Viral infections may trigger immune complex glomerulonephritisvia Toll-like receptors (TLR), as certain TLR trigger immunityupon recognition of viral nucleic acids. On the basis of previousfindings regarding viral double-stranded RNA and TLR3 in experimentallupus erythematosus, a similar role for TLR7 that recognizesviral single-stranded RNA was hypothesized. Immunostaining ofkidney sections of nephritic MRLlpr/lpr mice revealed TLR7 expressionin infiltrating ER-HR3–positive macrophages and few CD11c-positivedendritic cells but not in glomerular mesangial cells as observedfor TLR3. This finding was consistent with the distributionpattern of intravenously injected single-stranded RNA in nephriticMRLlpr/lpr mice. TLR7 ligation activated monocytes and dendriticcells, both isolated from MRLlpr/lpr mice, to secrete IFN-,IL-12p70, IL-6, and CCL2. In vivo, a single injection of theTLR7 ligand imiquimod increased serum levels of IL-12p70, IFN-,and IL-6. A course of 25 µg of imiquimod given every otherday from week 16 to 18 of age aggravated lupus nephritis inMRLlpr/lpr mice. This was associated with increased glomerularimmune complex deposits as well as interstitial expression ofCCL2 in imiquimod-treated MRLlpr/lpr mice. Different types ofviral nucleic acids seem to modulate systemic autoimmunity throughspecific interactions with their respective TLR. Different TLRexpression profiles on immune cell subsets and nonimmune parenchymalcell types determine the molecular mechanisms involved in viralinfection–associated exacerbation of lupus nephritis andpossibly other types of immune complex glomerulonephritis.
Viral infection can trigger disease activity in immune complexglomerulonephritis, e.g., lupus nephritis, but the molecularmechanisms remain poorly understood. It is believed that viralcompounds that induce immune responses also interfere with thecontrol of autoimmunity in susceptible individuals. Thus, pattern-recognitionreceptors that recognize such viral compounds may stimulateboth antiviral immunity and, under certain circumstances, autoimmunedisease activity.
Toll-like receptors (TLR) are a family of such pattern-recognitionreceptors, and a subgroup of TLR activates antiviral immunityupon recognition of viral compounds (1,2). For example, variousviral nucleic acids ligate a group of TLR on dendritic cellsand induce dendritic cell maturation characterized by the upregulationof MHC class II, induction of co-stimulatory molecules, andsecretion of type I interferons (3,4). In contrast to theirestablished role for pathogen control, the role of TLR in autoimmunityor lupus nephritis is less well defined (5,6). Systemic exposureto unmethylated CpG-DNA (ligand of TLR9), can aggravate theimmune complex glomerulonephritis induced by apoferritin (7),as well as the lupus-like immune complex glomerulonephritisof MRLlpr/lpr mice (8). In both disease models, aggravationwas associated with enhanced autoantibody production and intrarenalmonocyte activation, consistent with the expression of TLR9on B cells and monocytic antigen-presenting cells. However,heterogeneous expression patterns of single TLR on leukocytesubpopulations and the discovery of TLR-specific signaling pathwayssupport specific types of immune responses for specific ligand–TLRinteractions (9). For example, viral double-stranded RNA (dsRNA)activates TLR3, which is expressed by glomerular mesangial cellsand monocytic antigen-presenting cells but not by B cells (10,11).As a consequence, exposure to viral dsRNA aggravates lupus nephritisin MRLlpr/lpr mice by inducing mesangiolysis as well as enhancedlocal inflammatory tissue damage, but circulating DNA autoantibodiesremain unaffected (11). As another viral nucleic acid, single-strandedRNA (ssRNA) or synthetic guanine nucleoside analogs are recognizedby TLR7 and in humans possibly by TLR8 (12–14). WhereasTLR7 signals for antiviral immunity upon infection with ssRNAviruses, its role in autoimmunity remains unknown (15). On thebasis of the known expression profiles of TLR3, TLR7, TLR8,and TLR9 on immune cell subsets, TLR7 follows the distributionof TLR9 rather than TLR3 (16). Furthermore, whereas TLR7, TLR8,and TLR9 signal through the adaptor molecule myeloid differentiationfactor 88, TLR3 is the only known TLR that depends on Toll-IL-1receptor domain-containing adaptor inducing IFN- and RNA helicaseretinoic acid–inducible gene 1 (RIG-1) (17,18). Despitethis different signaling pathways, ligation of all of theseviral nucleic acid–specific TLR induces a robust inductionof IFN- (3,19,20). IFN- is a critical mediator of both antiviralimmunity and autoimmune tissue injury (21). The recognitionof viral ssRNA via TLR7 may represent an important mechanismof virus-induced autoimmunity, e.g., in lupus. Thus, we characterizedthe expression of TLR7 in experimental lupus and studied theeffects of TLR7 ligation on lupus disease activity as a modelfor intercurrent viral infection in systemic lupus erythematosus(SLE).
Animals and Experimental Protocol
Ten-week-old female MRLlpr/lpr mice were obtained from HarlanWinkelmann (Borchen, Germany) and kept in filter-top cages undera 12-h light and dark cycle. Water and standard chow (Sniff,Soest, Germany) were available ad libitum. All experimentalprocedures had been approved by the local government authorities.For assessing renal TLR mRNA expression, kidneys were obtainedfrom 5- and 20-wk-old female MRLlpr/lpr mice. In addition, 16-wk-oldfemale MRLlpr/lpr mice were distributed into three groups thatreceived intraperitoneal injections every other day as follows:(1) 25 µg of imiquimod, a compound of the imidazoquinadinefamily and TLR7 agonist (Sequoia Research Products Ltd, Oxford,UK) (13) in 100 µl of 10% DMSO (Sigma-Aldrich, Steinheim,Germany); (2) 100 µl of 10% DMSO; or (3) 100 µlof normal saline. All mice were killed by cervical dislocationat the end of week 18 of age. For assessing the renal distributionof ssRNA, 3'-rhodamine–labeled ssRNA40, known to ligatemTLR7 (12), was injected intravenously into MRLlpr/lpr miceat the age of 16 wk. Renal tissue was collected 2 h later andsubjected to further analysis as described below.
Evaluation of Glomerulonephritis
Blood and urine samples were collected from each animal at theend of the study period as described (8) to determine proteinuriaand creatinine using an automatic autoanalyzer (Integra 800;Roche Diagnostics, Mannheim, Germany). Serum DNA autoantibodieswere determined by ELISA using the anti-mouse IgG1 and IgG2aantibodies (Bethyl Laboratories Inc., Montgomery, TX; 1:100).From all mice, kidneys were fixed in 10% buffered formalin,processed, and embedded in paraffin. Five-micrometer sectionsfor silver and periodic acid-Schiff stains were prepared followingroutine protocols. The severity of the renal lesions was gradedusing the indices for activity and chronicity as described forhuman lupus nephritis (22).
Immunostaining
Immunostaining was performed on either paraffin-embedded orfrozen sections as described (8) using the following primaryantibodies: Anti-mouse TLR7 (1:50, IMG581; Imgenex, San Diego,CA), anti-mouse ER-HR3 (1:50, monocytes/macrophages; DPC Biermann,Bad Nauheim, Germany), anti-mouse CD11c (1:50, clone HL3, BDPharmingen, Heidelberg, Germany), anti-mouse CD3 (1:100, clone500A2; BD Pharmingen), anti-mouse smooth muscle actin (1:100,myofibroblasts, clone 1A4; Dako, Carpinteria, CA), anti-mouseCCL5 (1:50, clone VL1; Peprotech, Rocky Hill, NJ), anti-mouseCCL2/MCP-1 (1:50, polyclonal; Santa Cruz Biotechnology, SantaCruz, CA), anti-mouse IgG1 (1:100, clone H143.225.8; Dianova,Hamburg, Germany), anti-mouse IgG2a (1:100, clone R19–15;Dianova), and anti-mouse C3c (1:200, GAM/C3c/FITC; Nordic ImmunologicalLaboratories, Tilburg, Netherlands). Negative controls includedincubation with a respective isotype antibody. For quantitativeanalysis, glomerular cells were counted in 10 cortical glomeruliper section. Semiquantitative scoring of glomerular IgG andC3c deposits from 0 to 3 plus was performed on 15 cortical glomerularsections as described (8).
Cell Culture Conditions and Cytokine ELISA
Bone marrow–derived dendritic cells and plastic-adherentspleen monocytes were isolated from MRLlpr/lpr mice, processed,and cultured as described (23,24). Spleen monocytes were treatedwith medium control or imiquimod 3 µg/ml, RNA40 alongwith DOTAP (Roche, Mannheim, Germany) 30 µg/ml, and pI:CRNA 30 µg/ml after 24 h of incubation. TLR9 ligand CpG-ODNno. 1668 at a concentration of 1 µg/ml was used as a controlin selected cases. After a period of 24 h, culture supernatantswere collected for cytokine measurements and cells were preparedfor flow cytometric analysis. Dendritic cells and spleen monocyteswere stimulated as above for 24 h, and cells were harvestedfor RNA isolation as described previously (23). J774 mouse macrophages(American Type Culture Collection, Rockville, MD) were grownin RPMI 1640 that contained 1 mM HEPES, 10% heat-inactivatedbovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin(Biochrom KG, Berlin, Germany). A murine mesangial cell linewas maintained in DMEM (Biochrom KG, Berlin, Germany) supplementedwith 2.5% FCS and 1% penicillin-streptomycin 100 U/ml and 100µg/ml, respectively (25). Cells were incubated for 24h without serum supplements before stimulation. Cytokine levelswere determined using commercial ELISA kits following the protocolprovided by the manufacturers: IL-6, IL-12p70, CCL2 (all OptEiA,BD Pharmingen), and IFN- (PBL Biomedical Labs, Piscataway, NJ).
Flow Cytometry
Flow cytometry of cultured cells or splenocytes was performedas described previously (11). The following primary antibodieswere used to detect TLR on mesangial cells and macrophages:Anti-mouse TLR3 (1:50, IMG516; Imgenex, San Diego, CA), TLR7(1:50), and anti-mouse TLR9 (5G5, provided by Dr. Stefan Bauer,Technical University, Munich, Germany). A biotinylated rabbitanti-mouse IgG antibody and streptavidin-APC (BD Pharmingen)were used for detection, and a rabbit IgG (BD Pharmingen) wasused as isotype control.
Real-Time Quantitative (TaqMan) Reverse Transcription–PCR
Real-time reverse transcription–PCR (RT-PCR) on RNA thatwas isolated from renal tissue was performed as described previously(11). Controls that consisted of ddH2O were negative for targetand housekeeper genes. Oligonucleotide primer (300 nM) and probes(100 nM) were from Applied Biosystems (Darmstadt, Germany) andused as described: TLR3, no. AF355152, forward 5'-CGAAAGTTGGACTTGTCATCAAATC-3',reverse 5'-ACTTGCCAATTGTCTGGAAACAC-3', 6 FAM 5'-CACTTAAAGAGTTCTCCC-3';TLR7, no. AY035889, forward 5'-TGCCACC-TAATTTACTAGAGCTCTATCTTTAT-3',reverse 5'-TAGGTCAAGAACTTGCAACTCATTG-3', 6 FAM 5'-CCAAGAAAATGATTTTAATAAC-3';TLR9, no. NM 031178, forward 5'-CAATCTGACCTCCCTTCGAGTACTT-3',reverse 5'-GCCACATTCTATACAGGGATTGG-3', 6 FAM 5'-ATTGCCGTCGCTGCGACCATG-3'.Primers and probes for murine 18S rRNA were obtained as predevelopedassay reagents from Applied Biosystems.
Statistical Analyses
Data were expressed as mean ± SEM. Comparison betweengroups was performed using univariate ANOVA. Post hoc Bonferronicorrection was used for multiple comparisons. P < 0.05 wasconsidered to indicate statistical significance.
Expression of TLR7 in Lupus Nephritis of MRLlpr/lpr Mice
We first determined the expression pattern of TLR7 in kidneysand spleens of MRLlpr/lpr mice and compared TLR3, TLR7, andTLR9 mRNA expression levels at an early (week 5) and late (week20) stage of autoimmune disease using real-time RT-PCR. At 5wk of age, no structural abnormalities were detected in kidneyand spleen as observed using light microscopy (data not shown).At this time point, expression levels of TLR7 and TLR9 mRNAwere low as compared with that in spleen. By contrast, kidneyTLR3 mRNA levels were comparable to that in spleen of 5-wk-oldMRLlpr/lpr mice, consistent with the known TLR3 mRNA expressionby mesangial cells (Figure 1). At 20 wk, proliferative lupusnephritis was associated with increased renal TLR7 and TLR9mRNA expression as compared with week 5 (Figure 1). To localizethe source of renal TLR7 mRNA expression, we used a polyclonalantibody specific for murine TLR7 and performed double stainingfor either ER-HR3 or CD11c in renal sections of 16-wk-old MRLlpr/lprmice. Approximately 30% of interstitial ER-HR3 macrophages andCD11c dendritic cells (ratio 90%:10%, respectively) stainedpositive for TLR7 (Figure 2A). Staining for TLR7 appeared ina speckled pattern, indicating that TLR7 is localized in anintracellular compartment. Intrinsic renal cells were negativefor TLR7, and the macrophages that were found to be presentin the glomerular compartment were rarely positive for TLR7.Together these data suggest that in nephritic kidneys of MRLlpr/lprmice, TLR7 is expressed mainly by infiltrating interstitialmacrophages but not by intrinsic renal cells.
Figure 1. Toll-like receptor 7 (TLR7) expression in MRLlpr/lpr mice. Expression of TLR mRNA was assessed by real-time reverse transcription–PCR (RT-PCR) in duplicate using RNA isolated from spleens and kidneys from seven MRLlpr/lpr mice each at 5 and 20 wk of age as described in Materials and Methods. TLR mRNA expression is expressed as a ratio to the respective 18S rRNA mRNA expression ± SEM.
Figure 2. TLR7 immunostaining and uptake of labeled single-stranded RNA (ssRNA) in kidneys of MRLlpr/lpr mice. (A) An mTLR7-specific antibody was used on renal sections of 18-wk-old nephritic MRLlpr/lpr mice. A PE-labeled secondary antibody was used for detection. Positive signals co-localized with ER-HR3–positive macrophages or CD11c-positive dendritic cells, both detected by a FITC-labeled secondary antibody. (B) Rhodamine-labeled ssRNA40 was injected intravenously into 18-wk-old MRLlpr/lpr mice, and renal tissue was harvested 2 h later. Fluorescence imaging of frozen sections showed uptake of ssRNA40 (red) into ER-HR3–positive macrophages (green). (C) Co-staining of rhodamine-labeled cells (red) for TLR7 (green) demonstrates uptake of RNA40 into TLR7-positive cells. Magnification, x530.
Localization of Labeled ssRNA after Intravenous Injection in MRLlpr/lpr Mice
To examine whether circulating ssRNA localizes to nephritickidneys of MRLlpr/lpr mice, we injected rhodamine-labeled ssRNAintravenously into 20-wk-old MRLlpr/lpr mice. Consistent withTLR7 immunostaining in the kidney, the labeled ssRNA was foundin infiltrating cells in a granular intracellular staining pattern(Figure 2B). Double labeling with an ER-HR3-specific antibodyidentified these cells as renal macrophages (Figure 2B). Rhodaminethat was injected into MRLlpr/lpr mice did not localize in thekidney (data not shown). Double labeling for TLR7 confirmedthat injected ssRNA was taken up into TLR7-positive cells (Figure 2C).Taken together, in kidneys of MRLlpr/lpr mice, injectedssRNA co-localizes in an intracellular granular pattern withTLR7-positive cells, i.e., infiltrating mononuclear cells, butnot with intrinsic renal cells.
Cultured Mesangial Cells and Macrophages Respond to Nucleic Acids According to Their Respective TLR Expression Profile
To confirm the respective TLR7 expression, we used establishedmurine cell lines for macrophages and mesangial cells. Underbasal culture conditions, mesangial cells expressed TLR3 mRNA,whereas mRNA for TLR7 and TLR9 were not detected (Figure 3A).By contrast, J774 macrophages expressed all three receptors(Figure 3A). The subcellular localization of TLR3, TLR7, andTLR9 was assessed by flow cytometry. In mesangial cells, TLR3and in macrophages TLR3, TLR7, and TLR9 were expressed intracellularly,whereas surface expression was absent (Figure 3B). Next, wequestioned whether mesangial cells and macrophages respond tomicrobial nucleic acids that correspond to their specific TLRexpression profile. We stimulated both cell types with syntheticmimics of microbial nucleic acids: pI.C RNA (TLR3), imiquimodand RNA40 (TLR7), and CpG-ODN (TLR9). Consistent with theirrespective TLR expression profile, mesangial cells producedthe CC-chemokine CCL2 only after exposure to mimics of viraldsRNA, whereas J774 macrophages responded to all TLR agoniststested (Figure 3C). Together, these data indicate that monocytesbut not mesangial cells express TLR7 in an intracellular compartmentand produce CCL2 upon exposure to ssRNA or imiquimod in vitro.
Figure 3. Nucleic acids activate cultured mesangial cells and J774 macrophages consistent with their respective TLR expression profile. Murine mesangial cells and J774 macrophages were cultured as described in Materials and Methods. (A) Expression of TLR mRNA was assessed by real-time RT-PCR in duplicate using RNA that was isolated from cultured cell lines. TLR mRNA expression is expressed as a ratio to the respective 18S rRNA mRNA expression ± SEM (*P < 0.05). (B) Flow cytometry for TLR before and after permeabilization for intracellular staining was performed as indicated. Expression of TLR (solid line) is demonstrated by a fluorescence shift compared with the isotype control antibody (dotted line). (C) Cultured cells were incubated with pI:C RNA, imiquimod (IMI), RNA40, CpG-DNA, or standard medium without supplements for 24 h as indicated. CCL2 production was measured in supernatants by ELISA. Results shown are representative of two comparable experiments each performed in duplicate.
TLR7 Agonists Induce Production of Proinflammatory Mediators in Monocytes and Dendritic Cells Isolated from MRLlpr/lpr Mice
Because TLR7 seems to be expressed by ER-HR3–positivemacrophages and CD11c-positive dendritic cells in nephritickidneys of MRLlpr/lpr mice, we intended to characterize thefactors produced by these cells in response to agonists of TLR7in comparison with other nucleic acid–like TLR agonists.Spleen monocytes and bone marrow dendritic cells were isolatedfrom MRLlpr/lpr mice as described in Materials and Methods.The ER-HR3–positive group comprised approximately 90%of the spleen monocytes, which were found to be negative forCD11c. Likewise, approximately 85% of the bone marrow–deriveddendritic cells were found to be positive for CD11c, which werefound to be negative for ER-HR3 as determined by flow cytometry(data not shown). Both cell types were incubated with pI:C RNA,RNA40, imiquimod, and CpG-DNA. In spleen macrophages both RNA40and imiquimod induced production of IL-6, IL-12p70, IFN-, andCCL2, respectively (Figure 4). In bone marrow–deriveddendritic cells, the observed responses were similar exceptfor increased IL-12p70 and IFN- production upon exposure topI:C RNA (Figure 4) as compared with stimulation of spleen monocytes.These data suggest that TLR7 agonists induce the productionof IL-12p70, IL-6, CCL2, and IFN- in both macrophages and dendriticcells isolated from MRLlpr/lpr mice.
Figure 4. TLR7 agonists activate ER-HR+ monocytes and CD11c+ dendritic cells that were isolated from MRLlpr/lpr mice. ER-HR3+/CD11c– monocytes and ER-HR3–/CD11c+ dendritic cells (DC) were isolated from spleens and bone marrow (BM) of MRLlpr/lpr mice and incubated with pI:C RNA, RNA40, imiquimod, CpG-DNA, or standard medium for 24 h as indicated. IL-6 (A), IL-12p70 (B), CCL2/MCP-1 (C), and IFN- (D) were measured in supernatants by ELISA. Results shown are from one of three comparable experiments. For each experiment, cells were pooled from three mice. Values represent means ± SEM; n.d., nondetectable.
Imiquimod Increases Serum IFN-, IL-6, and IL-12p70 Levels in MRLlpr/lpr Mice
Circulating IFN-, IL-6, and IL-12p70 levels are markers of diseaseactivity in lupus. Thus, having demonstrated the effect of TLR7ligation on IFN-, IL-6, and IL-12p70 secretion in antigen-presentingcell subsets that were isolated from MRLlpr/lpr mice in vitro,we next studied serum levels of these factors 6 h after intraperitonealinjection of 25 µg of imiquimod, vehicle, or saline into16-wk-old MRLlpr/lpr mice. Injection of imiquimod significantlyincreased serum levels of IL-12p70, IL-6, and IFN- in MRLlpr/lprmice as compared with vehicle-injected controls (Figure 5).
Figure 5. Serum IFN-, IL-12p70, and IL-6 levels in MRLlpr/lpr mice. Serum was obtained from 16-wk-old MRLlpr/lpr mice 6 h after the first intraperitoneal injection of saline, vehicle, or 25 µg of imiquimod as indicated (n = 5 to 10). Serum levels were determined by ELISA. Data are means ± SEM. *P < 0.05 versus saline.
Imiquimod Aggravates Autoimmune Tissue Injury in MRLlpr/lpr Mice
From the above results, one would predict that ligation of TLR7would be associated with more severe autoimmune tissue injuryin MRLlpr/lpr mice. We therefore treated groups of lupus micewith intraperitoneal injections of 25 µg of imiquimod,vehicle, or saline on alternate days from weeks 16 to 18 ofage. Saline-treated MRLlpr/lpr mice had diffuse proliferativeglomerulonephritis with moderate mesangial hypercellularity,increase of mesangial matrix, and few periglomerular inflammatorycell infiltrates at week 18 (Figure 6A). Vehicle injectionsdid not alter these histopathologic findings. By contrast, imiquimodinjections increased mesangial matrix deposits with focal segmentalsclerosis in glomeruli and cellular crescent formation associatedwith marked periglomerular inflammatory cell infiltrates (Figure 6A).Mesangiolysis was not observed. Aggravation of renal diseasewas illustrated by an increase in proteinuria and the activityand chronicity scores of the lupus nephritis in imiquimod-treatedMRLlpr/lpr mice as compared with the other groups of mice (Table 1).There was a trend toward increased glomerular macrophagesand CD3 cells in imiquimod-treated MRLlpr/lpr mice, but thisdid not reach statistical significance (Table 1). In additionto the aggravation of glomerular damage, imiquimod injectionsinduced tubulointerstitial damage. Infiltrating ER-HR3 macrophagesand CD3 lymphocytes accumulated particularly in periglomerularfields and areas around glomerular crescents (Figure 6A, Table 1).On the basis of our in vitro studies with macrophages anddendritic cells, we hypothesized that TLR7 ligation would triggerlocal chemokine expression in nephritic kidneys of MRLlpr/lprmice. Thus, we performed immunostaining for CCL2. At 18 wk,single spots of CCL2 protein were noted within the glomerulartuft and along Bowman’s capsule of some glomeruli, aswell as in focal interstitial areas in kidneys of vehicle-treatedMRLlpr/lprmice (Figure 6B). By contrast, imiquimod-treated MRLlpr/lprmice showed marked CCL2 staining that co-localized with tubularepithelial cells, interstitial leukocytic cell infiltrates,and glomerular crescents. Taken together, imiquimod aggravatedautoimmune tissue injury in MRLlpr/lpr mice associated withincreased local expression of CCL2 in areas of inflammatorycell infiltrates and tissue damage in nephritic kidneys ofMRLlpr/lprmice.
Figure 6. Renal histopathology. (A) Renal sections of 18-wk-old MRLlpr/lpr mice from all groups were stained with periodic acid-Schiff (PAS) and antibodies for ER-HR3 (macrophages), CD3 (lymphocytes), and smooth muscle antigen for myofibroblasts (SMA) as indicated. Insert in PAS-stained sections of imiquimod-treated MRLlpr/lpr mice illustrates glomerular tuft necrosis and crescent formation not detected in mice of the other groups. Inserts in ER-HR3–and CD3-stained sections show respective glomeruli. Images are representative of eight to 10 mice in each group. (B) Renal sections of 18-wk-old vehicle- or imiquimod-treated MRLlpr/lpr mice were stained for CCL2/MCP-1. Arrows indicate CCL2-positive glomerular and interstitial cells. Note CCL2 positivity also in tubular epithelial cells in imiquimod-treated mice. Images are representative of five mice in each group. Magnification, x400 in A; x530 in A inserts; x400 in B.
Table 1. Serum, urinary, and histologic findings in MLRlpr/lpr micea
Imiquimod Increases Renal Immune Complex Deposition in MRLlpr/lpr Mice
We previously observed that the activation of TLR9 as well asTLR3 aggravated lupus nephritis in MRLlpr/lpr mice. Unlike TLR3,TLR9 activation induced a marked increase of DNA autoantibodyproduction and glomerular immune complex deposits (8,11). Thus,we investigated the effects of TLR7 ligation on serum dsDNAautoantibody levels and glomerular IgG deposits in MRLlpr/lprmice. Imiquimod somewhat increased serum total IgG as well asIgG1- and IgG2a-dsDNA autoantibodies as compared with vehicle-treatedMRLlpr/lpr mice, although this was not statistically significant(Figure 7A). However, imiquimod-treated MRLlpr/lpr mice showedincreased glomerular capillary and mesangial deposits of totalIgG, IgG1, and IgG2a (Figure 7B, Table 1). This was associatedwith increased glomerular capillary and mesangial deposits ofcomplement factor C3c in imiquimod-treated MRLlpr/lpr mice (complementdeposit score 2.4 ± 0.3 [imiquimod] versus 1.3 ±0.1 [vehicle]; P < 0.01; Figure 7C). Together, imiquimod-inducedaggravation of lupus nephritis in MRLlpr/lpr mice is associatedwith increased glomerular immune complex deposition.
Figure 7. DNA autoantibodies and immune complex deposits in MRLlpr/lpr mice. (A) Serum double-stranded DNA autoantibody levels were determined by ELISA (n = 5 to 10). Data are means ± SEM. P > 0.05 versus vehicle for IgG and both IgG isotypes. (B and C) Immunostaining for total IgG, IgG1, IgG2a, and complement factor C3c were performed on renal sections of MRLlpr/lpr mice as described in Materials and Methods. Magnification, x400.
Viral infections can aggravate disease activity in preexistingSLE, but the role of viral ssRNA in this context is hypothetical.We used the model of spontaneous lupus-like immune complex glomerulonephritisin MRLlpr/lpr mice to study the effects of intermittent exposureto imiquimod, a synthetic TLR7 ligand with immunostimulatoryeffects comparable to viral ssRNA (12–14). We provideevidence for an intracellular expression of TLR7 on renal macrophagesand dendritic cells in nephritic lesions of MRLlpr/lpr mice,which corresponds to the distribution of nucleic acid–specificTLR, i.e., TLR3 and TLR9 in these cell types (8,11). Spleenmonocytes and bone marrow–derived dendritic cells produceproinflammatory cytokines, chemokines, and type I interferonsupon ligation of TLR7. Obviously, the immunostimulatory effectsof ssRNA relates to the cell type–specific expressionpattern of TLR7, which clearly differs from the viral dsRNA-specificTLR3. These data may provide a new understanding of viral infection-inducedexacerbation of lupus nephritis as well as other types of glomerulonephritis.
TLR7 Is Expressed by Renal Macrophages in Experimental Lupus
Antiviral host defense requires activation of innate immunity,including the local production of type I interferons and chemokines(26). The finding that injected ssRNA localized to TLR7-positivemacrophages in kidneys of MRLlpr/lpr mice suggests a role forTLR7 in activating renal macrophages in the kidney. For example,our studies with spleen monocytes that were isolated from MRLlpr/lprmice argue in favor of TLR7 activation and its effect on tissuemacrophages contributing to the local production of proinflammatorymediators, including IL-12, IL-6, CCL2, IFN-, which are knownto contribute to the progression of lupus nephritis (27–31).As TLR7-positive macrophages were mainly detected in the renalinterstitium, the aggravation of glomerular injury in imiquimod-treatedMRLlpr/lpr mice should be secondary to increased glomerularimmune complex deposition. Furthermore, a low number of CD11c-positivedendritic cells were found to express TLR7 in the kidney. Arecent study has demonstrated the antigen-presenting phenotypeof this cell population in glomerulonephritis (32). In fact,TLR7 ligation is a potent trigger for dendritic cell maturation(14). The functional role of intrarenal CD11c-positive dendriticcells for the progression of nephritis in MRLlpr/lpr mice remainsunclear. However, our in vitro data with CD11c-positive bonemarrow–derived dendritic cells suggest that this celltype contributes to local production of proinflammatory mediators,(IL-6, IL-12, CCL2, and IFN-). For example, IL-6 that is derivedeither from renal macrophages or CD11c-positive dendritic cellsmay suppress regulatory T cells that control autoreactive Tcells (33). In fact, in our study, exposure to imiquimod increasedserum levels of the aforementioned mediators and aggravatedautoimmune tissue injury in kidneys and lungs of the MRLlpr/lprmice. This was associated with enhanced renal production ofthe chemokine CCL2 and increased interstitial macrophage andT cell infiltrates. On the basis of similar effects of TLR7,TLR3, and TLR9 ligands on cytokine and chemokine productionby monocytes and dendritic cells, these data add to the conceptof how nucleic acid–specific TLR on antigen-presentingcells can modulate autoimmunity, e.g., lupus nephritis (5,34).
TLR7 Is not Expressed by Intrinsic Renal Cells
Immunostaining for TLR7 revealed that TLR7 is not expressedby intrinsic renal cells. In addition, labeled ssRNA did notlocalize to intrinsic renal cells after injection. These observationswere consistent with our findings in cultured mesangial cells,which did not express TLR7 mRNA and did not respond to imiquimod.So far, TLR7 has not been reported to be expressed in any nonimmunecell type (35). Thus, the expression of TLR7 seems to be restrictedto antigen-presenting cells in mice and humans (16). These dataindicate that imiquimod-induced aggravation of nephritis inMRLlpr/lpr mice is unrelated to direct activation of intrinsicrenal cells.
Effect of TLR7 Ligation on Humoral Immunity in Experimental Lupus
Lupus is characterized by polyclonal autoantibody productionand B cell proliferation, the latter expressing TLR7 at intermediatelevels (16). TLR7 ligation with repeated injections of imiquimodin MRLlpr/lpr mice was associated with a trend toward increasedserum dsDNA autoantibody levels, a significant increase in glomerularimmune complex deposits, and complement activation. The lastmay explain why imiquimod aggravated glomerular injury in MRLlpr/lprmice despite having no effect on glomerular macrophage counts.The finding that imiquimod had only a moderate effect on autoantibodyproduction is supported by a recent study that showed that Bcells do not respond to TLR7 ligands unless their B cell sensitivityis enhanced by dendritic cell–derived IFN- (36). Obviously,TLR7 ligands alone cannot induce B cell activation because IFN-producingdendritic cells control TLR7 sensitivity of B cells. We assumethat imiquimod had a moderate effect on B cell–dependentproduction of dsDNA autoantibodies in MRLlpr/lpr mice becauseB cells localize in close proximity to IFN-producing dendriticcells in lymphoid organs and imiquimod injection increased serumIFN- levels in MRLlpr/lpr mice. In fact, imiquimod and ssRNAwere shown to induce IFN- production in bone marrow–deriveddendritic cells that were isolated from MRLlpr/lpr mice. UnlikeCpG-TLR9–mediated activation of B cells, TLR7 ligationrequires additional co-factors, e.g., IFN-, to mount B cellactivation and subsequent DNA autoantibody production. Thus,TLR7 ligation has a moderate effect on autoantibody productionin MRLlpr/lpr mice.
On the basis of this and previous studies, three different mechanismsby which nucleic acid–specific TLR can contribute to theexacerbation of immune complex glomerulonephritis seem to exist:Viral dsRNA activates production of proinflammatory mediatorsthrough TLR3 expressed by glomerular mesangial cells as wellas macrophages and dendritic cells (11). Viral dsRNA does notactivate autoantibody production in MRLlpr/lpr mice, becauseB cells lack TLR3 expression. TLR7 and TLR9 both are expressedon renal macrophages, dendritic cells, and B cells (8,37). However,here we show that TLR7 can activate B cells only in the presenceof additional co-factors, which result in an intermediate increaseof autoantibody production and glomerular immune complex deposition.In addition, all three nucleic acid–specific TLR inducedendritic cell maturation toward an antigen-presenting phenotypeas well as production of proinflammatory cytokines, chemokines,and type I interferons by renal macrophages. In summary, varioustypes of nucleic acids induce specific patterns of immune responsesthat relate to the cell type–specific expression and functionof the respective TLR. These findings contribute to the understandingof the broad clinical spectrum and the lack of universal serummarkers of infection-associated disease activity of lupus nephritisand possibly other types of immune complex glomerulonephritis.
Acknowledgments
The work was supported by grants from the Deutsche Forschungsgemeinschaft(AN372/4-1, GRK 1201) and the Fritz Thyssen Foundation to H.J.A.H.J.A. and D.S. were supported by a grant from the EU Networkof Excellence "MAIN" (FP6-502935). S.S. was supported by theElse-Kroener-Fresenius Foundation.
Parts of this project were prepared as a doctoral thesis atthe Faculty of Medicine, University of Munich, by R.D.P.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
, IL-12p70, IL-6, and CCL2. In vivo, a single injection of the TLR7 ligand imiquimod increased serum levels of IL-12p70, IFN-
and RNA helicase retinoic acid–inducible gene 1 (RIG-1) (17,18). Despite this different signaling pathways, ligation of all of these viral nucleic acid–specific TLR induces a robust induction of IFN-
