Lupus nephritis develops from a combination of genetic and environmentalfactors such as microbial infection. A role for microbial nucleicacids (e.g., via nucleic acid–specific Toll-like receptors[TLR]) was hypothesized, in this context, because microbialnucleic acids can trigger multiple aspects of autoimmunity invitro and in vivo. Eight-week-old MRLlpr/lpr and MRL wild-typemice received an injection of pI:C RNA (ligand to TLR-3), imiquimod(ligand to TLR-7), or CpG-DNA (ligand to TLR-9) on alternatedays for 2 wk. Only CpG-DNA triggered the onset of lupus nephritisin MRLlpr/lpr mice, as defined by diffuse proliferative glomerulonephritisassociated with glomerular IgG and complement C3 deposition,proteinuria, and glomerular macrophage infiltrates. None ofthe compounds caused DNA autoantibody production or glomerulonephritisin MRL wild-type mice. The role of CpG-DNA to trigger lupusnephritis in MRLlpr/lpr mice was found to relate to its potentimmunostimulatory effects at multiple levels: B cell IL12p40production, B cell proliferation, double-stranded DNA autoantibodysecretion, and dendritic cell IFN- production. The inductionof lupus nephritis by CpG-DNA is motif specific and could beprevented by co-injection of inhibitory DNA. In summary, amongthe ligands tested, CpG-DNA triggers lupus nephritis in geneticallypredisposed hosts. These data support the concept that systemiclupus erythematosus is triggered by pathogens that release CG-richDNA.
Systemic lupus erythematosus (SLE) usually develops from a combinationof multiple susceptibility genes (1,2). Because most monozygotictwins are discordant for the clinical manifestations of lupus,coincident environmental factors may play a role (3). Microbialinfections are suspected to trigger the onset of lupus, similarto their postulated role in the pathogenesis of rheumatic feveror type 1 diabetes (4). Attempts to identify a single causativepathogen have been unsuccessful, but molecular mimicry to asingle pathogen epitope is unlikely to cause polyclonal lymphoproliferationin lupus. By contrast, persistent viral replication may providea stimulus for chronic lymphoproliferation and SLE (5). Alternatively,several pathogens could trigger autoimmunity, if susceptibilitygenes tune the antimicrobial immune response toward a loss oftolerance (6). Thus, more general pathogen-associated molecularpatterns (PAMP) with immunostimulatory qualities may triggerthe onset of lupus in a genetically predisposed host.
The discovery of the Toll-like receptor (TLR) family has contributedsignificantly to our understanding of how PAMP recognition translatesinto innate and adaptive immune responses (7,8). TLR recognizePAMP that occur in all classes of microbes (e.g., diacetyl andtriacetyl lipoproteins [TLR-1, -2, -6], LPS [TLR-4], double-strandedRNA [dsRNA; TLR-3], single-stranded RNA (TLR-7, -8), or CpG-DNA(TLR-9). In the context of SLE, the nucleic acid–relatedPAMP are particularly attractive in view of their potentialto trigger autoimmune tissue injury. For example, exposure toPAMP that ligate TLR-3 or -7 is required to induce autoimmunediabetes in transgenic mice that harbor large numbers of pancreaticislet–reactive cytotoxic T cells (9). In this model, theTLR-induced local production of IFN- was identified to triggerthe recruitment of autoreactive T cell infiltrates into thepancreatic islet. In addition, the role of TLR-3 or -9 ligationfor dendritic cell maturation was also documented in a modelof autoimmune myocarditis in which dendritic cells that wereloaded with a heart-specific self-peptide could not induce Tcell–mediated myocarditis unless being pulsed with dsRNA,LPS, or CpG-DNA (10). After resolution of acute myocarditis,repetitive exposure to these PAMP resulted in relapse of disease.Because lymphoproliferation and autoantibody production aretypical characteristics of SLE, it is noteworthy that viralsingle-stranded RNA and CpG-DNA can uncouple the proliferationof autoreactive B cells and autoantibody production from anycontrol by T helper cells (11,12). In addition, chromatin-containingimmune complexes from patients with lupus have been identifiedas potent inducers of IFN- production, a marker of disease activityin lupus (13,14). Furthermore, CpG-DNA was shown to activatedendritic cells to produce IL-6, which inhibits the CD4+CD25+T cell–mediated suppression of autoreactive T cells (15).Thus, microbial nucleic acids may represent universal PAMP thatcould contribute to the onset of lupus through multiple mechanisms,including the induction of lymphoproliferation, autoantibodyproduction, cytokine, and type I IFN production as well as thecontrol of autoreactive B and T cell subset (12,15–17).In view of these in vitro studies, we questioned whether transientexposure to dsRNA, imiquimod, and CpG-DNA would trigger lupusnephritis in vivo. In exposing young lupus-prone MRLlpr/lprmice or nonautoimmune MRL wild-type mice to these compounds,we also addressed the role of the lpr mutation for TLR-mediatedinduction of autoimmune tissue injury in MRL mice.
Animals and Experimental Protocol
Five-week-old female MRLlpr/lpr or MRL wild-type mice were obtainedfrom Harlan Winkelmann (Borchen, Germany) and maintained throughoutin filter-top cages under a 12-h light/dark cycle. Autoclavedwater and standard chow (Sniff, Soest, Germany) were availablead libitum. At 8 wk of age, MRL and MRLlpr/lpr mice were distributedinto groups of five mice that received intraperitoneal injectionsof the following compounds on alternate days: pI:C RNA 50 µg(Sigma-Aldrich, Taufkirchen, Germany), 25 µg of imiquimod(Sequoia Research Products, Oxford, UK) in 100 µl of 0.25%sodium acetate (Merck, Darmstadt, Germany), 40 µg of CpG-DNA,40 µg of GpC-DNA, 40 µg of G-rich DNA, or a combinationof 40 µg of CpG-DNA and 40 µg of G-rich DNA (TIBMolbiol,Berlin, Germany). The sequences of the synthetic oligodeoxynucleotides(ODN) were as follows: CpG-DNA (ODN 1668) 5'-TCC ATG ACG TTCCTG ATG CT-3', GpC-DNA (ODN 1720) 5'-TCG ATG AGC TTC CTG ATGCT-3', and G-rich inhibitory DNA (ODN 2114) 5'-TCC TGG AGG GGAAGT-3'. All mice were killed by cervical dislocation at theend of week 10 of age. The experimental procedure had been approvedby the local government authorities.
Evaluation of Glomerulonephritis
Blood and urine samples were collected from each animal at theend of the study period as described (18). Urine albumin concentrationand serum dsDNA autoantibodies were determined by ELISA as describedpreviously (19). Urinary creatinine concentrations were determinedusing an automatic autoanalyzer (Integra 800; Roche Diagnostics,Mannheim, Germany). From all mice, kidneys were fixed in 10%buffered formalin, processed, and embedded in paraffin. Two-micrometersections for periodic acid-Schiff stains were prepared followingroutine protocols (data not shown). The severity of the renallesions was graded using the indices for activity and chronicityas described for human lupus nephritis (20).
Immunostaining
Immunostaining was performed on either paraffin-embedded orfrozen sections as described (18) using the following primaryantibodies: Anti-mouse Mac-2 (1:50, monocytes/macrophages; BDPharmingen, Hamburg, Germany), anti-mouse CD3 (1:100; Serotec,Oxford, UK), anti-mouse B220 (1:400, early Pro-B to mature Bcells, clone RA3–6B2; BD Pharmingen), anti-mouse Ki-67(1:100, cell proliferation; Dianova, Hamburg, Germany), anti-mouseIgG1 (1:100, M32015; Caltag Laboratories, Burlingame, CA), andanti-mouse IgG2a (1:100, M32215; Caltag), anti-mouse C3 (1:200,GAM/C3c/FITC; Nordic Immunological Laboratories, Tilburg, Netherlands).Negative controls included incubation with a respective isotypeantibody. For quantitative analysis, glomerular cells were countedin 15 cortical glomeruli per section. Semiquantitative scoringof complement C3 deposits from 0 to 3 was performed on 15 corticalglomerular sections as described (18).
Cell Culture Conditions and Cytokine ELISA
Bone marrow–derived dendritic cells and CD19-positiveB cells were isolated from MRL and MRLlpr/lpr mice, processed,and cultured as described (21). Cells were stimulated with 30µg/ml pI:C RNA, 3 µg/ml imiquimod, 1 µg/mlCpG-ODN, or medium control for 24 h, if not indicated otherwise.Cytokine levels were determined in cell supernatants using commercialELISA kits for IL-6, IL-12p40 (OptEiA; BD Pharmingen), and IFN-(PBL Biomedical Labs, Piscataway, NJ) following the protocolsprovided by the manufacturers.
B Cell Proliferation Assay
Proliferation of B cells was assessed using CellTiter 96 ProliferationAssay (Promega, Mannheim, Germany). In brief, 1 x 105 B cellswere incubated in 96-well plates in 100 µl of RPMI mediumthat contained 10% FCS, 100 units/ml penicillin, and 100 µg/mlstreptomycin (Biochrom KG, Berlin, Germany) with TLR ligandsas described previously for a period of 72 h. To each well,20 µl of CellTiter 96 Aqueous One Solution (Promega) wasadded and incubated at 37°C for 4 h. The optical densitywas measured at 492 nm using a spectrophotometric plate reader.
Statistical Analyses
Data were expressed as mean ± SEM. Comparison betweenTLR ligand groups was performed by one-way ANOVA using the PRISM4software (GraphPad Software, San Diego, CA). Post hoc Bonferronicorrection was used for multiple comparisons. The t test wasused to compare ODN 1668 + 2114 versus ODN 1668. P < 0.05indicated statistical significance.
Splenomegaly and Induction of DNA Autoantibodies in Young MRL and MRLlpr/lpr Mice
At 8 wk of age, no structural abnormalities were detected inkidney and spleens of MRL wild-type mice as observed using lightmicroscopy. Spleens of age-matched MRLlpr/lpr mice displayedlymph follicle hyperplasia with enlarged B cell zones. Renalmorphology did not show any abnormalities in MRLlpr/lpr miceof this age. In MRLlpr/lpr mice, the production of autoantibodiesprecedes the onset of lupus nephritis (22). Given the pathogenicrole of lymphoproliferation and autoantibody production forthe pathogenesis of lupus nephritis, we first tested whetherexposure to nucleic acid–like PAMP induces splenomegalyand the production of DNA autoantibodies in young MRL or MRLlpr/lprmice. In both mouse strains, spleen weight increased only withexposure to CpG-DNA (Figure 1A). Serum dsDNA autoantibodieswere determined by ELISA in all groups of mice at 10 wk of age.In saline-treated MRL wild-type mice, serum dsDNA antibodieswere absent and none of the compounds induced significant levelsof dsDNA IgG autoantibodies. By contrast, 10-wk-old saline-treatedMRLlpr/lpr mice had detectable levels of total IgG and IgG1dsDNA antibodies (Figure 1B). Exposure to CpG-DNA and imiquimodincreased serum levels of total IgG dsDNA antibodies. In addition,CpG-DNA increased the production of IgG2a and IgG2b dsDNA antibodiesas compared with saline-treated MRLlpr/lpr mice (Figure 1B).Hence, only the combination of the lpr mutation and CpG-DNAinduced both splenomegaly and DNA autoantibody production inMRL mice. However, the ELISA kits for detection of IgG2a andIgG2b dsDNA antibodies were observed to be more sensitive comparedwith the total IgG ELISA kit.
Figure 1. Lymphoproliferation and serum double-stranded DNA (dsDNA) autoantibodies in MRL and MRLlpr/lpr mice. (A) Spleen weights were assessed at 10 wk of age in MRL and MRLlpr/lpr mice treated with pI:C RNA, imiquimod, and CpG-DNA as indicated (n = 5). (B) Serum levels of dsDNA autoantibodies of the IgG, IgG1, IgG2b, and IgG2a isotypes were determined by ELISA. pIC, poly I:C RNA; Imi, imiquimod; CpG, CpG-ODN 1668. Data are means ± SEM. *P < 0.05 versus saline; #P < 0.05 versus MRL wild-type mice. n.d., not detectable.
B Cell Activation in MRL and MRLlpr/lpr Mice
The potential of microbial nucleic acids to induce autoantibodyproduction in MRLlpr/lpr mice may be linked to their potentialto activate B cells. The proliferation of cultured B cells thatwere isolated from medium-treated MRL and MRLlpr/lpr mice wascomparable (Figure 2A). CpG-DNA induced the proliferation ofCD19-positive B cells that were prepared from MRLlpr/lpr butnot from MRL wild-type mice. This was consistent with the markedexpansion of spleen follicular B220+ B cell areas in MRLlpr/lprmice that received a injection of CpG-DNA (Figure 2B). Consistentwith previously published data, the effect of imiquimod wasless prominent in the absence of additional co-stimuli, andpI:C RNA did not affect the proliferation of B cells of eithermouse strain. In fact, even 10-fold higher doses of imiquimodor pI:C RNA did not induce B cell proliferation in 8-wk-oldMRLlpr/lpr mice (data not shown). CpG-DNA but not pI:C RNA increasedthe production of IL-12p40 in B cells that were prepared fromboth MRL and MRLlpr/lpr mice (Figure 2C). Imiquimod-inducedIL-12p40 production was less potent, but in the presence of5000 µg/ml IFN-, imiquimod also markedly induced IL-12p40.Thus, by virtue of its effect on spleen size and autoantibodyproduction, CpG-DNA is a potent B cell mitogen in MRLlpr/lprmice.
Figure 2. B cells in MRL and MRLlpr/lpr mice. (A) CD19+ B cells were prepared from spleens of MRL and MRLlpr/lpr mice and incubated with pI:C RNA, imiquimod, CpG-DNA, or standard medium as indicated. After 72 h of incubation, B cell proliferation was assessed by CellTiter 96 proliferation assay. The OD was read at 490 nm. (B) Spleen sections were prepared from mice of all groups and stained for B220+ B cells. Images are representative of five mice in each group. (C) CD19+ B cells were prepared from spleens of MRL and MRLlpr/lpr mice and incubated with pI:C RNA, imiquimod, CpG-DNA, or standard medium in the presence or absence of 5000 µg/ml murine IFN- as indicated. IL-6 and IL-12p40 were measured in supernatants by ELISA after 24 h of incubation with the respective Toll-like receptor (TLR) ligands. Results shown are means ± SEM from three comparable experiments. *P < 0.05 versus medium. Magnification, x50.
Dendritic Cell Cytokine Release in MRL and MRLlpr/lpr Mice
Dendritic cells coordinate adaptive immune responses duringantimicrobial immunity as well as autoimmunity. Therefore, weexamined whether Ftl3 ligand–induced, CD11c-positive,ER-HR3–negative dendritic cells that were prepared fromMRL or MRLlpr/lpr mice release proinflammatory cytokines whenincubated with different dosages, pI:C RNA, imiquimod, or CpG-DNA.All three TLR agonists induced IL-12p40 and IL-6 in dendriticcells (Figure 3). Imiquimod and pI:C RNA were less potent ininducing IL-12p40 and IL-6 release in dendritic cells from MRLor MRLlpr/lpr mice, and higher dosages of imiquimod did notfurther increase cytokine release as a result of cytotoxicity.Imiquimod and CpG-DNA but not pI:C RNA induced IFN-. No differencesbetween dendritic cells from MRL and MRLlpr/lpr mice were detectedexcept for high dosages of CpG-DNA.
Figure 3. Activation of dendritic cells that were isolated from MRL and MRLlpr/lpr mice. ER-HR3–/CD11c+ dendritic cells were prepared from bone marrow of MRL and MRLlpr/lpr mice and incubated with standard medium or various dosages of pI:C RNA, imiquimod, or CpG-DNA for 24 h as indicated. IL-12p40, IL-6, and IFN- were measured in supernatants by ELISA. Results are means ± SEM from three comparable experiments. *P < 0.05.
Serum IL-12p40, IL-6, and IFN- Levels in Young MRL and MRLlpr/lpr Mice
Circulating IL-12p40, IL-6, and IFN- levels are markers of diseaseactivity in lupus. Therefore, we questioned pI:C RNA-, imiquimod-,and CpG-DNA–induced changes in the respective serum levelsin 10-wk-old MRL and MRLlpr/lpr mice. Serum IL-12p40, IL-6,and IFN- levels were determined at 3, 6, 12, and 24 h aftera single agonist injection in MRL and MRLlpr/lpr mice. Baselinelevels of all cytokines were low in saline-treated MRL and MRLlpr/lprmice (Figure 4). IL-12p40 was induced by imiquimod and CpG-DNAin both strains with a maximum at 6 h after injection (Figure 4).Similarly, IL-6 was induced by pI:C RNA and CpG-DNA in bothstrains (Figure 4). Serum IFN- levels were low in all groupsof mice and did not respond to the TLR agonists except thatCpG-DNA induced the 3-h levels in MRLlpr/lpr mice (Figure 4).These data suggest that IL-12p40, IL-6, and IFN- serum levelsare induced differently upon exposure to pI:C RNA, imiquimod,or CpG-DNA.
Figure 4. Serum IL-12p40, IL-6, and IFN- levels in MRL and MRLlpr/lpr mice. Serum was obtained from 10-wk-old MRL and MRLlpr/lpr mice at different time points after injection with pI:C RNA, imiquimod, and CpG-DNA as indicated (n = 5). Serum levels were determined by ELISA. Data are means ± SEM. *P < 0.05 versus saline; #P < 0.05 versus MRL wild-type mice.
Lupus Nephritis in Young MRL lpr/lpr and MRL Mice
The induction of DNA autoantibody production could be associatedwith respective renal immune complex deposition and subsequentlupus nephritis. Therefore, we quantified glomerular immuneIgG1 and IgG2a deposition by immunohistochemistry. In MRL wild-typemice of all groups, glomerular IgG1 and IgG2a deposits werenot detected. Saline-treated MRLlpr/lpr mice had mild glomerularIgG1 and IgG2a deposits, predominantly in a capillary stainingpattern (Figure 5). In MRLlpr/lpr mice injections of pI:C RNAand imiquimod did not significantly increase the amount of glomerularIgG1 and IgG2a deposits (Figure 5). By contrast, injection ofCpG-DNA was associated with a robust increase of capillary andmesangial staining of IgG1 and IgG2a (Figure 5, Table 1). Glomerularimmune complexes cause glomerular damage through local complementactivation. Only CpG-DNA caused marked glomerular C3 depositsin MRLlpr/lpr mice, whereas C3 staining was hardly detectablein pI:C RNA- or imiquimod-treated MRLlpr/lpr mice and absentin MRL wild-type mice (Table 1). Histologically, saline-treatedMRLlpr/lpr mice revealed few hyaline deposits in the mesangiumand focal mesangioproliferative lesions as compared with age-matchedMRL wild-type mice (Figure 5). Diffuse global proliferativelupus nephritis was detected only in CpG-DNA–treated MRLlpr/lprmice (Figure 5), consistent with increased numbers of Ki-67–positiveproliferating glomerular cells in these mice (Table 1). Chroniclesions such as fibrous crescents, glomerulosclerosis, interstitialfibrosis, and tubular atrophy were absent in all groups of mice.These findings also were indicated by application of the respectivehistologic score for disease activity of lupus nephritis (Table 1).Immunostaining for CD3 lymphocytes and Mac-2 macrophages inrenal sections revealed increased numbers of glomerular macrophagesin CpG-DNA–injected MRLlpr/lpr mice only (Figure 5, Table 1).CD3 lymphocytes were not found in significant numbers in kidneysof MRL and MRLlpr/lpr mice. Only CpG-DNA–treated MRLlpr/lprmice had proteinuria indicated by an elevated urinary albumin/creatinineratio (Table 1). These data indicate that exposure to pI:C RNA,imiquimod, or CpG-DNA does not induce lupus nephritis in MRLwild-type mice. By contrast, CpG-DNA but not pI:C RNA or imiquimodcan trigger lupus nephritis in MRLlpr/lpr mice, which is associatedwith enhanced cytokine production, B cell activation, dsDNAautoantibody production, glomerular immune complex deposition,and subsequent glomerular damage.
Figure 5. Renal histopathology in MRLlpr/lpr mice. Renal sections of 10-wk-old MRLlpr/lpr mice from all groups were stained with antibodies for IgG2a and Mac-2 (macrophages) as indicated. Glomeruli are encircled. Images are representative of five mice in each group. Magnification, x400.
Table 1. Histologic and urinary findings in MRL and MRLlpr/lpr micea
Inhibitory DNA Blocks the CpG-DNA–Induced Onset of Lupus Nephritis in MRLlpr/lpr Mice
The immunomodulatory effects of microbial DNA are thought tobe motif specific, and inhibitory DNA sequences have been shownto suppress the immunostimulatory effects of CpG-DNA (23). Wetherefore treated additional groups of 8-wk-old female MRLlpr/lprmice using the same injection protocol as before with saline,CpG-DNA (ODN 1668), GpC-DNA (ODN 1720), inhibitory DNA (ODN2114), or ODN 1668 + 2114. Co-injection of ODN 2114 inhibitedthe CpG-DNA–induced onset of lupus nephritis in youngMRLlpr/lpr mice. This was documented by a significant reductionof glomerular C3 deposits, activity index for lupus nephritis,glomerular macrophages, and albuminuria and glomerular Ki-67–positivecells (Table 2). This was consistent with a reduction of serumIL-6 and IL-12p40 levels and a trend toward reduced spleen sizeand IFN- levels in ODN 1668 + 2114–treated MRLlpr/lprmice as compared with mice that were treated with ODN 1668 (Table 2).Injections with the inhibitory ODN 2114 or the control GpC-ODNhad no effect on lupus nephritis of young MRLlpr/lpr mice. Thesedata show that the DNA-induced lupus nephritis is specific tothe CpG-motif. Furthermore, inhibitory DNA elements that aredelivered in synthetic ODN can prevent CpG-DNA–inducedonset of lupus nephritis by suppressing CpG-DNA–inducedlymphoproliferation, autoantibody production, and subsequentrenal injury.
In vitro studies suggest that microbial nucleic acids can modulateautoimmunity through multiple mechanisms, but their impact onthe onset of lupus nephritis in MRLlpr/lpr mice is not known(11–13,15,17,24–26). We used MRLlpr/lpr and MRLwild-type mice to study the effects of an intermittent exposureto pI:C RNA, imiquimod, and CpG-DNA on lymphoproliferation andautoantibody production in genetically predisposed and nonpredisposedhosts. None of the compounds caused DNA autoantibody productionor glomerulonephritis in MRL wild-type mice. By contrast, CpG-DNAbut not pI:C RNA or imiquimod triggered an early onset of lupusnephritis in MRLlpr/lpr mice.
CpG-DNA Triggers Lupus Nephritis in Young MRLlpr/lpr Mice
TLR-9 is not expressed by intrinsic renal cells (18). Hence,the potential of CpG-DNA to induce lupus nephritis in youngMRLlpr/lpr mice is more likely to mediate its modulatory effectson immune cells at extrarenal sites (e.g., in lymphoid organs).In fact, the onset of lupus nephritis is thought to arise fromglomerular immune complex deposition, complement activation,and subsequent glomerular inflammation (1). At 8 wk of age,MRLlpr/lpr mice are characterized by an early stage of lymphoproliferation,but serum DNA autoantibodies are still absent (22). Thus, youngMRLlpr/lpr mice represent a model of a genetically predisposedhost in which overt autoimmune tissue injury (i.e., proteinuriclupus nephritis) is absent. After exposure of MRLlpr/lpr miceto dsRNA, imiquimod, and CpG-DNA, prominent glomerular depositsof IgG, the complement factor C3, diffuse proliferative glomerulonephritis,and overt proteinuria were observed exclusively in CpG-DNA–treatedMRLlpr/lpr mice. Only CpG-DNA activated immunity at multiplelevels (B cell cytokine production, B cell proliferation, autoantibodyproduction, dendritic cell cytokine production, and elevationof serum IFN- levels). This is consistent with previous datadocumenting the immunostimulatory effects of CpG-DNA in otherautoimmune-prone mouse strains (27,28). By contrast, dsRNA andimiquimod did not induce renal disease in MRLlpr/lpr mice, despitethe potential of imiquimod to trigger dsDNA autoantibody production(11). dsRNA and imiquimod less potently triggered polyclonalB cell proliferation in young MRLlpr/lpr mice even at a 10-foldhigher dosage. TLR-7 ligands were shown previously to activatemore potently preactivated B cells (29) (e.g., in the presenceof IFN- [30]), an effect that we also observed in primary Bcells from MRLlpr/lpr mice. However, serum IFN- levels wereundetectable or low in 8-wk-old MRL and MRLlpr/lpr mice, respectively.Polyclonal B cell proliferation is critical to trigger the onsetof autoimmune tissue injury in MRLlpr/lpr mice and in humanSLE (31,32). Imiquimod also failed to induce the class switchto anti-dsDNA IgG2a, which are pathogenic in lupus nephritisand specifically induced by TLR-9 signaling (33). This may explainwhy dsRNA and imiquimod did not induce lupus nephritis despitetheir stimulatory effects on serum IL-6 and IL-12p40 levels(pI:C RNA) or anti-dsDNA total IgG production (imiquimod). Werecently showed that TLR-3, -7, and -9 ligation markedly aggravatesthe progression of advanced lupus nephritis to a comparableextent in 16-wk-old MRLlpr/lpr mice (18,19,21). This involvedtwo additional mechanisms that apply only to MRLlpr/lpr micein which a renal lesion is already present at the time of exposureto TLR ligands (1). Mesangial cell apoptosis that is inducedby the same dosages of pI:C RNA can cause mesangiolysis in activatedglomerular mesangial cells of nephritic MRLlpr/lpr mice (21,34).We did not observe mesangiolysis in young MRLlpr/lpr mice thatwere treated with pI:C RNA, indicating that the low basal expressionof TLR-3 in mesangial cells of nonnephritic kidneys of MRLlpr/lprmice does not support dsRNA-induced glomerular injury (34).In fact, increasing the activation state of glomerular macrophagesis a potent mechanism to exacerbate glomerular injury in experimentalglomerulonephritis (35–37). However, this mechanism couldnot operate in MRL wild-type mice or MRLlpr/lpr mice that werenot treated with CpG-DNA in which glomerular macrophages wereabsent.
Induction of Lupus Nephritis in MRLlpr/lpr Mice by DNA is Motif Specific
In this study, none of the ODN induced lupus nephritis in MRLlpr/lprmice except for CpG-DNA. CpG was identified to be the criticalmotif in bacterial DNA that mediated its immunostimulatory effectsthrough TLR-9 (25,38). Inhibitory DNA sequence elements counterbalancethe immunostimulatory effects of CpG-DNA, but they occur atdifferent frequencies in microbial and vertebrate DNA (39,40).Inhibitory ODN can block CpG-DNA–induced effects in vitro(12,21,23). Here we show that injections with inhibitory DNAprevent CpG-DNA–induced lupus nephritis in young MRLlpr/lprmice. This was associated with an inhibitory DNA-related suppressionof CpG-DNA–induced dsDNA autoantibody production and serumIL-6, IL-12p40, and IFN- levels. Thus, inhibitory DNA can antagonizethe multiple motif-specific immunostimulatory effects of CpG-DNAin young lupus-prone MRLlpr/lpr mice.
CpG-DNA Does Not Induce Lupus Nephritis in MRL Wild-Type Mice
MRL mice carry a number of susceptibility genes, of which someloci predispose to lymphoproliferation and DNA autoantibodyproduction, whereas others predispose to either lupus nephritisor arthritis (41,42). Here we show that CpG-DNA–inducedactivation of B cells and dendritic cells does not trigger theonset of lupus nephritis in MRL mice most likely because MRLmice still are capable of controlling the number of autoreactiveB cells and T cells via Fas-induced apoptotic cell death (43).Repeated injections with CpG-DNA can cause splenomegaly anddisruption of lymph follicles in nonautoimmune mice, but CpG-DNAcan trigger high-affinity DNA autoantibody production in normalmice only when injected together with mammalian DNA (44,45).
Among nucleic acids and nucleic acid–like PAMP, CpG-DNAhas a unique potential to trigger the onset of lupus nephritisin young autoimmune-prone MRLlpr/lpr mice. This relates to thepotent immunostimulatory effects of CpG-DNA at multiple levels:B cell cytokine production, B cell proliferation, autoantibodyproduction, dendritic cell cytokine production, and elevationof serum IFN- levels. However, CpG-DNA does not trigger theonset of lupus nephritis in the presence of Fas, despite numerousother autoimmune susceptibility genes in MRL mice. These dataconfirm the concept of a combination of a strong genetic predispositionin the host and external factors for the pathogenesis of lupusnephritis. In addition, these data support the concept of SLE’sbeing associated with chronic viral infections that continuouslyrelease CpG-DNA (e.g., Ebstein-Barr virus) rather than thosethat release RNA (e.g., hepatitis C virus) (5).
Acknowledgments
This work was supported by grants from the Deutsche Forschungsgemeinschaft(AN372/8-1, GRK 1202), the Fritz Thyssen Foundation, the EUNetwork of Excellence "MAIN" (FP6-502935), the EU IntegratedProject "INNOCHEM" (FP6-518167), and the Else Kröner-FreseniusFoundation. A.E. was supported by the Molecular Medicine programfrom the medical faculty of the University of Munich.
Parts of this project were prepared as a doctoral thesis atthe Faculty of Medicine, University of Munich, by A.E.
The technical support of Dan Draganovici and Ewa Radomska isgratefully acknowledged. We are grateful to Dr. Bruno Luckowfor the generous gift of dsDNA.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
production. The induction of lupus nephritis by CpG-DNA is motif specific and could be prevented by co-injection of inhibitory DNA. In summary, among the ligands tested, CpG-DNA triggers lupus nephritis in genetically predisposed hosts. These data support the concept that systemic lupus erythematosus is triggered by pathogens that release CG-rich DNA. 
