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*
Department of Medicine, Medical University of South Carolina and the Medical Research Service, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29425;
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110;
Department of Pathology, University of Miami School of Medicine, Miami, FL 33125; and
Department of Medicine, University of Colorado Health Science Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of C3 in the kidney and its deposition there in IC glomerulonephritis in humans suggests a pathogenic role for C3 in renal immunopathology (8). C3 expression and deposition has also been documented in IC-mediated glomerulonephritis, in both induced rodent models (e.g., Ag- or autoantibody-injected mice) as well as spontaneous lupus models (e.g., MRL/lpr and NZB/NZW F1 mice) (9, 10, 11). Deposition of C3 in the kidney is a key pathologic finding in lupus nephritis (10). A recent study of antiglomerular basement membrane (GBM)-mediated nephritis in C3- and C4-deficient nonlupus murine strains demonstrated decreased renal neutrophil infiltrate, glomerular capillary thrombosis, and proteinuria compared with wild-type mice (12). The extent of protection was greater in the C3-deficient than C4-deficient animals, suggesting that both classical and alternative complement pathways may be involved in the development of the disease. Further evidence for a role of complement in lupus nephritis was provided by the protection against renal disease provided by anti-C5 Ab treatment of NZB/NZW F1 mice (13). Based on these studies, C3 in the kidney is postulated to mediate disease by locally activating the inflammatory cascade and by direct complement damage to glomerular tissue through formation of the membrane attack complex.
Paradoxically, deficiencies of some components of the
classical complement pathway are associated with an increased incidence
of lupus and lupus-like disease, including IC glomerulonephritis in
humans and murine knockout strains (14, 15). Systemic
lupus erythematosus (SLE) develops in a high percentage of individuals
with homozygous deficiency of C1q (93%), C4 (75%), and C2 (
33%).
Deficiency of C3 is associated with membranoproliferative
glomerulonephritis in 8 of 22 individuals (36%), and with lupus-like
disease in 5 of 22 (23%), although the lupus-like disease is
relatively mild compared with that associated with C1q and C4
deficiency (14, 16, 17, 18). In B6/lpr mice that
produce low titer anti-DNA Abs, but do not develop renal disease,
C4 deficiency enhanced autoantibody production and the mice developed
proliferative renal disease. C3 deficiency in B6/lpr mice
had no apparent effect on Ab production and the mice did not develop
renal disease (19). These etiopathogenetic findings
strongly suggest that the classical complement pathway components,
excluding C3, protect against the development of systemic autoimmune
disease. Given these contrasting clinical and experimental results, the
role of C3 in lupus nephritis is unclear.
MRL/MpJ-Faslpr (MRL/lpr) mice spontaneously develop an autoimmune syndrome similar to human SLE, including autoantibody production and IC glomerulonephritis (20, 21). To gain further insight into the role of complement and C3 in autoimmune glomerulonephritis, B6/129 C3-deficient mice were backcrossed to MRL/lpr mice for eight generations. Serologic, clinical, and histologic disease activity were assessed over time in wild-type (MRL/lpr C3+/+), heterozygous (MRL/lpr C3+/-), and homozygous C3-deficient mice (MRL/lpr C3-/-).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MRL/MpJ-Faslpr (MRL/lpr) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The C3-deficient construct was generated by replacing 2.3 kb of the 5' flanking region and the first 105 bp of exon 1 of the C3 gene with the neomycin resistance gene as previously described (22). MRL/lpr mice deficient in C3 were derived by genetic backcrosses in which the MRL/lpr parental strain was initially mated to heterozygous (B6 x129)F1 C3-deficient mice. Heterozygous F1 offspring were backcrossed for seven generations with MRL/lpr mice, resulting in eight genetic backcrosses with the MRL/lpr genetic background. The eighth backcross generation was interbred to yield Fas-/-/C3+/+, Fas-/-/C3+/-, and Fas-/-/C3-/- mice. The mice were housed and bred under pathogen-free conditions initially in the Division of Comparative Medicine at the Washington University School of Medicine (St. Louis, MO) and subsequently at the animal facility of the Ralph H. Johnson Veterans Affairs Medical Center (Charleston, SC).
Genotyping of the mice by PCR
Primers for PCR for C3 were as follows: V789 (5'; C3) 5'-AGGGACCAG CCCAGGTTCAG-3', V788 (5'; Neo) 5'-TCGTCCTGCAGTTCATTCAG-3', and V787 (3'; C3) 5'-GATCCCCAGAGCTAATG-3'. DNA was isolated from tail snips (3- to 4-wk-old mice) using a QIAamp Tissue kit (Qiagen, Santa Clarita, CA). PCR was performed by adding 500 ng of genomic tail DNA into a 25-µl reaction mixture containing 1.5 mM MgCl2, 0.5 µM concentrations each of oligonucleotide mix, 0.2 mM concentrations each of dNTP mix, and 1.5 U of AmpliTaq Gold (Perkin-Elmer, Norwalk, CT). PCR was performed with a 10-min denaturation step of 94°C followed by 35 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min with a final extension of 10 min at 72°C in a Perkin-Elmer GeneAmp 9600. After PCR amplification, samples were electrophoresed in a 2% agarose gel and visualized by ethidium bromide staining.
Primers for PCR for Fas were as follows: Fas A (5'-AGGTTACAAAAGGTCA CCC-3'), Fas B (5'-GATACGAAGATCCTTTCCTGTG-3'), and Fas C (5'-CAAACGCAGTCAAATCTGCTC-3'). In brief, individual genomic tail DNA was isolated and used in PCR. PCR was performed with a 10-min denaturation step of 94°C followed by 40 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min with a final extension of 10 min at 72°C. After PCR amplification, samples were electrophoresed in a 2% agarose gel and visualized by ethidium bromide staining.
MHC typing
H-2 of the mice was determined by PCR RFLP analysis using the techniques of Peng and Craft (23) specifically designed to differentiate H-2k and H-2b. Briefly, tail DNA was amplified using the primers IAA1F 5'-GAAGACGACATTGAGGCCGACCACGTAGGC-3' and IAA1R 5'-ATTGGTAGCTGGGGTGGAATTTGACCTCTT-3'. PCR was performed with a 10-min denaturation step of 94°C followed by 35 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min with a final extension of 10 min at 72°C. The resultant PCR product was digested with HindIII (Promega, Madison, WI) and run on a 2.5% agarose gel. Amplified DNA of the H-2b genotype is digested as a 167-bp/96-bp doublet whereas DNA of the H-2k genotype is represented as a 263-bp single fragment, allowing differentiation by agarose gel electrophoresis.
Urine albumin excretion
Mice were placed in metabolic cages for 24-h urine collection every 2–4 wk beginning at 12 wk of age. To prevent bacterial growth, antibiotics (ampicillin, gentamicin (Life Technologies, Rockville, MD), and chloramphenicol (Sigma, St. Louis, MO) were added to collection tubes. Urinary albumin excretion was determined by ELISA using a standard curve of known concentrations of mouse albumin (Cappel, Durham, NC). Briefly, 96-well ELISA plates were coated with 2.5 µg/ml rabbit anti-mouse albumin (Cappel) overnight at 4°C. After washing with PBS-0.05% Tween 20 (PBS-T), PBS-0.25% gelatin (type A from porcine skin; Sigma) was added to each well and incubated for 1 h at room temperature (RT) to block nonspecific binding. Urine samples were added in serial dilutions, starting at a 1/10 dilution to each well, and incubated for 1 h at RT. After washing with PBS-T, HRP-conjugated rabbit anti-mouse albumin (Cappel) was added and incubated for 1 h. After additional washing, substrate solution containing 3,3',5,5'-tetramethylbenzidene (TMB; Sigma) was added in 0.1 M citrate buffer (pH 4.0) and 0.015% H2O2. After incubation for 35 min, absorption at A380 was determined on a Flow microtiter plate reader (Dynatech, McLean, VA) and reported as milligrams of albumin per mouse per day.
Measurement of anti-DNA Ab
Anti-DNA Ab levels were measured by ELISA as previously
described (24). Briefly, 96-well ELISA plates were coated
with 5 µg/ml double-stranded calf thymus DNA (Sigma) in sodium salt
citrate buffer at 37°C overnight. After washing with PBS-T, sera were
added in serial dilutions starting at 1/100 and incubated for 45 min at
RT. After washing with PBS-T, HRP-conjugated goat anti-mouse IgG Ab
(
-chain specific; Sigma) was added, followed by TMB for color
development. A380 was measured as
above. Results are shown as the A380
at a 1/100 dilution. dsDNA was derived by S1
nuclease (Sigma) treatment of phenol-purified calf thymus DNA.
Measurement of anti-GBM Ab
GBM Ab levels were measured by ELISA as previously described
(25). Briefly, 96-well ELISA plates were coated with 50
µl/well rat GBM in PBS (40 µg/ml) for 90 min at RT. After washing
with PBS-T, sera were added in serial dilutions, starting at 1/100, and
incubated for 90 min at RT. After washing with PBS-T, HRP-conjugated
goat anti-mouse IgG Ab (-chain specific; Sigma) was added,
followed by TMB for color development.
A380 was measured as above. Results
are shown as the A380 at a 1/100
dilution.
Isolation of cryoglobulins from sera
Cryoglobulins were isolated from sera as previously described (26). Briefly, the mice were bled and blood samples were immediately placed at 37°C for 2 h. After centrifugation at 300 x g at 37°C, supernatants were collected and incubated at 4°C for 72 h. After incubation, the samples were centrifuged at 2000 x g, supernatants were removed, and precipitates were washed five times with cold PBS. After being washed, they were resuspended in PBS at the same volume as the original sera. IgG content was then measured. The isolated cryoglobulins were placed at 37°C for 3 h before use. All assays of cryoglobulins and their activity were performed using warmed plates and reagents. Assays were conducted in a hot room maintained at 37°C.
Measurement of Igs
Total IgG Ab levels in sera or in cryoglobulins were determined
by ELISA using a standard curve of known concentrations of mouse IgG.
ELISA plates were coated with 1 µg/ml goat anti-mouse Ig
(
-chain specific; Southern Biotechnology Associates, Birmingham, AL)
overnight at 4°C and then warmed to 37°C for cryoglobulin assays.
After washing with PBS-T, sera or cryoglobulins were added in serial
dilutions starting at a 1/1000 dilution and incubated for 45 min at RT.
Color development was measured as described above by using a
HRP-conjugated goat anti-mouse IgG (-chain specific; Sigma) and
TMB substrate. The same method was used for measurement of serum Ig
isotype levels and total Ig in sera and in cryoglobulins, except
HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 or
HRP-conjugated goat anti-mouse H and L chains were used (Southern
Biotechnology Associates).
Measurement of circulating ICs, rheumatoid factor, and C3 levels in serum
Circulating ICs levels were determined by the C1q ELISA methods
previously described with some modifications (27). ELISA
plates were coated with 10 µg/ml human C1q (Sigma) in 0.1 M carbonate
buffer (pH 9.6), incubated for 48 h at 4°C, blocked for 2 h
at RT with 1% BSA in PBS, and washed with PBS. EDTA-treated sera
samples were added in serial dilutions starting at a 1/50 dilution and
plates were incubated for 1 h at RT and overnight at 4°C. The
assay was then performed as described above with HRP-conjugated goat
anti-mouse IgG (-chain specific; Sigma). Aggregated human
-globulin was used as a positive control. BALB/c mice sera (The
Jackson Laboratory) were used as a negative control.
Rheumatoid factor levels in serum were measured by ELISA as previously described (21). ELISA plates were coated with rabbit IgG (1 µg/ml) overnight at 4°C. After washing, sera were added in serial dilutions, starting at 1/100 dilution, and incubated for 45 min at RT. The assay was then performed as described above with HRP-conjugated rabbit anti-mouse IgG (Fc specific; Pierce, Rockford, IL) or rabbit anti-mouse IgM (µ-chain specific; Pierce).
C3 levels in serum were measured by ELISA. ELISA plates were coated with goat anti-mouse C3 (Cappel) and incubated overnight at 4°C. After washing and blocking with 5% BSA in PBS for 1 h, sera were added in serial dilutions, starting at 1/200 dilution, and incubated for 1 h at RT. The assay was then performed as described above with HRP-conjugated goat anti-mouse C3 (Cappel). BALB/c mice sera (The Jackson Laboratory) were used as a positive control.
Pathology
At the time of sacrifice (24 wk), the kidneys were removed. One kidney was fixed with 10% buffered Formalin, embedded in paraffin, and then sectioned before staining with hematoxylin and eosin. The other kidney was snap frozen in liquid nitrogen and placed in OCT medium. Frozen sections (4-µm thick) were stained with fluorescein-conjugated anti-mouse IgG or C3. The hematoxylin and eosin kidney slides were interpreted in a blinded fashion and graded for glomerular inflammation, proliferation, crescent formation, and necrosis. Interstitial and tubular changes were also noted. Scores from 0 to 4+ (0, none; 1+, mild; 2+, moderate; 3+, moderate-severe; 4+, severe; scores of crescent formation and necrosis were doubled) were assigned for each of these features and then added together to yield a final renal score. Changes were also assessed as to whether they were focal or diffuse. Vasculitis was judged as either being present or absent. Immunofluorescence slides were read blinded and graded 0–4+ (0, none; 1+, mild staining; 2+, moderate staining; 3+, moderate-high staining; 4+, high staining) for fluorescence intensity.
Statistics
The unpaired Student’s t test was used to test for
significant differences between groups. The Mann-Whitney two-tailed
U test or Kruskal-Wallis test was used to determine the
significance of changes in renal score (Table I
), glomerular C3
deposition (Table II), and glomerular IgG deposition (Fig. 6). A
p < 0.05 was considered to be statistically
significant.
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Results |
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The genotype of the mice was determined by PCR for C3 and Fas. After the eighth backcross to MRL/lpr mice, C3+/- mice were bred to generate double homozygous animals: C3+/+Fas-/- (MRL/lpr C3+/+), C3-/-Fas-/- (MRL/lpr C3-/-), and C3+/-Fas-/- (MRL/lpr C3+/-) mice. In the pups from these breedings, the C3-null gene was inherited in Mendelian fashion with no significant observable fetal loss. The MHC class II genotype of the mice was determined by PCR RFLP analysis. All mice selected for study in each group were H-2k/k. Twenty-five to 26 mice were randomly selected from each group for study purposes.
To verify that the genotype of the mice was expressed phenotypically,
serum C3 levels were measured in the study mice over time. As shown in
Fig. 1, C3 was undetectable in the serum
of the MRL/lpr C3-/- mice and was at
intermediate and highest levels in the C3+/- and
C3+/+ mice, respectively
(p < 0.05), indicating a gene dose effect on
serum C3 levels. There was a trend toward decreasing serum C3 levels in
the C3+/- and C3+/+ mice
with aging and disease expression.
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At the time of sacrifice of the mice at 24 wk of age, there were no differences in body weights or spleen weights among the three groups (data not shown). Splenocytes were assayed by flow cytometry and no significant differences were noted in cell number or percentage of CD19+, CD21+, CD4+, CD8+, IgM+, or CD3+ cells (data not shown).
Ig levels in sera or in cryoglobulins
Deficiency of C3 is known to have effects on overall IgG levels,
whereas complement receptor deficiency affects IgG isotype levels
(2, 30, 31). It is also known that MRL/lpr
renal disease and vasculitis are linked with IgG3 cryoglobulin
production (28, 29, 32, 33). We measured serum levels of
total IgG and IgG3 by ELISA and found no statistically significant
differences among the three groups (Fig. 2, A and B).
Measurements of the other IgG isotypes (IgG1, IgG2a, and IgG2b) and
total Ig also demonstrated no statistically significant differences
among the three groups, although the C3-/- mice
trended toward lower IgG2a levels (data not shown).
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C). Measurement of serum autoantibody levels
Production of anti-dsDNA Abs, a T cell-dependent autoimmune
response, is linked with renal disease in MRL/lpr mice, and
it is known that deficiency of C3 inhibits T cell-dependent B cell
responses (2, 21, 34, 35). To determine whether C3
deficiency had any effect on anti-DNA and/or anti-GBM Ab
production, serum levels of these autoantibodies were measured by
ELISA. As shown in Fig. 3, serum levels
of anti-dsDNA and anti-GBM Ab in the three groups increased as
the mice aged. However, there were no significant differences among the
three groups at any time point. Similar to the anti-DNA and
anti-GBM Ab responses, the lack of C3 did not affect IgG or IgM
rheumatoid factor production (data not shown).
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To determine the role of C3 on clearance of circulating ICs, we
measured circulating ICs by ELISA using the C1q binding assay. As shown
in Fig. 4, the mice in all three groups
developed increasing serum levels of ICs with aging, although there
were no significant differences among the three groups at any time
point. Sera from control BALB/c mice had no detectable circulating ICs
by this assay (data not shown).
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To determine the effect of C3 on autoimmune nephritis, we
measured 24-h urinary albumin excretion by ELISA beginning at 12 wk of
age. As shown in Fig. 5, the
MRL/lpr C3-/- mice developed
increasing albuminuria at 16 wk of age, while albuminuria remained
<0.1 mg/mouse/day in the other two groups. At 20 wk of age, the
C3+/- mice also started to develop albuminuria,
while the C3+/+ mice had a further delayed onset
and a lower level of albuminuria. At 24 wk of age, there was a
significant difference in albuminuria between the
C3-/- group and the other two groups. Due to
the death of the most severely affected C3-/-
mice between 24 and 30 wk, there was a decrease in albuminuria at 30 wk
in this group.
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A group of mice was sacrificed at the age of 24 wk (nine
C3+/+, four C3+/-, and
nine C3-/- mice). Despite the increased
albuminuria in the C3-/- mice, there were no
differences among the three groups of mice in the overall renal
pathology scores (Table I). In the
glomerular region, each of the three groups had marked glomerular
hypercellularity, mesangial expansion, inflammation, and epithelial
reactivity. In the interstitial region, all of the mice had chronic
interstitial inflammation with infiltration of inflammatory cells. None
of the kidneys from the three groups had significant tubular
changes.
Medium vessel vasculitis in the kidney is a pathologic feature of renal
disease in MRL/lpr mice (29). At 24 wk of age,
there was no significant difference in the incidence or severity of
vasculitis between the three groups (Table I).
Immunofluorescence analysis
To determine the role of C3 genotypes on glomerular IC deposition,
immunofluorescence analysis was performed. Frozen sections of the
kidneys from the three groups were stained with fluorescein-conjugated
anti-mouse C3 or IgG. As expected, there was no C3 detected in the
glomeruli of the C3-/- mice, while it was
readily evident in the glomeruli of the other two groups (Table II).
As shown in Fig. 6, there was
significantly greater IgG deposition overall in the
C3-/- mice compared with the wild-type
C3+/+ mice, although there was overlap in graded
values between the two groups. The small numbers of
C3+/- mice precluded statistical comparison of
IgG deposition to the other two groups, although the glomerular IgG
deposition in the C3+/- mice was overall similar
to the C3-/- mice.
Survival
MRL/lpr C3-/- and
C3+/- mice had increased early mortality
compared with the C3+/+ group with 57% mortality
at 30 wk compared with 22% of the mice in the
C3+/+ mice (Fig. 7). The difference in the survival
between the C3-/- and
C3+/- groups and the C3+/+
group, however, did not reach statistical significance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial hypothesis was that the genetic deficiency of C3 would likely be protective against the development of renal disease in MRL/lpr mice by preventing the inflammatory response and cellular damage mediated by complement activation. We were also aware, however, that C3 deficiency might worsen disease by altering IC clearance in a deleterious fashion. As described, genetic C3 deficiency in MRL/lpr mice had minimal effect on disease expression with the only significant differences being greater IC deposition in the kidneys and earlier onset of albuminuria in the C3-deficient mice.
Why C3 deficiency in MRL/lpr mice was not protective against renal disease is not clear and is in contrast with previously published reports of the effects of specific complement deficiencies on chronic inflammatory renal disease. Our laboratory recently described the beneficial effect of genetic deficiency of the alternative complement pathway component Factor B in MRL/lpr renal disease (36). In the Factor B-deficient mice, there was decreased C3 activation likely due to the lack of an amplification loop for C3 activation via Factor B. We postulated that this lack of C3 activation may partially explain the protection afforded by Factor B deficiency. It is also possible that MHC differences between the Factor B-deficient mice and the wild-type mice played a role in disease expression. The Factor B-deficient mice were H-2b/b whereas the wild-type mice were H-2k/k, perhaps affecting Ag presentation leading to differences in pathogenic Ab production. There is no published data to date suggesting a role of H-2 in disease in MRL/lpr mice; therefore, the mechanisms underlying renal protection in the Factor B-deficient mice warrants further investigation.
Further evidence for a critical role of complement activation in chronic inflammatory glomerulonephritis was demonstrated by blocking the activation of C5. Anti-C5 Ab treatment effectively decreased proteinuria and proliferative renal disease in NZB/NZW F1 mice, another murine model of SLE (13). Although we did not measure levels of C5a in our mice, we believe that levels of C5a would be low in mice deficient in C3 given that C3 activation is required for activation of C5. Thus, although the C3-deficient MRL/lpr mice were also likely deficient in activation of C5, they still developed renal disease.
Also in contrast to our results are studies of genetically or pharmacologically induced (e.g., cobra venom factor-treated mice) C3 deficiency in acute induced models of IC glomerulonephritis (12, 37, 38). In these models of glomerulonephritis, complement deficiency prevented the onset of renal disease. There are, however, significant differences between studying NZB/NZW F1 mice given anti-C5 mAb, cobra venom factor-treated mice with induced glomerulonephritis, and MRL/lpr mice genetically deficient in C3. For example, it is possible that the murine strains differ in the role of C3 in renal disease or that acute glomerulonephritis may be more complement dependent than chronic glomerulonephritis. More likely, however, the differential effect on renal disease reflects the other biologic effects of C3 beyond activation of C5 and its resultant mediation of cellular effector function. In addition, compensatory inflammatory pathways were likely activated in the MRL/lpr mice congenitally deficient in C3. In mice with an intact complement cascade, C5a is a key factor in recruitment of inflammatory cells to the kidney. In mice deficient in the ability to generate C5a from birth, other chemotactic factors, such as IL-8, likely compensate for the lack of C5a. Such compensation may not occur or may be delayed when C5 activation is blocked pharmacologically in an otherwise intact animal.
Our results, however, are in agreement with a recent study by Mitchell et al. (39). Using C1q-deficient mice that develop a lupus-like disease, these investigators determined the role of C3 in renal disease in this alternative murine model of SLE. Mice that are genetically deficient in Factor B and C2 were bred onto the C1q-deficient background. Lacking C2 and Factor B, these mice were unable to activate C3 by either the classical or alternative pathways and thus were functionally C3 deficient. Similar to our results, there was minimal to no effect of C3 deficiency on renal disease in the C1q-deficient mice, indicating that significant lupus-like renal disease can develop without the activation of C3.
The lack of effect of C3 deficiency on disease in MRL/lpr and C1q-deficient mice, we believe, reflects the multiple biologic effects of C3. C3 represents a double-edged sword for IC glomerulonephritis. Depending on the model of renal injury or whether disease is acute or chronic, C3 may be harmful due to its activation of inflammatory pathways or beneficial based on its effects in enhancing IC clearance.
It is well known that the activated complement components C4b and C3b bind covalently to ICs, which are then cleared from the circulation by the binding of C4b and C3b to complement receptors on the surface of erythrocytes in humans and platelets in rodents (3, 37, 40). One possible reason for the increased IC deposition in the C3-deficient mice glomeruli is from a lack of C3b. If this is the mechanism for the increased glomerular IC deposition in the C3-deficient mice, then serum IC levels should also be higher in C3-deficient mice, which they were not. The presence of C4b in the C3-deficient mice may have compensated for the lack of C3b in the clearance of circulating IC. It is also possible, although unlikely, that there were differences in serum levels of IC that we did not detect using the C1q assay.
Another possible reason for increased IC deposition in the glomerulus in the setting of C3 deficiency is altered IC transportation through the GBM. The complement system promotes transfer of IC across the GBM (41, 42). Recently, Sheerin et al. (43), by using an induced model of membranous glomerulonephritis in C3-deficient C57BL/6 mice, demonstrated that C3 deficiency retarded the passage of ICs across the GBM and led to an accumulation of ICs in the glomeruli. C3 and/or subsequent activation of the complement system in the glomeruli presumably alters the physiochemical characteristics of ICs in the subendothelial site that stimulates transmembrane passage. Our data in lupus-prone mice support this hypothesis regarding altered IC clearance in C3-deficient mice. The glomerulus is structurally adapted to filtering, causing it to be continually exposed to IC trapping; therefore, some mechanism is essential to remove ICs. The current data suggest that C3 is a critical mediator for removal of bound ICs in the glomerulus.
In the kidney, complement activation is regulated by a number of complement regulatory proteins such as decay accelerating factor (DAF), CD59, membrane cofactor protein (MCP), and CR1 (44). DAF, MCP, and CD59 are ubiquitously expressed on glomerular endothelial, epithelial, and mesangial cells, while CR1 is localized exclusively on podocytes. In rodents, a single membranous protein "Crry" possesses both DAF- and MCP-like functions and is expressed on the same resident glomerular cells as DAF and MCP (45, 46). Expression of these complement regulatory proteins is altered by complement attack itself and other factors such as cytokines (47, 48, 49, 50). In human lupus nephritis, expression of DAF and CD59 is increased in the glomerular capillary wall in epithelial and mesangial cells (47, 51, 52). Similarly, Crry expression is up-regulated in the kidneys of MRL/lpr mice as renal disease progresses (53). This up-regulation of complement regulatory proteins might be a protective response against the complement-mediated injury in this disease. Indeed, injection of soluble Crry and Crry-overproducing transgenic mice protects against injected Ab-induced renal disease, further supporting a deleterious role for complement in these models of glomerulonephritis (53, 54).
The complement system does not act in isolation in modulating
inflammation in glomerulonephritis. Other pro-and anti-inflammatory
pathways are activated by ICs. Mesangial cells are located in the
glomeruli and border directly on endothelial cells (capillaries), which
possess numerous microholes at the juxtacapillary region
(55). This histological structure allows ICs access to
FcRs expressed on mesangial cells (56, 57, 58). Mesangial
cells play an important role in inflammation in the kidney (59, 60). Interaction of ICs with mesangial cells via Fc receptors
triggers a cascade of renal injury characterized by cellular
proliferation, matrix protein accumulation, chemokine release, and
mononuclear cell recruitment (56, 61, 62, 63, 64, 65). A recent study
of lupus-prone mice (NZB/NZW F1 mice) deficient
for Fc receptor -chain I and III expression (FcRI and FcRIII)
indicated that the presence of FcRs was required for development of
proliferative glomerulonephritis (66). Despite the
glomerular deposition of ICs, including C3 and the activation of the
complement cascade, FcR-deficient mice developed significantly less
renal disease and had significantly increased survival compared with
control mice. These results suggest that C3 activation alone is
insufficient to induce renal disease without mesangial cell/macrophage
activation through Fc receptors. Complement activation, however,
clearly enhances immune responses mediated through Fc receptors and as
demonstrated by the Crry and anti-C5 experiments remains a viable
therapeutic target in treating glomerulonephritis.
In conclusion, our results demonstrate that 1) C3 and/or activation of C3 is not required for full expression of glomerulonephritis in MRL/lpr mice, and 2) C3 may in fact be beneficial in MRL/lpr mice through direct or indirect effects on the clearance of IC deposition in the glomeruli. These results are in contrast to a number of studies demonstrating beneficial effects of inhibiting complement factors in inflammatory glomerulonephritis. Additional studies are needed to define the factors that mediate the role of complement activation in glomerulonephritis, including the role of local vs systemic production and activation of complement. MRL/lpr C3-deficient mice provide a novel and useful model for dissecting the role of C3-dependent and C3-independent effects in autoimmune disease.
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Footnotes |
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2 Abbreviations used in this paper; IC, immune complex; GBM, glomerular basement membrane; TMB, 3,3',5,5'-tetramethylbenzidene; SLE, systemic lupus erythematosus; RT, room temperature; DAF, decay accelerating factor; MCP, membrane cofactor protein.
Received for publication January 2, 2001. Accepted for publication March 12, 2001.
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 R. J. Brownlie, K. E. Lawlor, H. A. Niederer, A. J. Cutler, Z. Xiang, M. R. Clatworthy, R. A. Floto, D. R. Greaves, P. A. Lyons, and K. G.C. Smith Distinct cell-specific control of autoimmunity and infection by Fc{gamma}RIIb J. Exp. Med., April 14, 2008; 205(4): 883 - 895. [Abstract] [Full Text] [PDF]
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C. Atkinson, F. Qiao, H. Song, G. S. Gilkeson, and S. Tomlinson Low-Dose Targeted Complement Inhibition Protects against Renal Disease and Other Manifestations of Autoimmune Disease in MRL/lpr Mice J. Immunol., January 15, 2008; 180(2): 1231 - 1238. [Abstract] [Full Text] [PDF]
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 T. Miwa, M. A. Maldonado, L. Zhou, K. Yamada, G. S. Gilkeson, R. A. Eisenberg, and W.-C. Song Decay-Accelerating Factor Ameliorates Systemic Autoimmune Disease in MRL/lpr Mice via Both Complement-Dependent and -Independent Mechanisms Am. J. Pathol., April 1, 2007; 170(4): 1258 - 1266. [Abstract] [Full Text] [PDF]
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 J Dong, Q-X Wang, C-Y Zhou, X-F Ma, and Y-C Zhang Activation of the STAT1 signalling pathway in lupus nephritis in MRL/lpr mice Lupus, February 1, 2007; 16(2): 101 - 109. [Abstract] [PDF]
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H. Sekine, K. L. Graham, S. Zhao, M. K. Elliott, P. Ruiz, P. J. Utz, and G. S. Gilkeson Role of MHC-Linked Genes in Autoantigen Selection and Renal Disease in a Murine Model of Systemic Lupus Erythematosus J. Immunol., November 15, 2006; 177(10): 7423 - 7434. [Abstract] [Full Text] [PDF]
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 J. J. Alexander, O.G.B. Aneziokoro, A. Chang, B. K. Hack, A. Markaryan, A. Jacob, R. Luo, M. Thirman, M. Haas, and R. J. Quigg Distinct and Separable Roles of the Complement System in Factor H-Deficient Bone Marrow Chimeric Mice with Immune Complex Disease J. Am. Soc. Nephrol., May 1, 2006; 17(5): 1354 - 1361. [Abstract] [Full Text] [PDF]
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N. S. Sheerin, K. Abe, P. Risley, and S. H. Sacks Accumulation of Immune Complexes in Glomerular Disease Is Independent of Locally Synthesized C3 J. Am. Soc. Nephrol., March 1, 2006; 17(3): 686 - 696. [Abstract] [Full Text] [PDF]
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S. E. Wenderfer, B. Ke, T. J. Hollmann, R. A. Wetsel, H. Y. Lan, and M. C. Braun C5a Receptor Deficiency Attenuates T Cell Function and Renal Disease in MRLlpr Mice J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3572 - 3582. [Abstract] [Full Text] [PDF]
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L. Bao, I. Osawe, M. Haas, and R. J. Quigg Signaling through Up-Regulated C3a Receptor Is Key to the Development of Experimental Lupus Nephritis J. Immunol., August 1, 2005; 175(3): 1947 - 1955. [Abstract] [Full Text] [PDF]
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J. J. Alexander, M. C. Pickering, M. Haas, I. Osawe, and R. J. Quigg Complement Factor H Limits Immune Complex Deposition and Prevents Inflammation and Scarring in Glomeruli of Mice with Chronic Serum Sickness J. Am. Soc. Nephrol., January 1, 2005; 16(1): 52 - 57. [Abstract] [Full Text] [PDF]
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X. K. Zhang, S. Gallant, I. Molano, O. M. Moussa, P. Ruiz, D. D. Spyropoulos, D. K. Watson, and G. Gilkeson Decreased Expression of the Ets Family Transcription Factor Fli-1 Markedly Prolongs Survival and Significantly Reduces Renal Disease in MRL/lpr Mice J. Immunol., November 15, 2004; 173(10): 6481 - 6489. [Abstract] [Full Text] [PDF]
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R L Rubin, M Teodorescu, E H Beutner, and R W Plunkett Complement-fixing properties of antinuclear antibodies distinguish drug-induced lupus from systemic lupus erythematosus Lupus, April 1, 2004; 13(4): 249 - 256. [Abstract] [PDF]
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R. J. Quigg Complement and the Kidney J. Immunol., October 1, 2003; 171(7): 3319 - 3324. [Full Text] [PDF]
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L. Bao, M. Haas, D. M. Kraus, B. K. Hack, J. K. Rakstang, V. M. Holers, and R. J. Quigg Administration of a Soluble Recombinant Complement C3 Inhibitor Protects Against Renal Disease in MRL/lpr Mice J. Am. Soc. Nephrol., March 1, 2003; 14(3): 670 - 679. [Abstract] [Full Text] [PDF]
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T. Miwa, M. A. Maldonado, L. Zhou, X. Sun, H. Y. Luo, D. Cai, V. P. Werth, M. P. Madaio, R. A. Eisenberg, and W.-C. Song Deletion of Decay-Accelerating Factor (CD55) Exacerbates Autoimmune Disease Development in MRL/lpr Mice Am. J. Pathol., September 1, 2002; 161(3): 1077 - 1086. [Abstract] [Full Text] [PDF]
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 S. Li, V. M. Holers, S. A. Boackle, and C. M. Blatteis Modulation of Mouse Endotoxic Fever by Complement Infect. Immun., May 1, 2002; 70(5): 2519 - 2525. [Abstract] [Full Text] [PDF]
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L. Bao, M. Haas, S. A. Boackle, D. M. Kraus, P. N. Cunningham, P. Park, J. J. Alexander, R. K. Anderson, K. Culhane, V. M. Holers, et al. Transgenic Expression of a Soluble Complement Inhibitor Protects Against Renal Disease and Promotes Survival in MRL/lpr Mice J. Immunol., April 1, 2002; 168(7): 3601 - 3607. [Abstract] [Full Text] [PDF]
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D. A. Mitchell, M. C. Pickering, J. Warren, L. Fossati-Jimack, J. Cortes-Hernandez, H. T. Cook, M. Botto, and M. J. Walport C1q Deficiency and Autoimmunity: The Effects of Genetic Background on Disease Expression J. Immunol., March 1, 2002; 168(5): 2538 - 2543. [Abstract] [Full Text] [PDF]
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S. Einav, O. O. Pozdnyakova, M. Ma, and M. C. Carroll Complement C4 Is Protective for Lupus Disease Independent of C3 J. Immunol., February 1, 2002; 168(3): 1036 - 1041. [Abstract] [Full Text] [PDF]
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