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Fc receptorindependent development of autoimmune glomerulonephritis in lupus-prone MRLlpr mice.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 2, February 2003, pp 486–494
DOI 10.1002/art.10813
© 2003, American College of Rheumatology
Fc Receptor–Independent Development of
Autoimmune Glomerulonephritis in
Lupus-Prone MRL/lpr Mice
Keiko Matsumoto,1 Norihiko Watanabe,1 Bunshiro Akikusa,2 Kazuhiro Kurasawa,1
Ryutaro Matsumura,3 Yasushi Saito,1 Itsuo Iwamoto,1 and Takashi Saito4
Objective. To determine the role of Fc receptors
(FcR), which play crucial roles in antibody and immune
complex–mediated inflammation and autoimmunity, including glomerulonephritis (GN), in the development of
autoimmune GN and vasculitis in MRL/lpr mice, one of
the most widely used lupus-prone mouse models.
Methods. FcR␥–/– MRL/lpr mice were generated
by backcrossing for 8 generations. The development of
GN and vasculitis of various sized vessels was analyzed
histopathologically in the kidney, lung, and skin. Autoantibody and immune complex levels were determined
biochemically at 16–24 weeks of age and compared with
the findings in FcR␥ⴙ MRL/lpr mice. The lifespan of the
mice was also recorded.
Results. Diffuse proliferative GN, with deposition
of IgG and C3, developed in both FcR␥–/– and FcR␥ⴙ
MRL/lpr mice. There was no difference in the survival
rate and degree of proteinuria between FcR␥ⴙ and
FcR␥–/– MRL/lpr mice. Regardless of the level of FcR
expression, there were no significant differences in the
levels of serum IgG, anti-DNA antibody, or circulating
immune complexes between the two types of mice.
Necrotizing vasculitis in medium-sized arteries of the
kidneys and lungs as well as small-vessel vasculitis in
the skin was observed in both in FcR␥ⴙ and FcR␥–/–
MRL/lpr mice. In contrast, the Arthus reaction was
induced in FcR␥ⴙ MRL/lpr mice, but not in FcR␥–/–
MRL/lpr mice.
Conclusion. Unlike (NZB ⴛ NZW)F1, the other
strain of lupus-prone mice that develops GN in an
FcR-dependent manner, the development of autoimmune GN and vasculitis in MRL/lpr mice was FcRindependent, implying heterogeneity of the contribution
of FcR to the development of autoimmune disease.
Systemic lupus erythematosus (SLE) is considered to be an autoimmune disease in which either the
deposition of immune complexes (ICs) or the autoantibodies themselves lead to the activation of complement
systems, ligation of Fc receptors (FcR), and subsequent
inflammation (1). SLE patients thus manifest multiple
inflammatory organ involvement, including IC-induced
glomerulonephritis (GN), central nervous system disorders, thrombocytopenic purpura, hemolytic anemia, polyserositis, and vasculitis. The MRL/lpr mouse is a wellestablished murine model of SLE, in which the disease is
characterized by GN with IC deposition, vasculitis,
splenomegaly, lymphadenopathy, hypergammaglobulinemia, and autoantibody production (2,3). However,
the contribution of complement systems and FcR to the
development of GN and damage to other organs in SLE
is still largely unknown.
In studies of FcR-deficient mice, it was recently
shown that FcR play crucial roles in antibody-mediated
(type II hypersensitivity) and IC-mediated (type III
hypersensitivity) inflammation (4–10); it has long been
accepted that type II and type III hypersensitivity reactions are mainly induced by the activation of comple-
Supported in part by grants from the Ministry of Education,
Culture, Sports, Science, and Technology and from the Ministry of
Health, Labor, and Welfare, Japan.
1
Keiko Matsumoto, MD, Norihiko Watanabe, MD, PhD,
Kazuhiro Kurasawa, MD, PhD, Yasushi Saito, MD, PhD, Itsuo
Iwamoto, MD, PhD: Graduate School of Medicine, Chiba University,
Chiba, Japan; 2Bunshiro Akikusa, MD, PhD: Matsudo City Hospital,
Matsudo, Japan; 3Ryutaro Matsumura, MD, PhD: Toho University
School of Medicine Sakura Hospital, Sakura, Japan; 4Takashi Saito,
PhD: Graduate School of Medicine, Chiba University, Chiba, Japan,
and RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.
Address correspondence and reprint requests to Takashi
Saito, PhD, Department of Molecular Genetics, Graduate School of
Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670,
Japan. E-mail: saito@med.m.chiba-u.ac.jp.
Submitted for publication July 17, 2002; accepted in revised
form November 6, 2002.
486
FcR-INDEPENDENT MECHANISM IN LUPUS NEPHRITIS
ment systems (11–13). FcR ␥-chain–deficient mice,
which lack expression of the high-affinity IgG receptor
Fc␥RI, the low-affinity IgG receptor Fc␥RIII, and the
high-affinity IgE receptor Fc␧RI, were unable to induce
IgG-mediated phagocytosis by macrophages and IgEmediated anaphylaxis by mast cells (4). More important,
it was shown that the Arthus reaction induced by specific
ICs was severely impaired in both FcR␥- and Fc␥RIIIdeficient mice (7–9). FcR␥-deficient mice also exhibited
reduced experimental hemolytic anemia and thrombocytopenia (5) as well as anti–glomerular basement membrane (anti-GBM) antibody–induced GN (6).
With regard to the pathogenesis of autoimmune
GN in SLE, a recent study by Clynes et al (14) demonstrated that autoimmune GN in (NZB ⫻ NZW)F1 mice,
a murine model of spontaneous lupus nephritis, was
diminished in mice that were deficient in FcR␥. Furthermore, it was reported that treatment with anti-C5 monoclonal antibody prevented autoimmune GN in (NZB ⫻
NZW)F1 mice (15). In contrast, we previously demonstrated that IgG3 anti-IgG2a autoantibody with cryoglobulin activity derived from MRL/lpr mice induced
GN in wild-type as well as FcR␥-deficient nonautoimmune mice (16). Thus, it remains to be determined
whether there is an underlying FcR-dependent mechanism that is responsible for the induction of autoimmune
GN in SLE.
To clarify the role of the FcR in the pathogenesis
of autoimmune GN and vasculitis in SLE, we analyzed
the development of GN and vasculitis in FcR␥-deficient
MRL/lpr mice. Our results indicate that an FcRindependent mechanism is crucial to the development of
GN and vasculitis in MRL/lpr mice.
MATERIALS AND METHODS
Mice. MRL/lpr/lpr and C57BL/6 mice were purchased
from Japan SLC (Shizuoka, Japan). FcR␥-deficient (FcR␥–/–)
mice were established with a C57BL/6 mouse background, as
described previously (6). FcR␥–/– MRL/lpr mice were generated by backcrossing FcR␥–/– mice with MRL/lpr/lpr mice for 8
generations. F8 FcR␥⫹/– MRL/lpr mice were intercrossed to
obtain FcR␥⫹/⫹, FcR␥⫹/–, and FcR␥–/– MRL/lpr mice for the
present experiments.
FcR␥ alleles were analyzed by polymerase chain reaction of tail DNA using a sense primer for exon 3 (5⬘GGAATTCGCTGCCTTTCGGACCTGGAT-3⬘) and 2 antisense primers: one for neor (5⬘-GCCAACGCTATGTCCTGATAG-3⬘) for the targeted allele, and the other for exon 4
(5⬘-GAAAATCGATGCTGTCCTGTTTTTGTA-3⬘) for the
wild-type allele. The lpr alleles were determined by polymerase
chain reaction of intron 2 of the Fas gene using a sense primer for
intron 2 (5⬘-GTAAATAATTGTGCTTCGTCAG-3⬘) and 2 antisense primers: one for ETn (5⬘-GTTGCGACACCAGTT-
487
ATGAA-3⬘) for the lpr allele, and the other for intron 2 (5⬘CAGGGAAATGTAGCAAGATG-3⬘) for the wild-type allele.
Arthus reaction. The Arthus reaction was induced as
described (8). Briefly, mice were injected intradermally in the
dorsal skin with 25 ␮l of rabbit anti-ovalbumin (anti-OVA)
serum (Sigma, St. Louis, MO) or normal rabbit serum (Cappel,
Durham, NC) containing 1 ␮g or 10 ␮g of IgG. Immediately
thereafter, mice were injected intravenously with 100 ␮l of
OVA (500 ␮g) in 1% Evans blue solution. The mice were
killed 3 hours later, and the reverse side of the skin around the
injection sites was analyzed for extravasation of the Evans blue
dye. Skin sections were also fixed in 10% buffered formalin
and stained with hematoxylin and eosin.
Histopathology. Kidneys, lungs, and ear skin were removed from the mice at 24 weeks of age, and sections were
stained with hematoxylin and eosin and with periodic–acid Schiff
for histopathologic examination. Sections were also studied for
deposition of IgG and C3 by direct staining with fluorescein
isothiocyanate–conjugated anti-mouse IgG or anti-mouse C3
antibody (Cappel).
The severity of glomerular lesions (⬎50 glomeruli) was
assessed by a pathologist (BA) in a blinded manner, and the
severity of proliferative lesions was graded. Glomerular cellularity was graded on a scale of 0–3⫹, where 0 ⫽ no or rare
small foci of glomerular lesions, 1⫹ ⫽ mild hypercellularity
involving ⬍50% of glomeruli, 2⫹ ⫽ moderate hypercellularity
involving ⬎50% of glomeruli, and 3⫹ ⫽ severe hypercellularity involving almost all glomeruli and/or cellular crescent
formation. The severity of glomerular deposits was also graded
on a 0–3⫹ scale, where 0 ⫽ none, 1⫹ ⫽ a few lesions with
minor deposition, 2⫹ ⫽ moderate deposition without “wireloop” lesions, and 3⫹ ⫽ severe deposition with “wire-loop”
lesions.
The severity of vasculitis in medium-sized vessels in the
kidneys and lungs (⬎10 interlobular arteries in kidneys; ⬎20
pulmonary arteries) was graded according to the extent of
perivascular leukocyte infiltration in the medium-sized arteries, where 0 ⫽ none, 1⫹ ⫽ mild, 2⫹ ⫽ moderate, and 3⫹ ⫽
severe, with or without vessel wall destruction. The severity of
skin vasculitis was judged according to the number of lesions
and the severity of perivascular leukocyte infiltration and
erythrocyte extravasation of ear skin, where 0 ⫽ none, 1⫹ ⫽
1–3 focal lesions with cell infiltration and erythrocyte extravasation, 2⫹ ⫽ ⬎4 focal lesions, and 3⫹ ⫽ generalized, severe
lesions.
Flow cytometry. Cells (1 ⫻ 106) from spleen and lymph
nodes were stained for 30 minutes at 4°C with antibodies
conjugated with fluorescein isothiocyanate, phycoerythrin, or
peridin chlorophyll protein. The antibodies used were CD4,
CD8, CD3, and B220 (PharMingen, San Diego, CA). Stained
cells were washed and analyzed by FACSCalibur using the
CellQuest program (Becton Dickinson, Mountain View, CA).
Measurement of serum IgG and anti-DNA antibody.
Serum levels of IgG, IgG1, IgG2a, and IgG3 were measured by
the radial immunodiffusion method using mouse Ig NL kits
(The Binding Site, Birmingham, UK). Serum anti–double
stranded DNA (anti-dsDNA) antibodies were detected by
enzyme-linked immunosorbent assay (ELISA) using the Mesacup DNA II test (MBL, Nagoya, Japan) with horseradish
peroxidase–conjugated anti-mouse IgG antibody (Cappel).
488
MATSUMOTO ET AL
Figure 1. Lymphoproliferation of CD4–,CD8–,B220⫹ T cells in the
spleen and lymph nodes of female FcR␥–/– MRL/lpr mice. Lymphocytes were prepared from the spleen and lymph nodes of 24-week-old
female FcR␥⫹/⫹, FcR␥⫹/–, and FcR␥–/– MRL/lpr mice. Cells were stained
with fluorescein isothiocyanate (FITC)–conjugated anti-CD4 and phycoerythrin (PE)–conjugated anti-CD8 monoclonal antibodies (top panels)
or with FITC-conjugated anti-CD3 and PE-conjugated anti-B220 monoclonal antibodies (bottom panels), and analyzed by FACSCalibur.
Measurement of circulating immune complexes. Circulating immune complexes (CICs) were determined by the
C1q binding ELISA. Briefly, EDTA-treated sera (diluted 1/10)
were added to C1q-coated plates, incubated for 1 hour at room
temperature, and then overnight at 4°C. After washing, horseradish peroxidase–conjugated anti-mouse IgG antibody (Cappel) was added to the plate. The plate was incubated for 1
hour, followed by incubation with ABTS (Zymed, San Francisco, CA) for 20 minutes, and the optical density (OD) at 405
nm was measured.
Measurement of urinary protein. Urine was collected
from individual mice in a metabolic cage. The protein content
was measured by the Bradford method.
Statistical analysis. The unpaired t-test was used for
statistical analysis. P values less than 0.05 were considered
significant.
RESULTS
Generation of FcR␥–/– MRL/lpr mice. To determine the role of FcR in the development of autoimmune
GN and vasculitis in lupus-prone MRL/lpr mice, we gen-
erated FcR␥–/– MRL/lpr mice by backcrossing FcR␥–/–
mice with MRL/lpr/lpr mice for 8 generations. The F8
FcR␥⫹/– MRL/lpr mice were then intercrossed to obtain
FcR␥⫹/⫹, FcR␥⫹/–, and FcR␥–/– MRL/lpr mice. The female FcR␥⫹/⫹ and FcR␥⫹/– mice (FcR␥⫹) and the female
FcR␥–/– MRL/lpr mice were used for further analysis.
MRL/lpr mice are known to show lymphoproliferation, particularly of CD4–,CD8–,B220⫹ T cells in
lymph nodes and spleen (17,18). Similar to MRL/lpr
mice, FcR␥–/– MRL/lpr mice exhibited a comparable
accumulation of CD4–,CD8–,B220⫹ T cells (Figure 1).
All 3 groups of mice showed similar weights of the lymph
nodes and spleen at 24 weeks of age. The mean ⫾ SD
spleen weights in the FcR␥⫹/⫹, FcR␥⫹/–, FcR␥–/–, and
C57BL/6 normal mice were 726 ⫾ 390, 852 ⫾ 471, 840 ⫾
266, and 105 ⫾ 7 mg, respectively.
Impaired Arthus reaction in FcR␥–/– MRL/lpr
mice. Because it has been shown that the IC-mediated
Arthus reaction is severely impaired in FcR␥–/– nonautoimmune mice (7,9), we first determined whether the
Arthus reaction was impaired in the FcR␥–/– MRL/lpr
mice. FcR␥⫹/⫹ and FcR␥–/– MRL/lpr mice were injected
intradermally with rabbit anti-OVA IgG, then injected
intravenously with OVA in 1% Evans blue, and extravasation was evaluated. In FcR␥⫹/⫹ MRL/lpr mice, extravasation was observed within 3 hours at sites injected
with anti-OVA IgG, with minimal extravasation at sites
injected with control IgG (Figure 2). In contrast, extravasation was not observed in FcR␥–/– MRL/lpr mice (Figure 2) or in FcR␥–/– mice (results not shown). Histologic
Figure 2. Arthus reaction in female FcR␥–/– MRL/lpr mice. Female
FcR␥⫹/⫹ and FcR␥–/– MRL/lpr mice were injected intradermally with
rabbit anti-ovalbumin antibody (1 or 10 ␮g of IgG) or normal rabbit Ig
(10 ␮g of IgG) and, thereafter, were injected intravenously with
ovalbumin (500 ␮g) in 1% Evans blue solution. Three hours later, the
reverse side of the skin around the injection sites was evaluated for
extravasation of the Evans blue dye. Left upper and right lower
quadrants show 10 ␮g and 1 ␮g of rabbit anti-ovalbumin IgG,
respectively; left lower and right upper quadrants show 10 ␮g of
normal rabbit IgG and saline, respectively. Color figure can be viewed
in the online issue, which is available at http://www.arthritisrheum.org.
FcR-INDEPENDENT MECHANISM IN LUPUS NEPHRITIS
489
Figure 3. Glomerulonephritis in female FcR␥–/– MRL/lpr mice. A, Kidneys were removed from 24-week-old
female FcR␥⫹/⫹, FcR␥⫹/–, and FcR␥–/– MRL/lpr and C57BL/6 mice, and sections were stained with hematoxylin
and eosin. B, Deposition of IgG and C3 in the glomeruli of FcR␥–/– MRL/lpr mice. The same renal sections were
stained with fluorescein isothiocyanate–labeled anti-IgG or anti-C3 antibodies. There are deposits of IgG and C3
in the glomeruli of FcR␥–/– MRL/lpr mice. (Original magnification ⫻ 400.) Color figure can be viewed in the
online issue, which is available at http://www.arthritisrheum.org.
examination revealed that extravasation of erythrocytes
and infiltration of polymorphonuclear leukocytes were
reduced in the skin of FcR␥–/– MRL/lpr mice (data not
shown). These results proved that, as in nonautoimmune
mice (7,9), the Arthus reaction is FcR-dependent in
MRL/lpr mice.
Glomerulonephritis in MRL/lpr mice. The
FcR␥–/– MRL/lpr mice developed diffuse proliferative
GN with mononuclear cell infiltration, mesangial and
endothelial cell proliferation, and crescent formation
after 16 weeks of age. In addition, subendothelial IC
deposition in the glomeruli (17,18) was identified. Proteinuria, followed by renal failure, developed in all
FcR␥–/– MRL/lpr mice. Diffuse proliferative GN developed in both FcR␥⫹ and FcR␥–/– MRL/lpr mice. Cell
infiltration, proliferation of mesangial cells and endothelial cells, and crescent formation were observed to
similar degrees in all 3 groups of MRL/lpr mice (Figure
3A). Subendothelial deposits forming “wire-loop” lesions were also observed in MRL/lpr mice regardless of
Table 1. Severity of glomerulonephritis in FcR␥⫺/⫺ MRL/lpr mice*
Genotype
⫹/⫹
FcR␥
FcR␥⫹/⫺
FcR␥⫺/⫺
C57BL/6
MRL/lpr mice
MRL/lpr mice
MRL/lpr mice
mice
No. of
mice
Proliferative
lesion
Deposition
10
20
13
6
1.60 ⫾ 0.70
1.60 ⫾ 0.82
1.62 ⫾ 0.77
0.00 ⫾ 0.00
1.30 ⫾ 0.48
1.40 ⫾ 0.60
1.70 ⫾ 0.85
0.00 ⫾ 0.00
* Kidneys were removed from 24-week-old female mice, and sections
were stained with hematoxylin and eosin and with periodic–acid Schiff.
The severity of glomerular proliferative lesions and depositions (⬎50
glomeruli/mouse) was graded as described in Materials and Methods.
Values are the mean ⫾ SD.
Figure 4. Proteinuria in female FcR␥–/– MRL/lpr mice. Urinary proteins were measured in female FcR␥⫹/⫹ (E; n ⫽ 10), FcR␥⫹/– (‚; n ⫽
20), and FcR␥–/– (䊐; n ⫽ 10) MRL/lpr mice and in female C57BL/6 mice
({; n ⫽ 10) at 8, 16, and 24 weeks of age. Values are the mean ⫾ SD.
490
MATSUMOTO ET AL
Figure 5. Development of vasculitis in female FcR␥–/– MRL/lpr mice. A, Kidneys and lungs were removed from 24-week-old female FcR␥⫹/⫹ and
FcR␥–/– MRL/lpr and C57BL/6 mice, and sections were stained with hematoxylin and eosin. Medium-sized vessel vasculitis is present in the kidneys
and lungs of the FcR␥–/– MRL/lpr mice. B, Ear skin was removed from 24-week-old female FcR␥⫹/⫹ and FcR␥–/– MRL/lpr and C57BL/6 mice, and
sections were stained with hematoxylin and eosin. Small vessel vasculitis is present in the skin of the FcR␥–/– MRL/lpr mice. (Original magnification ⫻ 200.)
Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
FcR␥ expression. There were no significant differences
in the severity scores for the glomerular proliferative
lesions and depositions between FcR␥⫹ and FcR␥–/–
MRL/lpr mice at 24 weeks of age (Table 1) or at 8, 12,
and 16 weeks of age (data not shown). In addition,
prominent mesangial IgG and C3 depositions were
observed in FcR␥⫹/⫹ and FcR␥–/– MRL/lpr mice (Figure
3B). Both FcR␥⫹ and FcR␥–/– MRL/lpr mice developed
proteinuria at 16 weeks of age, which became severe at
24 weeks; there was no significant difference in urinary
protein excretion between these two groups (Figure 4).
These results indicate that FcR are not centrally involved in the development of IC-induced autoimmune
GN in MRL/lpr mice.
Medium-sized vessel vasculitis in the kidneys
and lungs of FcR␥–/– MRL/lpr mice. Since MRL/lpr mice
develop necrotizing vasculitis in the medium-sized arteries of the kidneys, lungs, intestines and, occasionally, the
heart (17,18), we examined the development of medium-
sized vessel vasculitis in the kidneys and lungs of the
mice.
Medium-sized vasculitis in kidneys and lungs was
found to be comparable between the FcR␥⫹ and the
FcR␥–/– MRL/lpr mice. Necrotizing vasculitis with mononuclear cell infiltration, vessel wall destruction, and
occasional thrombosis was similarly observed in the
interlobular arteries of the kidneys of both FcR␥⫹/⫹ and
FcR␥–/– MRL/lpr mice (Figure 5A). Necrotizing vasculitis of the pulmonary arteries, with mononuclear cell
infiltration and vessel wall destruction, was also observed to a similar degree in FcR␥⫹/⫹ and FcR␥–/–
MRL/lpr mice (Figure 5A). There were no significant
differences in the severity scores for vasculitis of
medium-sized vessels in the kidneys and lungs of these
mice at 24 weeks of age (Table 2), or at 8, 12, and 16
weeks of age (data not shown).
Small vessel vasculitis in FcR␥–/– MRL/lpr mice.
In addition to vasculitis of medium-sized vessels, MRL/
FcR-INDEPENDENT MECHANISM IN LUPUS NEPHRITIS
Table 2. Severity of medium-sized vasculitis in kidneys and lungs of
FcR␥⫺/⫺ MRL/lpr mice*
Genotype
⫹/⫹
FcR␥
FcR␥⫹/⫺
FcR␥⫺/⫺
C57BL/6
MRL/lpr mice
MRL/lpr mice
MRL/lpr mice
mice
No. of mice
Kidney
Lung
10
20
13
6
1.20 ⫾ 0.57
1.32 ⫾ 0.84
1.50 ⫾ 0.89
0.00 ⫾ 0.00
1.70 ⫾ 0.67
1.40 ⫾ 0.68
1.69 ⫾ 0.85
0.41 ⫾ 0.37
* Kidneys and lungs were removed from 24-week-old female mice, and
sections were stained with hematoxylin and eosin. The severity of
medium-sized vessel vasculitis in the kidneys (⬎10 interlobular arteries) and lungs (⬎ 20 pulmonary arteries) was graded as described in
Materials and Methods. Values are the mean ⫾ SD.
lpr mice also develop small vessel vasculitis in the skin.
This is frequently visible as purpuric lesions on the ears
and tails. Small vessel vasculitis was observed in the skin
of both FcR␥⫹ and FcR␥–/– MRL/lpr mice. Histologic
examination of the skin of the ears revealed leukocytoclastic vasculitis with polymorphonuclear leukocyte infiltration and erythrocyte extravasation in FcR␥⫹ and
FcR␥–/– MRL/lpr mice (Figure 5B). There were no
significant differences in the severity scores for small
vessel vasculitis in the skin of these mice at 24 weeks of
age (Table 3).
Survival of FcR␥–/– MRL/lpr mice. MRL/lpr mice
exhibit early mortality due to renal failure. We therefore
compared the survival rates in the FcR␥⫹ MRL/lpr mice
with those in the FcR␥–/– MRL/lpr mice; no significant
difference was found (Figure 6). By 32 weeks of age,
50% of the FcR␥–/– mice had died (n ⫽ 8), whereas 44%
of the FcR␥⫹/⫹ (n ⫽ 9) and 42% of the FcR␥⫹/– (n ⫽
12) mice had died. The median survival rates were 34,
34, and 32 weeks of age in the FcR␥⫹/⫹, FcR␥⫹/–, and
FcR␥–/– MRL/lpr mice, respectively.
IgG, anti-DNA antibody, and circulating immune
complex levels in FcR␥–/– MRL/lpr mice. It has been
shown that autoantibody production, including antiDNA antibodies, and IC formation are linked with renal
diseases in MRL/lpr mice (17,18). It has also been
reported that FcR are involved in the clearance of ICs
491
(19,20). Therefore, we measured the levels of serum
IgG, anti-dsDNA antibody, and CICs in FcR␥⫹ and
FcR␥–/– MRL/lpr mice at 24 weeks of age.
Serum IgG levels in FcR␥⫹/⫹ (mean ⫾ SD 1,270 ⫾
501 mg/dl, n ⫽ 8) and FcR␥⫹/– (1,253 ⫾ 510 mg/dl, n ⫽ 19)
MRL/lpr mice were significantly elevated compared with
the level in control C57BL/6 mice (406 ⫾ 46 mg/dl, n ⫽ 6)
(P ⬍ 0.005) (Figure 7). IgG levels in FcR␥–/– MRL/lpr mice
were also elevated (1,243 ⫾ 510 mg/dl, n ⫽ 9) (Figure 7).
There were no differences in the serum levels of IgG1,
IgG2a, and IgG3 among the 3 groups of FcR␥ MRL/lpr
mice (data not shown). Anti-DNA antibody level in the
serum of FcR␥–/– MRL/lpr mice (mean ⫾ SD 0.619 ⫾
0.594 at OD405, n ⫽ 9) was increased to a similar degree as
the levels in the FcR␥⫹/⫹ (0.514 ⫾ 0.470, n ⫽ 8) and
FcR␥⫹/– (0.711 ⫾ 0.661, n ⫽ 19) MRL/lpr mice, while the
C57BL/6 mice showed background levels (0.030 ⫾ 0.008,
n
⫽
6)
(Figure 7).
CIC levels were significantly elevated in FcR␥⫹ and
–/–
FcR␥ MRL/lpr mice compared with control C57BL/6
mice (Figure 7); however, there were no significant differences among the FcR␥–/– (1.029 ⫾ 0.524 at OD405, n ⫽ 9),
FcR␥⫹/⫹ (1.201 ⫾ 0.661, n ⫽ 8), and FcR␥⫹/– (0.925 ⫾
0.439, n ⫽ 19) MRL/lpr mice, suggesting that the clearance
of ICs in FcR␥–/– MRL/lpr mice was not very different from
that in the FcR␥⫹ MRL/lpr mice.
DISCUSSION
In this study, we show that Fc receptors are not
essential for the development of autoimmune glomerulonephritis and vasculitis in lupus-prone MRL/lpr mice.
Furthermore, we suggest that an FcR-independent
Table 3. Severity of small vessel vasculitis in the skin of FcR␥⫺/⫺
MRL/lpr mice*
Genotype
⫹/⫹
FcR␥
FcR␥⫹/⫺
FcR␥⫺/⫺
C57BL/6
MRL/lpr mice
MRL/lpr mice
MRL/lpr mice
mice
No. of mice
Skin vasculitis
7
11
8
6
0.86 ⫾ 0.69
1.20 ⫾ 1.13
1.15 ⫾ 0.98
0.00 ⫾ 0.00
* Ear skin was removed from 24-week-old female mice, and sections
were stained with hematoxylin and eosin. The severity of skin vasculitis
was graded as described in Materials and Methods. Values are the
mean ⫾ SD.
Figure 6. Survival rate of female FcR␥–/– MRL/lpr mice. Survival
rates were compared among female FcR␥⫹/⫹ (n ⫽ 9), FcR␥⫹/– (n ⫽
12), and FcR␥–/– (n ⫽ 8) MRL/lpr mice and in female C57BL/6 mice
(n ⫽ 10).
492
MATSUMOTO ET AL
Figure 7. Serum levels of IgG, anti-DNA antibody, and circulating immune complexes (CICs) in female FcR␥–/–
MRL/lpr mice. Levels of IgG, anti-DNA antibody, and CIC were determined by immunodiffusion, enzyme-linked
immunosorbent assay, and C1q binding assay, respectively, in female FcR␥⫹/⫹ (n ⫽ 8), FcR␥⫹/– (n ⫽ 19), and
FcR␥–/– (n ⫽ 9) MRL/lpr mice and in female C57BL/6 mice (n ⫽ 6) at 24 weeks of age. Horizontal lines show the
mean for each group. Ab ⫽ antibody; OD405 ⫽ optical density at 405 nm.
mechanism plays an important role in the development
of IC-induced autoimmune GN and vasculitis in these
mice. We found that both FcR␥⫹ and FcR␥–/– MRL/lpr
mice developed diffuse proliferative GN with depositions of IgG and C3. The survival rates and degree of
proteinuria did not differ significantly between these two
groups of MRL/lpr mice. Moreover, there were no
significant differences in the levels of serum IgG, antiDNA antibody, and CIC between these mice, suggesting
that the generation of autoantibodies and the clearance
of ICs in MRL/lpr mice are independent of FcR␥. We
also found that necrotizing vasculitis in the mediumsized arteries of the kidneys and lungs as well as small
vessel vasculitis in the skin developed in both FcR␥⫹ and
FcR␥–/– MRL/lpr mice to almost the same extent. In
contrast, the Arthus reaction was not induced in FcR␥–/–
MRL/lpr mice, as has been observed in FcR␥–/– nonautoimmune mice (7,9). Collectively, these results indicate
that autoimmune GN and vasculitis develop in MRL/lpr
mice in an FcR-independent manner.
FcR␥–/– mice lack the expression of the activating
Fc␥ receptors (Fc␥RI and Fc␥RIII) but retain the
inhibitory Fc␥ receptor (Fc␥RII). The fact that the
development of GN and vasculitis was not altered in
FcR␥–/– MRL/lpr mice indicates that Fc␥RII alone does
not modulate these diseases. Together with the observation that Fc␥RIIB knockout mice exhibit augmented GN
(21), the inhibitory function may operate together with
the FcR␥-mediated inducing function.
It has been shown that most cases of GN are
induced by immune mechanisms. There are 2 representative types of immunologically induced GN: one occurs
via the emergence of anti-GBM antibody (anti-GBM–
induced GN), and the other occurs via the deposition of
ICs on the wall of glomerular capillaries (IC-induced
GN, including SLE). Our previous study using FcR␥–/–
mice revealed that FcR⫹ cells were required to initiate
inflammatory processes and tissue damage in antiGBM–induced GN (6). A recent study by Clynes et al
(14) demonstrated that IC-induced autoimmune GN in
(NZB ⫻ NZW)F1 mice was diminished in the FcR␥–/–
variety, suggesting that the inflammatory process
through the activation of FcR-positive cells is essential
for the induction of autoimmune GN in (NZB ⫻
NZW)F1 mice. In marked contrast, we demonstrate here
that FcR are not essential for the development of
autoimmune GN in MRL/lpr mice. This observation is
consistent with our previous finding that severe proliferative GN is induced even in FcR␥-deficient mice by
the administration of MRL/lpr-derived monoclonal antibody, which is an IgG3 anti-IgG2a autoantibody with
cryoglobulin activity (16).
Our results clearly indicate that an FcRindependent mechanism is responsible for the development of IC-induced autoimmune GN in MRL/lpr mice.
However, the molecular and cellular mechanisms involved in this process in this mouse are not well understood (17,18,22). Several studies have suggested the
important role played by the cryoglobulin activity of
IgG3 autoantibodies, which do not bind to Fc␥R (20,23),
in the development of GN in this mouse. It has been
shown that MRL/lpr mouse–derived IgG3 autoantibodies, which have a unique property of self-assembly
through nonspecific IgG3 Fc–Fc interactions and gener-
FcR-INDEPENDENT MECHANISM IN LUPUS NEPHRITIS
ate monoclonal cryoglobulins (24,25), induce “wireloop” lesions resembling those in GN in nonautoimmune mice (26–28). It has also been reported that
IgG3 antibody without cryoglobulin activity inhibits GN
induced by IgG3 antibody with cryoglobulin activity
(29). Therefore, IgG3 autoantibodies with cryoglobulin
activity may contribute to the development of GN in
MRL/lpr mice in an FcR-independent manner. Although we could not measure cryoglobulin levels, the
mean ⫾ SD serum IgG3 levels were not significantly
different among the FcR␥⫹/⫹ (313 ⫾ 127), FcR␥⫹/–
(306 ⫾ 123), and FcR␥–/– (346 ⫾ 109) MRL/lpr mice.
It remains to be clarified whether IC-mediated
medium-sized vessel vasculitis is induced by FcRdependent mechanisms. The present study demonstrated that FcR are not essential for the development of
medium-sized vessel vasculitis in MRL/lpr mice. In the
case of (NZB ⫻ NZW)F1 mice, unfortunately, Clynes et
al (14) did not address the role of FcR in this type of
vasculitis. Consistent with our results, Chan et al (30)
demonstrated that mutant MRL/lpr mice with defective
B cells lacking Ig secretion developed medium-sized
vessel vasculitis. Taking the findings together, antibodyand FcR-independent cellular mechanisms appear to be
essential for the development of medium-sized vessel
vasculitis in MRL/lpr mice.
We also demonstrated that an FcR-independent
mechanism plays an important role in the development
of small vessel vasculitis in MRL/lpr mice. This result
contradicts the findings of our previous study that
pointed to the involvement of FcR-dependent mechanisms in the development of skin vasculitis in an experimental system using MRL/lpr-derived autoantibody
(16). In that system, we found that skin vasculitis was
induced in FcR␥⫹ mice, but not in FcR␥–/– mice, by the
administration of IgG3 monoclonal autoantibody derived from MRL/lpr mice and possessing anti-IgG2a
rheumatoid factor activity (16).
It has previously been shown that IgG3 autoantibodies with both cryoglobulin and rheumatoid factor
activities induce small vessel vasculitis as well as GN,
whereas those with cryoglobulin activity alone induce
only GN and not vasculitis (31). Therefore, the amount
of autoantibodies (in vivo continuous production for an
FcR-independent mechanism versus exogenous administration for an FcR-dependent pathway), the nature and
specificity of autoantibodies (cryoglobulin activity versus
rheumatoid factor activity), the subclass of autoantibodies (IgG3 alone versus IgG3 plus other subclasses), and
the size and extent of IC accessibility of autoantibodies
to the tissue (glomerular capillaries versus skin vessels)
493
may contribute to the development of GN and skin
vasculitis in MRL/lpr mice. Indeed, the administration of
Ig-binding peptide, which also inhibits FcR binding in
vitro, was shown to decrease GN in MRL/lpr mice (32).
This could be attributed to the alteration of the positive
charge of ICs by peptide binding and possibly of Ig
binding on FcR (32).
Thus, our results indicate that the in vivo induction
of skin vasculitis in MRL/lpr mice is predominantly FcRindependent, although both FcR-independent and FcRdependent mechanisms exert effects on the development
of skin vasculitis in MRL/lpr mice. The FcR-dependent
regulation may operate under some limited conditions, the
administration of IgG3 monoclonal antibody with rheumatoid factor activity being one example (16).
With regard to the difference between our results
and the report by Clynes et al (14) of the FcR dependency on the induction of GN, in addition to the various
differences between MRL/lpr and (NZB ⫻ NZW)F1
mice, the possibility has not been formally excluded that
the NZW-based Sle-1 gene locus (33,34), which is critical for the development of SLE in (NZB ⫻ NZW)F1
mice but not in MRL/lpr mice and has very close linkage
with the Fc␥R locus, could be altered by homologous
recombination. A definitive answer requires further
analysis of this locus in our mice.
The present genetic analysis presents clear evidence that the induction of GN and vasculitis is mediated by both FcR-dependent and FcR-independent
mechanisms, probably depending on the nature of the
ICs. Cryoglobulin-type autoantibody with low affinity
for antigen may induce these diseases in an FcRindependent manner, and high-affinity IgG autoantibody produced in acquired immune responses may
trigger these diseases in an FcR-dependent way.
The FcR-independent mechanism responsible
for IC-induced autoimmune GN and vasculitis in MRL/
lpr mice remains to be elucidated. Because it has been
shown that mice deficient in complement C5a receptor
exhibit an impaired Arthus reaction (35), activation of
the complement system by IgG3 ICs may be involved in
the development of GN and vasculitis in MRL/lpr mice.
In addition, cryoglobulin deposits may be recognized as
a foreign body by phagocytes, including macrophages
and mesangial cells, and may induce the production of
various inflammatory mediators by the phagocytes (36).
In summary, we have shown that FcRindependent mechanisms play a dominant role in the
development of autoimmune GN and vasculitis in lupusprone MRL/lpr mice. Clarification of the cellular and
molecular mechanisms involved in FcR-independent
494
MATSUMOTO ET AL
tissue damage may lead to new strategies for the treatment of autoimmune GN and vasculitis in SLE.
ACKNOWLEDGMENT
We thank Dr. S. Izui for discussion and Ms H.
Yamaguchi for secretarial assistance.
19.
20.
21.
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