Development of Spontaneous Multisystem Autoimmune Diseaseand Hypersensitivity to Antibody-Induced Inflammation
in Fc� Receptor IIa–Transgenic Mice
Caroline Tan Sardjono,1 Patricia L. Mottram,1 Nicholas C. van de Velde,1 Maree S. Powell,1
David Power,1 Ronald F. Slocombe,2 Ian P. Wicks,3 Ian K. Campbell,3 Steven E. McKenzie,4
Mark Brooks,5 Andrew W. Stevenson,6 and P. Mark Hogarth1
Objective. The major human Fc receptor,Fc�RIIa, is the most widespread activating FcR. Ouraim was to determine the role of Fc�RIIa in a transgenicmouse model of immune complex–mediated autoimmu-nity and to characterize the development of spontaneousautoimmune disease.
Methods. Arthritis was induced in normal andFc�RIIa-transgenic mice by immunization with type IIcollagen (CII) or by transfer of arthritogenic anti-CIIantibodies. Also, mice that spontaneously developedautoimmune disease were assessed by clinical scoring ofaffected limbs, histology and serology, and measure-ment of autoantibody titers and cytokine production.
Results. Fc�RIIa-transgenic mice developedcollagen-induced arthritis (CIA) more rapidly than didarchetypal CIA-sensitive DBA/1 (H-2q) mice, while non-
transgenic C57BL/6 (H-2b) mice did not develop CIAwhen similarly immunized. Passive transfer of a singledose of anti-CII antibody induced a more rapid, severearthritis in Fc�RIIa-transgenic mice than in nontrans-genic animals. In addition, most immune complex–induced production of tumor necrosis factor � by acti-vated macrophages occurred via Fc�RIIa, not theendogenous mouse FcR. A spontaneous, multisystemautoimmune disease developed in aging (>20 weeks)transgenic mice (n � 25), with a 32% incidence ofarthritis, and by 45 weeks, all mice had developedglomerulonephritis and pneumonitis, and most hadantihistone antibodies. Elevated IgG2a levels were seenin mice with CIA and in those with spontaneous disease.
Conclusion. The presence of enhanced passiveand induced autoimmunity, as well as the emergence ofspontaneous autoimmune disease at 20–45 weeks of age,suggest that Fc�RIIa is a very important factor in thepathogenesis of autoimmune inflammation and a possi-ble target for therapeutic intervention.
Antibody-induced inflammation is a major com-ponent of several autoimmune diseases (1,2). The roleof cell surface receptors for antibodies, especially IgGFc� receptors (Fc�R), was recognized following amelio-ration of tissue destruction in type III hypersensitivityreactions after administration of soluble recombinanthuman Fc�RIIa in vivo (3). Subsequent studies withFcR-deficient mice (4) showed that Fc�R play signifi-cant roles in antibody-induced inflammatory diseasemodels such as collagen-induced arthritis (CIA) (5),passive antibody-induced arthritis (6), and intraarticularantigen-induced arthritis (7). However, rodents lack anortholog of Fc�RIIa, the most abundant and widespread
Supported by grants from the National Health and MedicalResearch Council and PrimaBiomed Ltd., Australia. Dr. Sardjono’swork was supported by PaperlinX Pty Ltd. Drs. Mottram and Powell’swork was supported by Nancy Prendergast fellowships from theArthritis Foundation, Australia.
1Caroline Tan Sardjono, PhD, Patricia L. Mottram, PhD,Nicholas C. van de Velde, BSc Hons, Maree S. Powell, PhD, DavidPower, PhD, P. Mark Hogarth, PhD: Austin Research Institute,Heidelberg, Victoria, Australia; 2Ronald F. Slocombe, PhD: Univer-sity of Melbourne, Melbourne, Victoria, Australia; 3Ian P. Wicks,PhD, Ian K. Campbell, PhD: Walter and Eliza Hall Institute, Parkville,Victoria, Australia; 4Steven E. McKenzie, PhD: Jefferson MedicalCollege, Philadelphia, Pennsylvania; 5Mark Brooks, MBBS: AustinHospital, Heidelberg, Victoria, Australia; 6Andrew W. Stevenson,PhD: Commonwealth Scientific Industrial Research Organization,Clayton South, Victoria, Australia.
Drs. Mottram, Powell, and Hogarth have stock options inPrimaBiomed. Dr. McKenzie has received consulting fees (less than$10,000 per year) from GlaxoSmithKline.
Address correspondence and reprint requests to P. MarkHogarth, PhD, Helen McPherson-Smith Laboratory, Austin ResearchInstitute, Studley Road, Heidelberg, Victoria 3084, Australia. E-mail:[email protected].
Submitted for publication September 24, 2004; accepted inrevised form June 30, 2005.
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activating FcR in higher primates. Fc�RIIa has uniquestructural, signaling, and biologic features (8–11). Un-like other FcR, Fc�RIIa can signal without the ho-modimeric FcR �-chain used by Fc�RI, Fc�RI, Fc�RIII,and Fc�RI, since both the ligand binding site and theimmunoreceptor tyrosine-based activation motif(ITAM) are in the same polypeptide (8). Moreover,Fc�RIIa is a dimer, with the ITAM-containing cyto-plasmic tails arranged in an FcR �-chain–like configu-ration (9,12).
Studies of Fc�RIIa, as well as other FcR, trans-fected into mouse or primate cells show that these FcRbehave identically in both ligand binding and activation/regulation (13,14). The interaction of Fc�RII ITAMsand immunoreceptor tyrosine-based inhibition motifs(ITIMs) was seen in both transfected mouse and humancell lines, and ITIM sequences in mice and humans arehighly conserved (15). Fc�RIIa in transgenic mice isexpressed under its own promoter and has the sameexpression pattern in mice and humans (16,17). Thus,Fc�RIIa can interact appropriately with intracellularsignaling pathways in mouse cells. Finally, genetic poly-morphisms of Fc�RIIa are associated with human auto-immune disease (1,18). In this study, we analyzed in-flammatory responses in transgenic mice expressingFc�RIIa and confirmed a role for this receptor inpassive, induced, and spontaneous autoimmune disease.
MATERIALS AND METHODS
Mice. DBA/1 (H-2q), C57BL/6 (H-2b), SJL/J (H-2s),(SJL � C57BL/6)F1 (H-2b/s), and Fc�RIIa-transgenic mice(H-2b) derived from (SJL � C57BL/6)F2 embryos (17) wereused. The Fc�RIIa-transgenic mice were inbred for �20generations and were homozygous for the transgene under thecontrol of its own promoter. They carried the high responder(Arg134) allele of Fc�RIIa, which binds mouse IgG2a, Ig2b,and Ig1, as well as human IgG1, IgG2, and IgG3 (11).
Induction of CIA. Mice were injected intradermally atthe base of the tail with 100 �l of 2 mg/ml type II collagen (CII)emulsion in Freund’s complete adjuvant (Difco, Detroit, MI)that contained 2.5 mg/ml heat-killed Mycobacterium tuberculo-sis H37Ra (Difco). Mice were immunized a second time 21days later (19). They were examined daily from days 1–60, andarthritis in each limb was graded on a scale of 0–3 (0 � normal,1 � mild swelling and redness, 2 � severe swelling/redness,and 3 � severe swelling and redness and joint rigidity). Themaximum possible score (arthritis index) was 12 for eachmouse.
Passively induced arthritis. Anti-CII monoclonal anti-body (mAb) M2139 (2 mg) (20) was injected intraperitoneallyinto Fc�RIIa-transgenic and nontransgenic C57BL/6 mice, andarthritis progression was monitored daily as described above.
Joint histology. Joints were preserved in 10% formalin/phosphate buffered saline (PBS), decalcified in 5% HCl, 3.5%
glacial acetic acid, 95% ethanol, and 12.5% (volume/volume)chloroform, and then were embedded in paraffin. Sections(4–6 �m) were stained with hematoxylin and eosin (H&E).
Enzyme-linked immunosorbent assay (ELISA) foranti-CII antibody. ELISA plates (96-well; Costar, Cambridge,MA) were coated with 50 �g/ml CII and blocked with 2%bovine serum albumin in PBS (1 hour at room temperature).Sera (serially diluted) were added, and antibody was detectedusing secondary horseradish peroxidase (HRP)–conjugatedsheep anti-mouse IgG F(ab�)2 fragments (Amersham, LittleChalfont, UK). Development was performed for 10 minuteswith ABTS (Boehringer Mannheim, Rockville, MD), andabsorbance was read at 405 nm. IgG isotypes were detectedwith specific antibodies (see below).
Bone marrow macrophage cultures. Bone marrowmacrophages were cultured in Dulbecco’s modified Eagle’smedium (DMEM) (CSL, Melbourne, Victoria, Australia) con-taining 2 mM glutamine, 50 mM 2-mercaptoethanol, 100units/ml penicillin, 100 �g/ml streptomycin, 10% (v/v) fetal calfserum, and 30% (v/v) L cell–conditioned medium at 37°C in10% CO2 for 5–7 days (21).
Peritoneal exudate macrophage (PEM) cultures. Micewere injected intraperitoneally with 0.5 ml of 4% (weight/volume) thioglycolate (CSL) 4 days prior to harvest. PEMswere cultured in DMEM, prepared as described above, for 1–3days.
Measurement of cytokine production. PEMs (1 �106/ml) were incubated with heat-aggregated IgG (HAGG; 50�g or 100 �g) or phorbol myristate acetate (PMA; 20 ng)(Sigma, St. Louis, MO) at 37°C for 24 hours. HAGG wasprepared from 8–10 mg/ml of human gamma globulin (San-doglobulin; Novartis, East Hanover, NJ) heated for 30 minutesat 63°C, then put on ice and brought to 1% (w/v) withpolyethylene glycol 6000 (Sigma) in PBS and kept on ice for 30minutes. The precipitated complexes were centrifuged(10,000g at 4°C for 10 minutes), the supernatant was discarded,and complexes were dissolved in PBS at 1 mg/ml.
Production of tumor necrosis factor � (TNF�) andinterleukin-10 (IL-10) by macrophages in the supernatant 2–24hours after stimulation with HAGG or PMA was detected byELISA using cytokine detection kits from eBioscience (SanDiego, CA). The specificity of induced TNF� secretion wasdetermined by preincubation of macrophages with anti-Fc�RIIa mAb IV.3 Fab (20 or 50 �g) at 4°C for 1 hour.
Detection of Fc�R by flow cytofluorometry. The fol-lowing Fc�R were detected: mouse Fc�RI using mAb X54-5/7.1 (mIgG1) (22), mouse Fc�RIIb using mAb Ly-17.2 (mIgG2;Cedarlane Laboratories, Hornby, Ontario, Canada), andhFc�RIIa using mAb IV.3 (mIgG2b) or mAb 8.7 (mIgG1).
Monitoring of spontaneous autoimmunity. Mice wereexamined from 12 weeks of age, and the index of arthritisseverity was assessed as described above. Organs were fixed in10% formalin/PBS, embedded in paraffin, and histologic sec-tions were stained with H&E. Immune complex deposition inkidney sections was detected with fluorescein isothiocyanate(FITC)–conjugated sheep anti-mouse IgG (Silenus Laborato-ries, Hawthorn, Victoria, Australia).
Transmission electron microscopy. Mouse kidneyswere cut into 1–2-mm cubes and fixed in 2–8% paraformalde-
ROLE OF Fc�RIIa IN AUTOIMMUNE INFLAMMATION 3221
hyde/2–5% glutaraldehyde in 0.15M cacodylate buffer (pH 7.4)for �6 hours at 4°C. Tissues were rinsed in cacodylate buffer andpostfixed in 1% osmium tetroxide in 0.15M cacodylate buffer (pH7.4) for 2 hours at room temperature. Samples were washed indistilled water, dehydrated in 10% incremental concentrations ofacetone, and then embedded in Procure–Araldite resin (ProSciTech, Thuringowa, Queensland, Australia). During the dehydra-tion, tissues were stained with 2% uranyl acetate in 70% acetone.Ultrathin sections were stained with 5% uranyl acetate in aqueoussolution for 30 minutes at room temperature and then werestained with Reynolds lead citrate for 10 minutes. Sections wereexamined using a Philips 300 electron microscope (Philips, Mah-wah, NJ) at 60 kV.
Radiographic analysis. Polychromatic hard radiogra-phy was performed (23) on mice, and images were recordedusing an FXE-225.20 microfocus x-ray source (Feinfocus,Stamford, CT) fitted with a tungsten target, operated at 60kVp. Projection geometry with a large sample-to-detectordistance resulted in high-resolution images. Exposures were�3 mA, and images were recorded with imaging plates (XRT,Port Melbourne, Victoria, Australia) scanned with a FujiBAS-5000 scanner (Fuji Photo Film, Tokyo, Japan).
Detection of antihistone antibodies. ELISA plateswere coated with 20 �g/ml purified histone (a mixture of H1,H2A, H2B, H3, and H4 from calf thymus) (Roche Laborato-ries, Basel, Switzerland). Serially diluted samples (50 �l) wereadded, incubated for 1 hour at room temperature, detectedwith HRP-conjugated sheep anti-mouse IgG F(ab�)2 fragments(Amersham), and then developed using ABTS.
Quantitation of IgG subclasses. Total serum IgGconcentrations were determined using ELISA plates coatedwith 50 �l (3 �g/ml) of rat anti-mouse IgG (BD PharMingen,San Diego, CA). Serially diluted serum samples (50 �l) wereadded and incubated for 1 hour at room temperature. Anti-body was detected with biotin-conjugated, isotype-specific ratanti-mouse IgG1, anti-mouse IgG2a, and anti-mouse IgG2bmAb (BD PharMingen) (1 hour at room temperature), thenwith streptavidin–HRP (1 hour at room temperature), anddeveloped with ABTS. Quantitation of IgG was by comparisonwith class-specific IgG standards.
Statistical analysis. Results were expressed as themean � SD. Statistical differences were analyzed using theMann-Whitney 2-sample rank test, correlation coefficients, orStudent’s t-test. All the statistical analyses were performedusing Microsoft Excel software (Microsoft, Redmond, WA). Pvalues less than 0.05 were considered statistically significant.
RESULTS
Expression of transgenic and endogenous FcR.The expression of Fc�R on bone marrow macrophagesfrom Fc�RIIa-transgenic and nontransgenic C57BL/6mice was analyzed by flow cytometry. Using mAb IV.3(anti-Fc�RIIa) (Figure 1A), Fc�RIIa was detected onmacrophages from Fc�RIIa-transgenic mice but not onmacrophages from nontransgenic mice. The expression
of the transgene did not significantly change the expres-sion of endogenous mouse FcR, since similar levels ofFc�RI (Figure 1B) and especially inhibitory Fc�RIIb(Figure 1C) were observed on Fc�RIIa-transgenic andnontransgenic macrophages. Similarly, expression ofFc�RIIa on neutrophils did not alter endogenous FcRexpression. Fc�RIIa was not expressed on B or T cells(data not shown), as expected (16,17).
Fc�RIIa confers susceptibility to CIA. In mice,CIA susceptibility is associated with the major histocom-patibility complex (MHC) genotype: H-2q and H-2r miceare highly susceptible, while H-2b, H-2d, and H-2s miceare less so (19,24). We compared CIA development inFc�RIIa-transgenic (H-2b) mice with the archetypalsusceptible strain DBA/1 (H-2q) and the less susceptibleC57BL/6 (H-2b) and (SJL � C57BL/6)F1 (H-2s/b) strains
Figure 1. Flow cytometric analysis of Fc� receptor (Fc�R) expressionin normal and human Fc�RIIa–transgenic mice. Bone marrow macro-phages from Fc�RIIa-transgenic mice (solid histograms) or nontrans-genic C57BL/6 mice (dotted line, open histograms) were stained for A,Fc�RIIa with monoclonal antibody (mAb) IV.3, B, Fc�RI with mAbX54-5/7.1, and C, Fc�RIIb with mAb Ly-17.2. Studies were performedwith fluorescein isothiocyanate–conjugated anti-mouse IgG (solid line,open histogram). FL-1 � fluorescence channel 1.
3222 SARDJONO ET AL
(Figure 2A). The Fc�RIIa-transgenic mice in this exper-iment were highly susceptible, with more rapid onset ofarthritis (day 18) compared with other mice (days 22 and24). Of the Fc�RIIa-transgenic mice, 15% developedarthritis after 1 immunization, whereas 2 immunizationswere always required for CIA development in DBA/1and (SJL � C57BL/6)F1 mice. No arthritis occurred inC57BL/6 mice under these conditions. By day 26, �90%of the Fc�RIIa-transgenic mice developed arthritis,compared with �10% of DBA/1 and (SJL � C57BL/6)F1 mice (P � 0.001).
Disease incidence and severity at �30 days weresimilar in Fc�RIIa-transgenic and DBA/1 mice. InFc�RIIa-transgenic mice treated with a limited course ofanti-Fc�RIIa F(ab�)2 antibody (100 �g/mouse intraperi-toneally on days 21, 24, 27, and 30), CIA was signifi-cantly reduced on day 35 (P � 0.05) compared withuntreated CIA in Fc�RIIa-transgenic mice. Althoughthis dose of antibody is unlikely to have blocked all in
vivo activity of Fc�RIIa, the data show that this FcRplays a role in disease severity in these mice (Figure 2B).
Histologic assessment of paws and ankle andknee joints from Fc�RIIa-transgenic mice on day 36 afterarthritis induction (when the maximal clinical index wasobserved) showed severe, destructive arthritis with pan-nus formation, infiltration of inflammatory cells (poly-morphonuclear cells and macrophages) into the synovialspace, and erosion of the cartilage (Figure 2C). No jointinflammation was seen in C57BL/6 controls (Figure 2D).
Correlation of increased levels of IgG2a with thearthritis index in transgenic mice. Despite the acceler-ated development of severe CIA, Fc�RIIa-transgenicmice had lower anticollagen antibody titers than suscep-tible DBA/1 mice, but similar to those of the lesssusceptible (SJL � C57BL/6)F1 mice (Figure 3A). Theanticollagen antibodies were predominantly IgG2, andanalysis of the serum IgG2 subclasses (Figure 3B)showed a significant increase in the IgG2a:IgG2b ratioin severely arthritic mice (P � 0.02) compared withmildly arthritic or unaffected Fc�RIIa-transgenic mice.There was a positive correlation between the arthritisindex and IgG2a levels (r � 0.57) and no correlationbetween arthritis index and IgG2b levels (r � �0.16).This suggests a dominant Th1 response in Fc�RIIa-transgenic mice following induction of CIA and is con-sistent with previous data obtained from DBA/1 mice(25). Thus, expression of Fc�RIIa, which binds mouseIgG2a avidly, may cause effector cells in the Fc�RIIa-transgenic mice to be sensitive to low levels ofautoantibody/immune complex activation, possibly trig-gering the early release of inflammatory mediators.
Production of TNF� by IgG-stimulated macro-phages from Fc�RIIa-transgenic mice. Because TNF� isa major inflammatory mediator in human RA andmouse CIA (24,26,27), we compared the production ofTNF� from immune complex–stimulated Fc�RIIa-transgenic and nontransgenic macrophages. HAGG-stimulated Fc�RIIa macrophages produced significantlymore TNF� (6.5 ng/ml) compared with nontransgenicmacrophages (1 ng/ml; P � 0.001) (Figure 3C). ElevatedTNF� production by Fc�RIIa macrophages was blocked(although not completely) in a dose-dependent mannerby anti-Fc�RIIa mAb IV.3 Fab to levels not significantlydifferent from those produced by nontransgenic macro-phages, indicating that immune complex–induced TNF�production was due principally to activation by Fc�RIIa,rather than through the endogenous mouse activatingFc�R. PMA stimulation of nontransgenic and transgenicmacrophages showed that these cells were equally re-sponsive (Figure 3C). No difference in IL-10 production
Figure 2. Analysis of collagen-induced arthritis (CIA) in Fc�RIIa-transgenic mice. A, Development of CIA in Fc�RIIa-transgenic mice(n � 41) compared with nontransgenic DBA/1 mice (n � 27), C57BL/6mice (n � 28), and (SJL � B6)F1 mice (n � 8). Pooled data from 5experiments are shown. B, Effects of treatment of arthritis withanti-Fc�RIIa F(ab�)2 mAb 8.7 (intraperitoneal injection of 100 �g ondays 21, 24, 27, and 30 versus treatment with phosphate buffered saline[PBS] alone [n � 8 mice per group]). The mAb 8.7 treatment causeda significant reduction in disease severity on day 40 (P � 0.05). Valuesin A and B are the mean � SD. C, A representative section showinghistopathologic features of an Fc�RIIa-transgenic mouse knee joint 36days after CIA induction (hematoxylin and eosin stained; originalmagnification � 100). D, Histopathologic features of a knee joint froma C57BL/6 mouse 36 days after collagen injection (hematoxylin andeosin stained; original magnification � 40). See Figure 1 for otherdefinitions.
ROLE OF Fc�RIIa IN AUTOIMMUNE INFLAMMATION 3223
was observed in HAGG-stimulated macrophages (datanot shown).
Exaggerated antibody hypersensitivity ofFc�RIIa-transgenic mice. The rapid CIA response inFc�RIIa-transgenic mice and increased sensitivity toHAGG implied an exaggerated response to pathologicantibodies. This possibility was tested using a passiveantibody-transfer arthritis model described by Holmdahland colleagues (20,28), wherein mice were normallygiven a single dose of a mixture of 2 anticollagenantibodies (4.5 mg each of M2139 and C1), followed by
50 �g of lipopolysaccharide (LPS) 5 days later. Theseantibodies have been tested for arthritis induction in anumber of mouse strains, including SJL, SJL � B6,BALB/c, and DBA/1 (28). None of these strains re-sponded without LPS. However, in Fc�RIIa-transgenicmice (Figure 3D), treatment with a single 2-mg dose ofmAb M2139 alone, without LPS, caused a rapid onset ofarthritis in 100% of mice, but had no effect in nontrans-genic controls.
Spontaneous development of autoimmunity inFc�RIIa-transgenic mice. Mice were housed for �1year, and we observed spontaneous progressive develop-ment of a systemic multiorgan autoimmune syndrome.
Development of destructive arthritis. A proportionof aging Fc�RIIa-transgenic mice spontaneously devel-oped severe, destructive, symmetric arthritis. In a groupof 25 Fc�RIIa-transgenic mice monitored for �1 year,none younger than 20 weeks of age developed arthritis,7 (28%) developed arthritis between 20–45 weeks of
Figure 3. Sensitivity of human Fc� receptor IIa (Fc�RIIa)–transgenicmice to antibody-mediated disease. A, Anticollagen antibody titers inFc�RIIa-transgenic (Tg) and nontransgenic (SJL � C57BL/6)F1 andDBA/1 mice (n � 5 per group), as determined on days 0, 21, and 36postimmunization. OD � optical density. B, Comparison of changes inIgG2 subclasses in Fc�RIIa-transgenic mice without collagen-inducedarthritis (CIA) (index 0), with severe arthritis (index 5–12; high CIA),and with mild arthritis (index 1–4; low CIA) after the second injectionof collagen (n � 5 mice per group). In the high CIA group, there wasa significant increase in the IgG2a:IgG2b ratio (P � 0.016 versus mildlyarthritic mice, by Student’s t-test), a positive correlation between thearthritis index and IgG2a levels (r � 0.57), and no correlation betweenthe arthritis index and IgG2b levels (r � �0.16). C, Comparison oftumor necrosis factor � (TNF�) production in heat-aggregated IgG(HAGG)–stimulated macrophages. Macrophages from Fc�RIIa-transgenic mice produced significantly higher levels of TNF� com-pared with nontransgenic C57BL/6 (B6) mice (P � 0.001) followingstimulation with HAGG. There was no significant difference (P �0.07) between Fc�RIIa-transgenic and C57BL/6 mouse macrophagestreated with HAGG following incubation with 20- or 50-�g doses ofmonoclonal antibody (mAb) IV.3. Responses to phorbol myristateacetate (PMA) stimulation were equivalent in both strains of macro-phages (P � 0.05). D, Arthritis index over time in Fc�RIIa-transgenicand (SJL � C57BL/6)F1 mice treated with 2 mg of mAb M2139 givenintraperitoneally on day 0 (n � 6 per group). Fc�RIIa-transgenic micetreated with mAb M2139 were hyperresponsive to anti–type II colla-gen. Values are the mean and SD.
Figure 4. Analysis of arthritis in mice with spontaneous autoimmunedisease. A, Cumulative percentage incidence in arthritis over time inhuman Fc� receptor IIa (Fc�RIIa)–transgenic mice (n � 25). Therewas no difference in arthritis incidence between males and females. Band C, Photograph and radiographic image of the paw of a 36-week-old Fc�RIIa-transgenic mouse with severe spontaneous arthritis (B)and a nonarthritic Fc�RIIa-transgenic age-matched control mouse(C). Radiography was performed for each mouse category, i.e.,nontransgenic, healthy Fc�RIIa-transgenic, Fc�RIIa-transgenic withmild arthritis, and Fc�RIIa-transgenic with destructive arthritis (n � 4per group) (results not shown). D–F, Representative sections showingthe histopathologic features of hematoxylin and eosin–stained mousejoints. D, Early active destructive arthritis in an ankle from a 28-week-old mouse, with polymorphonuclear-dominant inflammatory cell infil-tration. E, A later stage of destructive disease in a knee from a36-week-old mouse, with predominantly mononuclear cell infiltrationand advanced pannus. F, A normal ankle joint from an older (age 36weeks) transgenic mouse. ca � cartilage; p � pannus; i � inflamma-tory cells. (Original magnification � 100.)
3224 SARDJONO ET AL
age, and 1 of the 25 developed arthritis thereafter, witha cumulative incidence of 32% (Figure 4A). Of the 8mice with arthritis, 5 were severely affected (mean � SDindex 10 � 2.38), with profoundly swollen joints andsevere histologic and radiologic changes (Figure 4). Theother 3 Fc�RIIa-transgenic mice had less severe arthri-tis. Despite differences in arthritis severity, in bothsubsets of Fc�RIIa-transgenic mice, the number ofaffected paws was similar. Age-matched nontransgenicmice never developed spontaneous disease.
Radiologic examination of the affected limbs. TheFc�RIIa-transgenic mice with severe arthritis developedmarked radiologic changes that mirrored the floridedema and distortion of the foot seen clinically. Severeankylosis of the tibiotarsal and tarsophalangeal cartilagewas seen, with loss of joint space and prominent periar-ticular new bone formation (Figure 4B) compared withage-matched Fc�RIIa-transgenic mice that did not de-velop arthritis (Figure 4C). Limbs from mice with mildarthritis revealed minimal radiologic changes (resultsnot shown).
Histologic evaluation of the joints. Joint histologyof Fc�RIIa-transgenic mice compared with that of non-transgenic, age-matched controls (Figures 4D–F)showed that mice with spontaneous severe arthritis hadsynovial hyperplasia and proliferation, cartilage erosion,pannus formation, and joint space infiltrate. The laterstage of the disease showed more advanced destructionof bone and thinning of the cartilage, with the infiltratechanging from polymorphonuclear (PMN) cells in earlyactive disease (Figure 4D) to macrophages in moreadvanced cases (Figure 4E). There was no evidence ofdisease in age-matched C57BL/6 or (SJL � C57BL/6)F1controls (Figure 4F).
Serum IgG2a elevation in affected mice. The levelsof total IgG, IgG1, IgG2a, IgG2b, and IgG3 in all micewere determined by ELISA. Only the total level ofIgG2a antibody was elevated (Figure 5A), and only inFc�RIIa-transgenic mice that developed destructive ar-thritis (mean � SD 6.6 � 2.5 �g/ml). IgG2a levels inmice with mild arthritis (2.0 � 0.8 �g/ml) were similar tothose in Fc�RIIa-transgenic mice with no disease (2.6 �1.2 �g/ml) and in nontransgenic mice (1.7 � 1.3 �g/ml).Thus, there was a strong correlation between IgG2alevels and disease severity, particularly with the forma-tion of pannus (r � 0.63). The levels of the other IgGsubclasses were not altered (Figure 5).
Other features of systemic autoimmune diseasein Fc�RIIa-transgenic mice. The Fc�RIIa-transgenicmice were screened for further evidence of inflamma-tion and autoimmunity by histologic examination of
H&E-stained sections of skin, lymph nodes, gut, salivaryglands, kidneys, eyes, brain, lungs, spleen, liver, pan-creas, and heart at 14–60 weeks of age and comparedwith aged-matched, nontransgenic controls. Glomerulo-nephritis (GN) and pneumonitis were commonly ob-served in Fc�RIIa-transgenic mice, with the diseaseincidence increasing with age. By 50 weeks of age, allmice were affected (Figure 5B). No abnormalities werefound in other organs or in nontransgenic mice of anyage.
Glomerulonephritis. The time of onset and degreeof severity of GN in individuals varied considerably. Fewmice developed GN before 20 weeks of age, and up to80% had the disease by 40 weeks. However, all mice �40weeks of age had moderate to severe GN, implying anage-related progression to severe GN (Figure 5B). Mul-tifocal lymphoplasmacytic infiltrate in the renal intersti-tium, mainly around major arcuate vessels, was seen at25–30 weeks (Figure 6A), with mild mesangial matrixdeposition in the glomeruli and some tubular thickening.In mice ages �40 weeks, more advanced disease wasseen, with enlarged glomeruli, increased mesangial ma-trix (Figures 6B and C), and condensation of glomerulartufts. There were proliferative and sclerotic changes inBowman’s capsule, indicative of crescent formation, andmild tubular proliferative changes (Figure 6B), althoughtubulointerstitial infiltrates were not present. The dis-
Figure 5. Serologic findings and incidence of kidney and lung diseasein mice with spontaneous autoimmune disease. A, Levels of serumIgG2a in individual human Fc� receptor IIa (FcR�IIa)–transgenicmice with destructive arthritis (n � 8) compared with unaffectedFcR�IIa-transgenic mice (n � 7), FcR�IIa-transgenic mice with mildarthritis (n � 6), and normal age-matched nontransgenic mice (n � 6).Bars indicate the mean � SD concentration of IgG2a in each group. P� 0.0012 for IgG2a levels in mice with destructive disease versusunaffected nontransgenic mice. IgG2a levels correlated with diseaseseverity, particularly with the formation of pannus (r � 0.63). TotalIgG was increased in arthritic mice, and this correlated strongly withIgG2a levels (r � 0.96). The levels of the other Ig subclasses (IgM,IgG1, Ig2b, and Ig3) were not significantly different between the 4groups of mice (P � 0.05 for all comparisons). B, Incidence ofglomerulonephritis (GN) and pneumonitis (Pn) in Fc�RIIa-transgenicmice at ages �20 weeks (n � 8), 21–40 weeks (n � 14), and �40 weeks(n � 9).
ROLE OF Fc�RIIa IN AUTOIMMUNE INFLAMMATION 3225
ease was self-limiting, since mice ages �40 weeks, withup to 65% of glomeruli affected, remained healthy, withnormal urinary protein and serum creatinine levels(results not shown). Age-matched nontransgenic miceshowed no evidence of disease (results not shown).
FITC-conjugated anti-mouse IgG staining of kid-ney sections showed an accumulation of immune com-plexes within the glomeruli, which produced a dense,granular appearance (Figure 6C). In contrast, kidneys
from age-matched nontransgenic control mice showeddiffuse, low-level staining of the tubules and mesangium,with no concentration of immunoglobulin in the glomer-uli (results not shown). Transmission electron micros-copy also revealed immune complex deposition (Figure6D), with features similar to those of lupus nephritis inhumans, including small, electron-dense deposits form-ing wire-loop lesions in the subendothelial basementmembrane.
Lung histopathology. Pneumonitis was found in25% of mice between ages 12 and 40 weeks, increasingto 100% of older mice (Figure 5B), and was character-ized by patches of perivascular inflammation with cellu-lar aggregates of macrophages, lymphocytes, plasmacells, and numerous PMN cells within alveolar walls(Figure 6E). In severe cases, up to 50% of the normalarchitecture of the lungs was obliterated, but the diseasewas self-limiting, and older mice remained healthy.Lungs of age-matched nontransgenic mice showed noinflammation.
Analysis of antihistone and antinuclear antibodies(ANAs). The histologic features suggested that the tissuedamage seen in the spontaneous autoimmune disease inFc�RIIa-transgenic mice was mediated, at least in part,by autoantibodies. Therefore, we evaluated mice for thepresence of autoantibodies known to be associated withhuman autoimmune disease. Initial immunofluores-cence studies with sera from most Fc�RIIa-transgenicmice and many older nontransgenic C57BL/6 miceshowed homogeneous nuclear staining of Chinese ham-ster ovary cells (results not shown). This staining patternwas similar to that of antihistone antibody huPIA3 (29).Sera were then tested for antihistone antibodies byELISA, using a mixture of purified histones. In miceages �20 weeks, 13 of 23 had antihistone antibodiesabove background levels (Figure 6F). Histone antibodytiters did not correlate with GN or arthritis incidence (r�0.05). No antihistone antibodies above backgroundlevels were seen in age-matched C57BL/6 mice or(SJL � C57BL/6)F1 mice at ages �25 weeks. None ofthe mice had the other common autoantibodies, such asanti–double-stranded or anti–single-stranded DNA orrheumatoid factor (data not shown).
DISCUSSION
Since it was found that recombinant soluble FcRinhibited immune complex vasculitis (3), there has beenwidespread interest in the role of FcR in antibody-induced inflammation in autoimmune diseases (5,8).Although many studies have analyzed the role of mouse
Figure 6. Analysis of spontaneous autoimmune glomerulonephritis,pneumonitis, and antihistone antibodies in a group of older humanFc� receptor IIa (Fc�RIIa)–transgenic mice (n � 31). A, Kidneysection (hematoxylin and eosin [H&E] stained) taken at age 25 weeks,showing multifocal lymphoplasmacytic infiltration in the renal inter-stitium, mainly around major arcuate vessels. The glomerulus (arrow)shows mild mesangial matrix deposition. Tubules were thickened butotherwise normal (original magnification � 200). B, An H&E-stainedkidney section taken at 45 weeks, showing enlarged glomeruli (thinarrows), increased mesangial matrix, and condensation of glomerulartufts. There are proliferative and sclerotic changes in Bowman’scapsule (thick arrow) indicative of crescent formation, and mildtubular proliferative changes (original magnification � 100). C, Fluo-rescence staining of immune complexes in the glomerulus. Kidneysections were stained with anti-mouse IgG, directly conjugated withfluorescein isothiocyanate, and immune complexes within the glomer-ulus appeared granular (original magnification � 200). Diffuse, low-level staining with no glomerular concentration of IgG was seen inage-matched, nontransgenic control mice (results not shown). D,Transmission electron microscopy of glomeruli, showing immunecomplexes deposited (D) on the glomerular basement membrane (BM,arrow) above the uriniferous space (U) and below the endothelial layer(En) (original magnification � 50). E, H&E-stained Fc�RIIa-transgenic mouse lung section taken at 40 weeks, showing infiltrationof inflammatory cells (arrows) and local destruction of lung architec-ture (original magnification � 50). F, Enzyme-linked immunosorbentassay for the presence of antihistone antibodies in Fc�RIIa-transgenicand nontransgenic mouse sera at 36 weeks of age. Antihistoneantibodies were present in serum from many of the Fc�RIIa-transgenic mice at all ages, but were not present in serum from thenontransgenic mice. Horizontal line shows the cutoff for positivity.
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FcR, including Fc�RI, Fc�RIIb, and Fc�RIIIa, whichare common to both mice and humans, humans have aunique FcR, Fc�RIIa, which is absent from rodents andwas therefore not analyzed in rodent models of autoim-munity. Our data demonstrate that Fc�RIIa-transgenicmice are highly susceptible to passive antibody-inducedinflammation and to active (collagen-induced) and spon-taneous autoimmune disease.
In CIA, there is variable susceptibility in mice,which is linked to MHC type (24). In this study, expres-sion of Fc�RIIa conferred susceptibility to CIA instrains of mice with low-susceptibility MHC (H-2b andH-2b/s). Indeed, CIA in Fc�RIIa-transgenic mice devel-oped more rapidly than in the archetypal CIA-susceptible DBA/1 strain, with almost 15% of Fc�RIIa-transgenic mice developing CIA after a single dose ofcollagen. These findings establish a role for Fc�RIIa inenhancing inflammatory responses in CIA, especiallysince treatment with anti-Fc�RIIa mAb reduced diseaseseverity. The immune mechanisms involved in the de-velopment of CIA have been well described, with both Tcells and anticollagen antibodies known to play majorroles (30,31).
The observation that anti-CII antibodies werelower on day 21 after CIA induction in Fc�RIIa-transgenic mice compared with DBA/1 or nontransgenicanimals is evidence that Fc�RIIa plays a role in increas-ing the sensitivity of effector cells to activation byimmune complexes. This possibility was supported bydata from the anti-CII antibody transfer model, whereina single dose of anti-CII antibody M2139 induced dis-ease in 100% of the Fc�RIIa-transgenic mice. Thiscontrasts with other strains, in which a mixture of 2antibodies plus LPS was required to induce this level ofdisease (20). Although the use of 1 antibody to inducearthritis in susceptible DBA/1 mice has been reported(32), the highest incidence was only 50% after 2 doses ofM2139 (total dose 9 mg), compared with the 100%incidence after a single 2-mg dose in the Fc�RIIa-transgenic mice.
TNF� is a major clinically validated inflamma-tory mediator in human rheumatoid arthritis (RA) (33),and the majority of immune complex–induced TNF�production from transgenic macrophages could be at-tributed to Fc�RIIa activation. Endogenous mouse FcR(Fc�RIIIa and Fc�RI) were responsible for the balanceof TNF�, which was equivalent to that produced fromHAGG-stimulated nontransgenic macrophages. Thisalso indicates that the endogenous receptors are func-tionally intact and unaffected by the presence of thetransgenic receptor.
A surprising characteristic of the Fc�RIIa-transgenic mice was the spontaneous development ofdisease with features of RA and systemic lupus erythem-atosus (SLE). Approximately 40% of older transgenicmice had features of both RA and SLE, similar to the“rhupus” overlap syndrome described in humans (34).The remaining 60% of the mice had features of SLE,e.g., endoproliferative GN, with intraglomerular accu-mulation of deposits (i.e., wire-loop lesions). SomeFc�RIIa-transgenic mice also developed mild, nonero-sive inflammatory arthritis similar to that seen in SLE.
While most of the older Fc�RIIa-transgenic micehad antihistone antibodies, the most interesting sero-logic observation was elevated IgG2a levels in mice withspontaneous severe destructive arthritis. Elevated IgG2awas seen in other autoimmune models in mice (35,36),and may reflect the cytokine profiles involved in leuko-cyte activation (37). The antibodies detected in our studywere not directed against CII and, currently, the autoan-tigen remains undefined.
The phenotype of the Fc�RIIa-transgenic miceresembles that of mice deficient in the inhibitoryFc�RIIb. These mice show increased susceptibility toCIA (36), elevated levels of TNF� and IgG2a (38), and,on a specific MHC background, spontaneously developSLE-like symptoms, including ANAs to double-strandedDNA and DNA/histone complexes (39) and GN (40).However, there are some fundamental differences com-pared with Fc�RIIa-transgenic mice. The Fc�RIIb-deficient mice never developed spontaneous severe de-structive arthritis; they showed exaggerated antibodyresponses, with elevated anti-CII antibodies in the CIAmodel, whereas Fc�RIIa-transgenic mice had low anti-body levels. Also, Fc�RIIb-knockout mice have anti-DNA antibodies, but Fc�RIIa-transgenic mice devel-oped only antihistone antibodies. Thus, analysis ofFc�RIIb-deficient mice by other investigators suggeststhat their phenotype was due largely to dysregulation ofB cell activation and loss of B cell tolerance (36,40)arising from unbalanced ITAM/ITIM signaling (38).While we cannot rule out such an imbalance, it must berelatively subtle, since Fc�RIIa-transgenic mice hadboth activating and inhibitory receptors (Figure 1).
In contrast, Fc�RIIb-deficient mice and cellsentirely lacked the inhibitory Fc�RIIb, but retained afull complement of activating receptors. Furthermore,data from other studies (41) show that transfection ofactivating FcR into cells that already express both acti-vating and inhibitory receptors left the inhibitoryFc�RIIb still functional and potent. An alternative ex-planation for our observations is that Fc�RIIa lowers the
ROLE OF Fc�RIIa IN AUTOIMMUNE INFLAMMATION 3227
threshold of immune complex activation or qualitativelychanges the response induced by pathogenic antibodies.Indeed, evidence from human in vitro studies suggestssuch a role for Fc�RIIa in activated macrophages, whereFc�RI signals are partly dependent on Fc�RIIa (42).Nonetheless, future studies of mechanisms will be infor-mative in this regard.
The development of destructive arthritis involvesinterlinked immunologic and cytokine pathways (43,44).It is clear that TNF� and IL-1� are major factors inactive human disease (44), and recent clinical trials havesuggested a role for B cells and possibly for immunecomplexes (45,46). Although animal models have beenuseful in suggesting possible mechanisms in humandisease, analysis of the role of the major and uniqueactivating human FcR (Fc�RIIa) has been lacking. Ouranalysis strongly suggests a role for this receptor in RA.It is particularly interesting that Fc�RIIa-transgenicmice spontaneously develop destructive arthritis. This israre in mice, having been observed only in older males ofthe susceptible DBA/1 strain (47), in strains with alter-ations in T cell tolerance, selection, and/or activation(for example, K/BxN [6] and SKG [48] strains), or inmice with engineered cytokine disturbances, such asTNF�-transgenic (26) and IL-1Ra–knockout (49) mice.
These studies show that expression of the uniqueactivating human FcR, Fc�RIIa, is associated with spon-taneous autoimmune inflammation and exaggerated re-activity to induced, antibody-dependent inflammation.Fc�RIIa may lower the threshold for stimulation ofeffector cell function, resulting in hypersensitivity toimmune complexes in vivo, and inducing enhancedcytokine secretion. Thus, Fc�RIIa plays a critical role indetermining the sensitivity to autoimmunity and, assuch, provides an attractive target for the treatment ofautoimmune disease.
ACKNOWLEDGMENTS
We thank Liliana Tatarczuch for assistance with thetransmission electron microscopy, Bill Pispalliaris for usefuldiscussion of the antihistone antibody analysis, Dacho Gao forthe production of the radiographs, and Dr. Peck Sze Tan(ARI) for antibodies.
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