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of June 16, 2017.
Increased Susceptibility of
Decay-Accelerating Factor Deficient Mice to
Anti-Glomerular Basement Membrane
Glomerulonephritis
Hajime Sogabe, Masaomi Nangaku, Yoshitaka Ishibashi,
Takehiko Wada, Toshiro Fujita, Xiujun Sun, Takashi Miwa,
Michael P. Madaio and Wen-Chao Song
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:2791-2797; ;
doi: 10.4049/jimmunol.167.5.2791
http://www.jimmunol.org/content/167/5/2791
Increased Susceptibility of Decay-Accelerating Factor Deficient
Mice to Anti-Glomerular Basement Membrane
Glomerulonephritis1
Hajime Sogabe,* Masaomi Nangaku,2* Yoshitaka Ishibashi,* Takehiko Wada,*
Toshiro Fujita,* Xiujun Sun,† Takashi Miwa,† Michael P. Madaio,‡ and Wen-Chao Song2†
C
omplement is a form of natural immunity that plays an
important role in host defense (1). However, if not properly controlled, activated complement can also cause bystander injury to host tissues (1, 2). To prevent complement-mediated autologous attack, host tissues express a number of fluid
phase and membrane-bound inhibitors (3–5). These inhibitors
work at different steps of the complement activation cascade, and
collectively they ensure that inappropriate complement activation
does not occur within normal tissues. Some of the membranebound complement inhibitors act by inactivating C3/C5 convertases (3–5). In humans, membrane C3 convertase inhibitors include
decay-accelerating factor (DAF),3 membrane cofactor protein
*Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan; and †Center for Experimental Therapeutics and Department
of Pharmacology, and ‡Renal-Electrolyte and Hypertension Division, University of
Pennsylvania School of Medicine, Philadelphia, PA 19104
Received for publication February 21, 2001. Accepted for publication June 25, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI44970 (to
W.C.-S.) and 53088 (to M.P.M.) and Grant in Aid for Scientific Research 11671030
from the Ministry of Education, Science and Culture (to M.N.).
2
Address correspondence and reprint requests to Dr. Wen-Chao Song, Center for
Experimental Therapeutics, Department of Pharmacology, University of Pennsylvania School of Medicine,1351 BRBII/III, 421 Curie Boulevard, Philadelphia, PA
19104; or Dr. Masaomi Nangaku, Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan.
3
Abbreviations used in this paper: DAF, decay-accelerating factor; MCP, membrane
cofactor protein; CR1, complement receptor 1; PNH, paroxysmal nocturnal hemoglobinuria; GBM, glomerular basement membrane; BUN, blood urea nitrogen.
Copyright © 2001 by The American Association of Immunologists
(MCP), and complement receptor 1 (CR1) (3–5). DAF prevents
the formation and accelerates the decay of C3 convertases,
whereas MCP and CR1 serve as cofactors for factor I-mediated
cleavage of C3b (3– 6). CR1 also accelerates the decay of C3 convertases as well as serving as an immune adherence receptor (3, 4).
In rodents, a transmembrane protein known as Crry, which possesses both DAF and MCP activities. has also been identified (7–
9). In addition to regulation at the C3 cleavage step, autologous
complement damage can also be restricted at the terminal step by
the GPI-linked membrane protein CD59 (4, 10, 11).
The identification and study of human DAF have historically
been associated with the human hematological disorder paroxysmal nocturnal hemoglobinuria (PNH) (12–14). PNH is caused by
a combined deficiency of DAF and CD59 on the affected blood
cells of the patients (12). As a result of DAF and CD59 deficiency,
blood cells of PNH patients are not protected from autologous
complement attack. In addition to circulating blood cells, DAF is
also expressed prominently on many other cell types such as endothelial and epithelial cells (5, 15). For example, in the human
kidney DAF has been detected in the glomerulus on mesangial and
epithelial cells (16, 17).
In principle, DAF should also protect these cells from complement-mediated inflammatory damage. This may be particularly
true in an autoimmune disease setting in which either binding of
autoantibodies to specific tissue Ags or formation of immune deposits in vital organs, such as the kidney, activates the classical
complement pathway. Although in vitro studies have demonstrated
that DAF expressed on these cells is functional as a C3 convertase
inhibitor (17, 18), thus far little direct evidence is available to
0022-1767/01/$02.00
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To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in
mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although
DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF
in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated
the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout
mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout
mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 ⴞ 25.0 vs 10.0 ⴞ 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 ⴞ 68 vs 325 ⴞ 18 ⴛ 103 ␮m3 per glomerulus; p <
0.0001) and cellularity (47.1 ⴞ 8.9 vs 32.0 ⴞ 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed
no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 ⴞ 0.7 vs
0.02 ⴞ 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were
associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane
C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo. The
Journal of Immunology, 2001, 167: 2791–2797.
2792
Materials and Methods
Animals
GPI-DAF-deficient mice were generated as described previously (23). In
brief, the first three exons of the GPI-DAF gene were deleted and replaced
with the NEO gene. As expected, no GPI-DAF was expressed in the knockout mouse tissues because the proximal promoter sequence necessary for
RNA transcription and the first two short consensus repeats (encoded by
exons 2 and 3, respectively) were absent. This was confirmed by Northern
blotting analysis (23). In contrast to the total abrogation of GPI-DAF gene
expression, TM-DAF gene expression in the knockout mouse testis was
unaffected (23). These GPI-DAF-null mice could develop, grow, and reproduce normally (23). The original C57BL/6 –129J knockout mice were
backcrossed with C57BL/6 mice for four generations. Because the founder
knockout mice had ⬎50% C57BL/6 background (C57BL/6 blastocyst were
used in the generation of chimera, and C57BL/6 females were used in the
subsequent germline transmission breeding), the four times-backcrossed
mice should have a predominantly C57BL/6 background (⬎97%). Littermates of the backcrossed mice were intercrossed to obtain wild-type and
DAF knockout founder mice. Age- and sex-matched F1 mice were used in
all experiments. Mice were housed in a specific pathogen-free facility and
were confirmed to be negative for common murine viral pathogens by sera
analysis. Experiments were conducted by following established guidelines
for animal care and all protocols were approved by the appropriate institutional committees.
Preparation of rabbit anti-GBM serum
The anti-mouse GBM serum was prepared as described by Nagai et al.
(30). Briefly, glomeruli were isolated by differential sieving from the renal
cortex and were disrupted by sonication. The GBM fraction was collected
by centrifugation at 76,000 ⫻ g for 60 min. Anti-GBM serum was raised
in Japanese white rabbits by repeated (five times) immunization with purified mouse GBM. For the immunization step, 1 mg GBM protein was
emulsified with 1 ml CFA (Difco Laboratories, Detroit, MI) and was administered to the rabbit by s.c. injection.
Induction of anti-GBM nephritis
Anti-GBM nephritis was induced according to a previously described protocol by Nagai et al. (30). Mice were immunized i.p. with 0.5 mg rabbit
IgG per 20 g body weight emulsified with CFA. Five days after immunization, 0.05 ml anti-GBM serum per 20 g body weight, diluted with 5 parts
of saline, was administered intravenously through the orbital plexus. In
preliminary experiments, a dose-response curve was established to determine a dose that did not produce nephritis (see below) in normal mice
despite glomerular deposition of IgG.
Experimental design
Mice between 6 and 8 wk of age were used in this study. Preliminary
experiments indicated that male knockout mice were more sensitive than
the females to disease induction, presumably reflecting the higher level of
complement activity in male mice in general (39, 40). Accordingly, only
male mice were used in experiments. Mice were sacrificed either at 8 h
(knockout, n ⫽ 5; wild-type, n ⫽ 10) or on day 8 (knockout, n ⫽ 8,
wild-type, n ⫽ 5) after administration of a subnephritogenic dose of antiGBM serum. The kidneys were processed and evaluated as described below. Serum and urine samples were collected at day 8. For urine collection,
the mice were housed in individual metabolic cages with free access to tap
water.
Northern blot and RT-PCR analyses
Total tissue RNAs were extracted with Trizol reagent (Life Technologies,
Gaithersburg, MD), fractionated in a 1% agarose gel, and transferred to
Hybond-N⫹ nylon membranes. To detect GPI-DAF mRNAs, a 276-bp
3⬘-cDNA-specific fragment (22) was used as a probe for hybridization in
QuickHyb solution (Stratagene, La Jolla, CA). To detect TM-DAF mRNA,
the membrane was stripped and rehybridized with a 180-bp specific probe
corresponding to the 3⬘-cDNA of TM-DAF (22). Finally, the membrane
was stripped again and hybridized with a control probe (GAPDH) to confirm equal loading of RNAs. First strand cDNAs for RT-PCR were synthesized as previously described (22) using total RNAs from kidneys and
oligo(dT) as a primer. The following two primers were used to amplify
mouse
GPI-DAF
cDNA:
5⬘-CATACATGTTTAATAACCTTGA
CAGTTTTG-3⬘ (upstream) and 5⬘-AACAAACAACACTATTAAATT
TATTGTATCC-3⬘ (downstream). The following two primers were used to
amplify mouse Crry cDNA: 5⬘-CCAGCCCCATCACAGCTTCCTTCT-3⬘
(upstream); and 5⬘-CTTCCCTCTCGCATCAGTGTT-3⬘ (downstream).
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corroborate the expectation that DAF plays a protective role in
vivo on nonvascular cells from complement-mediated injury. In
humans, rare cases of complete DAF deficiency due to germline
mutation of the DAF gene (Inab serological phenotype) have been
identified (19, 20). These individuals differ from PNH patients in
that they lack DAF expression in all their tissues, whereas DAF
deficiency in PNH patients, resulting from somatic mutations of a
gene critical to GPI-anchor biosynthesis (21), is limited to affected
blood cells and occurs in conjunction with CD59 dysfunction (12).
Furthermore, although DAF gene mutation apparently did not lead
to PNH-like disease, two of the five individuals had an intestinal
inflammatory disorder (19, 20). However, due to the rare nature of
DAF gene mutation in the human population, it has not been possible to determine whether such individuals are more susceptible to
complement-mediated inflammatory tissue damage.
To address this issue and to aid the study of DAF biology in
vivo, we cloned the mouse homologue of human DAF (22) and
generated a knockout mouse that completely lacks the expression
of the GPI-DAF gene product (23). In the mouse, two DAF genes,
arranged in tandem on mouse chromosome 1, have been identified
(22, 24, 25). One DAF gene, termed GPI-DAF, is equivalent to
human DAF in that it encodes a GPI-anchored protein and is expressed broadly in mouse tissues (22, 24, 25). The second DAF
gene, termed TM-DAF, encodes a transmembrane DAF and is expressed only on mouse sperm (22, 24, 26). Additionally, the
mouse and the rat express in many of their tissues a rodentspecific transmembrane C3 regulator called Crry (7–9). Crry
was established to possess both DAF and MCP activities (7–9)
and may play the substituting role for MCP in most mouse and
rat tissues because the MCP gene is expressed only in the testes
in these two species (27, 28). The fact that GPI-DAF knockout
mice could survive and function normally (23) also suggests
that Crry may be able to compensate DAF function in regulating
spontaneous (alternative pathway) complement activation.
However, the relative role of DAF and Crry as membrane regulators in preventing classical pathway complement activation
in vivo is not clear.
In this study, we used the GPI-DAF knockout mice and investigated their susceptibility to complement-mediated inflammatory
damage in a well-established autoimmune disease model, antiglomerular basement membrane (GBM) Ab induced glomerulonephritis in mice (29, 30). The involvement of the complement system in anti-GBM glomerulonephritis has been well documented
(31, 32). For example, mice deficient in the complement components C3 and C4 were partially protected from anti-GBM glomerular injury (33), and depletion of complement by cobra venom
factor in rats and rabbits reduced the degree of inflammation and
disease progression (34, 35). Furthermore, a soluble form of
recombinant Crry, either administered as a fusion protein or
produced in vivo through transgenic expression, has been demonstrated to attenuate the development of Ab-induced glomerulonephritis in the mouse (36 –38). Nevertheless, there are complement-dependent and independent inflammatory responses initiated
by nephrotoxic Abs that may in part be related to the Ab dosage
(33, 34). We demonstrate in this study that a calibrated dose of
anti-GBM serum caused severe glomerulonephritis in GPI-DAF
knockout mice but not in wild-type controls. This result offers
direct evidence that DAF protects self-tissues from complement
attack in an autoimmune disease setting and suggests that activity
of membrane C3-regulatory proteins is a critical determinant for
complement susceptibility in autoimmune tissue injury.
DAF AND ANTI-GBM GLOMERULONEPHRITIS
The Journal of Immunology
2793
Western blot analysis
Statistical analysis
Membrane proteins of kidneys were solubilized as previously described
(41). In brief, kidneys were washed with PBS twice; homogenized in a
20⫻ volume of PBS, pH 7.2, containing 10 mM EDTA, 1% Nonidet P-40,
0.1 mM PMSF, 1 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin A with a Polytron
(Polytron, Paterson, NJ); and then solubilized for 30 min at 4°C. Nuclei and
cytoplasmic debris was then pelleted at 14,000 rpm using a microcentrifuge
for 10 min at 4°C.
SDS-PAGE was performed with 300 ␮g/lane of solubilized kidney samples under nonreducing condition. Western blot to detect Crry expression
was performed using 1F2 (10 ␮g/ml; PharMingen, San Diego, CA), a rat
anti-mouse Crry mAb (8). The bound Ab was then detected with alkaline
phosphatase-conjugated goat anti-rat IgG, (1/500; Promega, WI). 5-Bromo-4-chloro-3-indolyl phosphate/NBT (Sigma, St. Louis, MO) was used as
a substrate for alkaline phosphatase.
Values are presented as mean ⫾ SEM. Statistical comparisons and correlation analysis were performed with the StatView program (Abacus Concepts, Berkeley, CA) using the Mann-Whitney U test or Student’s t test as
appropriate. A p value of ⬍0.05 was considered statistically significant.
Histology
Immunofluorescence
To examine the deposition of C3, 4-␮m frozen sections were stained with
FITC-conjugated goat Abs against mouse C3 (1/100; Cappel, ICN Pharmaceuticals, Aurora, OH). For fibrinogen, FITC-conjugated goat Abs
against rat fibrinogen, which have cross-reactivity with the mouse fibrinogen (1/400; Cappel, ICN Pharmaceuticals) was used. For semiquantitative
analysis of rabbit IgG deposition in glomeruli, indirect immunofluorescence using biotinylated goat anti-rabbit IgG (1/400; Vector Laboratories,
Burlingame, CA) and NeutrAvidin-Oregon Green 488 conjugate (Molecular Probes, Eugene, OR) was used. Fluorescence-positive glomeruli were
counted, and the percentage of positive glomeruli in each sample was calculated. Deposition of autologous (mouse) IgG in the glomeruli was
assessed by using biotinylated goat anti-mouse IgG (1/400; Vector) and
NeutrAvidin-Oregon Green 488 conjugate.
Measurement of circulating IgG and IgM levels
Circulating levels of total IgG and IgM were determined with the Mouse
IgG ELISA kit and Mouse IgM ELISA kit (Bethyl Laboratories, Montgomery, TX) according to the manufacturer’s protocol. Circulating levels
of rabbit IgG-specific mouse IgG were measured by ELISA using the procedure detailed below. Ninety-six-well ELISA plates coated with rabbit
IgG (Organon Teknika, Durham, NC) were incubated with test plasma that
was diluted to 1/2000. After being washed extensively with PBS containing
0.05% Tween 20, the plates were incubated with HRP-conjugated antimouse IgG (Vector) diluted to 1/1000. For the development, the wells were
incubated with the reaction solution containing 3,3⬘,5,5⬘-tetramethylbenzidine (Sigma). The reaction was stopped by addition of H2SO4, and the
OD450 was determined and taken as a measurement of anti-rabbit IgG Abs.
Measurement of urinary albumin and blood urea
nitrogen (BUN)
Urinary albumin excretion was measured by a mouse albumin ELISA
quantitation kit (Bethyl Laboratories) according to the manufacturer’s protocol. BUN was measured by the urease-indophenol method with a Urea
NB kit (Wako Pure Chemical Industries, Tokyo, Japan).
Expression of GPI-DAF but not TM-DAF gene in the
mouse kidney
As alluded to earlier, two DAF genes are known to exist in the
mouse (22, 24). To examine the respective expression of the GPIDAF and TM-DAF genes in the wild-type mouse kidney and the
possibility that compensatory expression of the TM-DAF gene
might have occurred in the GPI-DAF knockout mouse kidney,
Northern blot analysis using cDNA probes specific to either the
GPI-DAF mRNA or the TM-DAF mRNA were performed. Fig. 1A
demonstrates that two GPI-DAF mRNA species were detected in
the wild-type but not in the GPI-DAF gene knockout mouse kidney. The pattern of two distinct mRNA species, presumably a result of alternative splicing, is similar to that observed in other
mouse tissues (23), although the relative abundance of these messages does vary from tissue to tissue (23). No TM-DAF mRNA
was detected in either the wild-type or the GPI-DAF gene knockout mouse kidney. This result suggests that the TM-DAF gene is
not expressed in the mouse kidney and that the GPI-DAF knockout
mouse is completely deficient of DAF expression in the kidney.
Comparison of Crry and GPI-DAF expression in wild-type and
knockout male and female mouse kidneys
To determine whether there was compensatory expression of Crry
in the DAF knockout mouse kidney, we performed Western blot
analysis of membrane protein extracts of kidneys using a rat antimouse Crry mAb. Fig. 1B shows that there is no significant difference in Crry protein levels between wild-type and knockout
mouse kidneys or between male and female mouse kidneys. Because we observed in our preliminary experiments a gender difference in the sensitivity of glomerulonephritis development, we
also investigated by RT-PCR whether there is a sex difference in
the expression of GPI-DAF. As shown in Fig. 1C, RT-PCR analysis revealed no appreciable difference in GPI-DAF expression
between the male and female kidneys. Consistent with the Western
blot data (Fig. 1B), no gender difference was detected by RT-PCR
in Crry expression between male and female kidneys (Fig. 1C).
DAF knockout mice are more susceptible to nephrotoxic
serum nephritis
Preliminary experiments established the minimum and subnephritogenic doses required for nephritis (0.05 ml/20g body weight).
We also observed in preliminary experiments that using this dosage, 72 ⫾ 10% of glomeruli in knockout male mice (n ⫽ 3)
showed damage, whereas 3 ⫾ 3% of glomeruli in wild-type male
mice (n ⫽ 3) were injured. Under the same conditions, 43 ⫾ 32%
of glomeruli in knockout female mice (n ⫽ 3) demonstrated damage, whereas 3 ⫾ 6% of glomeruli in wild-type female mice (n ⫽
3) were injured. Thus, although there was a trend of increased
sensitivity in the knockout mice of both sexes, knockout males
appeared to be more sensitive to nephritis development. Because
male mice have higher complement activity than female mice (39,
40), we decided to use male mice only in our experiments to simplify the study.
Eight days after anti-GBM serum administration, glomeruli of
DAF knockout mice appeared enlarged and showed global glomerular hypercellularity and segmental sclerosis at the periphery
of the tuft (Fig. 2). Furthermore, the knockout mice had significant
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Renal cortical tissue for light microscopy was fixed in methyl Carnoy’s
solution and embedded in paraffin. Sections of 4 ␮m were cut and stained
with periodic acid-Schiff and counterstained with hematoxylin. To accurately perform quantitative analysis, computer-assisted morphometry was
used (42). For each individual animal, nine photographs of randomly chosen areas of a cortex were taken at ⫻200 magnification. Each photograph
included one to four glomeruli, and its genotype was blinded to eliminate
potential experimental bias. The photographs were converted to picture
files using a scanner at a resolution of 300 dots per inch and were evaluated
on a video screen using Photoshop software (Adobe Systems, San Diego,
CA). Only equatorially sectioned glomeruli were evaluated. With this
method, total glomerular nuclear counts, percentage of glomeruli exhibiting glomerular injury, and glomerular tuft volume were determined. Glomerular injury was defined by evidence of segmental increases in glomerular matrix, segmental collapse and obliteration of capillary lumina and
accumulation of hyaline which was frequently associated with synechial
attachments to Bowman’s capsule. To measure the glomerular tuft volume,
mean planar glomerular area was first determined and was used to calculate
glomerular tuft volume by a previously described formula (43). The mean
planar glomerular area was determined by manually tracing the outer edges
of all glomerular tufts in each kidney section and then calculating and
summarizing the encircled areas by computerized morphometry (44).
Results
2794
DAF AND ANTI-GBM GLOMERULONEPHRITIS
accumulation of mesangial matrix (Fig. 2C), and in some cases
there were completely collapsed lobules and enlarged epithelial
cells (Fig. 2D). By contrast, the kidneys of the wild-type mice were
largely intact, with patent capillaries without increased matrix or
cellularity (Fig. 2A). Fig. 3 shows the results of quantitative anal-
ysis of the morphological changes. Both glomerular volume and
the number of cells per glomerulus were significantly increased in
the DAF knockout mice as compared with controls. Even more
striking is the increased percentage of glomeruli that showed signs
of injury in the knockout mice (Fig. 3C). Approximately 70% of
knockout glomeruli showed signs of injury whereas relatively few
in the wild-type sustained similar degree of damage (Fig. 3).
On day 8 after nephrotoxic serum administration, when histological evidence of nephritis was apparent in the knockout mice,
the BUN level remained normal in the knockout mice (26.4 ⫾ 1.0
and 27.6 ⫾ 1.6 mg/dl in knockout and wild-type, respectively), but
the urinary albumin level was elevated in the knockout mice
(1.4 ⫾ 0.7 mg/24 h for the knockout compared with 0.02 ⫾ 0.01
mg/24 h for the wild-type mice; Table I). No wild-type mice developed abnormal albuminuria (⬍0.05 mg/day). However, three of
the knockout mice with the most severe glomerular damage excreted ⬎2 mg/day of albumin, and there was a significant correlation between albuminuria and glomerular damage as assessed by
the percentage of glomeruli showing histological signs of injury
under light microscopy (r ⫽ 0.88, p ⫽ 0.0022).
Increased complement activation and fibrin deposition in DAF
knockout mouse kidneys
FIGURE 2. Representative light microscopy (periodic acid-Schiff) of
glomeruli from wild-type (A) and DAF knockout mice (B–D) 8 days after
administration of nephrotoxic serum. The wild-type mouse kidneys showed
minimal pathological change. By contrast, glomeruli from DAF knockout
mice were enlarged and hypercellular (B), and segmental sclerosis with
accumulation of mesangial matrix was evident (C). D, Inflamed glomerulus
with a completely collapsed lobule and enlarged epithelial cells from a
knockout mouse. Original magnification, ⫻200.
Potential mechanisms of increased nephritis were then explored.
Eight hours after anti-GBM serum treatment, there was prominent
deposition of rabbit as well as mouse IgG in the wild-type and
knockout kidneys (Fig. 4, top and bottom rows). By contrast, deposition of C3 in capillary loops of glomeruli was observed only in
the knockout mouse kidneys (Fig. 4, second row, and Table II).
This was associated with fibrinogen deposition and thrombus formation, which was detected only in the glomeruli of the knockout
mice (Fig. 4, third row, and Table II). Analysis of circulating total
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FIGURE 1. Analysis of GPI-DAF, TM-DAF, and
Crry gene expression in wild-type and GPI-DAF
knockout mouse kidneys. A, Northern blot analysis
of DAF expression in the wild-type (⫹/⫹) and GPIDAF knockout (⫺/⫺) mouse kidneys. GPI-DAF is
expressed in the wild-type but not the knockout
mouse kidney. TM-DAF is not expressed in either
the wild-type or the knockout mouse kidney. Equal
loading of total RNA in the two lanes is indicated by
using GAPDH cDNA as a control probe. The same
membrane was used in all three hybridizations. Left
ordinate, Positions of the 18S and 28S ribosomal
RNAs. B, Western blot analysis of Crry expression
in wild-type (lanes 1 and 3) and knockout (lanes 2
and 4) male (lanes 1 and 2) or female (lanes 3 and
4) kidneys. Left ordinate, Positions of molecular
mass markers. C, RT-PCR analysis of GPI-DAF and
Crry expression in male (M) or female (F) mouse
kidneys. Reactions were conducted either with (⫹)
or without (⫺) reverse transcriptase (RT) during the
first strand cDNA synthesis.
The Journal of Immunology
2795
IgG, IgM at day 1 and rabbit IgG-specific mouse IgG at day 7 after
disease induction (i.e., anti-GBM administration) also revealed no
significant difference between wild-type and knockout mice (total
IgG 0.47 ⫾ 0.11 mg/ml for wild-type, n ⫽ 5, 0.50 ⫾ 0.11 mg/ml
for knockout, n ⫽ 8; total IgM 0.21 ⫾ 0.02 mg/ml for wild-type, n ⫽
5, and 0.17 ⫾ 0.02 mg/ml for knockout n ⫽ 8; ELISA readings of
rabbit IgG-specific mouse IgG assay 480 ⫾ 201 OD450 for wild-type,
n ⫽ 5, and 589 ⫾ 442 OD450 for knockout, n ⫽ 8; p ⫽ 0.62).
Discussion
The complement system plays paradoxical roles in the pathogenesis and manifestations of autoimmune diseases (1, 2). Although
complement, particularly its early components, is recognized to
facilitate immune complex clearance (1, 2), full activation of the
complement system in an autoimmune disease setting can generate
inflammatory and cytolytic mediators (31, 32). Anti-GBM-induced
glomerulonephritis in rodents has been often used as an animal
model to dissect the roles of complement and other inflammatory
pathways in immune glomerulonephritis (29, 30, 46). However,
the role of complement-regulatory proteins has not been adequately addressed. In this study, we used GPI-DAF knockout mice,
generated in our laboratory (23), to evaluate the role of an endogenous membrane complement-regulatory protein, DAF, in the
pathogenesis of nephrotoxic serum glomerulonephritis. Our results
show that DAF knockout mice are more susceptible to nephrotoxic
serum glomerulonephritis than the wild-type controls. Injection of
a subnephritogenic (in normal mice) rabbit anti-mouse GBM serum caused significant nephritis in DAF knockout mice.
DAF is a central membrane complement regulator which in humans has been mostly studied in the context of PNH syndrome and
xenotransplantation experiments (12, 47). Although a number of
studies have examined the expression of DAF protein in both normal and diseased human kidneys (16, 17), the role of DAF in
immune glomerulonephritis has not been adequately addressed.
Table II. C3 and fibrinogen deposition in glomeruli of wild-type and
DAF knockout mice
Table I. Urinary albumin and BUN levels in wild-type and DAF
knockout mice
Urinary albumin (mg/24 h)
BUN (mg/dl)
Knockout (n ⫽ 8)
Wild-Type (n ⫽ 5)
1.4 ⫾ 0.7
26.4 ⫾ 1.0
0.02 ⫾ 0.01
27.6 ⫾ 1.6
C3 deposition (%)
Fibrinogen deposition (%)
Knockout
(n ⫽ 5)
Wild-Type
(n ⫽ 10)
18.1 ⫾ 15.4ⴱa
4.7 ⫾ 2.2ⴱ
0⫾0
0⫾0
ⴱ, p ⬍ 0.05 vs wild-type group (Mann-Whitney U test).
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FIGURE 3. Quantitative analysis of glomerulonephritis in wild-type
(n ⫽ 5) and DAF knockout (n ⫽ 8) mice after anti-GBM treatment. At least
nine sections, each containing one to four glomeruli, from each mouse
were examined. Compared with wild-type animals, DAF knockout mouse
kidneys had larger glomerular volumes (A, 516 ⫾ 68 vs 325 ⫾ 18 ⫻ 103
␮m3 per glomerulus), displayed higher cellularity (B, 47.1 ⫾ 8.9 vs 32.0 ⫾
3.1 cells per glomerulus) and contained more injured glomeruli (C, 68.8 ⫾
25.0 vs 10.0 ⫾ 3.5%). p ⬍ 0.05 for all.
FIGURE 4. Immunofluorescence staining of rabbit IgG, mouse C3, fibrinogen, and mouse IgG deposition in glomeruli of normal (nontreated
wild-type (column A), anti-GBM-treated wild-type (column B), or antiGBM-treated DAF knockout (column C) mice. Equal deposition of rabbit
IgG (8 h after treatment) and mouse IgG (7 days) was observed in the
treated mice regardless of the genotype (columns B and C, top and bottom
rows). However, C3 and fibrinogen deposition, mainly along the glomerular capillary loops was observed only in the treated knockout mouse glomeruli (8 h after treatment) (columns B and C, middle two rows; see also
Table II). In the middle two rows, background staining of C3 and fibrin in
the glomeruli is indicated by arrows and specific staining is indicated by
arrowheads. Background staining of C3 was distinct, mostly restricted to
Bowman’s capsules, and may indicate local C3 synthesis (45) or nonspecific activity of the anti-mouse C3 Ab used.
2796
that female mice may have less circulating complement activity
(39, 40). A similar gender bias in complement-dependent phenotypes was also observed in a CD59 gene knockout mouse (40).
The present results that DAF-deficient mice were more sensitive
to glomerulonephritis provide further evidence for the role of complement in immune-mediated glomerular damage. Although it has
been generally accepted based on clinical and animal experimental
data that complement plays a detrimental role in immune glomerular damage (31, 32), some recent experiments using Fc receptor
knockout mice have questioned the relevance of the complement
pathway in autoimmune glomerulonephritis (55, 56). In contrast,
studies of C3- and C4-deficient mice have clearly demonstrated a
role of complement in anti-GBM glomerulonephritis and showed
that the relative contribution in the disease pathogenesis by the
complement pathway is dependent on the dosage of the nephrotoxic Abs used (33). Results presented here suggest that the function of membrane complement-regulatory proteins may be another
critical variable in determining the degree of complement-mediated inflammatory damage in an immune disease setting.
Acknowledgments
We thank Dr. Takeshi Sugaya (Tanabe Seiyaku, Osaka, Japan) and Dr.
Norio Hanafusa (University of Tokyo School of Medicine, Tokyo, Japan)
for their technical support. We also thank Drs. Reiko Inagi, Toshio Miyata,
and Kiyoshi Kurokawa (Tokai University School of Medicine, Kanagawa,
Japan) for their generous support.
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Investigation using animal models has been hampered by the initial slow pace in identifying the animal orthologues of human DAF
in nonprimate species (7). It is now known, however, that both the
rat and the mouse contain DAF genes (4), as well as a related and
rodent-specific membrane complement regulator Crry (4). In fact,
two DAF genes, possibly resulting from a recent gene duplication
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that the GPI-DAF gene but not the TM-DAF gene is expressed in
the mouse kidney. Furthermore, although the TM-DAF gene is still
intact in the GPI-DAF knockout mouse (23), no compensatory
expression of TM-DAF in the GPI-DAF knockout mouse kidney
was observed. Thus, our GPI-DAF gene knockout mouse provides
an appropriate animal model to examine the consequence of DAF
deficiency in immune glomerulonephritis development.
A number of studies have demonstrated a protective role for two
other membrane regulators of complement activation, the membrane attack complex inhibitor CD59 and the rodent-specific C3
inhibitor Crry. For example, it has been shown that neutralization
of CD59 function exacerbated complement-mediated kidney injury in the rat (48). Similarly, in a rat model of thrombotic microangiopathy induced by anti-endothelial cell Abs, perfusion of
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related study, neutralization of rat Crry caused tubulointerstitial
injury in the kidney (50). Much evidence in support of a protective
role of Crry in immune glomerular damage has also accumulated
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In this regard, both administration of a recombinant, soluble Crry
systemically or overexpression of Crry in transgenic mice was protective from Ab-induced glomerular injury (36 –38). Thus, it is
especially remarkable that DAF knockout mice were more sensitive to nephrotoxic serum, despite normal Crry expression in the
DAF knockout mouse kidney (Fig. 1). Although our finding does
not exclude a protective role of membrane-anchored Crry in this
disease model, it does suggest that the function of DAF in the
mouse kidney cannot be completely compensated by Crry in this
model. The relative efficacy of protection by membrane-anchored
Crry and DAF in this disease model remains to be defined. However, recent in vitro assays have established that, at least in the
fluid phase, recombinant mouse DAF is more active on a molar
basis than recombinant Crry as a classical pathway complement
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