [4]. These studies have also been performed usingautologous rat serum albumin (RSA) that had been citrullinated [13]. Inthis study RSA was citrullinated and injected into animals in the pres-ence of FCA which produced autoantibodies and T-cell proliferations

425G.M. Thiele et al. / International Immunopharmacology 13 (2012) 424–431

directed against citrullinatedRSA. These data indicate that tolerance canbe broken by citrullinating a self‐protein.

Previous studies using the collagen‐induced arthritis model in ratshave shown that antibodies could be detected against citrullinatedfibrinogen following immunization, again underscoring the role ofcitrullinated antigens in RA pathogenesis [5]. As with the aforemen-tioned studies, this study also involved the immunization of the anti-gen type II collagen from calf articular cartilage in the presence ofFCA. The studies described above all used FCA to induce arthritis. Todate, there have been no animal studies that have used a citrullinatedself‐protein to induce arthritis in the absence of an adjuvant. This isimportant since a disease model that does not require an adjuvantcould lend added physiologic relevance to human disease. Therefore,it was the purpose of this study to evaluatewhether citrullinatedmousetype II collagen (Cit-Col) initiated an autoimmune collagen-inducedarthritis in the absence of an adjuvant and, if so, to characterize theresulting immune responses utilizing a number of complimentary sci-entific approaches.

2. Materials and methods

2.1. Animals

Male DBA/1 (H-2q) mice were purchased from Jackson Laboratory(Bar Harbor, Maine, U.S.A.) and maintained on a Purina breeder's diet[11]. Animals were allowed free access to their food and/or water upto 1 h prior to sacrifice. All procedures were approved by the AnimalSubcommittee of the Omaha VA Medical Center in accordance withthe National Institutes of Health Guidelines on the Use of LaboratoryAnimals.

2.2. Antigen preparation

Mouse type II collagen (Col), purchased from Chondrex (Redmond,WA, U.S.A.), was citrullinated using rabbit skeletal muscle PAD (Sigma,St. Louis, MO, U.S.A.) as previously described [13]. Verification of Cit-Colwas determined using an Anti-Citrulline (modified) Detection Kit fromMillipore (Temecula, CA, U.S.A.) as per manufacturer's directions.

2.3. Immunization of Cit-Col

In order to determine if Cit-Col initiates an RA-like disease in theabsence of FCA, mice were immunized with Col (Chondrex), Cit-Col orCol in the presence of Freund’s Complete Adjuvant (FCA) (Chondrex)as a positive control. Prior to immunization mice were pre-bled andtested for background levels of antibodies to Col, Cit-Col, bovine serumalbumin (BSA) or Cit-BSA. Antibody levels to BSA were subtractedfrom Cit-BSA levels to determine the amount of antibody specific forthe citrullinated epitope. Col or Cit-Col was immunized into DBA/1miceweekly for 4 weeks via injection at the base of the tail. As a positivecontrol mice were immunized with FCA-Col at week 1 and then Incom-plete Freund’s Adjuvant (IFA) at week 3. One week following the im-munization schedule, blood was collected using retro-orbital venouspuncture and serum frozen at−20 °C until analyzed.

2.4. Determination of arthritis

Mice were assessed for Cit-Col induced arthritis (CCIA) byassessing their paws both visually and through the use of caliper mea-surements [15]. For visual based assessment, digits were observedweekly for 5 weeks and photos were taken to demonstrate rednessand swelling in the hind paw. To confirm the visual observation of in-flammation, caliper measurements were done using a Vernier caliper[15]. This measurement was done by placing the caliper on the hindankle (medial/lateral). Each hind ankle was measured twice andthen averaged at weeks 0, 3, and 5. Histological assessment of CCIA

was done by evaluating H & E sections of the joints in the right hindpaw. Briefly, at week 5 mice were sacrificed and the hind limb wasextracted below the knee and placed in formalin for 48 h. Sampleswere then decalcified using 5 changes of 10% EDTA for 6 weeks, andthe presence of inflammatory cells, thinning of the panus, and boneerosions assessed.

2.5. HPMA copolymer detection of arthritis

Previous studies by Wang et al. demonstrated the use of an infra-red labeled co-polymer for the imaging and detection of inflamma-tion in a rat model of arthritis [16–18]. This was accomplished by con-jugating IRDye (800CWNHS Ester Infrared dye) from Li-Cor Biosciences(Lincoln, NE, U.S.A.) to N-(2-hydroxypropyl) methacrylamide (HPMA)copolymer. Mice were then intravenously injected via the tail veinwith 0.5 mg of HPMA copolymer and left in their cages for a 24‐hour pe-riod. Detection of the infrared signal of the copolymer was performedusing an XENOGEN IVIS 200 series imaging system (Hopkinton, MA,U.S.A.). Infrared signal intensity was analyzed using Living Image soft-ware from XENOGEN.

2.6. Micro CT analysis

Mice were injected with Col, Cit-Col and FCA-Col, their legs excisedabove ankle at sacrifice, and fixed in 10% formalin for 48 h. Similar toBarck et al. [19] the right hind paws obtained from experimental micewere scanned (perpendicular to their longitudinal axis) with a high-resolution Micro-CT system (SkyScan 1172; SkyScan, Aartselaar, Bel-gium) with a voxel size of 4.8 μm. The X-ray tube voltage was 60 kVand the current was 112 mA, with a 0.5 mm thick aluminum filter anda fixed exposure time of 530 ms. Six frames were averaged for eachrotation with a rotation step of 0.7° following an angle of 180°. Samplereconstructions were performed by the system reconstruction software(NRECON, Skyscan) and analyzed with a CT-Analyzer (CTAn, Skyscan).3D images were reconstructed using CT-Vox software (Skyscan) andCT-Vol software (Skyscan) to produce a visual representation of theresults.

For quantification, a core volume of interest (VOI) consisting of615 slices, each of 4.88 μm thickness was selected which includedthe middle three metatarsophalangeal (MTP) joints. The VOI con-sisted of 307 slices (4.88 μm each and total length of 1.5 mm) aboveand below the central slice which was taken as the center of thejoint, resulting in a total length of 3 mm which was sufficiently longto cover the MTP joints. The specific parameters measured and calcu-lated within the selected VOI were as follows: (1) Bone volume (BV)was calculated using polyhedrons corresponding to the enclosed vol-ume of the triangulated surface; [20] and, bone surface area (BS) wascalculated by the marching cubes method to triangulate the surface ofthe bone [21].

2.7. Determination of antibodies to collagen

Serumwas collected at weeks 0, 3, and 5 from animals immunizedwith Col, Cit-Col, or FCA-Col and were screened for the presence ofantibodies to Col, Cit-Col or Cit-BSA. For these experiments ELISAgrade Col or Cit-Col from Chondrex was coated on Immulon IV(Nunc, Fisher Scientific, St. Louis, MO, USA) microtiter plates at a con-centration of 2 μg/well. To determine the amount of anti-citrullineantibodies, BSA and Cit-BSA were also coated on the plates. Antigenswere incubated overnight, washed, blocked in 2% BSA, and incubatedwith the serum from mice in each of the different groups at a 1:50dilution. Bound mouse antibody was detected using an HRP-rabbitanti-mouse IgG antibody from Sigma Chemical Co. followed by theuse of TMB substrate. Color change was determined by an MRX IImicroplate reader (Dynatech, Chantilly, VA, USA) at 450 nm. Standardcurves were generated using known concentrations of mouse IgG

Fig. 1. Caliper measurements of ankle joints in Cit-Col induced arthritis (CCIA) mice.Data expressed are the percent change over control means±SEM of 10 animals.*P≤0.01 significantly different for times 3 and 5 in the Cit-Col and FCA-Col injectedas compared to the unmodified Col injected animals.

426 G.M. Thiele et al. / International Immunopharmacology 13 (2012) 424–431

(Sigma Chemical Co.) and extrapolating the concentrations in unknownsamples using Revelation Software (Dynatech). Serum at week 0 priorto immunization was subtracted from weeks 3 and 5 and data wasexpressed in nanograms per milliliter.

2.8. Anti-citrullinated protein antibodies

Serum from mice injected with Col, Cit-Col or FCA-Col was testedfor ACPA using the human Diastat Anti-Cyclic Citrullinated Protein(CCP) antibody kit from Axis-Shield Diagnostics Ltd. (Dundee, UK).For these studies serum was diluted 1:10 and an HRP rabbit anti-mouse IgG (Sigma Chemical Co.) was substituted for the anti-humanantibody in the kit. Plates were developed and color change measuredon the MRX II microplate reader. To maintain consistency in the kitthe human standard was used to determine the concentration of theanti-CCP antibodies in the serum as previously published by Kuhn etal. [5].

2.9. Proliferative responses

To determine the proliferative responses of T-cells from Col, Cit-Col,or FCA-Col immunizedmice, the antigens Col, Cit-Col, and Cit-BSAwereadded to 96-well flat bottom plates at concentrations ranging from 0.25to 2 μg/ml. Spleens frommice in each groupwere subjected tomechan-ical disassociation and T-cells purified using a pan T-cell isolation kit IIfrom Miltenyi Biotec (Auburn, CA, U.S.A.). Purified T-cells at a concen-tration of 1×105cells/well and irradiated antigen presenting cellswere incubated with the above antigens for 48 h at 37 °C in 5% CO2,pulsed with 1.0 μCi/well of [3H] thymidine (GE Healthcare, Piscataway,NJ, U.S.A.) for 16 h, and harvested on a 96-well harvester (Tomtec,Orange, CO, U.S.A.). Filter mats containing incorporated thymidinewere placed in scintillation fluid and counted on a 1450 MicrobetaScintillation Counter (Perkin Elmer Life Sciences, Waltham, MA, U.S.A.).T-cell proliferations were evaluated by subtracting background levelsfrom naïve mice and media in the proliferation plate to test samples.Data are expressed as stimulation index (SI) and calculated as previouslypublished [22].

2.10. T-cell cytokine levels

Supernatants from T-cell proliferation plates were saved prior toharvesting of T-cells and frozen at −70 °C until use. Frozen superna-tants were thawed on ice and tested for the presence of IL-6 (BD Bio-sciences, San Diego, CA, U.S.A.) and IL-23 (eBioscience, San Diego, CA,U.S.A.). Cytokine levels were determined as per manufacturer's direc-tions and expressed in pg/ml. Absorbance was detected at 450 nmusing an MRX II Microplate Reader (Dynatech) and data analyzedusing Revelations Software from Dynatech.

2.11. Statistics analysis

Results are expressed as means±SEM with group comparisonscompleted using Student's T-test or ANOVA. Statistical significancewas achieved if P values were less than 0.05. All statistical analyseswere performed using Sigma Stat Version 3.5 (Systat Software, Inc.,San Jose, CA, U.S.A.).

3. Results

To determine if Cit-Col would induce an RA like autoimmune disease(Citrullinated Collagen‐induced arthritis=CCIA),micewere immunizedwith Col (control), Cit-Col, and FCA-Col. CCIA was first assessed for in-flammation using the caliper method at weeks 0, 3, and 5 as describedin Materials and methods. As shown in Fig. 1, prior to injection of anti-gens, the average baseline ankle thickness was recorded and used to cal-culate percent change in the right ankle joint of the immunized animals.

At week 3 the ankle size was increased by 9.44% in Col, 29.67% in Cit-Col,and 43.23% in FCA-Col injected mice. A similar increase in size was ob-served in the animals at week 5; Col 5.83%, Cit-Col 57.19%, and FCA-Col53.43%with a significance of P≤0.01for the change in paw thickness inCit-Col and FCA-Col immunized mice compare to the Col injectedgroup. Visually (Fig. 2A), animals presented with redness and swellingin the hind paws from mice injected with Cit-Col and FCA-Col as com-pared to Col injected mice hind paws. The swelling wasmost profoundat week 5 following injection. However, after week 6 the inflammationwould diminish. Inflammation of the joints was also evident in histo-logical sections of the hind paws that were stained by H&E. As shownin Fig. 2B, infiltration by immune cells was seen in the joints of miceinjected with Cit-Col and FCA-Col as compared to the joints of Colinjected mice.

Inflammation was further characterized using an HPMA water-soluble co-polymer, previously used to identify sites of inflammationin the collagen‐induced arthritis rat model [16,18]. Mice immunizedwith Col, Cit-Col, and FCA-Col were injected with an IR-Dye labeledHPMA copolymer and examined for sites of inflammation using liveinfrared imaging. As shown in Fig. 2C mice immunized with Cit-Color FCA-Col showed an increase in the detectable levels of the HPMAcopolymer as assessed by in vivo IVIS imaging. Detectable levels werevisualized in at least three paws in the Cit-Col injected mice and twopaws in the FCA-Col injected mice. A slightly stronger signal was ob-served in the FCA-Col injectedmice compared to the Cit-Col immunizedmice.

Fig. 3A shows an anteroposterior radiographic image from microCT and the boxes show the middle three MTP joints which were cho-sen as the region of interest (ROI) for all micro CT studies. Panel 1 ofFig. 3B represents the analyzed VOI showing bone loss, joint erosion,loss of joint space, and irregular joint contours. Bone loss is a prominentpathological feature of adjuvant-induced arthritis in animal models[23]. To quantitatively evaluate this bone loss in theMTP joints, additionalmicro CT analysis was performed. Bone volume (BV) and bone surface(BS) have been widely used to document bone loss in arthritic rodentmodels [19,23–25]. As shown in Fig. 3C, mice immunized with Cit-Col(10.15% decline, P=0.033) and FCA-Col (9.89% decline, P=0.027) dem-onstrated a significantly lowermeanBS. Similar resultswere observed forBV for the Cit-Col (12.32%; p=0.006) and FCA-Col (8.71%; p=0.001)compared to Col immunized mice. The above mentioned loss in BS andBV, panels 2 and 3 (colored) of Fig. 3B, were assessed using the sameVOI as detailed above but at a higher threshold and are representativeof 3 mice per group.

Fig. 2. Inflammation induced in CCIA mice. (A) Redness and swelling are depicted in these representative photos at week 5. (B) H&E staining of joint tissue for CCIA mice. Inflam-mation and bone erosions are depicted by black arrows. Images were taken at 10× and represent the images from 10 animals in each group. (C) Detection of inflammation in CCIAmice using an IRDye-labeled HPMA co-polymer. A stronger signal is present in the paws of the Cit-Col and FCA-Col injected animals. Only background levels in the tail left over frominflammation caused by the immunization can be seen in all the injected animal groups.

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Serum collected from mice immunized with Col, Cit-Col, or FCA-Col was assayed by ELISA for the presence of circulating antibodiesto native Col, Cit-Col, or Cit-BSA at weeks 0, 3, and 5. As demonstratedin Table 1, at weeks 3 and 5, mice immunized with FCA-Col had sig-nificantly increased levels of serum antibodies to Col and Cit-Col ascompared to sera from mice injected with Col. Interestingly, serumantibody concentrations in mice injected with Cit-Col increased, yetnot significantly compared to sera from Col injected mice. Whensera from Cit-Col injected mice were screened against Cit-BSA, therewas a significantly increased level of antibody to the citrullinated epi-tope as compared to serum from Col injected mice. Antibody concen-trations in the sera of Cit-Col injected mice to Col, Cit-Col, and Cit-BSAwere relatively equivalent. This is most likely a result of the observa-tion that Col in its purified form has been shown to be citrullinatedduring the purification process [26]. Therefore, the response is mainlyto the citrullinated epitope and to some extent Col.

In order to determine the amount of anti-CCP antibodies in theseanimals, serum from mice immunized with Col, Cit-Col, or FCA-Colwere tested using the human anti-CCP assay kit as described above.As shown in Fig. 4, animals injected with Cit-Col had significantly in-creased levels of anti-CCP antibodies in their sera as compared to theCol injected mice. FCA-Col injected mice had significantly higher anti-CCP antibody concentrations than either the Col or Cit-Col immunizedmice.

T-cell proliferation assays using T-cells from Cit-Col and FCA Colinjected animals showed a significant increase in stimulation index(SI) to unmodified Col at concentrations of 0.25, 0.5, and 1 μg (Fig. 5A).

When T-cells from mice immunized with Col, Cit-Col or FCA-Col weretested against Cit-Col as the antigen, a significant increase in the SIwas only evident by the T cells from the Cit-Col injected animals at allconcentrations tested compared to the Col or FCA-Col injected mice(Fig. 5B). When T-cells were tested on Cit-BSA to look for the adductand not the carrier, a significant increase in proliferation was evidentat 0.25 and 0.5 μg concentrations only by T-cells from Cit-Col injectedanimals (Fig. 5C).

To begin examining the type of T-cell cell responses generated toCit-Col, supernatants from the proliferation assays were collectedand tested for cytokines using commercial ELISA kits. When T-cell su-pernatants from Cit-Col injected mice were tested for the presence ofIL-6 (Fig. 6A), a significant increase was detected in the in response tothe antigens; Col, Cit-Col, and Cit-BSA as compared to the superna-tants from Col or FCA-Col injected mice. When T-cell supernatantsfrom Cit-Col and FCA Col injected mice were tested for the presenceof IL-23 (Fig. 6B), a significant response was demonstrated to Cit-Colas the antigen when compared to T-cell supernatants from Col injectedmice. This response was increased, but not significantly, when Cit-BSAwas used as the stimulating antigen for T-cells from Cit-Col injectedmice. T cell supernatants from FCA-Col injected mouse T-cells had noIL-23 response to the Cit-BSA antigen indicating that the T-cell responsein this group of mice is to the Col epitope and not the citrullinated partof the antigen. While these data demonstrate an increase in IL-6 andIL-23, it is not clear if they are generated by the T-cells in the assay orthe irradiated feeder splenocytes. More work will need to be done todifferentiate this response.

Fig. 3.Micro-CT detection of bone erosions in CCIA mice. (A) Representative images of Col, Cit-Col, and FCA-Col injected mice hind paws. The white box indicates the area of interestused for calculation of bone erosion parameters. (B) Panel 1 depicts the region of interest zoomed in on the bone erosions in the joint tissue. Panel 2 depicts the region of interestreconstructed to 3D at a higher threshold (for representation purposes) showing the amount of bone loss and joint damage. Panel 3 depicts the region of interest reconstructedfrom panel 2 converted to a color image showing the amount of bone loss for visual interpretation. (C) Micro-CT analysis of mouse paws following induction of CCIA. Bone volumeand surface show significant loss in bone material as compared to controls. Data expressed are the percent change over control means±SEM of 3 animals. *P≤0.005 and #P≤0.03significantly different for Cit-Col and FCA-Col injected as compared to the unmodified Col injected animals.

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4. Discussion

Citrullination of self‐proteins has been suggested to play a significantrole in the pathogenesis of rheumatoid arthritis (RA). Recent studieshave used animal models to study these citrullinated self‐proteins andhow they interact to induce an RA like disease [4,5,13,14]. These studieshave systematically used robust inflammatory adjuvants (i.e. Freund’scomplete adjuvant) as the catalyst for induction of the disease. In con-trast, it was the purpose of this study to inject citrullinated mouse Col-lagen (Cit-Col) in the absence of any adjuvant, break tolerance, andinitiate RA in a mouse model system.

For the first time, we have shown that the injection of mice withcitrullinated mouse Type II Col (Cit-Col) as the antigen results in tol-erance loss, and the generation of an immune response culminatingin joint damage. Articular inflammation in these mice was detected byan increase in caliper scores, microscopic examination (H&E), and red-ness and swelling in the hind paws. Inflammation was also evident insome of the front paws as well. The presence of inflammation in thismodel was confirmed using an infrared-labeled copolymer that has

Table 1Antibody response in CCIA mice. Antibody data from mice immunized with Col, Cit-Col, an

Screening antigens

Injected Ag 3 Week Col 3 Week Cit-Col 3 Week Cit-

Col 40.72±6.09 41.62±5.69 31.28±8.9Cit-Col 142.77±36.01 118.31±14.10 195.53±35FCA-Col 1857.31±381.08* 331.45±127.85* 131.71±29

*Pb0.05 Compared to injected Col. All data expressed in ng/ml.

been previously reported as being capable of detecting sites of inflam-mation [16,18]. Unlike inflammation observed in the FCA-Col treatedanimals, where some animals either do not become inflamed, or only1 or 2 paws become inflamed, all of the Cit-Col injected animals hadevidence of paw redness and swelling.

To further confirm the presence of an RA like disease, right hindpaws were subjected to micro-CT and evaluated for erosions and/orbone loss. Previous studies have used micro-CT to confirm the pres-ence of bone erosion and periarticular osteopenia [24]. We used twoparameters of three-dimensional micro-CT to quantitatively investi-gate these alterations. Significant bone loss was evident in mice im-munized with both Cit-Col and FCA-Col, but was actually greater inthe Cit-Col injected mice than in the FCA-Col group. Taken together,these results show that this novel CCIAmodel demonstrated an arthritisphenotype that parallels that of the widely used CIA model.

Perhaps most noteworthy were our observations that the antibodyresponse in mice injected with Cit-Col showed only minimal antibodyagainst Col. Instead the antibody response was primarily directedagainst the citrullinated epitope as evidenced by the antibody reactivity

d FCA-Col and test against Col, Cit-Col or Cit-BSA.

BSA 5 Week Col 5 Week Cit-Col 5 Week Cit-BSA

6 136.20±43.01 54.24±17.25 146.87±47.49.85* 352.48±88.73 266.72±71.23 298.92±35.29.08 3971.48±707.01* 861.27±255.90 195.62±40.50

Fig. 5. T-cell proliferation assays in CCIA mice. (A) Depicts the stimulation index direct-ed against the Col antigen. *P≤0.05 significantly different from Col injection for boththe Cit-Col and FCA-Col injected animals. (B) Depicts the stimulation index directedagainst the Cit-Col antigen. *P≤0.01 significantly different from Col injection. No sig-nificance was demonstrated in the FCA-Col injected mice. (C) Depicts the stimulationindex directed against the Cit-BSA antigen. Data expressed are the means±SEM of10 animals. *P≤0.05 significantly different from Col injection. No significance wasdemonstrated in the FCA-Col injected mice.

Fig. 4. Anti-CCP antibody concentrations in serum from CCIA mice. Data expressed arethe means±SEM of 10 animals. *P=0.022 significantly different from Col injection.*P≤0.001 significantly different from Col injection.

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to Cit-BSA. In comparison the antibody response in the serumof FCA-Colinjected mice was primarily to Col with very little antibody to thecitrullinated epitope (Cit-BSA). This may appear to be contradicted bythe increased antibody levels detected in the serum from this group ofmice in theACPA assay (Fig. 4) as performed by Kuhnet al. [5]. However,it should be noted that the anti-CCP assay used in this study to detectACPA is thought to contain various citrullinated antigens, one of whichcould be Col [27]. So, the activity in this assay is most likely due to reac-tivity with the Col portion of the Cit-Col antigen used in the assay. It isalso possible that other potential proteins and/or peptides are beingcitrullinated in vivo and are being detected by the kit, as this has beenpreviously proposed [28]. These processes would most likely occur viamacrophage migration into sites of inflammation and processing theseantigens and in turn expressing PAD enzyme which would citrullinatemore self‐proteins expressed in the joint [1]. In fact, PAD-2 and PAD-4enzymes have been found to be highly expressed in lymphocytic cells,monocytic cells, and synovial tissue of RA patients. Interestingly, theseenzymes have been correlated with the intensity of inflammation, andhave been detected within the citrullinated fibrin deposits of the joints[29].

A significant increase in the proliferation to Col was observed forT-cells from both the Cit-Col and FCA-Col injected mice. However,when these T-cells were stimulated with Cit-Col there was a signifi-cantly increased response in the Cit-Col injected mice compared tothe FCA-Col immunized mice. In addition this response in our modelwas directed primarily against the citrullinated epitope as determinedby their reactivity to Cit-BSA (carrier antigen never exposed to themice). The ability to process and present Cit-Col to T-cells demonstratedthe ability to generate an autoreactive response in the absence of an ad-juvant (FCA). These T-cell responses are consistent with other reportsshowing that a self‐protein (citrullinated rat serum albumin [RSA])can induce an autoreactive T-cell response to both the carrier (RSA)and the modified epitope (CCP), but these studies were performed inthe presence of adjuvant [13]. Data generated from the supernatantsof the T-cell proliferation assays show increased secretion of both IL-23 and IL-6 in the Cit-Col stimulated cells as compared to Col stimulatedcells. Importantly, these two cytokines have been shown to differentiateCD4+ T-cells into Th17 cells, which are thought to be linked to theinduction of autoimmune diseases including RA [30,31]. To our knowl-edge, the data reported in this study is the first showing that citullinationof a self-protein initiates an arthritis-like disease in the absence of ad-juvants. Thus, these studies indirectly support a pathogenic role ofcitrullinated self-proteins in RA. The ability to produce these responseswithout the use of FCA provides a potential mechanism by which thecitrullination of these proteins initiates and drives disease-related in-flammation. It is important to recognize that FCA immunization alone

causes non-specific inflammation andhas been shown to induce arthritiseven in the absence of antigen [32]. The ability to induce these inflamma-tory responses without the use of adjuvant and the generation of T-cellresponses supports the physiologic relevance of this novel animalmodel, one that may more closely mimic human disease.

Fig. 6. Cytokine involvement from T-cell proliferation in CCIA mice. Supernatant wascollected and assay using commercial available kits for (A) IL-6 and (B) IL-23 cytokines.Data expressed are the means±SEM of 10 animals. *P≤0.05 significantly differentfrom Col injection.

430 G.M. Thiele et al. / International Immunopharmacology 13 (2012) 424–431

In conclusion, this study demonstrates for the first time the use ofa citrullinated self‐protein to generate an autoimmune response in theabsence of an adjuvant. Inflammation and damage to the joint tissue inthe CCIAmodelwere determined using several complimentarymethodsincluding: caliper scores, visual observation, IR polymer (shown to de-tect inflammation), micro-CT, and histology. Immunological confirma-tion was determined by the induction of antibody and T-cell responsesto both the Col and Cit-Col from these injected mice. The observationthat this model is characterized primarily by a T-cell mediated responsesuggests that this model may parallel the human disease. Finally, thedevelopment of this model is extremely useful as no adjuvant is used.Therefore, the evaluation of the immune response is not confoundedby the presence of adjuvants that non-specifically stimulate immuneresponses.

Abbreviations

RA Rheumatoid ArthritisFCA Freund's Complete AdjuvantIFA Incomplete Freund's AdjuvantPAD Peptidylargine DeiminaseCIA Collagen‐Induced ArthritisCCIA Citrullinated Collagen‐induced ArthritisRSA Rat Serum Albumin

BSA Bovine Serum AlbuminHPMA N-(2-hydroxypropyl)methacrylamideVOI Volume of InterestMTP MetatarsophalangealCCP Cyclic Citrullinated PeptideSEM Standard Error of the MeanBV Bone VolumeSI Stimulation IndexACPA Anti Citrullinated Protein Antigens

Competing interests

The authors have no competing interest to disclose for thismanuscript.

Author contributions

GMT contributed to the conception, design, interpretation of data,and preparation of the manuscript. MJD contributed to the conception,design, interpretation of data, and preparation of themanuscript. AD car-ried out the procedures, contributed interpretation of data and prepa-ration of the manuscript. CDH carried out the procedures, contributedinterpretation of data and preparation of themanuscript. JPL CDH carriedout the procedures, contributed interpretation of data and preparation ofthe manuscript. DRA contributed to the conception, design, interpreta-tion of data, and preparation of themanuscript. DWprovided intellectualknowledge for use of the infrared polymer and preparation of the manu-script. JRO contributed to the conception, design, interpretation of data,and preparation of the manuscript. TRM contributed to the conception,design, interpretation of data, and preparation of the manuscript. LWKcontributed to the conception, design, interpretation of data, and prepara-tion of the manuscript. All authors have read and approved of the finalmanuscript for submission.

Acknowledgments

The authors would like to thank the members of the ExperimentalImmunology Laboratory for their valuable support of this work. TheDepartment of Internal Medicine and the College of Medicine at UNMC,the American College of Rheumatology Research and Education Foun-dation within Our Reach award. Also, the Omaha VA Medical CenterResearch Services.

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