Development of spontaneous multisystem autoimmune disease and hypersensitivity to antibody-induced inflammation in Fcgamma receptor IIa-transgenic mice.
ABSTRACT The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease.
Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production.
FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease.
The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is likely that complement activation leads to neutrophil chemotaxis, but neutrophil activation via Fc gamma RII, which results in inflammatory mediator release, is inhibited. The data indicate that rsFc gamma RII is a potential therapeutic agent for the treatment of antibody or immune complex-mediated tissue damage.Journal of Experimental Medicine 12/1993; 178(5):1617-28. · 13.21 Impact Factor
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ABSTRACT: Arthritis spontaneously develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. We have backcrossed these mice onto the arthritis-susceptible DBA/1 background and found an acceleration of the onset of arthritis with successive generations of interbreeding. Bioactive TNF-alpha in primary synovial membrane cell cultures was significantly higher in the DBA/1 transgenic mice than in transgenic mice on the original background. Elevated levels of human TNF-alpha were accompanied by increases in synovial cell expression of murine IL-1beta and IL-6, but murine granulocyte-macrophage CSF, IFN-gamma, and IL-4 could not be detected. Interestingly, the anti-inflammatory cytokine IL-10 could be detected, but levels were not modulated by expression of the transgene. Analysis of the synovial membrane cell composition revealed that >50% of synovial cells were CD45-negative cells, presumably fibroblasts and endothelial cells, and the majority of CD45-expressing cells were neutrophils. Peritoneal macrophages and lymphocytes from the spleen, bone marrow, and lymph nodes required LPS stimulation to produce human TNF-alpha, indicating that, when activated, cells of these lineages were capable of expressing the transgene; however, few were found in synovial tissues. In contrast, fibroblasts derived from synovial tissue spontaneously released human TNF-alpha, and using immunohistochemical techniques, this cytokine was localized to fibroblast-like cells and chondrocytes. We propose that arthritis in DBA/1 human TNF-alpha transgenic mice is driven in part through the spontaneous expression of transgene by connective tissue cells, and there is little evidence of the participation of lymphocytes in this model.The Journal of Immunology 09/1997; 159(6):2867-76. · 5.52 Impact Factor
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ABSTRACT: By virtue of its ability to couple the BCR to an inhibitory pathway, FcgammaRIIB can potentially determine the fate of B cells upon IgG immune complex engagement. We now provide evidence for FcgammaRIIB as a component of a peripheral tolerance pathway with the observation that RIIB-/- mice develop autoantibodies and autoimmune glomerulonephritis in a strain-dependent fashion. Transfer of the autoimmune phenotype is associated with the presence of donor RIIB-/- B cells, with the RIIB+/+ myeloid cells primarily derived from the recipient. These results suggest that deficiency of RIIB on B cells leads to autoimmune disease in specific genetic backgrounds, thus identifying it as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.Immunity 09/2000; 13(2):277-85. · 19.80 Impact Factor
ARTHRITIS & RHEUMATISM
Vol. 52, No. 10, October 2005, pp 3220–3229
© 2005, American College of Rheumatology
Development of Spontaneous Multisystem Autoimmune Disease
and Hypersensitivity to Antibody-Induced Inflammation
in Fc? Receptor IIa–Transgenic Mice
Caroline Tan Sardjono,1Patricia L. Mottram,1Nicholas C. van de Velde,1Maree S. Powell,1
David Power,1Ronald F. Slocombe,2Ian P. Wicks,3Ian K. Campbell,3Steven E. McKenzie,4
Mark Brooks,5Andrew W. Stevenson,6and P. Mark Hogarth1
Objective. The major human Fc receptor,
Fc?RIIa, is the most widespread activating FcR. Our
aim was to determine the role of Fc?RIIa in a transgenic
mouse model of immune complex–mediated autoimmu-
nity and to characterize the development of spontaneous
Methods. Arthritis was induced in normal and
Fc?RIIa-transgenic mice by immunization with type II
collagen (CII) or by transfer of arthritogenic anti-CII
antibodies. Also, mice that spontaneously developed
autoimmune disease were assessed by clinical scoring of
affected limbs, histology and serology, and measure-
ment of autoantibody titers and cytokine production.
Results. Fc?RIIa-transgenic mice developed
collagen-induced arthritis (CIA) more rapidly than did
archetypal CIA-sensitive DBA/1 (H-2q) mice, while non-
transgenic C57BL/6 (H-2b) mice did not develop CIA
when similarly immunized. Passive transfer of a single
dose of anti-CII antibody induced a more rapid, severe
arthritis 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 the
endogenous mouse FcR. A spontaneous, multisystem
autoimmune disease developed in aging (>20 weeks)
transgenic mice (n ? 25), with a 32% incidence of
arthritis, and by 45 weeks, all mice had developed
glomerulonephritis and pneumonitis, and most had
antihistone antibodies. Elevated IgG2a levels were seen
in mice with CIA and in those with spontaneous disease.
Conclusion. The presence of enhanced passive
and induced autoimmunity, as well as the emergence of
spontaneous autoimmune disease at 20–45 weeks of age,
suggest that Fc?RIIa is a very important factor in the
pathogenesis 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 role
of cell surface receptors for antibodies, especially IgG
Fc? receptors (Fc?R), was recognized following amelio-
ration of tissue destruction in type III hypersensitivity
reactions after administration of soluble recombinant
human Fc?RIIa in vivo (3). Subsequent studies with
FcR-deficient mice (4) showed that Fc?R play signifi-
cant roles in antibody-induced inflammatory disease
models such as collagen-induced arthritis (CIA) (5),
passive antibody-induced arthritis (6), and intraarticular
antigen-induced arthritis (7). However, rodents lack an
ortholog of Fc?RIIa, the most abundant and widespread
Supported by grants from the National Health and Medical
Research Council and PrimaBiomed Ltd., Australia. Dr. Sardjono’s
work was supported by PaperlinX Pty Ltd. Drs. Mottram and Powell’s
work was supported by Nancy Prendergast fellowships from the
Arthritis Foundation, Australia.
1Caroline Tan Sardjono, PhD, Patricia L. Mottram, PhD,
Nicholas C. van de Velde, BSc Hons, Maree S. Powell, PhD, David
Power, PhD, P. Mark Hogarth, PhD: Austin Research Institute,
Heidelberg, Victoria, Australia;2Ronald F. Slocombe, PhD: Univer-
sity of Melbourne, Melbourne, Victoria, Australia;
PhD, Ian K. Campbell, PhD: Walter and Eliza Hall Institute, Parkville,
College, Philadelphia, Pennsylvania;
Hospital, Heidelberg, Victoria, Australia;
PhD: Commonwealth Scientific Industrial Research Organization,
Clayton South, Victoria, Australia.
Drs. Mottram, Powell, and Hogarth have stock options in
PrimaBiomed. Dr. McKenzie has received consulting fees (less than
$10,000 per year) from GlaxoSmithKline.
Address correspondence and reprint requests to P. Mark
Hogarth, PhD, Helen McPherson-Smith Laboratory, Austin Research
Institute, Studley Road, Heidelberg, Victoria 3084, Australia. E-mail:
Submitted for publication September 24, 2004; accepted in
revised form June 30, 2005.
3Ian P. Wicks,
4Steven E. McKenzie, PhD: Jefferson Medical
5Mark Brooks, MBBS: Austin
6Andrew W. Stevenson,
activating FcR in higher primates. Fc?RIIa has unique
structural, 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 the
immunoreceptor 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-
Studies of Fc?RIIa, as well as other FcR, trans-
fected into mouse or primate cells show that these FcR
behave identically in both ligand binding and activation/
regulation (13,14). The interaction of Fc?RII ITAMs
and immunoreceptor tyrosine-based inhibition motifs
(ITIMs) was seen in both transfected mouse and human
cell lines, and ITIM sequences in mice and humans are
highly conserved (15). Fc?RIIa in transgenic mice is
expressed under its own promoter and has the same
expression pattern in mice and humans (16,17). Thus,
Fc?RIIa can interact appropriately with intracellular
signaling 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 expressing
Fc?RIIa and confirmed a role for this receptor in
passive, 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)F2embryos (17) were
used. The Fc?RIIa-transgenic mice were inbred for ?20
generations and were homozygous for the transgene under the
control 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 at
the 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 21
days later (19). They were examined daily from days 1–60, and
arthritis 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). The
maximum possible score (arthritis index) was 12 for each
Passively induced arthritis. Anti-CII monoclonal anti-
body (mAb) M2139 (2 mg) (20) was injected intraperitoneally
into Fc?RIIa-transgenic and nontransgenic C57BL/6 mice, and
arthritis 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) for
anti-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 detected
using secondary horseradish peroxidase (HRP)–conjugated
sheep anti-mouse IgG F(ab?)2fragments (Amersham, Little
Chalfont, UK). Development was performed for 10 minutes
with ABTS (Boehringer Mannheim, Rockville, MD), and
absorbance was read at 405 nm. IgG isotypes were detected
with specific antibodies (see below).
Bone marrow macrophage cultures. Bone marrow
macrophages were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (CSL, Melbourne, Victoria, Australia) con-
taining 2 mM glutamine, 50 mM 2-mercaptoethanol, 100
units/ml penicillin, 100 ?g/ml streptomycin, 10% (v/v) fetal calf
serum, and 30% (v/v) L cell–conditioned medium at 37°C in
10% CO2for 5–7 days (21).
Peritoneal exudate macrophage (PEM) cultures. Mice
were injected intraperitoneally with 0.5 ml of 4% (weight/
volume) thioglycolate (CSL) 4 days prior to harvest. PEMs
were cultured in DMEM, prepared as described above, for 1–3
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 was
prepared from 8–10 mg/ml of human gamma globulin (San-
doglobulin; Novartis, East Hanover, NJ) heated for 30 minutes
at 63°C, then put on ice and brought to 1% (w/v) with
polyethylene glycol 6000 (Sigma) in PBS and kept on ice for 30
minutes. 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?) and
interleukin-10 (IL-10) by macrophages in the supernatant 2–24
hours after stimulation with HAGG or PMA was detected by
ELISA using cytokine detection kits from eBioscience (San
Diego, CA). The specificity of induced TNF? secretion was
determined 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), and
hFc?RIIa using mAb IV.3 (mIgG2b) or mAb 8.7 (mIgG1).
Monitoring of spontaneous autoimmunity. Mice were
examined from 12 weeks of age, and the index of arthritis
severity was assessed as described above. Organs were fixed in
10% formalin/PBS, embedded in paraffin, and histologic sec-
tions were stained with H&E. Immune complex deposition in
kidney sections was detected with fluorescein isothiocyanate
(FITC)–conjugated sheep anti-mouse IgG (Silenus Laborato-
ries, Hawthorn, Victoria, Australia).
Transmission electron microscopy. Mouse kidneys
were cut into 1–2-mm cubes and fixed in 2–8% paraformalde-
ROLE OF Fc?RIIa IN AUTOIMMUNE INFLAMMATION3221
hyde/2–5% glutaraldehyde in 0.15M cacodylate buffer (pH 7.4)
for ?6 hours at 4°C. Tissues were rinsed in cacodylate buffer and
postfixed in 1% osmium tetroxide in 0.15M cacodylate buffer (pH
7.4) for 2 hours at room temperature. Samples were washed in
distilled water, dehydrated in 10% incremental concentrations of
acetone, and then embedded in Procure–Araldite resin (ProSci
Tech, Thuringowa, Queensland, Australia). During the dehydra-
tion, tissues were stained with 2% uranyl acetate in 70% acetone.
solution for 30 minutes at room temperature and then were
stained with Reynolds lead citrate for 10 minutes. Sections were
examined 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 recorded
using an FXE-225.20 microfocus x-ray source (Feinfocus,
Stamford, CT) fitted with a tungsten target, operated at 60
kVp. Projection geometry with a large sample-to-detector
distance resulted in high-resolution images. Exposures were
?3 mA, and images were recorded with imaging plates (XRT,
Port Melbourne, Victoria, Australia) scanned with a Fuji
BAS-5000 scanner (Fuji Photo Film, Tokyo, Japan).
Detection of antihistone antibodies. ELISA plates
were 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) were
added, incubated for 1 hour at room temperature, detected
with HRP-conjugated sheep anti-mouse IgG F(ab?)2fragments
(Amersham), and then developed using ABTS.
Quantitation of IgG subclasses. Total serum IgG
concentrations were determined using ELISA plates coated
with 50 ?l (3 ?g/ml) of rat anti-mouse IgG (BD PharMingen,
San Diego, CA). Serially diluted serum samples (50 ?l) were
added and incubated for 1 hour at room temperature. Anti-
body was detected with biotin-conjugated, isotype-specific rat
anti-mouse IgG1, anti-mouse IgG2a, and anti-mouse IgG2b
mAb (BD PharMingen) (1 hour at room temperature), then
with streptavidin–HRP (1 hour at room temperature), and
developed with ABTS. Quantitation of IgG was by comparison
with class-specific IgG standards.
Statistical analysis. Results were expressed as the
mean ? SD. Statistical differences were analyzed using the
Mann-Whitney 2-sample rank test, correlation coefficients, or
Student’s t-test. All the statistical analyses were performed
using Microsoft Excel software (Microsoft, Redmond, WA). P
values less than 0.05 were considered statistically significant.
Expression of transgenic and endogenous FcR.
The expression of Fc?R on bone marrow macrophages
from Fc?RIIa-transgenic and nontransgenic C57BL/6
mice was analyzed by flow cytometry. Using mAb IV.3
(anti-Fc?RIIa) (Figure 1A), Fc?RIIa was detected on
macrophages from Fc?RIIa-transgenic mice but not on
macrophages from nontransgenic mice. The expression
of the transgene did not significantly change the expres-
sion of endogenous mouse FcR, since similar levels of
Fc?RI (Figure 1B) and especially inhibitory Fc?RIIb
(Figure 1C) were observed on Fc?RIIa-transgenic and
nontransgenic macrophages. Similarly, expression of
Fc?RIIa on neutrophils did not alter endogenous FcR
expression. 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-2qand H-2rmice
are highly susceptible, while H-2b, H-2d, and H-2smice
are less so (19,24). We compared CIA development in
Fc?RIIa-transgenic (H-2b) mice with the archetypal
susceptible strain DBA/1 (H-2q) and the less susceptible
C57BL/6 (H-2b) and (SJL ? C57BL/6)F1(H-2s/b) strains
Figure 1. Flow cytometric analysis of Fc? receptor (Fc?R) expression
in 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 mAb
X54-5/7.1, and C, Fc?RIIb with mAb Ly-17.2. Studies were performed
with fluorescein isothiocyanate–conjugated anti-mouse IgG (solid line,
open histogram). FL-1 ? fluorescence channel 1.
3222SARDJONO ET AL
(Figure 2A). The Fc?RIIa-transgenic mice in this exper-
iment were highly susceptible, with more rapid onset of
arthritis (day 18) compared with other mice (days 22 and
24). Of the Fc?RIIa-transgenic mice, 15% developed
arthritis after 1 immunization, whereas 2 immunizations
were always required for CIA development in DBA/1
and (SJL ? C57BL/6)F1mice. No arthritis occurred in
C57BL/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)F1mice (P ? 0.001).
Disease incidence and severity at ?30 days were
similar in Fc?RIIa-transgenic and DBA/1 mice. In
Fc?RIIa-transgenic mice treated with a limited course of
anti-Fc?RIIa F(ab?)2antibody (100 ?g/mouse intraperi-
toneally on days 21, 24, 27, and 30), CIA was signifi-
cantly reduced on day 35 (P ? 0.05) compared with
untreated CIA in Fc?RIIa-transgenic mice. Although
this dose of antibody is unlikely to have blocked all in
vivo activity of Fc?RIIa, the data show that this FcR
plays a role in disease severity in these mice (Figure 2B).
Histologic assessment of paws and ankle and
knee joints from Fc?RIIa-transgenic mice on day 36 after
arthritis induction (when the maximal clinical index was
observed) showed severe, destructive arthritis with pan-
nus formation, infiltration of inflammatory cells (poly-
morphonuclear cells and macrophages) into the synovial
space, and erosion of the cartilage (Figure 2C). No joint
inflammation was seen in C57BL/6 controls (Figure 2D).
Correlation of increased levels of IgG2a with the
arthritis index in transgenic mice. Despite the acceler-
ated development of severe CIA, Fc?RIIa-transgenic
mice had lower anticollagen antibody titers than suscep-
tible DBA/1 mice, but similar to those of the less
susceptible (SJL ? C57BL/6)F1mice (Figure 3A). The
anticollagen antibodies were predominantly IgG2, and
analysis of the serum IgG2 subclasses (Figure 3B)
showed a significant increase in the IgG2a:IgG2b ratio
in severely arthritic mice (P ? 0.02) compared with
mildly arthritic or unaffected Fc?RIIa-transgenic mice.
There was a positive correlation between the arthritis
index and IgG2a levels (r ? 0.57) and no correlation
between 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 mouse
IgG2a avidly, may cause effector cells in the Fc?RIIa-
transgenic mice to be sensitive to low levels of
autoantibody/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? is
a major inflammatory mediator in human RA and
mouse CIA (24,26,27), we compared the production of
TNF? from immune complex–stimulated Fc?RIIa-
transgenic and nontransgenic macrophages. HAGG-
stimulated Fc?RIIa macrophages produced significantly
more TNF? (6.5 ng/ml) compared with nontransgenic
macrophages (1 ng/ml; P ? 0.001) (Figure 3C). Elevated
TNF? production by Fc?RIIa macrophages was blocked
(although not completely) in a dose-dependent manner
by anti-Fc?RIIa mAb IV.3 Fab to levels not significantly
different 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 activating
Fc?R. PMA stimulation of nontransgenic and transgenic
macrophages 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/6
mice (n ? 28), and (SJL ? B6)F1mice (n ? 8). Pooled data from 5
experiments are shown. B, Effects of treatment of arthritis with
anti-Fc?RIIa F(ab?)2mAb 8.7 (intraperitoneal injection of 100 ?g on
days 21, 24, 27, and 30 versus treatment with phosphate buffered saline
[PBS] alone [n ? 8 mice per group]). The mAb 8.7 treatment caused
a significant reduction in disease severity on day 40 (P ? 0.05). Values
in A and B are the mean ? SD. C, A representative section showing
histopathologic features of an Fc?RIIa-transgenic mouse knee joint 36
days after CIA induction (hematoxylin and eosin stained; original
magnification ? 100). D, Histopathologic features of a knee joint from
a C57BL/6 mouse 36 days after collagen injection (hematoxylin and
eosin stained; original magnification ? 40). See Figure 1 for other
ROLE OF Fc?RIIa IN AUTOIMMUNE INFLAMMATION3223
was observed in HAGG-stimulated macrophages (data
Exaggerated antibody hypersensitivity of
Fc?RIIa-transgenic mice. The rapid CIA response in
Fc?RIIa-transgenic mice and increased sensitivity to
HAGG implied an exaggerated response to pathologic
antibodies. This possibility was tested using a passive
antibody-transfer arthritis model described by Holmdahl
and colleagues (20,28), wherein mice were normally
given a single dose of a mixture of 2 anticollagen
antibodies (4.5 mg each of M2139 and C1), followed by
50 ?g of lipopolysaccharide (LPS) 5 days later. These
antibodies have been tested for arthritis induction in a
number 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-transgenic
mice (Figure 3D), treatment with a single 2-mg dose of
mAb M2139 alone, without LPS, caused a rapid onset of
arthritis in 100% of mice, but had no effect in nontrans-
Spontaneous development of autoimmunity in
Fc?RIIa-transgenic mice. Mice were housed for ?1
year, and we observed spontaneous progressive develop-
ment of a systemic multiorgan autoimmune syndrome.
Development of destructive arthritis. A proportion
of aging Fc?RIIa-transgenic mice spontaneously devel-
oped severe, destructive, symmetric arthritis. In a group
of 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)–transgenic
mice to antibody-mediated disease. A, Anticollagen antibody titers in
Fc?RIIa-transgenic (Tg) and nontransgenic (SJL ? C57BL/6)F1and
DBA/1 mice (n ? 5 per group), as determined on days 0, 21, and 36
postimmunization. OD ? optical density. B, Comparison of changes in
IgG2 subclasses in Fc?RIIa-transgenic mice without collagen-induced
arthritis (CIA) (index 0), with severe arthritis (index 5–12; high CIA),
and with mild arthritis (index 1–4; low CIA) after the second injection
of collagen (n ? 5 mice per group). In the high CIA group, there was
a significant increase in the IgG2a:IgG2b ratio (P ? 0.016 versus mildly
arthritic mice, by Student’s t-test), a positive correlation between the
arthritis index and IgG2a levels (r ? 0.57), and no correlation between
the arthritis index and IgG2b levels (r ? ?0.16). C, Comparison of
tumor 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) following
stimulation with HAGG. There was no significant difference (P ?
0.07) between Fc?RIIa-transgenic and C57BL/6 mouse macrophages
treated with HAGG following incubation with 20- or 50-?g doses of
monoclonal antibody (mAb) IV.3. Responses to phorbol myristate
acetate (PMA) stimulation were equivalent in both strains of macro-
phages (P ? 0.05). D, Arthritis index over time in Fc?RIIa-transgenic
and (SJL ? C57BL/6)F1mice treated with 2 mg of mAb M2139 given
intraperitoneally on day 0 (n ? 6 per group). Fc?RIIa-transgenic mice
treated 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 autoimmune
disease. A, Cumulative percentage incidence in arthritis over time in
human Fc? receptor IIa (Fc?RIIa)–transgenic mice (n ? 25). There
was no difference in arthritis incidence between males and females. B
and 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 with
mild arthritis, and Fc?RIIa-transgenic with destructive arthritis (n ? 4
per group) (results not shown). D–F, Representative sections showing
the histopathologic features of hematoxylin and eosin–stained mouse
joints. 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 a
36-week-old mouse, with predominantly mononuclear cell infiltration
and advanced pannus. F, A normal ankle joint from an older (age 36
weeks) 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, with
a cumulative incidence of 32% (Figure 4A). Of the 8
mice with arthritis, 5 were severely affected (mean ? SD
index 10 ? 2.38), with profoundly swollen joints and
severe histologic and radiologic changes (Figure 4). The
other 3 Fc?RIIa-transgenic mice had less severe arthri-
tis. Despite differences in arthritis severity, in both
subsets of Fc?RIIa-transgenic mice, the number of
affected paws was similar. Age-matched nontransgenic
mice never developed spontaneous disease.
Radiologic examination of the affected limbs. The
Fc?RIIa-transgenic mice with severe arthritis developed
marked radiologic changes that mirrored the florid
edema and distortion of the foot seen clinically. Severe
ankylosis of the tibiotarsal and tarsophalangeal cartilage
was seen, with loss of joint space and prominent periar-
ticular new bone formation (Figure 4B) compared with
age-matched Fc?RIIa-transgenic mice that did not de-
velop arthritis (Figure 4C). Limbs from mice with mild
arthritis revealed minimal radiologic changes (results
Histologic evaluation of the joints. Joint histology
of Fc?RIIa-transgenic mice compared with that of non-
transgenic, age-matched controls (Figures 4D–F)
showed that mice with spontaneous severe arthritis had
synovial hyperplasia and proliferation, cartilage erosion,
pannus formation, and joint space infiltrate. The later
stage of the disease showed more advanced destruction
of bone and thinning of the cartilage, with the infiltrate
changing from polymorphonuclear (PMN) cells in early
active disease (Figure 4D) to macrophages in more
advanced cases (Figure 4E). There was no evidence of
disease in age-matched C57BL/6 or (SJL ? C57BL/6)F1
controls (Figure 4F).
Serum IgG2a elevation in affected mice. The levels
of total IgG, IgG1, IgG2a, IgG2b, and IgG3 in all mice
were determined by ELISA. Only the total level of
IgG2a antibody was elevated (Figure 5A), and only in
Fc?RIIa-transgenic mice that developed destructive ar-
thritis (mean ? SD 6.6 ? 2.5 ?g/ml). IgG2a levels in
mice with mild arthritis (2.0 ? 0.8 ?g/ml) were similar to
those 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 IgG2a
levels and disease severity, particularly with the forma-
tion of pannus (r ? 0.63). The levels of the other IgG
subclasses were not altered (Figure 5).
Other features of systemic autoimmune disease
in Fc?RIIa-transgenic mice. The Fc?RIIa-transgenic
mice were screened for further evidence of inflamma-
tion and autoimmunity by histologic examination of
H&E-stained sections of skin, lymph nodes, gut, salivary
glands, kidneys, eyes, brain, lungs, spleen, liver, pan-
creas, and heart at 14–60 weeks of age and compared
with aged-matched, nontransgenic controls. Glomerulo-
nephritis (GN) and pneumonitis were commonly ob-
served in Fc?RIIa-transgenic mice, with the disease
incidence increasing with age. By 50 weeks of age, all
mice were affected (Figure 5B). No abnormalities were
found in other organs or in nontransgenic mice of any
Glomerulonephritis. The time of onset and degree
of severity of GN in individuals varied considerably. Few
mice developed GN before 20 weeks of age, and up to
80% had the disease by 40 weeks. However, all mice ?40
weeks of age had moderate to severe GN, implying an
age-related progression to severe GN (Figure 5B). Mul-
tifocal lymphoplasmacytic infiltrate in the renal intersti-
tium, mainly around major arcuate vessels, was seen at
25–30 weeks (Figure 6A), with mild mesangial matrix
deposition in the glomeruli and some tubular thickening.
In mice ages ?40 weeks, more advanced disease was
seen, with enlarged glomeruli, increased mesangial ma-
trix (Figures 6B and C), and condensation of glomerular
tufts. There were proliferative and sclerotic changes in
Bowman’s capsule, indicative of crescent formation, and
mild tubular proliferative changes (Figure 6B), although
tubulointerstitial infiltrates were not present. The dis-
Figure 5. Serologic findings and incidence of kidney and lung disease
in mice with spontaneous autoimmune disease. A, Levels of serum
IgG2a in individual human Fc? receptor IIa (FcR?IIa)–transgenic
mice with destructive arthritis (n ? 8) compared with unaffected
FcR?IIa-transgenic mice (n ? 7), FcR?IIa-transgenic mice with mild
arthritis (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 versus
unaffected nontransgenic mice. IgG2a levels correlated with disease
severity, particularly with the formation of pannus (r ? 0.63). Total
IgG was increased in arthritic mice, and this correlated strongly with
IgG2a levels (r ? 0.96). The levels of the other Ig subclasses (IgM,
IgG1, Ig2b, and Ig3) were not significantly different between the 4
groups of mice (P ? 0.05 for all comparisons). B, Incidence of
glomerulonephritis (GN) and pneumonitis (Pn) in Fc?RIIa-transgenic
mice at ages ?20 weeks (n ? 8), 21–40 weeks (n ? 14), and ?40 weeks
(n ? 9).
ROLE OF Fc?RIIa IN AUTOIMMUNE INFLAMMATION3225
ease was self-limiting, since mice ages ?40 weeks, with
up to 65% of glomeruli affected, remained healthy, with
normal urinary protein and serum creatinine levels
(results not shown). Age-matched nontransgenic mice
showed 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 showed
diffuse, 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 (Figure
6D), with features similar to those of lupus nephritis in
humans, including small, electron-dense deposits form-
ing wire-loop lesions in the subendothelial basement
Lung histopathology. Pneumonitis was found in
25% of mice between ages 12 and 40 weeks, increasing
to 100% of older mice (Figure 5B), and was character-
ized by patches of perivascular inflammation with cellu-
lar aggregates of macrophages, lymphocytes, plasma
cells, and numerous PMN cells within alveolar walls
(Figure 6E). In severe cases, up to 50% of the normal
architecture of the lungs was obliterated, but the disease
was self-limiting, and older mice remained healthy.
Lungs of age-matched nontransgenic mice showed no
Analysis of antihistone and antinuclear antibodies
(ANAs). The histologic features suggested that the tissue
damage seen in the spontaneous autoimmune disease in
Fc?RIIa-transgenic mice was mediated, at least in part,
by autoantibodies. Therefore, we evaluated mice for the
presence of autoantibodies known to be associated with
human autoimmune disease. Initial immunofluores-
cence studies with sera from most Fc?RIIa-transgenic
mice and many older nontransgenic C57BL/6 mice
showed homogeneous nuclear staining of Chinese ham-
ster ovary cells (results not shown). This staining pattern
was similar to that of antihistone antibody huPIA3 (29).
Sera were then tested for antihistone antibodies by
ELISA, using a mixture of purified histones. In mice
ages ?20 weeks, 13 of 23 had antihistone antibodies
above background levels (Figure 6F). Histone antibody
titers did not correlate with GN or arthritis incidence (r
?0.05). No antihistone antibodies above background
levels were seen in age-matched C57BL/6 mice or
(SJL ? C57BL/6)F1mice at ages ?25 weeks. None of
the mice had the other common autoantibodies, such as
anti–double-stranded or anti–single-stranded DNA or
rheumatoid factor (data not shown).
Since it was found that recombinant soluble FcR
inhibited immune complex vasculitis (3), there has been
widespread 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 human
Fc? receptor IIa (Fc?RIIa)–transgenic mice (n ? 31). A, Kidney
section (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 but
otherwise normal (original magnification ? 200). B, An H&E-stained
kidney section taken at 45 weeks, showing enlarged glomeruli (thin
arrows), increased mesangial matrix, and condensation of glomerular
tufts. There are proliferative and sclerotic changes in Bowman’s
capsule (thick arrow) indicative of crescent formation, and mild
tubular proliferative changes (original magnification ? 100). C, Fluo-
rescence staining of immune complexes in the glomerulus. Kidney
sections were stained with anti-mouse IgG, directly conjugated with
fluorescein 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 in
age-matched, nontransgenic control mice (results not shown). D,
Transmission electron microscopy of glomeruli, showing immune
complexes 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 infiltration
of inflammatory cells (arrows) and local destruction of lung architec-
ture (original magnification ? 50). F, Enzyme-linked immunosorbent
assay for the presence of antihistone antibodies in Fc?RIIa-transgenic
and nontransgenic mouse sera at 36 weeks of age. Antihistone
antibodies were present in serum from many of the Fc?RIIa-
transgenic mice at all ages, but were not present in serum from the
nontransgenic mice. Horizontal line shows the cutoff for positivity.
3226 SARDJONO ET AL
FcR, including Fc?RI, Fc?RIIb, and Fc?RIIIa, which
are common to both mice and humans, humans have a
unique FcR, Fc?RIIa, which is absent from rodents and
was therefore not analyzed in rodent models of autoim-
munity. Our data demonstrate that Fc?RIIa-transgenic
mice are highly susceptible to passive antibody-induced
inflammation 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 in
strains of mice with low-susceptibility MHC (H-2band
H-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 of
collagen. These findings establish a role for Fc?RIIa in
enhancing inflammatory responses in CIA, especially
since treatment with anti-Fc?RIIa mAb reduced disease
severity. The immune mechanisms involved in the de-
velopment of CIA have been well described, with both T
cells and anticollagen antibodies known to play major
The observation that anti-CII antibodies were
lower on day 21 after CIA induction in Fc?RIIa-
transgenic mice compared with DBA/1 or nontransgenic
animals is evidence that Fc?RIIa plays a role in increas-
ing the sensitivity of effector cells to activation by
immune complexes. This possibility was supported by
data from the anti-CII antibody transfer model, wherein
a single dose of anti-CII antibody M2139 induced dis-
ease in 100% of the Fc?RIIa-transgenic mice. This
contrasts with other strains, in which a mixture of 2
antibodies plus LPS was required to induce this level of
disease (20). Although the use of 1 antibody to induce
arthritis in susceptible DBA/1 mice has been reported
(32), the highest incidence was only 50% after 2 doses of
M2139 (total dose 9 mg), compared with the 100%
incidence after a single 2-mg dose in the Fc?RIIa-
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 balance
of TNF?, which was equivalent to that produced from
HAGG-stimulated nontransgenic macrophages. This
also indicates that the endogenous receptors are func-
tionally intact and unaffected by the presence of the
A surprising characteristic of the Fc?RIIa-
transgenic mice was the spontaneous development of
disease with features of RA and systemic lupus erythem-
atosus (SLE). Approximately 40% of older transgenic
mice 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). Some
Fc?RIIa-transgenic mice also developed mild, nonero-
sive inflammatory arthritis similar to that seen in SLE.
While most of the older Fc?RIIa-transgenic mice
had antihistone antibodies, the most interesting sero-
logic observation was elevated IgG2a levels in mice with
spontaneous severe destructive arthritis. Elevated IgG2a
was 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 study
were not directed against CII and, currently, the autoan-
tigen remains undefined.
The phenotype of the Fc?RIIa-transgenic mice
resembles that of mice deficient in the inhibitory
Fc?RIIb. These mice show increased susceptibility to
CIA (36), elevated levels of TNF? and IgG2a (38), and,
on a specific MHC background, spontaneously develop
SLE-like symptoms, including ANAs to double-stranded
DNA 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 antibody
responses, with elevated anti-CII antibodies in the CIA
model, 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 of
Fc?RIIb-deficient mice by other investigators suggests
that their phenotype was due largely to dysregulation of
B 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 be
relatively subtle, since Fc?RIIa-transgenic mice had
both activating and inhibitory receptors (Figure 1).
In contrast, Fc?RIIb-deficient mice and cells
entirely lacked the inhibitory Fc?RIIb, but retained a
full complement of activating receptors. Furthermore,
data from other studies (41) show that transfection of
activating FcR into cells that already express both acti-
vating and inhibitory receptors left the inhibitory
Fc?RIIb still functional and potent. An alternative ex-
planation for our observations is that Fc?RIIa lowers the
ROLE OF Fc?RIIa IN AUTOIMMUNE INFLAMMATION3227
threshold of immune complex activation or qualitatively
changes the response induced by pathogenic antibodies.
Indeed, evidence from human in vitro studies suggests
such a role for Fc?RIIa in activated macrophages, where
Fc?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 involves
interlinked immunologic and cytokine pathways (43,44).
It is clear that TNF? and IL-1? are major factors in
active human disease (44), and recent clinical trials have
suggested a role for B cells and possibly for immune
complexes (45,46). Although animal models have been
useful in suggesting possible mechanisms in human
disease, analysis of the role of the major and unique
activating human FcR (Fc?RIIa) has been lacking. Our
analysis strongly suggests a role for this receptor in RA.
It is particularly interesting that Fc?RIIa-transgenic
mice spontaneously develop destructive arthritis. This is
rare in mice, having been observed only in older males of
the susceptible DBA/1 strain (47), in strains with alter-
ations in T cell tolerance, selection, and/or activation
(for example, K/BxN  and SKG  strains), or in
mice with engineered cytokine disturbances, such as
TNF?-transgenic (26) and IL-1Ra–knockout (49) mice.
These studies show that expression of the unique
activating 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 of
effector cell function, resulting in hypersensitivity to
immune complexes in vivo, and inducing enhanced
cytokine secretion. Thus, Fc?RIIa plays a critical role in
determining the sensitivity to autoimmunity and, as
such, provides an attractive target for the treatment of
We thank Liliana Tatarczuch for assistance with the
transmission electron microscopy, Bill Pispalliaris for useful
discussion of the antihistone antibody analysis, Dacho Gao for
the production of the radiographs, and Dr. Peck Sze Tan
(ARI) for antibodies.
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