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Mesangial cell complement receptor 1-related protein y limitscomplement-dependent neutrophil accumulation in immune complex
glomerulonephritis
Introduction
The complement system is tightly regulated by more than
30 plasma and cell membrane proteins to protect self
tissue from injury.1 In humans, decay-accelerating factor
(DAF; CD55) and membrane cofactor protein (MCP;
CD46) are the main membrane-bound intrinsic regulators
of C3 activation. In rodents, complement receptor 1
(CR1) related gene/protein y (Crry) has combined decay-
accelerating and factor I cofactor activity for C3b and
C4b; therefore, it combines functionalities of both DAF
and MCP.2
It has been shown using transgenic and recombinant
protein techniques that Crry is protective in different
immune complex-mediated kidney diseases.3–6 Deficiency
of Crry led to early embryonic lethality as a result of
unrestricted complement activation and concomitant pla-
cental inflammation, which could be averted by coexisting
C3 deficiency in Crry)/) C3)/) mice.7 Because of the
resultant C3 deficiency, the role of Crry in protecting
Lihua Bao,1 Ying Wang,1 Peili
Chen,1 Menaka Sarav,1 Mark Haas,2
Andrew W. Minto,1 Miglena
Petkova1 and Richard J. Quigg1
1Section of Nephrology, The University of
Chicago, Chicago, IL, and 2Department of
Pathology, Johns Hopkins University School
of Medicine, Baltimore, MD, USA
doi:10.1111/j.1365-2567.2009.03102.x
Received 16 January 2009; revised 6 March
2009; accepted 11 March 2009.
Correspondence: L. Bao, MD, Section of
Nephrology, The University of Chicago, 5841
S. Maryland Avenue, MC5100 Chicago, IL
60637, USA. Email:
Senior author: Richard J. Quigg,
email: [email protected]
Summary
The absence of complement receptor 1 (CR1) related gene/protein y
(Crry) leads to embryonic lethality as a result of unrestricted complement
activation and concomitant neutrophil infiltration. Here we used Crry)/)
C3+/) mice to investigate the role of Crry in the pathogenesis of immune
complex glomerulonephritis (GN). After 3 weeks of immunization with
horse spleen apoferritin, six of nine Crry)/) C3+/) mice and none of the
six control C3+/) mice developed proliferative GN (P = 0�010). After
5 weeks of immunization, GN scores in Crry)/) C3+/) mice were
0�67 ± 0�22 mean ± standard error of the mean (SEM), compared with
0�32 ± 0�16 in C3+/) mice. Glomerular hypercellularity was attributable to
neutrophil infiltration in mice with GN (1�7 ± 0�3/glomerulus) compared
with those without GN (0�4 ± 0�1/glomerulus) (P = 0�001). Absent stain-
ing for a-smooth muscle actin and proliferating cell nuclear antigen sug-
gested that mesangial cell proliferation did not play a significant role in
this model. Serum C3 levels in Crry)/) C3+/) mice were approximately
20% and 30% those of wild-type mice and C3+/) mice, respectively. To
determine whether this acquired hypocomplementaemia was relevant to
this GN model system, Crry)/) C3+/) mouse kidneys were transplanted
into wild-type mice followed by immunization with apoferritin for 1 or
2 weeks. Surprisingly, none of the Crry)/) C3+/) mouse kidneys developed
GN at these early time-points, indicating that increasing circulating C3
levels several-fold did not increase susceptibility to GN. Renal expression
of decay-accelerating factor was not different among any of the groups
studied. Thus, our data indicate that mesangial cell Crry limits comple-
ment activation and subsequent neutrophil recruitment in the setting of
local immune complex deposition.
Keywords: complement; inflammation; knockout mice
Please cite this article in press as: Bao L. et al. Mesangial cell complement receptor 1-related protein y limits complement-dependent
neutrophil accumulation in immune complex glomerulonephritis, Immunology (2009) doi: 10.1111/j.1365-2567.2009.03102.x
Abbreviations: CR, complement receptor; Crry, CR1-related gene/protein y; GN, glomerulonephritis; hpf, high-power field; IF,immunofluorescence.
� 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904 e895
I M M U N O L O G Y O R I G I N A L A R T I C L E
kidneys from complement activation cannot be readily
determined in unmanipulated DKO (Crry)/)C3)/)) mice.
We previously generated a Crry-deficient mouse kidney
model by transplanting Crry)/) C3)/) kidneys into Crry-
and C3-sufficient wild-type mice. Without the potent
complement-regulating functions of Crry, spontaneous
unrestricted complement activation occurred in the
tubulointerstitium of Crry-deficient kidneys, which
quickly led to fibrosis and renal failure.8
Other groups have utilized creative approaches to gen-
erate Crry-deficient mice, exploiting the fact that fetal
lethality is attributable to maternal alternative pathway
activation and formation of proinflammatory C5 prod-
ucts.9–11 In one such instance, it was interesting that
Crry)/) C3+/) mice showed substantial alternative path-
way turnover which led to reduced C3 concentration sys-
temically, suggesting that Crry has a critical role in
maintaining homeostasis of the complement system.10
While both Crry- and factor H-deficient mice have con-
siderable systemic complement consumption, the former
do not develop de novo renal pathology as do factor
H-deficient mice, suggesting either functional differences
between these two important complement regulators in
restricting complement activation within the kidney, and/
or fundamental differences between the two strains.11,12
The current study focuses on the role of Crry deficiency
in the pathogenesis of glomerulonephritis (GN).
In addition to the tubulointerstitium of the kidneys,
Crry is also present in the mouse glomerular mesan-
gium.13,14 In distinct contrast to the tubulointerstitium
Crry, the mesangial cell Crry appears to be of little con-
sequence in normal mice.8 To examine its relevance when
there are immune complex deposits, we utilized a chronic
serum sickness model induced with horse spleen apoferr-
itin in native Crry)/) C3+/) mice,15,16 as well as Crry)/)
C3+/) kidneys transplanted into wild-type mice.
Materials and methods
Animal work protocols
Crry+/) and C3+/) mice were kindly provided by Dr Hec-
tor Molina (Washington University School of Medicine,
St Louis, MO). The C57BL/6 mice used in the backcross-
ing procedure were purchased from Harlan Sprague Daw-
ley (Indianapolis, IN). Crry+/) and C3+/) mice had been
backcrossed onto the C57BL/6 background for at least
eight generations. Crry+/) and C3+/) mice were inter-
crossed to generate Crry)/) C3)/) mice, followed by cross-
ing with C3+/) mice to generate the Crry)/) C3+/)
animals employed in these studies. As controls, C3+/)
mice on a C57BL/6 background were used.
Experimental serum sickness was induced by daily
intraperitoneal injections of 4 mg of horse spleen apoferr-
itin (Calzyme Laboratories, Inc., San Luis Obispo, CA).
Crry)/) C3+/) mice were studied after 3 weeks (n = 9)
and 5 weeks (n = 9), and control C3+/) mice received the
same injection schedule for 3 weeks (n = 6) and 5 weeks
(n = 8). At the termination of the study, all mice were
killed and the kidneys were harvested for further studies.
These animal experiments were approved by the Univer-
sity of Chicago Animal Care and Use Committee.
Kidneys from a separate group of Crry)/) C3+/) mice
were transplanted into normal C57BL/6 mice (n = 9)
according to a protocol described previously.17 In brief,
donor mice were anaesthetized and the donor left kidney
was removed with artery, vein and ureter attached, and
preserved in cold saline on ice. The recipient was then
anaesthetized and the left kidney was excised. End-to-side
anastomoses of the donor renal vein, artery and ureter to
the recipient inferior vena cava, aorta and bladder, respec-
tively, were performed. Mice were allowed to recover
from the surgery for about 1 week. Serum sickness was
then induced for 1 week (n = 3) and 2 weeks (n = 3), as
described above. Controls were similarly transplanted
mice receiving saline instead of apoferritin (n = 3).
Measurements of blood urea nitrogen (BUN) andurinary albumin
BUN concentrations were detected with a Beckman
Autoanalyzer (Beckman Coulter, Fullerton, CA). Urinary
albumin concentrations were measured with a mouse
albumin enzyme-linked immunosorbent assay (ELISA) kit
(Bethyl Laboratories, Montgomery, TX) and normalized
to urinary creatinine as described previously.18
Measurements from tissue
To evaluate renal pathological changes, kidney tissues
were fixed in 4% paraformaldehyde and embedded in
paraffin. Four-lm sections were stained with periodic
acid-Schiff and examined by a renal pathologist (MH) in
a blinded manner. For each slide, the severity of GN,
glomerulosclerosis, interstitial nephritis, and arteritis was
graded in a semiquantitative (0–4) manner as previously
described.4
For immunofluorescence (IF) microscopy, 4-lm cryo-
stat sections were fixed in ether-ethanol and stained with
fluorescein isothiocyanate (FITC)-conjugated antibodies
to mouse C3 and immunoglobulin G (IgG) (Cappel Phar-
maceuticals, Inc., Aurora, OH). A semiquantitative score
of glomerulus staining intensity and distribution from 0
to 4 was given in a blinded manner as described previ-
ously.18 To visualize the co-localization of these different
targets, FITC-conjugated anti-mouse C3 (shown in green)
and tetramethyl rhodamine isothiocyanate (TRITC)-con-
jugated anti-mouse IgG (shown in red) (Cappel Pharma-
ceuticals) were used on the same renal sections from
different groups of mice.
e896 � 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904
L. Bao et al.
To assess different renal-infiltrating cells, immunohisto-
chemistry was performed on 4% paraformaldehyde-fixed
and paraffin-embedded kidney sections. Antigen was
retrieved by heating slides in a citrate buffer (pH 6�0) in
a microwave oven for 10 min. Endogenous peroxidases
and biotin were blocked using 0�3% hydrogen peroxide
and the Avidin/Biotin Blocking Kit (Vector Laboratories,
Burlingame, CA). Normal goat serum (10%, volume/vol-
ume) was also used as a separate blocking step. The slides
were then incubated with rat anti-mouse neutrophil
(AbD Serotec, Oxford, UK), rat anti-mouse B220 (BD
Pharmingen, San Jose, CA) for B cells, rat anti-mouse
Thy-1�2 (AbD Serotec) for T cells, or rat anti-mouse F4/
80 (AbD Serotec) for macrophages/monocytes, followed
by mouse anti-rat IgG (BD Pharmingen) and streptavi-
din-peroxidase (Sigma, St Louis, MO). Specifically bound
antibodies (Abs) were detected using the 3, 30-diamino-
benzidine (DAB) Substrate Kit (Vector Laboratories). To
quantify each type of leucocyte, at least 20 glomeruli or
20 high-power fields (hpfs) (·400) of the renal cortex in
each mouse kidney section were examined in a blinded
manner.
Proliferating cell nuclear antigen (PCNA) and
a-smooth muscle actin (a-SMA) were used as markers of
mesangial cell proliferation and activation.19,20 These were
identified in tissues stained for neutrophils as follows.
Paraffin-embedded slides were first stained with anti-
mouse neutrophil Abs, as described above. Following
DAB development (generating a brown colour), the per-
oxidases in the first staining were blocked with 0�3%
hydrogen peroxide. Slides were subsequently incubated
with rabbit anti-PCNA (Santa Cruz Biotechnology, Santa
Cruz, CA), or anti-a-SMA (US Biological, Swampscott,
MA). Ab staining was then detected with the ImmPress
Reagent (Vector Laboratories) according to the manufac-
turer’s instructions. The VIP Substrate Kit (Vector Labo-
ratories) (generating a purple colour) was used as a
substrate. All sections were counter-stained with methyl-
green (Vector Laboratories).
Serum C3 ELISA
Serum C3 levels were determined by ELISA. Plates were
first coated with goat anti-mouse C3 (Cappel Laborato-
ries, Durham, NC). Serially diluted serum samples were
added followed by horseradish peroxidase (HRP)-goat
anti-mouse C3 (Cappel). Serum C3 levels from normal
C57BL/6 (n = 9), C3)/) (n = 9), C3+/) (n = 9) and
Crry)/) C3+/) (n = 9) mice were measured. To evaluate
whether systemic consumption of C3 occurred during
development of serum sickness, serum samples at the end
of the respective experiments were also measured. Results
are expressed as relative optical density (OD450) values
adjusted for standard wild-type mouse serum samples
included in all ELISA plates.
Western blotting
Western blotting was used to detect DAF in kidney tissue.
Frozen renal cortical tissue protein was obtained and
quantified as described previously.21 Equal amounts of
protein samples were reduced and separated by sodium
dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS-PAGE) following electrophoretic transfer to a poly-
vinylidene difluoride membrane (Millipore, Bedford,
MA). Membranes were blocked with 5% non-fat milk for
1 hr at room temperature, and incubated with rabbit
anti-DAF Ab (Santa Cruz Biotechnology) followed by
HRP-anti-rabbit Ab (Cell Signaling Technology, Danvers,
MA). Renal tissue samples from DAF-deficient mice were
used as negative controls. Membranes were also probed
with anti-actin antibody (Sigma) to confirm equal loading
of samples.
Statistical analyses
All data are expressed as mean ± standard error of the
mean (SEM) and were analysed using MINITAB software
(Minitab Inc., State College, PA). Two-sample t-tests and
Kruskal–Wallis tests were used for parametric and non-
parametric data analysis of differences between two
groups. For comparisons for data obtained from different
groups and at different time-points, analysis of variance
(ANOVA) followed by Tukey’s pairwise comparisons was
used. The v2 test was used to analyse the difference of the
incidences between the two groups. Potential correlations
among variables were examined using the Pearson prod-
uct moment correlation coefficient.
Results
In normal C57BL/6 mice, repetitive active immunization
with heterologous horse spleen apoferritin leads to gener-
ation of chronic serum sickness and the progressive
glomerular deposition of immune complexes primarily
within mesangial regions. Because Crry is the primary
complement regulator in mesangial cells, we were inter-
ested in whether its absence would affect pathological
manifestations of glomerular disease. Thus, we induced
chronic serum sickness in Crry)/) C3+/) mice. As the
most appropriate controls for these animals, a group of
C3+/) mice was studied concurrently. Because of the time
dependence of disease manifestation, mice were studied
after either 3 or 5 weeks of disease.
As expected, C3+/) mice had IgG (presumably in
immune complexes) in mesangial regions after 3 weeks of
apoferritin immunization; although the amounts of IgG
were high, they were even higher after 5 weeks. C3 depo-
sition tracked with mesangial immunoglobulin deposits,
and increased similarly with time (Fig. 1). Despite the
apparent complement activation, none of the six mice at
� 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904 e897
Crry in IC glomerulonephritis
the 3 weeks and three of the eight mice at 5 weeks devel-
oped evidence of GN.
Crry)/) C3+/) mice developed mesangial deposition of
IgG after 3 and 5 weeks of apoferritin administration,
and this deposition did not differ from that found in
C3+/) mice (Fig. 1a). At 3 weeks, C3 deposition was
greater in Crry)/) C3+/) mice than in C3+/) mice
(P = 0�022), at which time six of nine Crry)/) C3+/)
mice developed histological features of GN (Fig. 2a)
(P = 0�01 versus C3+/) mice). After 5 weeks of immuni-
zation, C3 staining was further increased in Crry)/)
C3+/) mice (Fig. 1b), with six of nine Crry)/) C3+/)
mice developing GN (Fig. 2a). The conclusion that C3
deposition was relevant to the development of GN was
supported by the correlation between C3 staining and
GN scores (r = 0�558, P = 0�001). Consistent with the
lack of significant tubulointerstitial or glomerular capil-
lary wall/podocyte disease, there was no significant ele-
vation or difference in BUN and urinary albumin
values among the groups.
C3 IgG Merge
Crry –/–C3 +/–
Crry–/–C3+/–
(3 weeks, n = 9)
Crry–/–C3+/–
(5 weeks, n = 9)
(3 weeks, n = 6)
C3+/–
(5 weeks, n = 8)C3+/–
C3+/–
C3 C3IgG IgG
3 weeks 5 weeks
0
1
2
3
4
0·5
1·5
2·5
3·5
*
IF in
tens
ity
(a)
(b)
Figure 1. Immunopathological features in
complement receptor 1 (CR1)-related gene/
protein y (Crry)-deficient mice with serum
sickness. (a) Representative immunofluores-
cence (IF) photomicrographs show co-localiza-
tion of glomerular deposits containing mouse
C3 and immunoglobulin (IgG) in Crry)/) C3+/)
mice compared with control C3+/) mice. (b)
Semiquantitative data for IF intensity of glomer-
ular C3 and IgG staining are also shown. Magni-
fication, ·600. *P = 0�022 versus C3+/) mice.
e898 � 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904
L. Bao et al.
The scores for GN were based on increased numbers of
cells within glomeruli, without specifically determining
their origins. As these cells could have included intrinsic
glomerular cells and extrinsic blood-derived inflammatory
cells, we enumerated these cell types in glomeruli. There
was no evidence for intrinsic glomerular cell proliferation
or injury (i.e. PCNA+ or a-SMA+ cells, respectively) in
any of the C3+/) and Crry)/) C3+/) mice immunized with
apoferritin. Furthermore, there were few B or T lympho-
cytes or F4/80+ monocytic cells within glomeruli or the
tubulointerstitium in any of the groups of animals. In
contrast, there were significant numbers of neutrophils in
glomeruli (Fig. 2a); glomerular neutrophil numbers were
highly correlated with GN scores (r = 0�879, P = 0�000),
consistent with the premise that this was an exudative
GN model. At 3 weeks, glomerular neutrophil numbers
were significantly greater in Crry)/) C3+/) mice compared
with C3+/) mice (Fig. 2b). These data demonstrate that
the lack of complement regulation by absent mesangial
cell Crry allowed a greater influx of neutrophils in glome-
ruli in the presence of potentially inflammatory immune
complexes.
We were next interested in the systemic complement
status of the two groups of mice. It was previously
reported that Crry)/) C3+/) mice had a systemic C3 level
that was about 20% that of wild-type mice;10,11,22 we
extended these results by evaluating the C3+/) controls
used here. As shown in Fig. 3, C3+/) mice had C3 levels
approximately half those of wild-type controls. Crry)/)
C3+/) mice had even greater reductions in C3 levels to
approximately 20% and 30% those of wild-type and
C3+/) mice, respectively. Serum C3 levels were affected by
neither the gender of the mice nor the presence or
absence of apoferritin immunization.
In studies to determine whether an increase in systemic
C3 levels would affect development of GN in Crry)/)
C3+/) mice, we transplanted kidneys from Crry)/) C3+/)
mice into C3-sufficient wild-type animals. In our previous
studies of Crry-deficient kidneys transplanted into wild-
Serum sickness 3 weeks 5 weeks
PAS
Neutrophils+
a-SMA
GN = 0
GN = 1
GN = 2
GN = 0·5
0
1
2 *
3
4
5
Neu
trop
hils
/glo
mer
ulus
C3 +/– Crry–/–C3 +/–
C3+/– (n = 9) C3+/– (n = 9)Crry–/–C3+/– (n = 6) Crry–/–C3+/– (n = 8)
3 weeks
C3 +/– Crry –/–C3+/–
5 weeks
(a)
(b)
Figure 2. Complement receptor 1 (CR1)-
related gene/protein y (Crry) deficiency
exacerbates development of proliferative
glomerulonephritis (GN) and glomerular
inflammation with neutrophils. (a) Representa-
tive histology and double-stain immunohisto-
chemistry showing the development of GN and
neutrophil influx (stained brown), but not
a-smooth muscle actin (a-SMA)-positive mes-
angial cells (stained purple), in glomeruli in
different groups of mice. (b) Quantitative data
for glomerular neutrophils relative to GN score
in individual mice from each group are shown.
There were significantly more glomerular neu-
trophils in Crry)/) C3+/) mice compared with
control C3+/) mice. *P = 0�024 versus C3+/)
mice. PAS, periodic acid Schiff.
0
0·05
0·1
0·15
*
*
0·2
0·25
Ser
um C
3 (O
D45
0)
Serum sickness (week) 0 0 3 5 0 3 5
WT
(n = 9) (n = 23) (n = 26)C3+/– Crry–/–3+/–
Figure 3. C3 levels are significantly reduced in complement receptor
1 (CR1)-related gene/protein y (Crry))/) C3+/) mice. C3 serum levels
were measured by enzyme-linked immunosorbent assay (ELISA) in
wild-type (WT), C3+/) and Crry)/) C3+/) mice. C3 levels from mice
of different genders and with or without serum sickness were com-
parable. *P < 0�01 versus the other two groups.
� 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904 e899
Crry in IC glomerulonephritis
type hosts, we observed a marked tubulointerstitial
inflammation which led to failure of the majority of kid-
neys within 3 weeks of transplantation.8 Notably, in these
kidneys, there was no evident glomerular pathology in
spite of the absence of Crry. Thus, we studied kidneys
after 1 and 2 weeks of chronic serum sickness, with the
hypothesis that their exposure to roughly fivefold higher
circulating C3 levels would represent a further challenge
to mesangial cells lacking Crry, and lead to severe GN.
This reasonable hypothesis turned out to be incorrect, as
none of the Crry)/) C3+/) kidneys developed GN after 1
or 2 weeks of apoferritin immunization (n = 3 each),
although tubular complement activation and damage were
evident (Fig. 4a). Consistent with our previous studies,
there were also no glomerular disease features in trans-
planted kidneys in control animals receiving saline
(n = 3). The native wild-type kidneys left in situ were also
analysed as controls for the transplanted Crry)/) C3+/)
kidneys. After 1 or 2 weeks of immunization, native wild-
type and transplanted Crry)/) C3+/) kidneys had compa-
rable glomerular IgG and C3 deposition (Fig. 4b),
although this deposition was at lesser extend compared
with that found at 3 weeks, as described above. As in
transplanted Crry)/) C3+/) kidneys, none of the native
wild-type kidneys developed GN after 1 or 2 weeks of
immunization.
Control (n = 3) 1 week (n = 3) 2 weeks (n = 3)
C3
IgG
PAS
0
1
2
3
0·5
1·5
2·5
3·5
IF In
tens
ity
C3 IgG
1 week
C3 IgG
2 weeks
Transplanted Crry–/–C3+/– kidney
Native WT kidney
(a)
(b)
Figure 4. Normalizing serum C3 levels induced tubular complement activation and damage but did not increase susceptibility to serum sickness-
induced glomerulonephritis (GN) in complement receptor 1 (CR1)-related gene/protein y (Crry))/) C3+/) mouse kidneys transplanted into wild-
type (WT) mice. (a) Prominent tubular deposits of C3 were observed in Crry)/) C3+/) mouse kidneys (white arrows) transplanted into wild-type
mice, which led to inflammation and tubular damage (black arrows). After 1 or 2 weeks of apoferritin immunization, none of the Crry)/) C3+/)
mouse kidneys developed proliferative GN. (b) Semiquantitative data showing immunofluorescence (IF) staining intensity for C3 and immuno-
globulin G (IgG) in glomeruli from transplanted Crry)/) C3+/) kidneys as well as the native wild-type kidneys. PAS, periodic acid Schiff.
e900 � 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904
L. Bao et al.
As described previously, Crry)/) C3+/) mouse kidneys
transplanted into wild-type mice had spontaneous com-
plement activation in the tubulointerstitium and consid-
erable tubular injury and inflammatory cell infiltration
(Fig. 4a); a composite ‘injury’ score for these variables in
the nine transplanted kidneys (2–3 weeks post-transplant)
was 1�2 ± 0�1. This was lower than we had previously
found in Crry)/) C3)/) kidneys transplanted into wild-
type mice (e.g. 1�7 ± 0�4 within 1 week after trans-
plantation). Although these data sets are not directly
comparable, it is possible that the presence of C3 in
Crry)/) C3+/) mice may have conferred relative resistance
to complement-mediated kidney injury when transplanted
into C3-sufficient hosts.
One plausible explanation for why Crry)/) C3+/) mouse
kidneys transplanted into wild-type hosts were relatively
resistant to both GN and tubulointerstitial disease was
compensation by another complement regulator. As DAF
is the other cell-bound C3 regulator in mice, we examined
its protein abundance in the various kidneys. However,
DAF protein quantities were not increased in Crry-
deficient kidneys from unmanipulated animals (Fig. 5),
nor in the setting of chronic serum sickness, in which
there was probably generation of complement activation
products capable of up-regulating DAF expression.23
Thus, a compensatory increase in cellular complement
regulation was an unlikely explanation for these results.
Discussion
Complement activation has long been considered as
pathogenic in many different forms of immune complex-
mediated GN diseases, including lupus nephritis,
membranoproliferative GN, postinfectious GN and mem-
branous nephropathy.24 Consistent with such a premise,
inhibition of complement activation by the provision of
exogenous soluble Crry, either transgenically expressed or
administered as a recombinant protein, has been shown
to be protective in murine lupus nephritis 3,4,21 and nephro-
toxic serum nephritis.5,6 These findings are consistent
with the premise that complement activation in these dis-
ease models is sufficiently robust to overcome intrinsic
complement regulation. By providing exogenous comple-
ment regulators, the balance is brought back towards suc-
cessful restriction of complement activation.
In serum sickness, various glomerular pathologies can
result which depend on the species/strain being studied,
as well as the immunization schemes. Here we used the
protocol as originally described by Stilmant, Couser and
Cotran,15 and subsequently modified by Iskandar, Eman-
cipator and colleagues,16 of active immunization of mice
with apoferritin. In this study, we were interested in
mesangial cell Crry, given the significant deposition of
immune complexes (ICs) within the mesangium.
Crry deficiency is embryonic lethal in mice, unless there
is maternal C3 deficiency.7 Here we crossed Crry)/) C3)/)
females with Crry)/) C3+/) males, thereby generating
Crry)/) C3+/) mice with absent Crry but a single wild-
type C3 allele. These mice became susceptible to develop-
ing GN early in the chronic serum sickness model,
illustrating the relevance of mesangial cell Crry in protect-
ing the C57BL/6 mouse from immune complex-mediated
complement activation and its downstream effects.
To examine whether the combined inherited and
acquired hypocomplementaemia in Crry)/) C3+/) mice
affected glomerular disease features in these studies, we
transplanted Crry)/) C3+/) mouse kidneys into syngeneic
C3-sufficient wild-type mice and then induced serum sick-
ness. Under these circumstances, glomeruli with immune
complex deposition were exposed to normal circulating C3
levels. In our previous work, in which Crry)/) C3)/)
mouse kidneys were transplanted into wild-type hosts,
Crry-deficient kidneys rapidly developed pathology in the
tubulointerstitial compartment, ultimately leading to organ
failure.8 Thus, like the placenta, the tubulointerstitial
compartment relies upon Crry as its critical complement
regulator. Under these conditions, spontaneous comple-
ment activation did not occur in Crry-deficient glomeruli,
and there was no evident glomerular pathology. Because of
the spontaneous complement activation occurring in the
kidney outside the glomerulus, it was necessary to use an
abbreviated disease model. Nonetheless, given the approxi-
mately fivefold increase in circulating C3 levels in the
recipient animals, this experimental design did allow us to
evaluate whether normal C3 levels but absent glomerular
Crry conferred upon transplanted Crry)/) C3+/) kidneys a
greater degree of susceptibility to this immune complex-
mediated GN. As anticipated following renal transplanta-
tion, Crry)/) C3+/) mouse kidneys developed complement
activation and injury within the tubulointerstitial compart-
ment. In contrast, they appeared resistant to developing
immune complex-mediated GN; an explanation for this
somewhat unexpected result is not obvious. The renal
transplantation protocol itself does not prevent develop-
ment of immune complex-mediated GN in this model, as
evidenced by past studies of wild-type kidneys transplanted
into complement factor H Cfh)/) mice.25 Clearly, the
DAF
Intensity
a-actin
ratio 0·94 0·92 0·90 0·88 0·84 0·86 0·88 0·88
WT C3+/– Crry–/–C3+/–
Figure 5. Renal expression of decay-accelerating factor (DAF) was
unaffected by the absence of complement receptor 1 (CR1)-related
gene/protein y (Crry). Western blot analysis showed that renal DAF
expression was comparable in wild-type (WT), C3+/) and Crry)/)
C3+/) mice. Shown are representative immunoblots from at least
three different experiments.
� 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904 e901
Crry in IC glomerulonephritis
transplanted kidneys are exposed to systemic complement
(and do not rely upon intrinsic C3 synthesis), as shown by
marked complement activation in the tubulointerstitium
in the current and previous studies.8 It may be that the
Crry)/) C3+/) mouse (and kidney) is able to compensate
for the lack of Crry from conception until the transplant
procedure. This theory is supported by the lower amounts
of C3 activation and tubulointerstitial damage that were
evident in Crry)/) C3+/) kidneys relative to Crry)/) C3)/)
kidneys in our previous studies.8
DAF is another complement regulator expressed in
normal mouse kidneys,26 with a similar main function to
Crry, namely regulation of C3 activation. Although it only
dissociates the C3 (and C5) convertase, and does not
facilitate complement factor I-mediated C3b inactivation,
it is an effective classical pathway C3 convertase inhibi-
tor,27 which is relevant to the current studies. Hence, we
considered whether a compensatory up-regulation of DAF
would compensate for the absence of Crry in these stud-
ies. However, there was no evidence for an increase in
DAF total protein, making this compensation an unlikely
explanation for the relative resistance of Crry)/) C3+/)
kidneys to immune complex-mediated GN (or spontane-
ous complement activation in the tubulointerstitium).
The different distributions of DAF and Crry in normal
rodent kidneys may also explain the lack of compensation
by DAF when Crry is absent and indicate their distinct
functions under different pathological circumstances. Crry
is normally present in the mesangium,13,14 while DAF is
localized in rodent glomerular capillary walls,26 as a result
in particular of its presence on podocytes.28
In the absence of systemic Cfh or renal Crry, unre-
stricted complement activation occurs in glomeruli 12 and
the tubulointerstitium,8 respectively. In immune complex-
mediated disease, Cfh is clearly relevant to limit further
glomerular complement activation, and prevent inflam-
matory GN.29 In the present studies, we show that glo-
merular Crry does have a role in limiting complement
activation by immune complexes within the mesangial
region. A third member of this group in mice is DAF. As
is true in humans, DAF may be relatively dispensable
compared with Cfh and Crry (or MCP in humans), inso-
far as there is no evidence for spontaneous complement
activation in animals lacking DAF. Yet, it does appear to
have a role in limiting Ab-directed complement activa-
tion. This has been shown by studies of nephrotoxic
serum nephritis in DAF)/) Crry)/) C3+/) mice (i.e. defi-
cient in both DAF and Crry) and our studies with
immune complex-mediated GN in DAF)/) mice.22,30 In
the former setting of Ab-directed complement activation
in the glomerular capillary wall, mice have exacerbated
proteinuria, yet comparable C3 deposition and glomerular
pathology between DAF)/) Crry)/) C3+/) and control
DAF)/) Crry+/) C3+/) mice.22 The finding that there is
little overlap in the sites of complement activation clearly
shows that, in addition to differences in specific comple-
ment regulatory functions, the distributions of the C3
regulators have substantial effects on where complement
can be activated and on the resultant disease expression
within the kidney. This concept extends further to differ-
ent disease models, in which different sites of comple-
ment activation occur which can be limited locally by the
presence of specific complement regulators.31
In this study, the histopathological expression of me-
sangial proliferative GN could be attributed almost exclu-
sively to an influx of neutrophils into glomeruli. This was
most predominant in Crry)/) C3+/) mice, and occurred
early in the disease course; however, it was also seen in a
few C3+/) mice after 5 weeks of disease. Such a neutro-
phil predominance was also observed in the inflammation
occurring in the Crry-deficient embryo.7 It is worth not-
ing that the Schlondorff group previously reported that
macrophages were the major infiltrating cell type in a
similar model induced in wild-type mice on the BALB/c
background, although neutrophils were not specifically
examined.32–34 Thus, even in the same apoferritin-
induced chronic serum sickness disease model in mice,
there can be considerable differences in phenotypic out-
come which depend on many factors, including the fun-
damental genetic background and the site and tempo of
complement activation.
Given the fact that extensive complement activation
occurred in the glomerular mesangial area, even in the
mice with intact Crry, one would assume that excessive
production of anaphylatoxins C3a and C5a played a
significant role in the glomerular recruitment of neutro-
phils. The seven membrane-spanning G protein-coupled
receptors for C3a and C5a are both expressed on neu-
trophils and monocytes,35,36 and C5a is chemotactic to
neutrophils.37 More importantly, a large body of evi-
dence supports the functional significance of C3aR and
C5aR signalling in neutrophil recruitment in many dif-
ferent experimental disease models. Blockade of C3aR
signalling with a specific antagonist significantly reduced
neutrophil influx in a murine focal cerebral ischemia
model,38 while preventing C5aR activation with a spe-
cific antagonist or neutralizing Ab significantly inhibited
neutrophil recruitment in rodent models of zymosan-
induced hyper-nociception,39 monarticular arthritis,40
neurodegeneration,41 immune-complex peritonitis42 and
renal ischemia-reperfusion.43 In our hands, we observed
a significant reduction of neutrophil infiltration in the
murine lupus model using C3aR and C5aR antago-
nists.44,45 Considering that C3a and C5a have broad
effects in both myeloid and lymphoid cells,46 and, as
mentioned earlier, variants of the very same chronic
serum sickness model (i.e. in wild-type BALB/c) generate
a monocytic inflammatory GN,32–34 the fundamental
basis for the neutrophil predominance remains to be
determined in the current model.
e902 � 2009 Blackwell Publishing Ltd, Immunology, 128, e895–e904
L. Bao et al.
In summary, here we provide evidence that glomerular
Crry limits experimental immune complex-mediated pro-
liferative GN. The proliferative nature of this disease can
be attributed to neutrophil infiltration into glomeruli,
and is progressive over time. These results add to our
understanding of the important role of effective local
complement regulation within the kidney, in this case to
limit complement activation brought about by tissue-
deposited immune complexes. These data support the
conclusion that complement inhibition represents a viable
approach to the treatment of the commonly occurring
clinical condition of immune complex-associated GN.
Acknowledgements
Grant support: National Institutes of Health grant
R01DK041873.
Disclosures
All authors declare no financial conflict of interest.
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