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Rheumatoid Arthritisand Progression of a Murine Model of Essential Role of Neutrophils in the Initiation

Brian T. Wipke and Paul M. Allen

http://www.jimmunol.org/content/167/3/1601doi: 10.4049/jimmunol.167.3.1601

2001; 167:1601-1608; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Essential Role of Neutrophils in the Initiation and Progressionof a Murine Model of Rheumatoid Arthritis 1

Brian T. Wipke and Paul M. Allen 2

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent oftheir role in the inductive phase of joint inflammation is unknown. In the K/B3N mouse RA model, transfer of autoreactive Igfrom the K/B3N mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA.We observed that after K/B3N serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with thepresence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophilsin the early inflammatory response to transferred arthritogenic serum from the K/B3N transgenic mouse. Mice were treated witha neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflam-mation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant tothe inflammatory effects of K/B3N serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days afterserum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogenor oxygen radicals (inducible NO synthase 2or gp91phox genes, respectively) gave normal inflammatory responses, indicating thatneither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating andmaintaining inflammatory processes in the joint. The Journal of Immunology,2001, 167: 1601–1608.

Rheumatoid arthritis (RA)3 in humans is a debilitating andchronic autoimmune disease characterized by chronic in-flammation of the distal joints. Affected joints display

hyperplasia of the synovia with increased synovial fluid volume,large cellular infiltrates of several cell types (neutrophils, macro-phages, fibroblasts, T cells, and dendritic cells) in the synovial andperiarticular regions, complement deposition, high levels of proin-flammatory cytokine expression, and eventual erosion and remod-eling of the cartilage and bone of the joint (reviewed in Refs. 1 and2). However, it has been difficult to study the initial stages ofdisease because afflicted individuals are normally diagnosed afterthe onset of severe and chronic joint inflammation. Small animalmodels of arthritis in which disease induction is synchronized andpredictable allow investigators to determine the contributions ofspecific cell types and effector molecules to the multiple steps ofdisease induction and pathogenesis.

The most extensively used small animal model of RA is type IIcollagen-induced arthritis (CIA) (3–7), in which immunization ofmice with heterologous type II collagen in adjuvant induces across-reactive immune response to murine type II collagen. Thisautoimmune reaction is mediated by immune complex formationand shares many characteristics with human RA (reviewed in Ref.8). Synovial inflammation (synovitis) can be induced with single

mAbs specific for type II collagen (9), and mixtures of type IIcollagen-specific mAbs against specific regions of collagen cannotonly induce synovitis, but also cause clinical arthritis (10), indi-cating that anti-collagen Ab deposition in the joints triggers thecomplete range of arthritic symptoms. The CIA model has alsobeen useful for studies of cytokine expression in the synovium andsynovial fluid compartments (8), determination of the relative rolesof complement (6) and Ig Fc receptors in disease initiation andpathogenesis (reviewed in Ref. 11), and development of effectiveanti-inflammatory therapeutics such as neutralizing Abs to IL-1(12, 13) and TNF-a (14–16).

Recently, a spontaneous murine disease with most of the char-acteristics of human RA was fortuitously discovered by breedingthe KRN transgenic TCR, specific for bovine RNase (42–56)/I-Ak,mouse to the nonobese diabetic (NOD) background (17). The F1

generation between KRN and NOD (abbreviated K/B3N) spon-taneously develops a progressive joint-specific autoimmune dis-ease between 3 and 5 wk of age, characterized by rapid symmet-rical onset of peripheral joint inflammation that is restrictedprimarily to the joints of the front and rear limbs. Pathology ofdisease in K/B3N mice is similar to human RA, with pannusformation, synovial hyperplasia, increased synovial fluid volume,cellular infiltrates, and chaotic remodeling of cartilage and bone inthe distal joints in later stages. The K/B3N mouse model alsoexhibits elevated expression of proinflammatory cytokines, hyper-gammaglobulinemia, and autoreactive Ab production (18), all ofwhich can be found in human RA patients. One difference betweenthe two diseases is the absence of detectable rheumatoid factor inK/B3N mice (17), although;20–30% of human RA patients arealso negative for serum rheumatoid factor (19). Immune com-plexes of rheumatoid factor-IgG and potentially other Ab-Ag im-mune complexes can be found in the joints of human RA patients(20), but their role in joint pathology and disease progression hasyet to be ascertained.

In the K/B3N model, KRN TCR transgenic cells recognize amouse (self)-derived peptide bound to I-Ag7, presented by B cellsand other MHC class II-positive APCs (18). The autoantigen for

Department of Pathology and Immunology, Washington University School of Med-icine, St. Louis, MO 63110

Received for publication January 10, 2001. Accepted for publication May 18, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Research Grant AI 31238.2 Address correspondence and reprint requests to Dr. Paul M. Allen, Department ofPathology and Immunology, Washington University School of Medicine, 660 SouthEuclid Avenue, Campus Box 8118, St. Louis, MO 63110. E-mail address:allen@immunology.wustl.edu3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-inducedarthritis; GPI, glucose-6-phosphate isomerase; IL-1ra, IL-1 receptor antagonist;iNOS, inducible NO synthase; NOD, nonobese diabetic.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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both K/B3N arthritogenic Ig and KRN T cells was recently iden-tified (21) as glucose-6-phosphate isomerase (GPI), an ubiquitouscytoplasmic enzyme that catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate during glycolysis. Our lab-oratory has recently established the molecular basis for the dualability of the KRN TCR to recognize RNase (42–56)/I-Ak and GPI(282–294)/I-Ag7 (22, 23). A working model of K/B3N diseaseinitiation postulates that GPI-specific B cells endocytose GPI viasurface Ig receptors and present the I-Ag7-restricted epitope to in-completely tolerized CD41 KRN T cells, which in turn providespecific help in maturation and Ig isotype switching, leading toautoantibody production (17, 18, 21) and subsequent induction ofjoint inflammation.

Transfer of serum or purified Ig from arthritic K/B3N miceinduces a synchronized joint-specific inflammatory reaction thatmimics the K/B3N disease (18), indicating that arthritogenic Igcan induce synovitis and rheumatoid-like arthritic disease. The dis-ease induced by a single administration of serum eventually re-solves, unless arthritogenic Ig is repeatedly transferred into recip-ients (18). The development of an easily inducible model of RAwith a rapid, synchronized onset facilitates the study of the patho-genic mechanisms involved in the initiation of joint autoimmunity.

In this study, we found that neutrophils play an indispensablerole in disease initiation in the K/B3N serum transfer model ofRA. We used a mAb to deplete neutrophils in recipient mice andfound that this treatment completely blocked the acute joint-spe-cific inflammatory response normally induced by K/B3N serumtransfer, and caused rapid and profound reversal of joint inflam-mation in diseased mice. Furthermore, we determined that neitherthe inducible NO synthase (iNOS)2norgp91phoxgene products arerequired for the serum-induced joint inflammation.

Materials and MethodsMice

NOD mice (6–8 wk old) were obtained from Taconic Farms (German-town, NY). KRN TCR transgenic mice on a B6 background (K/B, thegenerous gift of D. Mathis and C. Benoist, Harvard Medical School, Bos-ton, MA) were bred to NOD mice to generate K/B3N mice. Mice deficientfor iNOS2(24) orgp91phox (25) gene expression, C57BL/6J, B6.AKR (H-2k), CB.17-SCID, and B6.AKR-RAG12/2 mice were obtained from TheJackson Laboratory (Bar Harbor, ME), and bred and housed under specificpathogen-free conditions in the animal colony at Washington University(St. Louis, MO). All mice were sex matched and age matched for exper-iments, and were between 6 and 10 wk of age.

Arthritic serum

Serum was separated from blood obtained from arthritic K/B3N mice(6–12 wk old) and frozen at270°C. To prepare larger pools for in vivoexperiments, frozen serum samples were thawed, centrifuged at 12,000rpm for 10 min, pooled and sterile filtered, and frozen in aliquots at270°C.Before injection, serum aliquots were thawed, centrifuged, and diluted withPBS. Each batch of pooled serum was titrated in B6.AKR mice (groups of3–5 mice per dose) for its ability to transfer joint-specific disease and todetermine an effective dose of serum for subsequent serum transfer exper-iments. A dose of 250ml per mouse was selected, as it consistently gavedisease induction in 100% of the mice.

Antibodies

The rat IgG2b mAb RB6-8C5 (26) was purified using protein G-Sepharose4 Fast Flow affinity matrix (Pharmacia, Piscataway, NJ) from ascites pro-duced in SCID mice. GK1.5 mAb (27) against mouse CD4 (rat IgG2b) waspurified from ascites by saturated ammonium sulfate precipitation (45%final) and dialysis against PBS, pH 7.4, before being stored at270°C.

Ab depletion in vivo

For in vivo neutrophil depletion, 250mg RB6-8C5 mAb was diluted to 0.5ml with PBS and injected i.p. at 3-day intervals beginning 1 day beforeserum transfer, except for the experiment described in Fig. 4. This dose ofAb is equal to or greater than has been shown previously to be efficacious

in completely eliminating neutrophils in vivo (28–30). We determined inearly experiments that a single injection of RB6-8C5 is not sufficient tocompletely block joint inflammation for more than;5–6 days, as dem-onstrated by the appearance of clinical signs of inflammation and measur-able thickening of the ankle joints. We attribute this to clearance of theRB6-8C5 Ab from the bloodstream and tissues and the maturation andrelease of new neutrophils from the bone marrow. Previous studies ofperipheral blood cell types in RB6-8C5-treated mice have established thatmacrophage, NK, and T and B lymphocyte populations are not signifi-cantly affected by such methods (31–37). Several studies have demon-strated that RB6-8C5 Ab fails to bind to mature macrophages, monocytes,or splenic B and T lymphocytes by either flow cytometry analysis (32, 36)or immunofluorescent microscopy (33). CD81 T cells have been previ-ously reported to be moderately affected by RB6-8C5 treatment (36, 38,39), but the effect on CD81 cells occurs several days after neutrophils aredepleted (38), and would be of no consequence in this model, as T cell-deficient RAG1 mice are equally susceptible to disease (see Fig. 2,D andE). For the control anti-CD4 Ab, 50ml ammonium sulfate-precipitatedGK1.5 mAb was diluted to 0.5 ml with PBS and injected i.p. Depletion wasmonitored by flow cytometry using biotinylated RB6-8C5 and GK1.5(PharMingen, San Diego, CA) and streptavidin-R-PE (Caltag, Burlingame,CA) using standard procedures. A minimum of 20,000 total events wascollected using a FACSCalibur and analyzed using CellQuest software(BD Biosciences, Mountain View, CA). Gates and histograms were setusing untreated B6.AKR peripheral blood stained with RB6-8C5 or GK1.5.

K/B3N serum transfer model

Serum as prepared above was injected i.p., and clinical scores were deter-mined daily based upon evidence of redness and swelling, using a scaleranging from 0 to 4, as described previously (17). Physical measurement ofankle thickness was performed using a Fowler Metric Pocket ThicknessGauge (Ralmikes Tool-A-Rama, Middlesex, NJ). Ankle measurementswere made above the footpad, axially across the ankle joint, and roundedoff to the nearest 0.05 mm. Data are presented as the mean of individualankle thicknesses within a group of mice (3–6 mice per group). Meanpercent inhibition of ankle swelling due to RB6-8C5 depletion was calcu-lated using the following formula: 12 (experimental mean ankle thick-ness2 experimental baseline mean ankle thickness)/(PBS mean anklethickness2 PBS baseline mean ankle thickness). Studentt tests for paireddata were performed using Kaleidagraph software (Synergy Software,Reading, PA) on calculated values from data of three identical experi-ments, with a total of 12 mice per experimental condition.

Histology

Tissue samples were prepared by fixing tissues 24–48 h in 10% phosphate-buffered Formalin (J. T. Baker, Phillipsburg, NJ). Fixed joints were decal-cified by treatment with Decal Overnight Bone Decalcifier solution (DecalChemical, Congers, NY) for 2 days with gentle rocking and daily replace-ment of the Decal solution. Samples were then washed with PBS, dehy-drated with a series of ethanol washes (50% ethanol, followed by 70%ethanol), and embedded in paraffin. Sections of tissue 4mm thick werestained with H&E. Representative sections from individual mice withingroups of three to six mice were selected to illustrate the general state ofeach group’s ankle joints.

ResultsPolymorphonuclear neutrophils were found in periarticular andsynovial regions of ankle joints within 48 h of serum transfer

To determine the timing of inflammatory cell recruitment to af-fected joints following K/B3N serum transfer, we performed his-tological studies on ankle joints over time. Injection of serum re-producibly resulted in rapid bilateral ankle swelling (Fig. 1A) andincreased clinical index scores (Fig. 1B) in B6.AKR mice within24–48 h post transfer. Ankles continued to increase in size up to7 days post transfer.

Sagittal sections of hind ankles through the center of the joint12 h after serum transfer showed no change in the size or conditionof the ankle and metatarsal joints compared with control mice(data not shown). Twenty-four hours after serum transfer, the an-kle joint (Fig. 1,A andB) and wrist joint (data not shown) wereinflamed, with redness (erythema), tenderness, and measurable an-kle swelling. Histological examination at 24 h (Fig. 1D, 350 mag-nification) revealed a slight increase in the size of the articular

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space contained within the synovium, as evidenced by a larger gapbetween the talus and the synovial membrane, consistent with ob-served edema in the region. The synovial fluid space was clear andfree of cellular infiltrate, and the synovial membrane appeared nor-mal (data not shown). Thirty-six to forty hours after serum trans-fer, ankle joints continued to increase in thickness and clinicalindex severity, with nearly all of the extremities involved (Fig. 1,A and B). Histology of the ankle joints at this time showed anincreased synovial fluid volume and the presence of neutrophils inthe synovial fluid surrounding the talus, calcaneus, and distal tibia/fibula (Fig. 1,E, 350 magnification, andF, 3500 magnification).Neutrophils also began to accumulate in the loose connective tis-sue posterior to the ankle joint between the Achilles tendon and thetalus, and also in the tissues surrounding the synovium on thelateral and anterior portions of the ankle joint.

At 48 h, all of the mice showed significant outward signs of anklejoint inflammation and swelling, with mean ankle sizes increased;20% in diameter (Fig. 1A). Histological analysis at 48 h showedinitial signs of synovial membrane expansion surrounding the articu-lar spaces of the joint, with neutrophils and other infiltrating cells inclose proximity to the joint spaces and in the synovial fluid (Fig. 1,Gand H, 3500 magnification). Neutrophil numbers were greatly in-

creased, infiltrating the low density connective tissue found posteriorto the ankle joints, and also on the sides of the ankle joint just belowthe malleoli of the tibia and fibula (data not shown).

Neutrophil depletion in vivo dramatically blocked the ability ofK/B3N arthritogenic serum to induce joint-specificinflammatory reactions in serum transfer recipients

To further investigate the role of neutrophils in the serum transfermodel, we used a depleting mAb (RB6-8C5) specific for a neu-trophil-restricted surface marker (Ly-6G), which has been used innumerous unrelated studies to deplete neutrophils in vivo (31–37).B6.AKR mice were treated with purified RB6-8C5 rat mAb orvehicle alone (PBS) on days21, 2, and 5 by i.p. injection (250mgper injection), and K/B3N serum was injected i.p. on day 0. Clin-ical score and ankle thickness were monitored for 7 days afterserum transfer, and the efficiency of neutrophil depletion in pe-ripheral blood was monitored by flow cytometry. Data from twosuch RB6-8C5 depletion experiments and their associated FACSmonitoring of peripheral blood are shown in Fig. 2. In the firstexperiment (Fig. 2,A–C), B6.AKR mice were treated with eitherthree doses of RB6-8C5, PBS, or the isotype-matched CD4-de-pleting rat Ab GK1.5 beginning 1 day before serum transfer (day

FIGURE 1. Joint inflammation occurs within48 h of serum transfer, and neutrophils are found inankle joints at the onset of inflammation.A andB,Kinetics of joint inflammatory disease caused bytransfer of K/B3N serum. A representative group ofmice (n5 3) received a single injection of 250mlK/B3N serum i.p., and were monitored for changesin ankle thickness and clinical index score. Detect-able swelling and inflammation occur within 48 h.Results shown are from one experiment and are rep-resentative of more than five experiments.C–H, His-tological time course of cellular infiltration into peri-articular and intraarticular spaces following serumtransfer. Mice received a single injection of 250mlK/B3N serum i.p., and ankle joints were harvestedat 12- to 24-h increments, treated for histology asdetailed inMaterials and Methods, and stained withH&E. Shown are ankle cross-sections of a normalB6.AKR mouse (C), and serum recipient mice at24 h (D), 40 h (EandF), and 48 h (GandH), whichare representative of the general inflammatory re-sponse in multiple mice for each time point. Photo-graphs of representative ankle joint sections areshown at magnifications of350 (C–E) and3500(F–H).

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21). Mice treated with RB6-8C5 showed no signs of clinical dis-ease through day 7 post transfer (Fig. 2,A andB). We found thatthere was no observable difference between groups treated witheither PBS or GK1.5 in either the severity of ankle swelling (Fig.2A) or clinical index scores (Fig. 2B). There also was no differencein the time of onset for inflammation, as both groups of miceshowed measurable swelling at 2 days post transfer and peaked on

days 6–7. The extremities of the PBS- and GK1.5-treated groupsbecame reddened and inflamed within 48 h, while the ankles andwrists of RB6-8C5-treated mice showed no signs of inflammatoryresponse and were indistinguishable from those of control micethat did not receive serum.

Depletion of RB6-8C5-positive peripheral blood leukocytes wasessentially complete 1 day after treatment and remained extremelylow (,1% of total PBL) throughout the experiment (Fig. 2C).Additionally, analysis of FACS plots of forward light scatter vsside light scatter demonstrated the near-total absence of cells in theneutrophil region of the plot, which in normal mice is.95% pos-itive for RB6-8C5 staining (data not shown). CD41 T cells werecompletely absent from the peripheral blood of GK1.5-treatedmice by FACS analysis (data not shown), yet the progression andseverity of serum-transferred disease were unaffected, showingthat the protective effect was specific for the depletion ofneutrophils.

In addition to its effect on neutrophils, RB6-8C5 mAb treatmenthas been reported to lead to a slow reduction in the number ofCD81 T cells over the course of several days (36, 38, 40). Toensure that the protective effect of RB6-8C5 mAb treatment wasnot due to depletion of CD81 T cells, serum transfer disease wasevaluated in RAG1-deficient mice (RAG12/2), which lack B andT cells. There was no significant difference between RAG1-defi-cient B6.AKR and wild-type B6.AKR mice in relation to the onsetof inflammation, progression, or severity of disease following se-rum transfer, demonstrating that T and B lymphocytes were dis-pensable in the serum transfer model. Furthermore, RB6-8C5 mAbtreatment protected RAG1-deficient mice from disease induction(Fig. 2, D and E). Collectively, the results demonstrated thatK/B3N Ig were unable to induce this joint-specific reaction inrecipient mice in the absence of a normal neutrophil compartment.This indicates that RB6-8C5-positive neutrophils play an impor-tant role in initiating this Ab-mediated disease.

Protection against K/B3N serum-induced joint swelling by RB6-8C5 neutrophil depletion correlates with normal anklemorphology and absence of cellular infiltrates

The ankles of RB6-8C5-treated mice showed remarkably normalmorphology at 5 and 7 days postserum transfer, virtually indistin-guishable from that of a normal mouse ankle. Ankles from RB6-8C5-treated mice 5 days after serum transfer showed no signs ofinflammatory infiltrate or synovial hyperplasia (Fig. 3,C andD).In contrast, ankles of PBS-treated mice demonstrated massive in-flammatory infiltrate, increased synovial fluid volume, and syno-vial hyperplasia with large numbers of neutrophils present in thesynovial fluid (Fig. 3,A andB). The lack of detectable infiltrationand synovial hypertrophy in these histological studies of neutro-phil-depleted mice correlates with the absence of measurableswelling (Fig. 2A) or visible inflammation (Fig. 2B), indicating thatthe presence and infiltration of neutrophils are obligatory for thecharacteristic changes induced by transfer of K/B3N serum.Therefore, neutrophils must possess properties or functions that areimportant or even indispensable for the inflammatory responsetriggered by K/B3N Ig transfer.

Testing of mice deficient for generation of reactive nitrogenspecies and reactive oxygen species in the K/B3N serumtransfer model

To begin to determine the mechanism(s) by which neutrophils mayact to induce joint disease, we assessed the responses of micedeficient for iNOS2 (iNOS2knockout mice), which are unable togenerate NO (24), andgp91phox-deficient mice that are unable togenerate hydrogen peroxide by the NADPH-dependent pathway

FIGURE 2. Depletion of neutrophils by administration of RB6-8C5 Abrenders mice resistant to K/B3N serum-induced joint inflammation. Datafrom two similar experiments are shown inA–C and D–F, respectively.Groups of five mice were treated with either PBS (0.5 ml), RB6-8C5 (250mgeach) or GK1.5 mAb (50ml purified ascites each,A–Conly) by injection i.p.on days21, 2, and 5 as indicated by arrows on thex-axis. On day 0, eachmouse received 250ml K/B3N serum i.p.A andD, Depletion of neutrophils,but not CD41 T cells prevents joint inflammation. Mean ankle thicknesseswere determined daily for each group of mice by measuring the width ofankles across the medial-lateral axis of the joint. SDs for mean ankle thicknessare indicated by error bars.B andE, Neutrophil-depleted mice show no clinicalsigns of joint inflammation. Clinical index scores correlate closely with anklethickness changes in control groups of mice. Clinical index scores were de-termined daily and are presented as the mean clinical score for each group ofmice, with SDs indicated by error bars.C andF, Percentage of PBL stainingpositively with RB6-8C5 Ab was determined on days 2, 5, and 7 for eachgroup of mice by Ab staining and flow cytometry, as described inMaterialsand Methods. Data are presented as the mean percentage of total PBL stainingpositively with RB6-8C5 mAb. SD values are indicated by the presence oferror bars. The mean percentage for the RB6-8C5-treated group never ex-ceeded 2% of total PBL in either experiment. RB6-8C5 staining for GK1.5-treated mice was nearly identical with that of PBS-treated mice (data notshown). These experiments with GK1.5 mAb depletion as a control (A–C) andRAG1-deficient recipients (D–F) were performed twice with identical results.In addition, neutrophil depletion by RB6-8C5 Ab treatment (days21, 2, and5) has been used in a total of six experiments and.30 wild-type mice withsimilar protection from disease.

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(25). Results from one of three experiments are shown in Fig. 4, inwhich we found that relative to congenic B6 mice,gp91phox-deficient mice andiNOS2knockout mice developed arthritis withsimilar kinetics (Fig. 4,A andB). In two of three experiments, weobserved slightly enhanced inflammation iniNOS2knockout micerelative to B6 andgp91phox-deficient mice. Neither strain proved tobe more resistant than B6 mice to the inflammation induced byK/B3N serum transfer. Histological analysis of ankle joints fromthese strains of mice failed to reveal any gross differences in jointmorphology (data not shown), suggesting that neither reactive in-termediate pathway is critical for the neutrophil-dependent inflam-matory response to K/B3N serum.

Reversal of K/B3N serum transfer inflammation with neutrophildepletion via RB6-8C5 treatment

The depletion of neutrophils before serum transfer prevents dis-ease. It was next important to determine whether neutrophil de-pletion could affect disease progression after the transfer of serum.Groups of B6.AKR mice were treated with a single i.p. injection of250 mg RB6-8C5 on days21, 0, 1, 2, or 3 days after K/B3Nserum was transferred. This protocol differed from our previousones, in that we were only administering a single dose ofRB6-8C5.

A single injection of RB6-8C5 was equally effective in com-pletely preventing joint inflammation for;6 days, regardless ofwhether it was administered 1 day before serum transfer (day21)or at the same time as serum (day 0, Fig. 5,B andC). At 1 day afterserum transfer, the remaining untreated groups of mice demon-strated visible signs of inflammation similar to mice in Figs. 1 and2 (data not shown) and only slightly elevated ankle measurements(Fig. 5,D–G). Treatment with RB6-8C5 beginning 1 day after se-rum transfer (day11) immediately and effectively blocked inflam-

mation and clinical manifestations of the disease within 24 h oftreatment (Fig. 5D). Treatment with RB6-8C5 2 and 3 days afterserum transfer quickly reversed the joint-specific inflammatory re-action, as measured by ankle swelling and clinical scores. Micethat received RB6-8C5 on day 4 (G) and day 5 (H) also showed arapid decline in ankle thickness beginning;1 day after treatment,indicating that at this time the inflammation is also reversible or atleast can be ameliorated by neutrophil depletion. All of the treatedgroups showed significant benefit from neutrophil depletion, asdetermined by reduction in erythema, puffiness, and joint thickness(data not shown). Inhibition of ankle swelling by neutrophil de-pletion became less effective when initiated after day 3 of serumtransfer (Fig. 5I). Mice were analyzed for the extent of neutrophildepletion on days 0, 2, 4, and 7 by FACS analysis. By day 5–6after RB6-8C5 mAb injection, neutrophils began to reappear inperipheral blood due to clearance of Ab from the bloodstream andtissues and maturation and release of neutrophils from the bonemarrow. There is a clear correlation between the time of recoveryof RB6-8C51 peripheral neutrophils and the onset or resumptionof joint inflammation (data not shown). These studies revealed thatthe early stages of experimental immune complex-induced arthri-tis, up to day 3, could be completely reversed by depletion ofneutrophils, and disease severity was ameliorated up through day5. These experiments indicate that not only are neutrophils criti-cally involved during the first 3–5 days of the disease, but they arealso required for the continuation and progression of the immunecomplex-mediated inflammatory state.

DiscussionTransfer of K/B3N serum Ig into recipient mice induces the syn-chronized and rapid onset of joint inflammation. We used thismodel to determine the importance of neutrophils in the initiation,

FIGURE 3. Histological examination of ankle cross-sections from RB6-8C5-treated and PBS-treated mice 5 days after serum transfer reveals cleardifferences in joint pathology. Ankles from mice in Fig. 2A–Cwere harvested, fixed, and decalcified. Paraffin-embedded samples were cross-sectionedthrough the center of the ankle joint and stained with H&E. Representative portions of tissue cross-sections are presented inA–D.A, Low power magnifiedview (350) of ankle joints from PBS-treated mouse on day 5. The area shown encompasses a portion of the calcaneus, talus, and fibula/tibia bones. Thesynovial fluid and pannus show massive cellular infiltrate. Infiltrating cells are visible in synovial fluid spaces of joints and as masses of cells in theconnective tissue area between ankle joint and the Achilles tendon.B, High power magnified view (3500) of boxed area indicated inA. Infiltratingneutrophils are visible in synovial fluid spaces and synovium.C, Low power (350) magnified view of ankle cross-section from day 5 RB6-8C5-treatedmouse in same region as inA. Note normal appearance of synovium and complete absence of infiltrating cells in surrounding tissues.D, High powermagnified view (3500) of boxed region inC from RB6-8C5-treated mouse on day 5 post transfer. Synovium appears normal in size and morphology, andjoint is free of cellular infiltrate, with normal synovial fluid volume. These results are representative of five separate experiments.

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progression, and maintenance of inflammatory joint disease in thisexperimental mouse model of rheumatoid-like polyarthritis. Wefound that Ab-induced neutrophil depletion caused a completeblock in the ability of K/B3N serum to induce joint swelling inrecipient mice, and this protection from inflammation could bemaintained for periods of at least 7 days with repeated RB6-8C5Ab treatment. The foot and ankle joint spaces remained free ofinfiltrating neutrophils by histology, and the synovia of the ankleremained free of cellular infiltrate and were of normal size andmorphology. When the neutrophil compartment was allowed torecover by termination of RB6-8C5 treatment, the repopulation ofthe peripheral blood neutrophil population (by maturation fromimmature precursor cells) correlated closely with the observed on-set of joint inflammation, indicating an active role for neutrophilsin the early stages of this disease rather than as late arrivals towell-established sites of inflammation. Joint inflammation could becompletely blocked or reversed by neutrophil depletion if initiatedby 3 days postserum transfer, but became progressively less ef-fective when initiated at later time points, indicating the probableinvolvement of other cell types at later stages in the K/B3N serumtransfer model. Formation of scar tissue and fibroblast proliferationmay also be contributing factors to residual ankle thickening afterprolonged inflammation.

This model disease can be transferred to normal mice with pu-rified GPI-specific IgG from arthritic mice (21), strongly suggest-ing that the initial triggering event is the formation and/or depo-

sition of GPI-IgG immune complexes in the joint spaces.Therefore, it is likely to share features with other immune com-plex-triggered autoimmune models (11, 41, 42). Local mononu-clear cells and possibly mast cells (43) become activated by im-mune complex-activated complement fragments (44, 45), tissuedamage, and/or FcgR cross-linking (11, 46), and release proin-flammatory cytokines such as IL-1b and TNF-a (43, 47) in or nearthe affected tissue (e.g., joints), which induces some relatively lowlevel of neutrophil recruitment. These few initial neutrophils be-come activated, extravasate, and home to the joints, where theyproceed to help create a proinflammatory cytokine milieu that isnecessary to maintain and expand the joint inflammatory response.In a rat model of arthritis unrelated to the K/B3N model, repeatedinjection of streptococcal cell wall extract causes joint inflamma-tion, which has also been shown to be neutrophil dependent andrequires P-selectin, ICAM-1, and macrophage-inflammatory pro-tein-2 (48), suggesting these molecules are likely to be important

FIGURE 4. Mice deficient iniNOS2andgp91phox gene expression de-velop joint inflammation with kinetics similar to control B6 mice. Micewere injected with K/B3N serum (200ml i.p.) and monitored daily forchanges in ankle thickness (A) and clinical index score (B). Representativeresults from one of three identical experiments are shown. There were noinstances in whichiNOS2knockout orgp91phox knockout mice were sig-nificantly more resistant to serum-induced joint inflammation than B6 con-trol mice, and in two experiments iNOS2 knockout mice appear to haveslightly enhanced inflammation relative to B6 mice.

FIGURE 5. Reversal of K/B3N serum-induced joint inflammation byneutrophil depletion via RB6-8C5 treatment. Groups of mice were treatedwith PBS or a single dose of RB6-8C5 (250mg i.p.) before, concurrentwith, or following serum transfer on day 0. Mice were treated with eitherPBS(A), or RB6-8C5 mAb on day21 (B), day 0 (C), day11 (D), day12(E), day 13 (F), day 14 (G), or day 15 (H). The time of RB6-8C5injection is marked with an arrow on thex-axis of each panel. Each panelrepresents a separate group of four mice, and these results are representa-tive of three identical experiments. All groups receiving RB6-8C5 showedeither significant protection from joint disease or dramatic reduction ofdisease severity following treatment.I, Mean percent inhibition of ankleswelling on day 6 after serum transfer was calculated for each experimentalgroup of mice, as described inMaterials and Methods, with PBS controlgroup ankle thickness set to 0% inhibition. Values depicted inI are themean of three identical experiments totaling 12 mice per experimentalgroup, with SD values indicated by error bars. Statistical significance wasdetermined by paired Studentt test analysis (p,p , 0.006; †,p , 0.013).Ab depletion of neutrophils becomes progressively less effective at revers-ing disease after sustained joint inflammation.

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in the K/B3N model for neutrophil recruitment. In the CIA model,it has been clearly demonstrated that autoreactive Abs to type IIcollagen initiate inflammation by binding to articular cartilage andcausing activation of complement, C3 deposition (44), and even-tual cleavage of C5 (45). Although the K/B3N RA model is alsoautoantibody mediated and shares pathology with CIA, the Ag(GPI) is not joint specific (21), and deposition of GPI-autoantibodyimmune complexes in the joints of mice has yet to be demon-strated, and remains an intriguing question.

Potential neutrophil effector mechanisms that may be critical forthe induction and progression of joint inflammation in the murineK/B3N serum transfer model include release of granules contain-ing degradative enzymes, and the production and release of proin-flammatory cytokines. Neutrophil granule contents such as myelo-peroxidase, elastase, matrix metalloproteinases, and collagenase(41, 49) can cause further damage to the tissue and amplify theneutrophil response, which has been observed in an Ab-mediatedmodel of bullous pemphigoid (50). Alternatively, activated neu-trophils are also capable of releasing proinflammatory cytokinessuch as TNF-a, IL-1, IL-6, and TGFb (51, 52), potentially affect-ing the activities of both neutrophils and other cell types, such asresident mononuclear cells and chondrocytes. TNF-a is at the apexof a proposed cascade model of proinflammatory cytokines inrheumatoid synovial tissue (2), and the role of TNF-a as a dom-inant proarthritogenic cytokine has been demonstrated with micetransgenic for human TNF-a (53). Neutrophils may also interferewith the balance of IL-1 and endogenous IL-1 receptor antagonist(IL-1ra) activity in the joint (54), either by direct secretion of IL-1or through TNF-a-mediated IL-1 induction. Mice that are deficientin the endogenous IL-1ra develop a chronic, progressive joint in-flammation disease on certain mouse backgrounds, illustrating theimportance of a balance between IL-1 and IL-1ra in normal jointphysiology and homeostasis of inflammatory cytokines.

Finally, TGFb is another proinflammatory cytokine found insynovial tissue and fluid (55) and is a powerful neutrophil che-moattractant (56). As with TNF-a and IL-1, neutrophils can bothproduce and respond to TGFb, providing a potential amplificationmechanism for continued neutrophil activation and recruitment. Incombination with their capacity to make TNF-a and IL-1, one canpropose a model for K/B3N serum transfer in which initial mono-nuclear cell activation and cytokine release in the joint tissuesinduces some level of initial neutrophil recruitment. The initialwave of recruited neutrophils would then be largely responsible foramplifying and sustaining recruitment of neutrophils to the in-flamed tissues through the continued generation and release ofproinflammatory cytokines such as TNF-a, IL-1, and TGFb.

In contrast to cytokine secretion and degranulation of proen-zymes that are usually directed toward extracellular targets, theoxidative species produced by the iNOS pathway (e.g., NO) andthe NADPH oxidase pathway (hydrogen peroxide) are usually di-rected intracellularly against phagocytosed bacteria or particlescontained within a phagosome. However, it has recently been pro-posed (49) that adherent neutrophils could release such reactivespecies into small pockets of synovial fluid, leading to inappropri-ate activation of proenzymes such as elastase or metallothioneinproteases, and damage of the synovial fluid hyaluronan or carti-lage. In several model systems of immunopathologic disease, in-hibition of either NADPH or iNOS pathways or both together hasyielded a reduction in the severity of inflammation or disease (29,57, 58). Unexpectedly, we found that mice deficient for eithergp91phox or iNOS2 activity presented similar disease phenotypesas wild-type control mice. There was no significant reduction inthe severity of disease induced by K/B3N serum transfer, nor wasthere any significant alteration in the time of disease onset for these

mutant strains of mice. Interestingly,iNOS2knockout mice dem-onstrated enhanced swelling relative to B6 mice in two of threeexperiments. This suggests that neither of these two pathways areobligatory for immune complex-triggered joint inflammation in theK/B3N serum transfer model, although one might expect that theyare likely to be involved in tissue damage once a joint becomesinflamed. Indeed, other studies have shown that overproduction ofNO and its reaction product, peroxynitrate, contributes to thepathophysiology of RA and joint inflammation (59, 60).

To summarize, it has been hypothesized, but not directly shown,that neutrophils are an important component of inflammatory re-sponses to immune complex deposition in the joints because theyare found there in high numbers. This study convincingly demon-strates that neutrophils are an essential component of the K/B3Nautoreactive IgG transfer model of rheumatoid-like arthritis, andthat an induced state of neutropenia confers protection from joint-specific inflammation. These results are important because theydemonstrate for the first time that neutrophils play an essentialinductive role in the generation of joint-specific inflammation inthe K/B3N serum transfer model (e.g., one or more of the prop-erties of neutrophils are responsible for early stages of inflamma-tion in the K/B3N). The mechanism by which neutrophils arerecruited specifically to the joint in the K/B3N model, and whichneutrophil characteristics are important in this location, are ques-tions that bear further consideration and will be the subject offuture experiments. Further studies to elucidate the exact operativemechanisms exerted by neutrophils may point to potential ap-proaches for pharmacological intervention in other inflammatorydiseases such as human RA, to help ameliorate or limit the severityand scope of joint disease.

AcknowledgmentsWe thank Diane Mathis and Christophe Benoist for providing us with theK/B3N mouse strain, and Emil Unanue, Eric Brown, Laura Mandik-Nayak, and Aleksandra Colic for critical reviews of this manuscript. Wealso thank Emil Unanue for the RB6-8C5 Ab and hybridoma. Darren Krea-malmeyer’s expert management of our mouse colony was invaluable. Wealso thank the Histology Core Facilities of the Departments of Pathologyand Molecular Microbiology & Pharmacology for their aid in preparinghistological sections for this body of work, and greatly appreciate the sec-retarial efforts of Jerri Smith in the preparation of this manuscript.

References1. Feldmann, M., F. M. Brennan, and R. N. Maini. 1996. Role of cytokines in

rheumatoid arthritis.Annu. Rev. Immunol. 14:397.2. Brennan, F. M., R. N. Maini, and M. Feldmann. 1998. Role of proinflammatory

cytokines in rheumatoid arthritis.Springer Semin. Immunopathol. 20:133.3. Loutis, N., P. Bruckner, and A. Pataki. 1988. Induction of erosive arthritis in mice

after passive transfer of anti-type II collagen antibodies.Agents Actions 25:352.4. Stuart, J. M., M. A. Cremer, A. S. Townes, and A. H. Kang. 1982. Type II

collagen-induced arthritis in rats: passive transfer with serum and evidence thatIgG anticollagen antibodies can cause arthritis.J. Exp. Med. 155:1.

5. Trentham, D. E., A. S. Townes, and A. H. Kang. 1977. Autoimmunity to type IIcollagen: an experimental model of arthritis.J. Exp. Med. 146:857.

6. Watson, W. C., P. S. Brown, J. A. Pitcock, and A. S. Townes. 1987. Passivetransfer studies with type II collagen antibody in B10.D2/old and new line andC57BL/6 normal and beige (Chediak-Higashi) strains: evidence of importantroles for C5 and multiple inflammatory cell types in the development of erosivearthritis.Arthritis Rheum. 30:460.

7. Wooley, P. H., H. S. Luthra, J. M. Stuart, and C. S. David. 1981. Type II col-lagen-induced arthritis in mice. I. Major histocompatibility complex (I region)linkage and antibody correlates.J. Exp. Med. 154:688.

8. Myers, L. K., E. F. Rosloniec, M. A. Cremer, and A. H. Kang. 1997. Collagen-induced arthritis, an animal model of autoimmunity.Life Sci. 61:1861.

9. Holmdahl, R., K. Rubin, L. Klareskog, E. Larsson, and H. Wigzell. 1986. Charac-terization of the antibody response in mice with type II collagen-induced arthritis,using monoclonal anti-type II collagen antibodies.Arthritis Rheum. 29:400.

10. Terato, K., K. A. Hasty, R. A. Reife, M. A. Cremer, A. H. Kang, and J. M. Stuart.1992. Induction of arthritis with monoclonal antibodies to collagen.J. Immunol.148:2103.

11. Kleinau, S., P. Martinsson, and B. Heyman. 2000. Induction and suppression ofcollagen-induced arthritis is dependent on distinct Fcg receptors.J. Exp. Med.191:1611.

1607The Journal of Immunology

by guest on September 5, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

12. Geiger, T., H. Towbin, A. Cosenti-Vargas, O. Zingel, J. Arnold, C. Rordorf,M. Glatt, and K. Vosbeck. 1993. Neutralization of interleukin-1b activity in vivowith a monoclonal antibody alleviates collagen-induced arthritis in DBA/1 miceand prevents the associated acute-phase response.Clin. Exp. Rheumatol. 11:515.

13. Butler, D. M., A.-M. Malfait, R. N. Maini, F. M. Brennan, and M. Feldmann.1999. Anti-IL-12 and anti-TNF antibodies synergistically suppress the progres-sion of murine collagen-induced arthritis.Eur. J. Immunol. 29:2205.

14. Piguet, P. F., G. E. Grau, C. Vesin, H. Loetscher, R. Gentz, and W. Lesslauer.1992. Evolution of collagen arthritis in mice is arrested by treatment with anti-tumor necrosis factor (TNF) antibody or a recombinant soluble TNF receptor.Immunology 77:510.

15. Thorbecke, G. J., R. Shah, C. H. Leu, A. P. Kuruvilla, A. M. Hardison, andM. A. Palladino. 1992. Involvement of endogenous tumor necrosis factora andtransforming growth factorb during induction of collagen type II arthritis inmice.Proc. Natl. Acad. Sci. USA 89:7375.

16. Williams, R. O., M. Feldmann, and R. N. Maini. 1992. Anti-tumor necrosis factorameliorates joint disease in murine collagen-induced arthritis.Proc. Natl. Acad.Sci. USA 89:9784.

17. Kouskoff, V., A.-S. Korganow, V. Duchatelle, C. Degott, C. Benoist, andD. Mathis. 1996. Organ-specific disease provoked by systemic autoimmunity.Cell 87:811.

18. Korganow, A.-S., H. Ji, S. Mangialaio, V. Duchatelle, R. Pelanda, T. Martin,C. Degott, H. Kikutani, K. Rajewsky, J.-L. Pasquali, C. Benoist, and D. Mathis.1999. From systemic T cell self-reactivity to organ-specific autoimmune diseasevia immunoglobulins.Immunity 10:451.

19. Chen, P. P., S. Fong, and D. A. Carson. 1987. Rheumatoid factor.Rheum. Dis.Clin. N. Am. 13:545.

20. Winchester, R. J., V. Agnello, and H. G. Kunkel. 1970. Gammaglobulin complexesin synovial fluids of patients with rheumatoid arthritis: partial characterization andrelationship to lowered complement levels.Clin. Exp. Immunol. 6:689.

21. Matsumoto, I., A. Staub, C. Benoist, and D. Mathis. 1999. Arthritis provoked bylinked T and B cell recognition of a glycolytic enzyme.Science 286:1732.

22. Basu, D., S. Horvath, I. Matsumoto, D. H. Fremont, and P. M. Allen. 2000.Molecular basis for recognition of an arthritic peptide and a foreign epitope ondistinct MHC molecules by a single TCR.J. Immunol. 164:5788.

23. Basu, D., S. Horvath, L. O’Mara, D. Donermeyer, and P. M. Allen. 2001. TwoMHC surface amino acid differences distinguish foreign peptide recognition fromautoantigen specificity.J. Immunol. 166:4005.

24. Laubach, V. E., E. G. Shesely, O. Smithies, and P. A. Sherman. 1995. Micelacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.Proc. Natl. Acad. Sci. USA 92:10688.

25. Pollock, J. D., D. A. Williams, M. A. Gifford, L. L. Li, X. Du, J. Fisherman,S. H. Orkin, C. M. Doerschuk, and M. C. Dinauer. 1995. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superox-ide production.Nat. Genet. 9:202.

26. Hestdal, K., F. W. Ruscetti, J. N. Ihle, S. E. Jacobsen, C. M. Dubois, W. C. Kopp,D. L. Longo, and J. R. Keller. 1991. Characterization and regulation of RB6-8C5antigen expression on murine bone marrow cells.J. Immunol. 147:22.

27. Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loken,M. Pierres, and F. W. Fitch. 1983. Characterization of the murine T cell surfacemolecule, designated L3T4, identified by monoclonal antibody GK1.5: similarityof L3T4 to the human Leu-3/T4 molecule.J. Immunol. 131:2445.

28. Rogers, H. W., and E. R. Unanue. 1993. Neutrophils are involved in acute, non-specific resistance toListeria monocytogenesin mice. Infect. Immun. 61:5090.

29. Andrews, D. M., V. B. Matthews, L. M. Sammels, A. C. Carrello, andP. C. McMinn. 1999. The severity of murray valley encephalitis in mice is linkedto neutrophil infiltration and inducible nitric oxide synthase activity in the centralnervous system.J. Virol. 73:8781.

30. Verdrengh, M., and A. Tarkowski. 1997. Role of neutrophils in experimentalsepticemia and septic arthritis induced byStaphylococcus aureus. Infect. Immun.65:2517.

31. Conlan, J. W. 1997. Critical roles of neutrophils in host defense against experi-mental systemic infections of mice byListeria monocytogenes,Salmonella ty-phimurium, andYersinia enterocolitica. Infect. Immun. 65:630.

32. Conlan, J. W., and R. J. North. 1994. Neutrophils are essential for early anti-Listeriadefense in the liver, but not in the spleen or peritoneal cavity, as revealedby a granulocyte-depleting monoclonal antibody.J. Exp. Med. 179:259.

33. Pedrosa, J., B. M. Saunders, R. Appelberg, I. M. Orme, M. T. Silva, andA. M. Cooper. 2000. Neutrophils play a protective nonphagocytic role in sys-temic Mycobacterium tuberculosisinfection of mice.Infect. Immun. 68:577.

34. Saunders, B. M., and C. Cheers. 1996. Intranasal infection of beige mice withMycobacterium aviumcomplex: role of neutrophils and natural killer cells.Infect.Immun. 64:4236.

35. Tateda, K., T. A. Moore, J. C. Deng, M. W. Newstead, X. Zeng, A. Matsukawa,M. S. Swanson, K. Yamaguchi, and T. J. Standiford. 2001. Early recruitment ofneutrophils determines subsequent T1/T2 host responses in a murine model ofLegionella pneumophilapneumonia.J. Immunol. 166:3355.

36. Tumpey, T. M., S.-H. Chen, J. E. Oakes, and R. N. Lausch. 1996. Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 in-fection of the murine cornea.J. Virol. 70:898.

37. Kielian, T., B. Barry, and W. F. Hickey. 2001. CXC chemokine receptor-2 li-gands are required for neutrophil-mediated host defense in experimental brainabscesses.J. Immunol. 166:4634.

38. De Oca, R. M., A. J. Buendia, L. Del Rio, J. Sanchez, J. Salinas, andJ. A. Navarro. 2000. Polymorphonuclear neutrophils are necessary for the re-cruitment of CD81 T cells in the liver in a pregnant mouse model ofChlamy-dophila abortus(Chlamydia psittaciserotype 1) infection.Infect. Immun. 68:1746.

39. Goossens, P. L., H. Jouin, and G. Milon. 1991. Dynamics of lymphocytes andinflammatory cells recruited in liver during murine listeriosis: a cytofluorimetricstudy.J. Immunol. 147:3514.

40. Barteneva, N., I. Theodor, E. M. Peterson, and, L. M. de la Maza. 1996. Role ofneutrophils in controlling early stages of aChlamydia trachomatisinfection.Infect. Immun. 64:4830.

41. Witko-Sarsat, V., P. Rieu, B. Descamps-Latscha, P. Lesavre, andL. Halbwachs-Mecarelli. 2000. Neutrophils: molecules, functions and pathophys-iological aspects.Lab. Invest. 80:617.

42. Heller, T., J. E. Gessner, R. E. Schmidt, A. Klos, W. Bautsch, and J. Kohl. 1999.Cutting edge: Fc receptor type I for IgG on macrophages and complement me-diates the inflammatory response in immune complex peritonitis.J. Immunol.162:5657.

43. Zhang, Y., B. F. Ramos, and B. A. Jakschik. 1992. Neutrophil recruitment bytumor necrosis factor from mast cells in immune complex peritonitis.Science258:1957.

44. Stuart, J. M., and F. J. Dixon. 1983. Serum transfer of collagen-induced arthritisin mice.J. Exp. Med. 158:378.

45. Wang, Y., S. A. Rollins, J. A. Madri, and L. A. Matis. 1995. Anti-C5 monoclonalantibody therapy prevents collagen-induced arthritis and ameliorates establisheddisease.Proc. Natl. Acad. Sci. USA 92:8955.

46. Blom, A. B., P. L. van Lent, H. van Vuuren, A. E. Holthuysen, C. Jacobs,L. B. van De Putte, J. G. van De Winkel, and W. B. van Den Berg. 2000. FcgRexpression on macrophages is related to severity and chronicity of synovial in-flammation and cartilage destruction during experimental immune-complex-me-diated arthritis (ICA).Arthritis Res. 2:489.

47. Warren, J. S. 1992. Relationship between interleukin-1b and platelet-activatingfactor in the pathogenesis of acute immune complex alveolitis in the rat.Am. J. Pathol. 141:551.

48. Schimmer, R. C., D. J. Schrier, C. M. Flory, J. Dykens, D. K. Tung,P. B. Jacobson, H. P. Friedl, M. C. Conroy, B. B. Schimmer, and P. A. Ward.1997. Streptococcal cell wall-induced arthritis: requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory pro-tein-2.J. Immunol. 159:4103.

49. Pillinger, M. H., and S. B. Abramson. 1995. The neutrophil in rheumatoid ar-thritis. Rheum. Dis. Clin. N. Am. 21:691.

50. Liu, Z., S. D. Shapiro, X. Zhou, S. S. Twining, R. M. Senior, G. J. Giudice,J. A. Fairley, and L. A. Diaz. 2000. A critical role for neutrophil elastase inexperimental bullous pemphigoid.J. Clin. Invest. 105:113.

51. Fava, R. A., N. J. Olsen, A. E. Postlethwaite, K. N. Broadley, J. M. Davidson,L. B. Nanney, C. Lucas, and A. S. Townes. 1991. Transforming growth factorb1(TGF-b1) induced neutrophil recruitment to synovial tissues: implications forTGF-b-driven synovial inflammation and hyperplasia.J. Exp. Med. 173:1121.

52. Cassatella, M. A., S. Gasperini, and M. P. Russo. 1997. Cytokine expression andrelease by neutrophils.Ann. NY Acad. Sci. 832:233.

53. Butler, D. M., A.-M. Malfait, L. J. Mason, P. J. Warden, G. Kollias, R. N. Maini,M. Feldmann, and F. M. Brennan. 1997. DBA/1 mice expressing the humanTNF-a transgene develop a severe, erosive arthritis: characterization of the cy-tokine cascade and cellular composition.J. Immunol. 159:2867.

54. Horai, R., S. Saijo, H. Tanioka, S. Nakae, K. Sudo, A. Okahara, T. Ikuse,M. Asano, and Y. Iwakura. 2000. Development of chronic inflammatory arthrop-athy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficientmice.J. Exp. Med. 191:313.

55. Wahl, S. M., J. B. Allen, H. L. Wong, S. F. Dougherty, and L. R. Ellingsworth.1990. Antagonistic and agonistic effects of transforming growth factor-b and IL-1in rheumatoid synovium.J. Immunol. 145:2514.

56. Brandes, M. E., U. E. Mai, K. Ohura, and S. M. Wahl. 1991. Type I transforminggrowth factor-b receptors on neutrophils mediate chemotaxis to transforminggrowth factor-b. J. Immunol. 147:1600.

57. Kakimoto, K., Y. Kojima, K. Ishii, K. Onoue, and H. Maeda. 1993. The sup-pressive effect of gelatin-conjugated superoxide dismutase on disease develop-ment and severity of collagen-induced arthritis in mice.Clin. Exp. Immunol.94:241.

58. Kubo, H., D. Morgenstern, W. M. Quinian, P. A. Ward, M. C. Dinauer, andC. M. Doerschuk. 1996. Preservation of complement-induced lung injury in micewith deficiency of NADPH oxidase.J. Clin. Invest. 97:2680.

59. Connor, J. R., P. T. Manning, S. L. Settle, W. M. Moore, G. M. Jerome,R. K. Webber, F. S. Tjoeng, and M. G. Currie. 1995. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase.Eur.J. Pharmacol. 273:15.

60. McCartney-Francis, N., J. B. Allen, D. E. Mizel, J. E. Albina, Q. W. Xie,C. F. Nathan, and S. M. Wahl. 1993. Suppression of arthritis by an inhibitor ofnitric oxide synthase.J. Exp. Med. 178:749.

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