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Staging the Initiation of -Induced Arthritis: A Critical Role for Immune Complexes

This information is current as Brian T. Wipke, Zheng Wang, Wouter Nagengast, David E. of September 25, 2021. Reichert and Paul M. Allen J Immunol 2004; 172:7694-7702; ; doi: 10.4049/jimmunol.172.12.7694 http://www.jimmunol.org/content/172/12/7694 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Staging the Initiation of Autoantibody-Induced Arthritis: A Critical Role for Immune Complexes1

Brian T. Wipke,2 Zheng Wang,† Wouter Nagengast,† David E. Reichert,† and Paul M. Allen3*

In the K/B؋N mouse model of arthritis, against glucose-6-phosphate isomerase cause joint-specific inflammation and destruction. We have shown using micro-positron emission tomography that these glucose-6-phosphate isomerase-specific autoantibodies rapidly localize to distal joints of mice. In this study we used micro-positron emission tomography to delineate the stages involved in the development of arthritis. Localization of Abs to the joints depended upon mast cells, , and FcRs, but not on C5. Surprisingly, anti-type II collagen Abs alone did not accumulate in the distal joints, but could be induced to do so by coinjection of irrelevant preformed immune complexes. Control Abs localized to the joint in a similar manner. Thus, immune complexes are essential initiators of arthritis by sequential activation of neutrophils and mast cells to allow Abs access to the joints,

where they must bind a target Ag to initiate inflammation. Our findings support a four-stage model for the development of Downloaded from arthritis and identify checkpoints where the disease is reversible. The Journal of Immunology, 2004, 172: 7694–7702.

uman (RA)4 is a debilitating, FcRs are important for disease induction, with the low affinity chronic characterized by progres- Fc␥RIII, present on , neutrophils, and mast cells, be- H sive inflammation and destruction of distal joints of the ing the critical player (21, 22). The alternative pathway of com- hands and feet (reviewed in Refs. 1Ð3). A variety of small animal plement is also required, including the C5a receptor (21, 23). Many http://www.jimmunol.org/ models of human RA have been described, including Ag-induced of the same innate components have also been arthritis (4), type II collagen-induced arthritis (CIA) (5Ð9), and the shown to be important in the CIA arthritis model (reviewed in K/BϫN model of arthritis (10). In the K/BϫN model, mice ex- Refs. 24Ð26). A fascinating aspect of the K/BϫN model is the pressing the KRN TCR transgene and class II MHC I-Ag7 spon- exquisite joint specificity of the disease despite the GPI autoanti- taneously develop polyarthritis by 4Ð5 wk of age with many of the gen being ubiquitously expressed. hallmarks of human RA, including synovial hyperplasia, cellular We previously described a novel detection system for determin- infiltration, bone and cartilage erosion and remodeling, and loss of ing the localization patterns of arthritogenic anti-GPI IgG in the joint function (10, 11). KRN cells escape deletion in the thymus joints of normal healthy mice, using rodent-scale positron emission

(10) and are activated by a peptide of glucose-6-phosphate isomer- tomography (microPET). The microPET R4 scanner permits dy- by guest on September 25, 2021 ase (GPI) bound to I-Ag7 in the periphery (12, 13). They subse- namic noninvasive high resolution imaging of radiolabeled GPI- quently provide help to GPI-reactive B cells, resulting in the pro- specific IgG in mice at multiple time points. Using this system, we duction of GPI-specific Abs that cause joint inflammation (11). observed that anti-GPI IgG rapidly localized within minutes to Arthritis can also be induced in most strains of mice by the transfer distal joints of the front and rear limbs (27) and remained there for of anti-GPI Abs (11, 12). Anti-GPI Abs can be detected in some at least 24 h. These kinetics were consistent with direct Ab rec- RA patients (14, 15). This adoptive transfer model has greatly ognition of GPI in the joints, and in support of this model, extra- facilitated elucidation of the effector molecules/mechanisms in- cellular GPI was shown to be present on the cartilaginous surfaces volved in the induction of arthritis. It has now been shown that the of normal joints in naive mice (28). IL-1 and TNF-␣ are essential for disease development The present studies were undertaken to delineate the order of (16, 17), as are mast cells, neutrophils, and macrophages (18Ð20). involvement of these critical cells, receptors, and molecules in the development of arthritis induced by autoantibodies. Using micro-

*Department of Pathology and Immunology and †Department of Radiology, Wash- PET, we demonstrate that neutrophils, mast cells, and immune ington University School of Medicine, St. Louis, MO 63110 complexes (ICs) are essential for getting the Ab into the joints, Received for publication November 20, 2003. Accepted for publication April 9, 2004. whereas C5 acts at a later stage. From these results we were able The costs of publication of this article were defrayed in part by the payment of page to propose a four-stage model for how autoantibody-induced ar- charges. This article must therefore be hereby marked advertisement in accordance thritis occurs. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from National Institutes of Health (P50CA94056 and P01AI031238), National Cancer Institute Grant R24CA86307, and the Pfizer- Materials and Methods Washington University Scientific Partnership Program. Mice 2 Current address: Elan Pharmaceuticals, Inc., 800 Gateway Boulevard, Building 800, Room 154B, South San Francisco, CA 94080. KRN TCR Tg mice (C57BL/6 background) were provided by D. Mathis and C. Benoist (Harvard Medical School, Boston, MA). Nonobese diabetic 3 Address correspondence and reprint requests to Dr. Paul M. Allen, Department of Pathology and Immunology, Washington University School of Medicine, Campus mice were obtained from Taconic Farms (Germantown, NY), and BALB/c Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: mice were obtained from National Cancer Institute (Frederick, MD). [email protected] K/BϫN mice were obtained by breeding homozygous KRN transgenic mice to nonobese diabetic mice in our mouse colony (Washington Uni- 4 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced /Ј Љ ٞ versity, St. Louis, MO). Fc⑀R1␥ deficient (FcRKO), B6/129F1, WBB6F1 arthritis; CII, type II collagen; DOTA, 1,4,7,10-tetraazacyclododecane-N,N ,N ,N - W W-v ϩ ϩ tetraacetic acid; FcRKO, Fc⑀R1␥ deficient; GPI, glucose-6-phosphage isomerase; IC, J-Kit /Kit , and WBB6F1/J-Kit /Kit mice were obtained from The immune complex; mPAP, mouse anti-peroxidase immune complex; PET, positron Jackson Laboratory (Bar Harbor, ME). All animals were housed in accor- emission tomography. dance with National Institutes of Health and American Association for

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 7695

Accreditation of Laboratory Animal Care regulations, and animal protocols MicroPET data collection were reviewed and approved by the Washington University animal studies committee. The microPET-R4 rodent scanner (Concorde Microsystems, Knoxville, TN) provides a 10 ϫ 8-cm field of view with a reconstructed resolution of 2.25 mm in the central 40 mm of the field of view. Images are recon- Measurement of ankle thickness structed using Fourier rebinning, followed by two-dimensional, filtered Mice were examined for clinical signs of inflammation, and ankle thickness back projection. Mice were anesthetized with 1Ð2% vaporized isofluorane, was determined with a pocket thickness gauge (Ralmikes Tool-A-Rama, and a microcatheter (Harvard Apparatus, Holliston, MA) was inserted into Middlesex, NJ) by measuring the ankle across the malleoli as previously the external jugular vein. Pairs of mice were immobilized in a supine po- sition upon custom-built support beds with attached anesthetic gas nose described (18). Ankle thickness measurements were rounded to the nearest 64 0.05 mm and reported as the average of a group of identically treated cones for data collection in the microPET-R4 scanner. Cu-labeled Abs in ␮ mice Ϯ 1 SD. 150 l of PBS were injected via a jugular vein catheter simultaneously with initiation of data collection, and the lines were flushed with saline to insure complete delivery of Abs. Data were collected continuously for Serum and polyclonal Abs 30Ð45 min. The delivered activity of each dose was determined by count- K/BϫN serum was harvested and processed as described previously (18). ing each sample syringe before and after injection using a dose calibration Polyclonal IgG from K/BϫN serum was purified by protein G-Sepharose instrument (Radioisotope Calibrator CRC-12; Capintec, Ramsey, NJ). For 4 Fast Flow (Amersham Pharmacia Biotech, Piscataway, NJ). Bound IgG later time points, mice were reanesthetized, immobilized, and scanned as was washed extensively with PBS, eluted using 0.1 M glycine HCl, pH 2.8, described above. Quantitation of regions of interest was performed by and immediately neutralized with 1 M Tris, pH 8.0, before dialysis against viewing regions of interest over the selected tissues and averaging the PBS. GPI-specific polyclonal Abs (anti-GPI IgG) were purified from pro- activity concentration over the contained voxels. Images were recon- tein G-purified total K/BϫN IgG on affinity columns of recombinant poly- structed with AsiPro (Concorde Microsystems, Knoxville, TN), and region

histidine-tagged mouse GPI conjugated to cyanogen bromide-activated of interest analysis was performed with Analyze AVW 3.0 (Biomedical Downloaded from Sepharose 4B (Sigma-Aldrich, St. Louis, MO), eluted, and dialyzed as Imaging Resource, Mayo Foundation, Rochester, MN). described above. Abs were concentrated with Centricon-10 concentration devICs (Millipore, Bedford, MA), quantitated by optical absorption mea- Biodistribution of labeled Abs ␮ sured at 280 nm, and sterile-filtered through 0.22- m pore size filters. The The biodistribution of 64Cu-DOTA-labeled anti-GPI IgG was determined arthrogen/CIA arthritis-inducing mAb mixture was purchased from Chemi- in age- and sex-matched groups of BALB/c mice treated with either GK1.5 con International (Temecula, CA). A mixture of control mAbs composed anti-CD4 mAb or RB6.8C5 anti-GR-1 mAb (250 ␮g was injected i.p. into of the same proportion (3:1 ratio of IgG2a:IgG2b) as the arthrogen/ each mouse 48 h before experimental use) and untreated FcRKO, http://www.jimmunol.org/ CIA mixture was created by mixing two irrelevant Abs. mAb 4E12 (IgG2a, B6129PF/1, WBB6F1W/Wv, and WBB6F1ϩ/ϩ mice. After tail vein in- anti-3.L2 TCR clonotype) was purified from ascites over protein G-Sepha- jection of the labeled Abs (250 ␮g for anti-GPI and 2 mg each for anti- rose, and mAb GIR94 (IgG2b) was donated by Drs. K. Sheehan and R. collagen and isotype control Abs), the animals were allowed free access to Schreiber (Washington University). For in vivo depletion, the anti- food and water. At the specified time point postadministration, the mice rat IgG2b mAb RB6.8C5 was purified using protein G-Sepha- were sacrificed, and the organs and tissues of interest were removed by rose 4 Fast Flow affinity matrix (Amersham Pharmacia Biotech) from as- dissection and weighed. The lower limbs were removed mid-femur and cites produced in SCID mice. The isotype control Ab GK1.5 mAb against divided into two pieces: the knee sections (from mid-tibia/fibula to mid- mouse CD4 (rat IgG2b) was purified from ascites by saturated ammonium femur) and ankle/paw sections (mid-tibia/fibula and entire rear paw) and sulfate precipitation (45% final concentration) and dialyzed against PBS, were counted separately. The upper limbs were counted as a single unit Ϫ pH 7.4, before being stored at 70¡C. after transecting them mid-humerus. The amount of radioactivity present in each sample was quantified by counting using an automatic well-type by guest on September 25, 2021 Arthritic serum transfer challenge counter (␥ 8000; Beckman Coulter, Fullerton, CA). The percent injected dose per organ was calculated by comparison with a weighed and counted Before injection, serum aliquots were thawed, centrifuged, and diluted with sample of the injectate. SDs are indicated with error bars. Student’s t tests ␮ PBS as described previously (18). All mice received 250 l i.p. because it were performed, and values for anti-GPI IgG samples, which significantly consistently produced disease induction in 100% of C57BL/6 and differ from normal IgG mouse values, are indicated with an asterisk in the BALB/c mice. figures ( p Ͻ 0.01). Ab depletion in vivo Preformed ICs For in vivo neutrophil depletion, 250 ␮g of RB6.8C5 mAb was diluted in Soluble murine peroxidase-anti-peroxidase (mPAP) ICs consisting primar- PBS and injected i.p. 48 h before use in microPET and biodistribution ily of two HRP molecules bound to three anti-peroxidase IgG (31) were experiments. Control GK1.5 mAb (50 ␮l of ascites) was diluted and in- obtained from DAKO (Carpinteria, CA), dialyzed extensively against PBS jected in the same manner. These doses of Ab were previously shown to to remove sodium azide, and sterile-filtered. A dose of 50 ␮l (250 ␮gof completely eliminate neutrophils or CD4ϩ T cells for 3Ð5 days (18). total protein) was selected for these experiments based upon pilot studies in which the minimum dose necessary to cause detectable changes in vas- -Conjugation of 1,4,7,10-tetraazacyclododecane-N,NЈ,NЉ,Nٞ- cular permeability was determined using Evans Blue dye injected system ically (data not shown). For microPET experiments, mPAP ICs were in- tetraacetic acid (DOTA) linker to Abs jected during dynamic scanning via the external jugular catheter 15 min All Abs were conjugated to DOTA using the modified N-hydroxysuccin- after labeled Abs were injected, and scanning was continued for an addi- imide method (29) as previously described (27). After conjugation, the tional 30 min. For biodistribution experiments with mPAP, the ICs were reaction mixtures were centrifuged repeatedly through a Centricon-30 with injected via tail vein 15 min after labeled Ab injection i.v. 30 mM ammonium citrate buffer, pH 6.5, to eliminate unconjugated small molecules. The purified conjugates were concentrated to 2 mg/ml in PBS Results buffer and stored at Ϫ70¡C until further use. The concentrations of Ab Anti-GPI Abs fail to localize in mice that are deficient in FcRs, conjugates were determined by UV spectrophotometer. Addition of DOTA mast cells, and neutrophils, but localize in C5-deficient mice did not affect the biological function or distribution of the Abs (27). We wanted to identify the steps involved in the localization of 64Cu preparation and radiolabeling of conjugated Abs anti-GPI Abs to the affected joints and the subsequent development 64 64 Ͼ of arthritis. To this end, we used mice deficient in FcR, C5, mast Cu ( CuCl2 in 0.1 M HCl; radionuclide purity, 99%) was produced with an in-house cyclotron from enriched 64Ni targets by a previously cells, and neutrophils, which previously had been shown to be described method (30). Typically, 100 ␮g of DOTA-conjugated Ab and 1 resistant to disease induction, and examined using microPET the 64 mCi of Cu were incubated in 30 mM ammonium citrate, pH 6.5, at 43¡C localization of anti-GPI Abs. Mice with a targeted disruption of the for 45 min. The reaction was terminated by addition of 5 ␮l10mMdi- ⑀ ␥ ␥ ␥ ethylenetrinitrilopenta-acetic acid solution. Labeled IgG was separated common Fc RI -chain (32) lack expression of Fc RI and Fc RIII from unincorporated label by a size-exclusion Bio-Spin6 column (Bio-Rad, (hereafter referred to as FcRKO mice) and are completely resistant Hercules, CA). to joint inflammation after transfer of anti-GPI serum (21, 22) (Fig. 7696 STAGES IN THE INITIATION OF Ab-INDUCED ARTHRITIS

1A). FcRKO mice exhibited a complete block in localization of termined that FcRs, mast cells, and neutrophils were all critical anti-GPI IgG to the front and rear limb joints compared with wild- players in the initial localization of anti-GPI IgG to the joints. C5 type B6129F1 control mice (Fig. 1, E and I), indicating that FcR(s) was not involved in this localization, placing its essential role in are critical for proximal events leading to localization of arthrito- the disease at a subsequent stage. genic Abs in the joints. The Fc␥RIII was the FcR that involved in this localization, as shown by complete lack of joint accumulation in Fc␥RIII-deficient mice (data not shown). Differences in Ab joint localization identified by microPET An opposite pattern was observed for mice deficient in C5, analysis are confirmed by classical biodistribution studies which have also been shown to be completely resistant to arthritis To confirm that Ab localization and disease resistance were dis- induction (21, 33) (Fig. 1B). The anti-GPI Abs localized to the tinct events and to test larger numbers of mice, we analyzed Ab joints identically to the control mice (Fig. 1, F and J). This ob- localization by biodistribution studies following a similar protocol servation is consistent with a previous report showing by immu- as that used in the microPET experiments. Mice were injected i.v. nohistology of the localization of anti-CII Abs to the cartilage with anti-GPI IgG or control IgG and were killed after 30 min, and surfaces in C5-deficient mice (8). Thus, C5 is not involved in the the front and rear limbs and knees were collected and measured in localization of Abs to the joints and must therefore be acting at a a gamma counter. The amount of localized Ab was determined by downstream stage. Mast cells and neutrophils have also been comparison with known standards of labeled Ab to yield the per- shown to be critical for disease induction (18, 20) (Fig. 1, C and centage of input dose per organ. Thirty minutes after injection of D). Mast cell-deficient W/Wv mice (Fig. 1, G and K) and neutro- the Abs, groups of FcRKO, W/Wv, and RB6-depleted mice all phil-depleted mice (Fig. 1, H and L) each showed dramatically exhibited decreased localization in the entire front and rear limbs Downloaded from reduced levels of accumulation of anti-GPI IgG in the front and relative to control mice ( p Ͻ 0.01; Fig. 2). The amount of Ab hind limbs. There still was some accumulation above baseline, the present in the knee joints of all the mice was equivalent. C5-de- levels of which were reminiscent of the lower levels seen when ficient mice were not tested by biodistribution, as they showed no subarthritogenic doses of anti-GPI IgG were followed by micro- defect in localization by microPET. These studies allowed us to PET (18). These findings suggest that each cell type is critical for confirm statistically our microPET findings, but lack the latter’s

localization of the Abs to the joints, but there are some overlapping ability to focus precisely on the specific areas that develop disease. http://www.jimmunol.org/ functions. It was a formal possibility that the anti-neutrophil- This highlights the power of the microPET in measuring more depleting Ab also affected mast cells. We eliminated this possibil- precise areas of Ab localization within the joints. Thus, FcRKO, ity by demonstrating that anti-neutrophil Ab treatment has no ob- mast cell-deficient, and neutrophil-deficient mice all show a dra- servable effect on either the number or the location of mast cells in matic reduction in anti-GPI IgG localization by both microPET the synovial regions (data not shown). From these studies we de- and biodistribution analysis. by guest on September 25, 2021

FIGURE 1. Disease resistance is distinct from localization of anti-GPI IgG to distal joints of front and rear limbs. Mice were tested for disease susceptibility by K/BϫN serum transfer and localization of anti-GPI IgG by microPET. AÐD, Ankle thickness measurements for control and experimental mice were determined daily for groups of three to five animals injected i.p. with 250 ␮l of pooled K/BϫN serum. Results represent one of three or more identical experiments with similar results. EÐL, Region of interest analysis of ankle joints of control mice (E) and mice deficient in FcR, C5, mast cells, and neutrophils (Œ) were determined from microPET scans as described in Materials and Methods. Mice were i.v. injected with 250 ␮gof64Cu-labeled anti-GPI IgG and dynamically scanned for 30 min. Each data curve represents the combined average concentration of 64Cu activity within the region of interest for the left and right ankles of an individual mouse and is presented as the percentage of maximum 64Cu activity within the region of interest (for each experiment. Each plot is a separate experiment, and each strain of mouse was individually tested three or four times. The Journal of Immunology 7697

bind to these complexes; and 3) the anti-GPI Abs form ICs in the serum, which facilitate entry of the Abs into the joints. Because GPI is present in both serum and joints, we could not distinguish the precise role of FcRs and ICs. Type II collagen, in contrast, is relatively sequestered; it is found only in joint, tracheal and bron- chial cartilage and in the vitreous humor of the eye. More impor- tantly, it is not detected in the circulation and therefore is pre- cluded from forming ICs in the serum. Immunization with type II collagen induces arthritis in the well-characterized model of CIA. In addition, a commercial mixture of four mAbs against distinct portions of murine type II collagen (34) (Arthrogen-CIA), can in- duce joint inflammation very similar to that induced by transfer of GPI-specific Abs (e.g., edema, synovitis, inflammation, cellular infiltrates, and eventual erosion of cartilage and bone) (11, 12, 34). To determine the trafficking and localization of the anti-collagen Abs, pairs of BALB/c mice were injected with an arthritogenic dose of 2 mg of labeled anti-collagen mAb mixture or an isotype control mixture of irrelevant Abs and dynamically scanned for 1 h

on the microPET. Surprisingly, no joint localization was observed Downloaded from in either mouse within the first hour or at 24 h (data not shown). Thus, the presence of circulating joint-specific Abs in the serum was not sufficient to induce joint localization and subsequent dis- ease without some additional trigger. http://www.jimmunol.org/ IC administration facilitates anti-collagen and control Ab localization to the joints Because anti-GPI IgG is capable of localizing to the joints without additional triggers, the presence of low levels of circulating GPI Ag (28, 35) may be an important factor in the localization of anti- GPI IgG. Injection of anti-GPI IgG could potentially provoke the formation of small numbers of ICs in the circulation, and forma- tion of these ICs could play a significant role in modifying the distribution and accessibility of specific Abs to the joints. To test by guest on September 25, 2021 this hypothesis, the microPET/anti-CII experiments were repeated with the addition of irrelevant ICs after establishing a baseline scan for the labeled anti-collagen and control Abs. Fifteen minutes after beginning the scan and simultaneous injection of anti-collagen and control Abs, soluble preformed mPAP (31) were injected i.v. dur- ing continued scanning. Representative results from average ankle FIGURE 2. Accumulation of arthritogenic anti-GPI IgG in distal limbs ROI analysis of three pairs of mice (Fig. 3) demonstrate that ir- is blocked in mice deficient for FcRs, mast cells, and neutrophils. Biodis- relevant mPAP complexes can exert dramatic effects on the local tribution of 250 ␮gof64Cu-labeled anti-GPI IgG in different organs and joint concentration of labeled Ab. Approximately 5 min after tissues was measured in mice at 30 min postinjection i.v. by dissection and mPAP injection, there was a rapid increase in the concentration of quantitation in a gamma counter against dilutions of the identical labeled labeled Ab within the ankle regions for both anti-collagen and Ab. The percent input dose of labeled Ab per organ was calculated as control Abs (4- to 5-fold over baseline levels before mPAP injec- described in Materials and Methods. The mean value of two to four ex- tion). Ab accumulation reached plateau levels 10Ð15 min after periments with a total of six to eight mice per group is shown with the mPAP injection (25Ð30 min into the scans) and remained stable SEM, except for A, which is the sum of four experiments totaling 20 mice thereafter. The timing and kinetics of Ab accumulation in the an- of each type. The statistical significance of the data was determined by kles in response to systemic mPAP injection are almost identical p Ͻ 0.01). No significant differences were found in other ,ء) Student’s t test surveyed tissues. with those observed with anti-GPI IgG without any additional ICs (27), implying that a similar underlying mechanism is responsible. We next wanted to ascertain whether this joint localization fol- lowing mPAP administration was specific for the anti-collagen Arthritogenic anti-type II collagen Abs fail to localize to distal Abs. There was no difference in the kinetics or overall level of Ab joints localization for specific (anti-collagen) and irrelevant (control) Our studies had shown that FcR were critical for the localization of Abs (Fig. 3). These findings are consistent with soluble ICs induc- anti-GPI Abs to the joints. GPI is found in three locations in the ing an overall change in vascular permeability that nonspecifically mouse: in the cytoplasm of all cells, in the circulation, and on the permits all Abs entry into the joint tissues. This could be occurring articular cartilage surface. We reasoned that FcR could be involved through either complement-mediated or FcR-mediated changes in in the joint localization of the Abs in three different ways: 1) the vascular permeability. The maximal density of labeled anti- anti-GPI Abs are binding to FcR-bearing cells in the blood, and collagen and control Ab in the ankle region was lower than that for these cells then bring the complexes into the joints; 2) the anti-GPI anti-GPI IgG (ϳ0.015 vs 0.03 ␮Ci/␮l, on the average), and this Abs directly bind GPI on the articular cartilage, and FcR cells then may be due either to the difference in the radiospecificity of the 7698 STAGES IN THE INITIATION OF Ab-INDUCED ARTHRITIS

PET experimental protocol. Mice were injected i.v. with anti- collagen or control IgG, and separate groups of animals for each Ab type were treated with either PBS or mPAP at 15 min to par- allel the microPET experiments. Animals were sacrificed after 45 min or 24 h, and a panel of organs and tissues was collected and measured in a gamma counter. As in our previous studies (27), biodistribution results (Fig. 4A) closely correlated with earlier mi- croPET findings (Fig. 3), with greatly enhanced joint localization for both Ab types at 45 min compared with PBS-treated mice that received either anti-collagen or control Abs. Mock-treated mice (PBS at 15 min) lacked specific joint localization regardless of whether they received anti-collagen or control Abs (Fig. 4A) and did not show any difference either in the initial scan (data not shown) or the 24 h postinjection scan (Fig. 4B). For these studies, we used a 2-mg dose of anti-collagen Ab, which is the minimal dose required to induce disease in susceptible strains and requires LPS to be given additionally. It was possible that this dose falls below a threshold for joint localization, analo-

gous to the variable and significantly reduced localization of sub- Downloaded from arthritogenic doses of anti-GPI IgG observed in previous studies (27). To determine whether increasing the dose of anti-collagen would enhance joint localization in the first 24 h, we analyzed the biodistribution of 4-mg doses of anti-collagen Ab in the absence of http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Anti-collagen mAbs fail to localize to distal joints without external modulation and rapidly localize to distal joints upon systemic administration of irrelevant preformed ICs. Labeled anti-collagen mAbs or control IgG mixture (2 mg total) were injected i.v., and mice were scanned continuously for 45 min. At 15 min postinjection, preformed mPAP ICs were injected i.v. (ϳ250 ␮g). Region of interest quantitation for ankle joints is presented as the average of both ankles for each of three identical experiments, and data are representative of five identical experiments (10 mice total). two Abs injected (ϳ350 mCi/2.0 mg of 64Cu-anti-collagen com- pared with ϳ350 mCi/250 ␮gof64Cu-anti-GPI IgG) or the in- volvement of other factors that may augment localization of anti- GPI IgG. In our initial microPET studies, we reported that control Abs did not localize to the joints. Based on these findings with ICs, it is now clear that the lack of localization was due to the absence of ICs, not the lack of a target Ag in the joint. Thus, by studying the localization of anti-collagen Abs using microPET, we made the FIGURE 4. Injection of systemic preformed ICs enhances joint local- important observation that soluble ICs in the blood are required for ization of both specific (anti-collagen) and nonspecific control Abs in bio- arthritogenic Abs to gain access to their target organs. distribution studies. Groups of mice were injected i.v. with 2 mg of labeled anti-collagen or isotype control mAb mixture and 15 min later received Preformed ICs modulate Ab access to the distal joints either PBS or 50 ␮l of mPAP ICs (murine peroxidase-anti-peroxidase ICs) regardless of Ab specificity i.v. Mice were killed at 45 min or 24 h postinjection, and biodistribution of Abs was determined as described in Materials and Methods. Mean per- To confirm the distribution and kinetics of anti-collagen localiza- centages of input dose per organ are presented for groups of three mice tion with larger groups of mice and by a different method, we again each; error bars indicate SEM values for each group. The significance of conducted classical biodistribution studies with the same 2-mg results (mPAP-treated group vs PBS group) was determined by Student’s p Ͻ 0.01). No significant differences were noted in other surveyed ,ء) dose of anti-collagen Abs or an isotype control mixture in BALB/c t test mice. Separate groups of three animals for each Ab type were tissues. These results are from one experiment with groups of three mice treated with either PBS or mPAP at 15 min to parallel the micro- per condition and time point. The Journal of Immunology 7699

IC modulation (mPAP). The higher 4-mg dose of anti-collagen, Four-stage model of how autoantibodies induce arthritis which is arthritogenic in many strains of mice including BALB/c From our studies we have been able to experimentally delineate without the addition of LPS (36), failed to give specific accumu- three stages involved in the initiation of arthritis by autoantibodies. lation or retention of anti-collagen relative to 4 mg of isotype con- We propose a four-stage working model in which we have added trol mAb and did not significantly alter the percentage of the total one additional stage. Some of the effector molecules and cells in dose of anti-collagen that accumulated in front or rear limb joints this model are most likely involved in multiple stages. For exam- relative to 2-mg doses (data not shown). Preliminary microPET ple, neutrophils can be involved in stages 1 and 3. Importantly, the analysis of mice injected with 4 mg of anti-collagen also failed to progression to these various stages is reversible, thus allowing sev- show any significant joint localization (data not shown). eral places where progression to arthritis can be prevented or re- Identification of stages subsequent to Ab localization to the joint versed. The details of the stages are given below and are dia- grammed in Fig. 5. The microPET and biodistribution studies clearly demonstrated that simply getting an Ab into the joint via IC-mediated increased Stage 1: Access of Ab to the joint. Our studies have shown that permeability was not sufficient to cause disease, as control Abs FcR, mast cells, neutrophils, and inflammatory stimuli such as ICs entered the joints to the same level as the arthritogenic anti- are absolutely required to get autoantibodies into the joints. Two collagen Abs, but did not cause arthritis (data not shown). Thus, a observations must be incorporated into a model of autoantibody second stage in arthritis development is that once the Abs enter the trafficking into the joints. First, mast cells are essential for auto- joints, they must subsequently recognize a target Ag on the joint to access the joint in these studies, yet are located surface, such as collagen II or bound GPI to the articular cartilage. perivascularly in the mouse ankle, and most likely would have Downloaded from A third stage must also exist after the autoantibodies enter the little, if any, direct access to circulating ICs. A blood neutrophil, in joint and bind to their Ag, which involves activation of the innate contrast, would be readily accessible to such ICs. Second, one immune system and inflammation. We obtained evidence for this must account for the apparent saturation (plateau) of the amount of stage when we determined that CII Abs along with ICs injected anti-GPI Abs that enter the joints, despite large amounts of labeled into BALB/c mice did not result in any arthritis symptoms (data anti-GPI IgG still present in the blood. We propose a sequence of not shown) despite efficient localization in the joints (Figs. 3 and events that occur upon injection of purified anti-GPI IgG or whole http://www.jimmunol.org/ 4). Control mice, which received CII Abs plus LPS instead of ICs, K/BϫN serum and involve distinct roles for mast cells and neu- did develop arthritis. Thus, there is another series of steps required trophils, culminating in the rapid localization of Ab in the joints for arthritis to develop, which LPS is capable of triggering. LPS is (Fig. 5, stage 1). a pleiotropic mediator of inflammation and can activate several Initially, the injected Ab encounters Ag in the circulation, forms different molecules/pathways that could be directly involved, small soluble ICs, and engages Fc␥RIII on neutrophils in the including IL-1, TNF, and Toll-like receptor-4. Thus, we have bloodstream, thus activating the neutrophils (37). The neutrophils, identified a third stage in disease induction, which involves the through the release of cytokines or other soluble mediators, cause activation of innate effector mechanisms and inflammation. a small change in the local vasculature, allowing ICs to access the by guest on September 25, 2021

FIGURE 5. A four-step model of autoantibody-induced arthritis. Stage 1: Access of Ab into the joint. a, Injected anti-GPI Abs (dark gray) bind to GPI present in the serum (squares) and form soluble ICs; b, ICs bind to Fc␥RIII on neutrophils; c, triggering the release of vasoactive mediators such as TNF-␣ (oval), resulting in a local increase in vascular permeability, thereby allowing the GPI anti-GPI ICs to enter the perivascular space where they encounter a mast cell; d, the GPI-anti-GPI ICs bind to the mast cell via the Fc␥RIII, causing them to degranulate, resulting in a large increase in vascular leakage; e, allowing anti-GPI Abs (dark gray), nonspecific Abs (light gray), and other serum proteins (not shown) to enter the joint. The increased vascular permeability is a transient event, and the vascular integrity is restored within 20 min. Stage 2: Recognition of target Ag in the joint. f, Anti-GPI Abs bind to GPI present on the cartilage surface; g, but nonspecific Abs have no target to bind and are cleared and/or degraded. Stage 3: Activation of innate effector mechanisms and inflammation. h, Anti-GPI Abs bound to the cartilage surface activate the alternative pathway of complement (C3CЈ), producing the , C5a; i, C5a then activates multiple cell types (neutrophils, mast cells, macrophages, and endothelial cells) to produce proinflammatory molecules (i.e., TNF-␣, IL-1, and chemokines), thereby causing inflammation. Stage 4: Chronic disease. After prolonged inflammation, chronic changes in the joint occur such as: j, synovial hyperplasia; k, pannus formation with inflammatory infiltrates comprising of T cells, B cells, macrophages, mast cells, and neutrophils; and l, activation of osteoclasts, resulting in bone erosion. 7700 STAGES IN THE INITIATION OF Ab-INDUCED ARTHRITIS mast cells found in close proximity to the microvasculature in the are stopped, the paws return to normal. Thus, stage 3 involves innate synovium. Next, the Fc␥RIII ligation on mast cells causes the de- immune system activation, inflammation, and arthritis development. granulation and release of mediators such as histamine and TNF-␣ Stage 4: Chronic disease. Once stages 1Ð3 have occurred, the that are capable of causing rapid and more widespread changes in chronic phase would occur in stage 4 (Fig. 5). This stage involves vascular permeability (38Ð42). Increased permeability amplifies macrophages, synoviocytes, and . The pathology in the vascular change through increased mast cell triggering, and Ab the joints would include synovial hyperplasia, pannus formation enters the joints through mass flow of fluid phase proteins. After a and bone/cartilage erosion. Some of the same effector molecules short period (i.e., 20 min), vascular integrity is restored through involved in earlier stages could also be operating in this stage, i.e., exhaustion of the preformed mast cell contents and/or a refractory TNF, whereas, other molecules may be unique to this stage, i.e., phase for the vasculature. Restoration of the vascular integrity MMPs. Once this stage has occurred, the disease is not reversible, would thus explain the observation that the amount of Ab levels off but some of the on-going inflammatory processes can be amelio- after 20 min (27). In the CII model, LPS administration must result rated with treatments such as TNF inhibitors. in production and/or release of some vasoactive molecules, leading to the anti-CII Abs gaining access to the joint. There is most likely Discussion some low level trafficking of Abs into a normal joint, the level of A key finding from this study was that systemic addition of soluble which normally falls below that needed for any observable pathol- small ICs containing an irrelevant protein Ag could specifically ogy. In the case of the administration of the 4-mg dose of anti-CII modulate access of labeled serum Abs to the joint regions. Inter- or in susceptible strains such as DBA/1J mice, there is sufficient estingly, addition of preformed mPAP ICs (at the dose used in trafficking of the Abs into the joints to account for the observed these studies) did not globally alter vascular permeability to the Downloaded from arthritis. This movement most likely occurs at a slower rate than same extent as observed in the front and hind limbs, as the lungs what we observed with the ICs. What the ICs do essentially is did not show any change in Ab accumulation within the first 45 greatly facilitate the rapid influx of the Abs into the joints above a min or at 24 h, and the change in skin localization was much threshold level. Thus, in stage 1, ICs cause entry of Abs into the smaller in magnitude than in the extremities. The systemic dose of joints through sequential interaction with PMNs and mast cells. mPAP ICs used in this study was titrated in vivo using i.v. Evans

blue dye to detect the lowest dose of mPAP necessary to trigger http://www.jimmunol.org/ Stage 2: Recognition of target Ag in the joints. The second es- dye extravasation into the epithelium of the ears (data not shown). sential stage involves the binding of the Abs to target autoantigens Therefore, the front and rear limbs of mice appear to be particu- in the joint (Fig. 5, stage 2). Once inside the joint, the anti-GPI and larly sensitive to the presence of circulating ICs. anti-CII Abs can bind to GPI or CII on the articular cartilage sur- Our findings also may provide a link between infection and face, respectively. The nonspecific Abs would not bind to any autoimmune disease. The process of postinfectious arthritis can be specific Ags, and would be cleared from the joints. It is possible to explained by our findings. An individual could contain circulating have some transient arthritis in the absence of a target Ag in the Abs against joint-specific Ags such as collagen, which under nor- joint, when a high level of Abs get into the joint, as can be seen mal circumstances do not have access to the joints or cause dis-

with . Stage 2 provides an important checkpoint for by guest on September 25, 2021 ease. During infection, non-joint-specific ICs are generated, which the control of arthritis and shows that getting autoantibodies into could increase the joint vascular permeability, thus permitting the the joints is necessary, but not sufficient, for arthritis to occur. autoantibodies to gain access to the target tissue (stage 1). The Stage 3: Activation of innate effector mechanisms and inflam- autoantibodies would bind their target Ag (stage 2), provoking a mation. Once the Abs have bound to their respective Ags, they transient inflammatory process that under many circumstances re- then would serve as substrates for activation of the innate immune solves spontaneously (stage 3). In some other individuals with system (Fig. 5, stage 3). This would involve both activation of the higher levels of autoantibodies or other factors, however, the dis- alternative pathway of complement, and potential activation of ease could become chronic rheumatoid arthritis (stage 4). In this PMNs, NK cells, mast cells, and macrophages through the FcRs. manner, nonspecific ICs could initiate an autoimmune process. A The activation of these pathways and cells would result in the similar mechanism may be responsible for the accumulation of ICs production of proinflammatory cytokines (IL-1 and TNF-␣) and in the joints of patients suffering from serum sickness. Resolution chemokines (e.g., inflammatory protein-1␣ and of the temporary arthritis observed in serum sickness occurs chemoattractant protein-1). This is the step that LPS through clearance of the ICs that lack specific targets in the joints. most likely acts upon in the CII model, as supported by the ob- Our inability to observe localization of monoclonal anti-CII to servation that either IL-1␤ or TNF-␣ is sufficient to induce arthritis the joints without the addition of ICs is in contrast to a previous with anti-CII Abs in the absence of LPS (43). ICs alone in the report by Stuart and Dixon (48). In that study they tracked the blood must not activate the to produce a localization of coinjected 125I-labeled, affinity-purified, polyclonal threshold level of these cytokines/chemokines. After the produc- anti-CII and 131I-labeled normal IgG in DBA/1J mice. They ob- tion of the proinflammatory cytokines and chemokines, a strong served that at 30 min there was specific localization of the anti-CII inflammatory response then ensues in the joints. This would occur to the hind feet, as determined by the CII/nIgG ratio, which peaked around 24Ð48 h, at which time the joint swelling is observed, and at 1 h. At 30 min, there was, however, a significant accumulation neutrophils are detectable histologically (18). The initiation of the of nIgG in the joints, ϳ50% of the anti-CII. There are several inflammatory response would also cause long term changes in vas- possible explanations for the differences in our two studies. The cular permeability and improved access of Abs into the joint, as most likely explanation involves the mouse strains used. DBA/1J has been found in infectious foci of inflammation (44Ð47), con- mice are the most susceptible strain to collagen-induced arthritis tributing to continued influx of autoantibodies and potential via immunization or transfer of antibodies. In our studies we tested spreading. Arthritis at this stage can also be reversed, as BALB/c mice, which are less susceptible. In the anti-CII transfer observed in the anti-GPI serum transfer model. The acute arthritis model, DBA/1J mice also do not require LPS to induce disease, that develops after a single injection of anti-GPI Abs wanes over whereas all other strains do. Thus, one component of this enhanced time, and by 14 days no inflammation is evident. Continual injec- susceptibility of DBA/1J mice may involve the enhanced ability of tion of Abs will maintain the arthritis; however, once the injections Abs to gain access to the joints via an unidentified mechanism, The Journal of Immunology 7701 thereby, accounting for their observed localization. Another, but localization of - complexes to collagenous tissues. J. Exp. Med. not mutually exclusive, possibility relates to a difference in traf- 135:323. 5. Loutis, N., P. Bruckner, and A. Pataki. 1988. Induction of erosive arthritis in mice ficking to the joints between a pool of monoclonal and polyclonal after passive transfer of anti-type II collagen antibodies. Agents Actions 25:352. anti-CII Abs. Future studies on the trafficking differences of Abs in 6. Stuart, J. M., M. A. Cremer, A. S. Townes, and A. H. Kang. 1982. Type II DBA/1J and other strains may provide important insights into ba- collagen-induced arthritis in rats: passive transfer with serum and evidence that IgG anticollagen antibodies can cause arthritis. J. Exp. Med. 155:1. sis for the genetic susceptibility of DBA/1J to collagen-induced 7. Trentham, D. E., A. S. Townes, and A. H. Kang. 1977. to type II arthritis. collagen: an experimental model of arthritis. J. Exp. Med. 146:857. Why only distal joints are targeted by the formation of ICs in the 8. Watson, W. C., P. S. Brown, J. A. Pitcock, and A. S. Townes. 1987. Passive transfer studies with type II collagen antibody in B10.D2/old and new line and serum remains to be determined. Several possibilities exist, includ- C57BL/6 normal and beige (Chediak-Higashi) strains: evidence of important ing the number or location of mast cells surrounding the joint roles for C5 and multiple inflammatory cell types in the development of erosive arthritis. Arthritis Rheum. 30:460. vasculature, the presence of activated neutrophils in the vascula- 9. Wooley, P. H., H. S. Luthra, J. M. Stuart, and C. S. David. 1981. Type II col- ture of the joints, or a fundamental difference in the joint endo- lagen-induced arthritis in mice. I. Major histocompatibility complex (I region) thelium itself. However, determination of these factors may allow linkage and antibody correlates. J. Exp. Med. 154:688. 10. Kouskoff, V., A.-S. Korganow, V. Duchatelle, C. Degott, C. Benoist, and for development of successful interventions for various joint in- D. Mathis. 1996. Organ-specific disease provoked by systemic autoimmunity. flammation involving soluble ICs, such as, fol- Cell 87:811. lowing bacterial or viral infection, serum sickness, systemic 11. Korganow, A.-S., H. Ji, S. Mangialaio, V. Duchatelle, R. Pelanda, T. Martin, C. Degott, H. Kikutani, K. Rajewsky, J.-L. Pasquali, et al. 1999. From systemic erythematosus, and RA. self-reactivity to organ-specific autoimmune disease via immunoglobulins. There is a large body of data implicating mast cells in rapid and 10:451. dramatic alterations of vascular integrity and recruitment of in- 12. Matsumoto, I., A. Staub, C. Benoist, and D. Mathis. 1999. Arthritis provoked by Downloaded from linked T and recognition of a glycolytic enzyme. Science 286:1732. flammatory cells in response to FcR-mediated signals, including 13. Basu, D., S. Horvath, I. Matsumoto, D. H. Fremont, and P. M. Allen. 2000. asthma (49), (42), and peritonitis models (50, Molecular basis for recognition of an arthritic peptide and a foreign epitope on 51). This occurs through the release of preformed granule contents, distinct MHC molecules by a single TCR. J. Immunol. 164:5788. ␣ 14. Matsumoto, I., D. M. Lee, R. Goldbach-Mansky, T. Sumida, C. A. Hitchon, including histamine, serotonin, TNF- , and proteases such as P. H. Schur, R. J. Anderson, J. S. Coblyn, M. E. Weinblatt, M. Brenner, et al. tryptase (52Ð55). Cross-linking of Fc␥RIII on mast cells has been 2003. Low prevalence of antibodies to glucose-6-phosphate isomerase in patients with rheumatoid arthritis and a spectrum of other chronic autoimmune disorders. shown to cause degranulation (56) and changes in vascular per- http://www.jimmunol.org/ Arthritis Rheum. 48:944. meability (albeit in tissues other than the distal joints). More re- 15. Schaller, M., D. R. Burton, and H. J. Ditzel. 2001. Autoantibodies to GPI in cently, mast cells were implicated in the K/BϫN serum transfer rheumatoid arthritis: linkage between an animal model and human disease. Nat. model (20), although their precise role was not described. Neutro- Immunol. 2:746. 16. Ji, H., A. R. Pettit, K. Ohmura, A. Ortiz-Lopez, V. Duchatelle, C. Degott, phils are not typically implicated in mediating rapid changes in E. M. Gravallese, D. Mathis, and C. Benoist. 2002. Critical roles for interleukin vascular permeability, but the microPET and biodistribution data 1 and tumor factor ␣ in antibody-induced arthritis. J. Exp. Med. 196:77. 17. Choe, J.-Y., B. Crain, S. R. Wu, and M. Corr. 2003. Interleukin 1 receptor de- in neutrophil-depleted mice clearly indicated a proximal role in Ab pendence of serum transferred arthritis can be circumvented by Toll-like receptor localization. This could occur through the release of soluble me- 4 signaling. J. Exp. 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and progression of a murine model of rheumatoid arthritis. J. Immunol. 167:1601. by guest on September 25, 2021 tiple cell types play specific and partially overlapping roles in the 19. Bruhns, P., A. Samuelsson, J. W. Pollard, and J. V. Ravetch. 2003. Colony- changing vascular permeability of the joints leading to autoanti- stimulating factor-1-dependent macrophages are responsible for IVIG protection body accessing the joints. in antibody-induced autoimmune disease. Immunity 18:573. 20. Lee, D. M., D. S. Friend, M. F. Gurish, C. Benoist, D. Mathis, and M. B. Brenner. Overall, from our studies we were able to provide direct evi- 2002. Mast cells: a cellular link between autoantibodies and inflammatory arthri- dence in support of a four-stage model for the development of tis. Science 297:1689. arthritis. This model positions ICs, neutrophils, and mast cells in 21. Ji, H., K. Ohmura, U. Mahmood, D. M. Lee, F. M. A. Hofhuis, S. A. Boackle, K. Takahashi, V. M. Holers, M. J. Walport, C. Gerard, et al. 2002. Arthritis an essential initial stage of the joint localization of Abs and pro- critically dependent on innate immune system players. Immunity 16:157. vides a conceptual framework for the design and interpretation of 22. Corr, M., and B. Crain. 2002. The role of Fc␥R signaling in the K/B ϫ N serum therapeutic interventions of Ab-mediated types of RA. transfer model of arthritis. J. Immunol. 169:6604. 23. Solomon, S., C. Kolb, S. Mohanty, E. Jeisy-Walder, R. Preyer, V. Scho¬llhorn, and H. Illges. 2002. Transmission of antibody-induced arthritis is independent of Acknowledgments complement component 4 (C4) and the complement receptors 1 and 2 (CD21/35). Eur. J. Immunol. 32:644. We thank Laura Mandik-Nayak, Fei Shih, Christine Pham, and 24. Holmdahl, R., R. Bockermann, J. Backlund, and H. Yamada. 2002. The molec- Sheeram Akilesh for their valuable comments, discussions, and experimen- ular pathogenesis of collagen-induced arthritis in mice: a model for rheumatoid tal assistance, and John Engelbach, Lynne Jones, and Nichole Mercer for arthritis. Ageing Res. Rev. 1:135. their assistance with animal handling in the microPET and biodistribution 25. Ravetch, J. V., and R. A. Clynes. 1998. Divergent roles for Fc receptors and studies. Stephen Horvath provided help with Ab purification, and complement in vivo. Annu. Rev. Immunol. 16:421. 26. Myers, L. K., E. F. Rosloniec, M. A. Cremer, and A. H. Kang. 1997. Collagen- David Donermeyer assisted in cloning and sequencing the recombinant induced arthritis, an animal model of autoimmunity. Life Sci. 61:1861. GPI-His protein. Richard Laforest provided data analysis for this project, 27. Wipke, B. T., Z. Wang, J. Kim, T. J. McCarthy, and P. M. Allen. 2002. Dynamic and Darren Kreamalmeyer provided essential support in serum banking visualization of a joint-specific autoimmune response through positron emission and management of the mouse colony. Jerri Smith assisted in preparation tomography. Nat. Immunol. 3:366. 28. Matsumoto, I., M. Maccioni, D. M. Lee, M. Maurice, B. Simmons, M. Brenner, of the manuscript for submission. We acknowledge Michael J. Welch for D. Mathis, and C. Benoist. 2002. How antibodies to a ubiquitous cytoplasmic 64 his support, and in particular the production of Cu at Washington Uni- enzyme may provoke joint-specific autoimmune disease. Nat. Immunol. 3:360. versity School of Medicine. 29. Lewis, M. R., A. Raubitschek, and J. E. Shively. 1994. A facile, water-soluble method for modification of proteins with DOTA: use of elevated temperature and optimized pH to achieve high specific activity and high chelate stability in ra- References diolabeled immunoconjugates. Bioconj. Chem. 5:565. 1. Feldmann, M., F. M. Brennan, and R. N. Maini. 1996. Rheumatoid arthritis. Cell 30. McCarthy, D. W., R. E. Shefer, R. E. Klinkowstein, L. A. Bass, 85:307. W. H. Margeneau, C. S. Cutler, C. J. Anderson, and M. J. Welch. 1997. Efficient 2. Brennan, F. M., R. N. Maini, and M. Feldmann. 1998. Role of pro-inflammatory production of high specific activity 64Cu using a biomedical cyclotron. Nucl. cytokines in rheumatoid arthritis. Springer Semin. Immunopathol. 20:133. Med. Biol. 24:35. 3. Han, Z., L. Chang, Y. Yamanishi, M. Karin, and G. S. Firestein. 2002. Joint 31. Sternberger, L. A., P. H. Hardy, Jr., J. J. Cuculis, and H. G. Meyer. 1970. The damage and inflammation in c-Jun N-terminal kinase 2 knockout mice with pas- unlabeled antibody enzyme method of immunohistochemistry: preparation and sive murine collagen-induced arthritis. Arthritis Rheum. 46:818. properties of soluble antigen-antibody complex (horseradish peroxidase-anti- 4. Cooke, T. D., E. R. Hurd, M. Ziff, and H. E. Jasin. 1972. The pathogenesis of horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. chronic inflammation in experimental antigen-induced arthritis. II. Preferential Cytochem. 18:315. 7702 STAGES IN THE INITIATION OF Ab-INDUCED ARTHRITIS

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