|Home | About | Journals | Submit | Contact Us | Français|
The development and progression of systemic lupus erythematosus (SLE) is strongly associated with complement activation and deposition. The anaphylatoxin C3a is a product of complement activation with immunomodulatory properties, and the receptor for C3a (C3aR) is not only expressed by granulocytes and antigen presenting cell populations, but it is also strongly up-regulated in lupus prone mice with active nephritis. In order to characterize the role of the C3aR in inflammatory nephritis, we bred C3aR knock-out mice onto the MRL/lpr genetic background (C3aR KO MRL). Compared to control MRL/lpr mice, C3aR KO MRL mice had elevated autoantibody titers and an earlier onset of renal injury. At 8 weeks, renal expression of a wide range of chemokines and chemokine receptors was increased in C3aR KO MRL kidneys compared to controls. Only the expression of MCP-1 was significantly decreased in the C3aR KO MRL mice. The increased chemokine and chemokine receptor expression seen in the C3aR KO MRL mice was associated with a more rapid rise in serum creatinine and the acceleration of renal fibrosis. However, loss of the C3aR had little impact on long-term kidney injury and did not alter survival. These findings suggest that activation of the C3aR plays a protective, not pathologic, role in the early phase of inflammatory nephritis in the MRL/lpr model of SLE.
Systemic lupus erythematosus is an autoimmune disorder where production of auto-antibodies, circulating immune complexes, systemic complement activation, and auto-reactive T-cells are associated with multi-system injury including nephritis, arthritis, serositis, dermatitis, and blood dyscrasias. Lupus nephritis is mediated in part by deposition and local production of immune complexes and complement activation products. Complement proteins participate at several levels in disease pathogenesis (Liszewski, 1989). Genetic deficiencies in the early components of the classical complement pathway, C1 inhibitor, C1q/r/s, C2, or C4, confer increased risk for developing disease (Manderson, 2004). While complement activation enhances the clearance of pathological immune complexes, the deposition of complement activation products contributes to inflammation, fibrosis, and local tissue injury (Couser, 1985).
The complement system is a cascade of enzymatic reactions with multiple immunologic effects, including cell lysis, B-cell activation, leukocyte recruitment, and clearance of apoptotic cells and immune complexes. Anaphylatoxins, C3a and C5a, are generated during complement activation. Activation of complement leads to the formation of C3b and C3a, and in turn C3b production leads to the generation of C5a and the membrane attack complex. Traditionally, C3a has been thought to be pro-inflammatory, enhancing cytokine production, mediating eosinophil chemotaxis, leukocyte degranulation, histamine release, and increased vascular permeability (Fischer, 1999; Wetsel, 1995). The effects of C3a require interaction with a specific receptor (C3aR), a 55-kDa protein of the rhodopsin family of G protein-coupled seven-transmembrane receptors. C3aR is expressed on leukocytes including, mast cells, eosinophils, dendritic cells, and some B-lymphocytes (Fischer, 1997; Kirchhoff, 2001; Werfel, 2000; Zwirner, 1999). In the kidney, C3aR expression is limited to glomerular podocytes and proximal tubular epithelial cells (Bao, 2005a; Braun, 2004; Peake, 1999). C3a/C3aR interactions on proximal tubular epithelial cells result in an increase in TGF-β and type I collagen production (Braun, 2004; Peake, 1999).
The MRL/MpJ-Tnfrsf6lpr (MRL) mouse is a widely used and extensively studied mouse strain which develops a severe spontaneous autoimmune disease similar to SLE (Hicks, 2006). The lpr mutation, a retroviral transposon insertion in the FAS gene, results in loss of FAS function and thus a defect in FAS mediated apoptosis (Kono, 2000; Nose, 2000), massive lymphoproliferation with the generation of auto-reactive T-cells, autoantibody formation, and circulating immune complexes (Hicks, 2006). The ensuing autoimmune disease is characterized by lymphadenopathy, complement activation, severe immune complex renal disease, and 50% lethality by 20 to 24 wks (Andrews, 1978). C3aR levels have been reported to be up-regulated in the kidneys of MRL mice as early as 6 wks, long before the development of nephritis (Bao, 2005a).
Based on the hypothesis that C3a acting via the C3aR may have a major functional role mediating disease progression in SLE, mice with a targeted deletion of the C3aR gene were bred onto the MRL/lpr genetic background (here after referred to as C3aR KO MRL). Comparative analyses of immunologic responses and indices of renal injury were then performed between MRL/lpr control (CTRL MRL) and C3aR KO MRL mice. Experimental data contained in this report suggest that loss of the C3aR results in accelerated onset, but not increased severity, of renal injury; thus, the activation of the C3aR is more likely to be protective than pathologic in the MRL/lpr model.
MRL mice (Jackson Laboratories, Bar Harbor, ME) and C3aR KO C57BL/6 mice (Kildsgaard, 2000a) maintained in our animal colony were employed for backcrossing. The gene encoding the C3aR maps to chromosome 6 (64.8cM) (Hollmann, 2007), a region which is not known to contain epigenetic modifiers for autoantibody formation, lymphoproliferation, or nephritis (Kono, 2006; Nguyen, 2002). F9 generation C3aR+/−MRL mice were then intercrossed to obtain homozygous C3aR−/− MRL/lpr (C3aR KO MRL) mice and C3aR+/+ MRL/lpr controls (CTRL MRL). Genotyping was confirmed by PCR for all F9 mice used in this study (data not shown). Only female mice were used for the studies. These studies were approved by the UTHSC-H Animal Welfare Committee.
Leukocytes were obtained for FACS analysis from spleens (16 and 20 wks), peripheral blood (20 wks), and cervical lymph nodes (20 wks). Cell populations were characterized with the following markers (eBiosciences, San Diego, CA): CD3 (clone 145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD19 (6D5), CD25 (PC61.5), (CD45/RB220 (RA3-6B2), and CD62L (MEL-14), and GR-1(Ly-6G). A minimum of 10,000 events were collected and analyzed on a FACSCaliber using CellQuest software (BD Biosciences, San Diego, CA).
Serum levels of C3 and titers of antibodies specific for double stranded DNA were measured by ELISA as previously reported (Wenderfer, 2005). For the non-quantitative C3 ELISA, goat polyclonal antisera specific for mouse C3 (Cappel/MP Biomedical, Solon, OH) was used for both capture and for detection, and results were compared between sera from C3aR KO MRL mice, CTRL MRL mice and pooled serum from non-autoimmune C57BL/6 mice. For autoantibody responses, end-point titers were measured by serial dilutions. Results are shown as fold differences in A450 between C3aR KO MRL and CTRL MRL serum at a 1/100 dilution.
Serum and urine was obtained from mice at 20 wks immediately prior to histologic analysis. Serum and urine creatinine was determined using a modified alkaline picrate method (Exocell, Philadelphia, PA); Urinary protein concentration was determined by BCA assay (Thermo Scientific, Rockford, IL) and normalized for urinary creatinine concentration.
Renal tissue was fixed in PBS buffered 4% formalin, dehydrated and embedded in paraffin. Four micron sections were stained with Periodic Acid Schiff (PAS) or Sirius red/picric acid stain (Grimm, 2003). Glomerular and tubular injury was scored as previously described (Wenderfer, 2005), except that tubulointerstitial disease in the renal cortex was also quantified by measuring the cortical fractional interstitial fibrosis volume of Sirius red staining in 10 high powered fields (hpf) per mouse (excluding glomeruli) (Grimm, 2003) using Image Pro software (Media Cybernetics, Inc., Bethesda, MD).
OCT embedded snap-frozen 4 micron sections were stained with the following antibodies: FITC conjugated anti-mouse C3 (ICN, Aurora, OH), IgG (Jackson ImmunoResearch, West Grove, PA), CD8, and CD4 (BD Biosciences). Control staining was also performed using matched isotypes or IgG (data not shown). A minimum of 10 glomeruli and 10 hpfs were scored per animal in a blinded manner as described previously (Wenderfer, 2005).
Quantitative mRNA expression analysis of chemokines and their receptors was performed with the mouse chemokine and receptor RT2 profiler PCR array (SA Bioscience Corporation, Frederick, MD). Kidney cortexes of different groups of mice (n=3/group) were collected after cardiac perfusion with HBSS. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) and followed by Turbo DNA-free treatment (Ambion, Austin, TX) and RNeasy Mini Kit clean up (Qiagen, Valencia, CA). Equal amounts of RNA from each sample were then converted to cDNA using RT2 First Strand Kit (SABiosciences, Frederick, MD). Quantitative real-time PCR was performed according to the manufacturer’s protocol using RT2 profiler PCR array PAMM-022 (SA Biosciences) on the Applied Biosystems 7900. Data were analyzed using ΔΔCt method to determine the expression level of each gene of interest normalized to the expression level of housekeeping gene controls. Fold differences in gene expression was calculated by normalizing expression in C3aR KO MRL mice to CTRL MRL mice to as 2(−ΔΔCt).
Data were analyzed using Sigma Stat software version 3.0 (Jandel Scientific, San Rafael, CA). Survival analysis was performed using log-rank analysis. Data in tables are presented as means ± standard error. Comparisons between groups were performed using the Student’s t test. A gene-wise, two-sample, t-test was done for each transcript to identify statistical differences in expression between C3aR KO MRL mice to CTRL MRL mice in vivo. P-values < 0.05 were assumed to be statistically significant.
Survival analysis was performed for up to 30 wks to study the effect of progressive renal disease in these strains. Mean survival in the C3aR KO MRL mice was 21.4 wks, compared to 23.7 wks for CTRL MRL mice. Kaplan-Meier analysis revealed a trend towards decreased survival in C3aR KO MRL mice (Figure 1); however, this did not reach statistical significance.
Both C3aR KO MRL and CTRL MRL mice developed systemic inflammation with multi-organ involvement. Inflammatory infiltrates in the lungs, liver, and intestines were assessed at 20 wks and were equivalent in C3aR KO MRL mice and their CTRL MRL littermates (data not shown). C3aR KO MRL mice developed dermatitis at rates comparable to CTRL MRL mice. Serum C3 levels were also measured using a semi-quantitative ELISA. C3aR KO MRL mice and CTRL MRL littermates had equivalent levels of circulating C3, with no apparent depletion compared to levels seen in pooled mouse serum (data not shown).
Characterization of splenic leukocytes at 16 wks and 20 wks revealed no significant differences between C3aR KO MRL and CTRL MRL mice in absolute splenocyte number or T-cell subsets (Table 1). The expression of T-cell activation markers as well as the proportion of CD4+CD25+ Treg cells was also similar between groups (data not shown). With respect to auto-antibody production, C3aR KO MRL mice developed slightly higher anti-double stranded DNA antibody titers, which was predominantly IgG2a (Figure 2).
The renal injury seen in MRL mice is a chronic progressive disease characterized by abnormal proteinuria and renal failure. At 20 wks, C3aR KO MRL mice had a 20% increase in proteinuria compared to CTRL MRL littermates (5.5 mg/mg vs. 4.6 mg/mg creatinine respectively)(Table 2). In addition, serum creatinine concentrations were 50% higher in C3aR KO MRL mice (1.5 mg/dl) compared to control mice (1.0 mg/dl).
Histologically, there were clear differences in the patterns of renal injury between the two strains (Table 3). At 16 wks, C3aR KO MRL mice had more glomerular crescents and more severe intra-renal vasculitis compared to CTRL MRL mice (Figure 3). While there were no significant differences in the severity of tubulo-interstitial infiltrates, the C3aR KO MRL mice had significantly more interstitial fibrosis than control mice with the greatest increase in Sirius red staining surrounding superficial rather than deep cortical glomeruli (Figure 4) By 20 wks, the differences in renal injury between the two strains became non-significant, although there was a trend toward more injury in the C3aR KO MRL mice in regard to both glomerular crescents and glomerulosclerosis. At 20 wks, there were no differences in the pattern or the intensity of glomerular IgG or C3 staining between the mouse strains, nor were there difference in CD4+ or CD8+ T-cell infiltrates (Table 4).
In order to explore the mechanism of the early onset of renal injury seen in C3aR KO MRL mice, chemokine and chemokine receptor expression was analyzed using qRT-PCR. Expression was measured at 8 wks, prior to onset of overt renal injury, and at 14 wks, just before histological differences were greatest between strains. These data are summarized in Figure 5. At 8wks, only a single chemokine (Ccl2/MCP-1) was significantly down-regulated at the mRNA level in C3aR KO MRL kidneys compared to controls. Conversely, significant increases in expression were detected at 8 weeks in four Ccl (Ccl5/RANTES, Ccl7, Ccl8, and Ccl19), three Cxcl (Cxcl5, Cxcl13, and Cxcl15), and one Xcl (Xcl1) class chemokines. There were also significant increases in five chemokine receptors (Ccr1l1, Ccr6, Ccr7, Ccr8, and Cxcr5). The differences in expression detected at 8wks disappeared by 14wks, at which time the trend was towards decreased mRNA expression in the C3aR KO MRL kidney. This was the result of relatively static levels of chemokine and chemokine receptor expression in the C3aR KO MRL mice with concurrent increases in expression in the CTRL MRL mice.
The pathogenic role of complement in the MRL model is complex. While human data strongly suggested a pathogenic role for C3 in lupus nephritis, MRL mice deficient in C3 paradoxically develop slightly more severe disease (Sekine, 2001). The absence of C3 was not associated with changes in renal pathology, the degree of lymphoproliferation, the titers of autoantibodies, or the levels of circulating immune complexes. Survival at 24 wks was similar, but there was a trend toward decreased survival in C3 deficient MRL mice. There was also increased proteinuria and glomerular IgG deposition in the C3 deficient MRL mice, which was thought to be due to the loss of C3 dependent clearance of circulating immune complexes and apoptotic bodies.
Cleavage of C3 not only generates products involved to IC clearance, but also the anaphylatoxin C3a. The anaphylatoxins are one of the primary effector mechanisms of complement activation, and the major complement anaphylatoxins C3a and C5a exert their effects via binding to the specific receptors. Previously, we described attenuated disease in C5aR KO MRL mice (Wenderfer, 2005). Here, we report that C3aR KO MRL mice develop accelerated onset of renal disease compared to CTRL MRL mice. Similar to C3 deficient MRL mice (Sekine, 2001), C3aR KO MRL mice had an earlier onset of renal injury compared to CTRL MRL mice and a trend toward decreased survival. C3 deficient MRL mice and C3aR KO MRL mice both developed exacerbated tubulo-interstitial fibrosis. While the glomerular immune complex deposition typical for the MRL model is unaffected by C3a/C3aR interactions, the loss of C3aR resulted in earlier onset of perivascular infiltrates and chronic tubulo-interstitial changes. As the complement split product C3a is the only known ligand for the C3aR, the results suggest that C3a generation is protective in the MRL mouse.
The opposing effects of the anaphylatoxin receptors C3aR and C5aR in disease progression in MRL mice is not unexpected, based on reports in other models using C3aR and C5aR KO mice. C3aR KO mice have exacerbated endotoxemia from LPS treatment (Kildsgaard, 2000b) and worse outcome after gram-negative bacteremia (Hollmann, 2007), whereas C5aR KO mice have attenuated responses to cecal ligation and puncture (Riedemann, 2004) as well as gram-negative bacteremia and endotoxic shock (Hollmann, 2007). The receptors also play opposing roles in Aspergillus-induced airway inflammation (Drouin, 2002; Kohl, 2006), Pseudomonas pneumonia (Hopken, 1996; Mueller-Ortiz, 2006), and contact dermatitis (Kawamoto, 2004; Tsuji, 2000). Opposing effects could be mediated either by differential expression of C3aR or C5aR on individual cell types, separate signaling pathways within the same cell, or via counter-regulation of anaphylatoxin receptor expression (Sayah, 2003; Schraufstatter, 2002;Melendi, 2007 Settmacher, 1999).
Previous studies have suggested that complement anaphylatoxins might regulate fibrosis (Addis-Lieser, 2005; Bao, 2003; Boor, 2007). The accelerated tubulo-interstitial fibrosis in C3aR KO MRL mice, combined with previous studies demonstrating reductions in renal fibrosis with loss of C5aR function (Bao, 2003; Welch, 2002; Wenderfer, 2005), suggest that chronic complement activation modulates the development of renal fibrosis, with the C5aR activation driving fibrosis and C3aR activation limiting its progression.
Serum autoantibody titers were elevated in C3aR KO MRL mice, and this increase was more prominent for the IgG2a isotype. In contrast, C5aR KO MRL mice were previously shown to have elevated IgG1 and depressed IgG2a anti-dsDNA antibody titers. Disease in lupus-prone mice is thought to be a Th1-mediated disease (Balomenos, 1998; Schwarting, 1999; Takahashi, 1996; Yin, 2002), where the IgG2a subtype is more prevalent. Therefore, the isotype predominance may simply reflect the overall disease activity in this model.
The results described here differ from those published previously showing that MRL mice treated with a C3a analog, SB290157, had preservation of renal function, delayed onset of proteinuria, and decreased tubulo-interstitial leukocyte infiltrates (Bao, 2005a). Bao et al treated MRL mice continuously from 13 to 19wks of age with 60mcg/gm/day of SB290157. At the time, SB290157 was thought to be a specific antagonist of C3aR (Ames, 2001). However, both agonist and antagonist activity of SB290157 has been described in vitro (Mathieu, 2005; Purwar, 2006). SB290157 appears to act as an antagonist when receptor density is low, while it has agonist activity in cell lines expressing higher levels of the C3aR, suggesting that it is a partial agonist (Mathieu, 2005). This may be particularly relevant in the MRL mouse, where renal C3aR expression is markedly increased early in the disease process (Bao, 2005a). Paradoxical effects of SB290157 have been reported in other disease models as well (Baelder, 2005; Bautsch, 2000; Drouin, 2002; Humbles, 2000). In addition to the partial agonist affects, SB290157 can lead to dose dependent neutropenia (Proctor, 2004). While the MRL mice treated with SB290157 were not tested for neutropenia, significant reductions in the numbers of renal infiltrating neutrophils were reported (Bao, 2005a). Thus it is possible that prolonged exposure to SB290157 may result in chronic neutropenia and improved survival independent of C3aR inhibition.
The data on patterns of chemokine and chemokine receptor expression were consistent with the pathologic findings in this study. At 8 wks, there were significant increases in transcript expression of several pro-inflammatory chemokines and chemokine receptors in C3aR KO MRL mice compared to CTRL MRL mice. The differences of expression level disappeared at 14 wks. The reduction in Ccl2/MCP-1 in the C3aR KO kidney’s is consistent with prior reports that MCP-1 induction is in part C3a dependent (Ahamed, 2004; Venkatesha, 2005). However, as the C3aR is thought to play a major role in granulocyte chemotaxis, it was surprising that the granulocyte chemotactic proteins, Cxcl5 (Wuyts, 1996) and Cxcl15 (Rossi, 1999) and the granulocyte receptor Cxcr1/IL8Ra (Fan, 2007; Moepps, 2006) were up-regulated early in C3aR KO MRL kidneys. This suggests that there may be an intrinsic compensatory response mediating granulocyte chemotaxis in the C3AR KO mice.
There were two sets of chemokine and cognate receptor pairs up-regulated (Ccl19/Ccr7 and Cxcl13/Cxcr5). CCR7 is expressed in many lymphoid tissues and activates B- and T-lymphocytes. Ccl19/Ccr7 interaction enhances migration of dendritic cells and T-cells to and within lymphoid tissues (Davalos-Misslitz, 2007; Debes, 2005), promotes renal fibrosis in the unilateral ureteral obstruction model (Sakai, 2006), and also drives production of bronchial-associated lymphoid tissue (Kocks, 2007). A similar mechanism may explain the large perivascular lymphoid infiltrates seen in the MRL kidney and accelerated in the C3aR KO MRL kidney. Cxcl13/Cxcr5 interaction has been implicated in human lupus and animal models of inflammatory nephritis (Adalid-Peralta, 2008; Duan, 2008; Steinmetz, 2008). Ccr6 and Ccr8 were also up-regulated, and are more restricted to cells of the adaptive immune response: CCR6 on dendritic cells, inactivated memory T-cells, and Th17 cells (Acosta-Rodriguez, 2007; Schutyser, 2003), CCR8 on monocytes and activated T-cells (Cosmi, 2001), and CXCR5 on follicular dendritic cells, T- and B-cells. Cell migration via CCR6, CCR7, CCR8, and CXCR5 is important in germinal center formation and delayed type hypersensitivity responses (Arnold, 2007; Hardtke, 2005; Jakubzick, 2006; Qu, 2004; Steinmetz, 2008; Varona, 2001) and may contribute to formation of lymphoid follicles in inflamed MRL kidneys. Further investigation into regulation of chemokine and chemokine receptor expression by anaphylatoxins will be required to determine whether these are direct or indirect effects.
Compared to CTRL MRL littermates, C3aR KO MRL mice have acceleration in the onset of renal injury and more severe interstitial fibrosis. This is associated with a significant broadly based increase in chemokines and chemokine receptor expression at 8 wks. However, the enhanced expression of chemokines and chemokine receptors declined markedly by 14 wks and by 20 wks the differences in glomerular injury were modest. Based on these data, it appears that activation of the C3aR in the MRL/lpr mouse functions primarily to delay the onset of chronic renal inflammation and fibrosis. The C3aR is neither sufficient nor required as a pathogenic mediator as the loss of C3aR expression had little overall impact on the severity of auto-immunity or long-term survival in the MRL/lpr model of lupus nephritis.
The authors would like to thank Todd Triplett for his technical assistance and Dr. Irma Gigli for her guidance and review of the manuscript. Supported in part by NIH grants DK61929 (SEW), AI025011, HL074333, AI068795 (RAW); DK071057, DK072322, and DK062197 (MCB).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.