Spiegelmer Inhibition of CCL2/MCP-1 Ameliorates Lupus Nephritis in MRL-(Fas)lpr Mice
Onkar Kulkarni*,
Rahul D. Pawar*,
Werner Purschke,
Dirk Eulberg,
Norma Selve,
Klaus Buchner,
Volha Ninichuk*,
Stephan Segerer*,
Volker Vielhauer*,
Sven Klussmann and
Hans-Joachim Anders*
* Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University, Munich, and NOXXON Pharma AG, Berlin, Germany
Correspondence: Dr. Hans-Joachim Anders, Medizinische Poliklinik der LMU, Pettenkoferstrasse 8a, 80336 Munich, Germany. Phone: +49-89-218075846; Fax: +49-89-218074860; E-mail: hjanders{at}med.uni-muenchen.de
Received for publication December 13, 2006.
Accepted for publication May 16, 2007.
The monocyte chemoattractant protein CCL2 is crucial for monocyteand T cell recruitment from the vascular to the extravascularcompartment at sites of inflammation. CCL2 is expressed in humanlupus nephritis and was shown to mediate experimental lupus;therefore, CCL2 antagonists may be beneficial for therapy. Thisstudy describes the l-enantiomeric RNA oligonucleotide mNOX-E36,a so-called Spiegelmer that binds murine CCL2 with high affinityand neutralizes its action in vitro and in vivo. The mirrorimage configuration of the Spiegelmer confers nuclease resistanceand thus excellent biostability. mNOX-E36 does not induce typeI IFN via Toll-like receptor-7 or cytosolic RNA receptors, asrecently shown for certain synthetic d-RNA. Autoimmune-proneMRLlpr/lpr mice that were treated with a polyethylene glycolform of mNOX-E36 from weeks 14 to 24 of age showed prolongedsurvival associated with a robust improvement of lupus nephritis,peribronchial inflammation, and lupus-like inflammatory skinlesions. Thus, mNOX-E36–based inhibition of CCL2 representsa novel strategy for the treatment of autoimmune tissue injury,such as lupus nephritis.
Blocking proinflammatory mediators has become a successful approachfor the treatment of chronic inflammation.1 In addition to TNFand IL, CC-chemokines are important candidates for specificantagonism because CC-chemokines mediate leukocyte recruitmentfrom the intravascular space to sites of inflammation.2,3 Thereis strong evidence that CCL2, formerly known as monocyte chemoattractantprotein-1, and its respective chemokine receptor CCR2 have acrucial role in autoimmune tissue injury such as the clinicalmanifestations of systemic lupus erythematosus.4 For example,MRLlpr/lpr mice deficient either for the Ccl2 or the Ccr2 geneare protected from lupus-like disease manifestations.5,6 Hence,the CCL2/CCR2 axis may represent a promising therapeutic target(e.g., for lupus nephritis). In fact, delayed gene therapy ortransfer of transfected cells, both resulting in in situ productionof an NH2-truncated CCL2, markedly reduced autoimmune tissueinjury in MRLlpr/lpr mice.7,8 However, such experimental approachescannot be used in humans because of irrepressible antagonistproduction and tumor formation.7,8
The biologic functions of proteins can be blocked by aptamers(three-dimensional nucleic acid structures that can be designedto bind to target molecules conceptually similar to antibodies9,10).However, the therapeutic use of oligonucleotide-based antagonistsis limited by their susceptibility to nuclease digestion resultingin low biostability. Spiegelmers represent a new class of nuclease-resistantRNA oligonucleotides. Spiegelmers are l-enantiomeric aptamersin which the mirror-image configuration of the ribonucleotidesprevents nuclease degradation.11 Hence, Spiegelmers show excellentbiostability without any further chemical modifications, whichrenders then very well suited for in vitro and in vivo applications.12–15We therefore hypothesized that Spiegelmer-based blockade ofCCL2 would be suitable for the treatment of lupus nephritisand other disease manifestations of systemic lupus erythematosus.Here we report the identification of the Spiegelmer mNOX-E36that specifically inhibits murine CCL2 (mCCL2) in vitro in theabsence of type I IFN induction in dendritic cells, recentlydescribed for natural and synthetic RNA.16–19 We furthershow that late onset (i.e., nonprophylactic therapy) with CCL2Spiegelmer effectively improves lupus nephritis, autoimmuneperibronchitis, and lupus-like skin disease in MRLlpr/lpr mice,independent of any previous problem associated with therapeuticCCL2/CCR2 blockade such as tumor formation or irrepressibleantagonist production in situ.7,8
Identification and Characterization of mNOX-E36
High-affinity aptamers to d-mCCL2 were identified after 11 roundsof in vitro selection. Alignment of the aptamers revealed onefamily of relatives that differed by point mutations. As determinedby comparative binding analysis, the most prevalent sequencewas also the best binding molecule. Sequential deletions fromthe 5' end and 3' end, respectively, enabled the truncationto the final candidate mNOX-E36. A secondary structure predictionusing the software mfold proposes three helical regions,20 aswell as three bulges and a large central loop spanning positions18 to 27 (Figure 1A). The dissociation constant of d-mNOX-E36and biotinylated d-mCCL2 (1 to 76) was determined in a pull-downassay at 37°C and calculated to 157 ± 48 pM (Figure 1B).Migration of mCCL2-stimulated THP-1 cells was functionally preventedby mNOX-E36 with an IC50 of approximately 3 nM (Figure 1C).Whereas the polyethylene glycol (PEG)ylated molecule mNOX-E36–3'PEGhad very similar inhibitory efficiency, the control Spiegelmerwas not inhibitory up to a concentration of 1 µM (datanot shown). The therapeutic use of Spiegelmers would be hamperedby induction of type 1 IFN via innate RNA recognition receptors.16–19To exclude this mechanism plasmacytoid dendritic cells (pDC)were prepared from MRLlpr/lpr mice and incubated with the anti-CCL2Spiegelmer and various synthetic RNA. d- but not l-enantiomericRNA40 induced IFN- production in pDC (Figure 2). Furthermore,IFN- release was absent upon exposure to control d- and l-RNA42as well as high concentrations of the anti-CCL2 Spiegelmer.Hence, Spiegelmers do not induce IFN- production in pDC.
Figure 1. mCCL2-binding aptamer/Spiegelmer mNOX-E36. (A) Secondary structure prediction of mCCL2 binding sequence mNOX-E36 as determined by the software mfold.28 (B) Affinity of mNOX-E36 to mCCL2 determined in a pull-down assay with radiolabeled aptamer mNOX-E36. The dissociation constant (157 ± 48 pM) was calculated (GRAFIT) using a 1:1 stoichiometry. (C) Inhibition of mCCL2-stimulated chemotactic cell migration by mNOX-E36. The resulting percentage of cell migration was measured, and the IC50 (3 nM) was deduced at the Spiegelmer concentration of half-maximal chemotaxis.
Figure 2. Spiegelmers do not induce IFN- release in plasmacytoid dendritic cells. Plasmacytoid dendritic cells were generated from bone marrow dendritic cells of MRLlpr/lpr mice by incubation with Ftl-3 ligand and incubated with 10 µg each of d- and l-enantiomeric phosphorothioates RNA40 (TLR7 agonistic sequence), RNA42 (control sequence) in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and various concentrations of mNOX-E36. IFN- production was measured by ELISA. Data are means ± SEM of two independent experiments.
Pharmacokinetics of Spiegelmer Plasma Levels in MRLlpr/lpr Mice
For monitoring drug exposure in MRLlpr/lpr mice, Spiegelmerplasma levels were determined at weekly intervals. The medianplasma levels of mNOX-E36 and mNOX-E36–3'PEG were approximately300 nM and 1 µM throughout the study, respectively (Figure 3).Thus, PEGylation increased the plasma levels of mNOX-E36 andthe progressive kidney disease of MRLlpr/lpr mice did not modulatethe pharmacokinetics of both Spiegelmers. In addition, neitherdrug accumulation nor metabolic induction or reduction was obvious.
Figure 3. Pharmacokinetics of anti-mCCL2 Spiegelmers in chronic kidney disease. Concomitant pharmacokinetic studies were performed for mNOX-E36 and mNOX-E36-PEG in female MRLlpr/lpr mice during the course of treatment from 14 to 24 wk of age. Plasma sampling was carried out 3 and 24 h after the first weekly injection for the Spiegelmers. Spiegelmer plasma levels were determined by modification of a sandwich hybridization procedure.22
Anti-CCL2 Spiegelmer Blocks the Emigration of Monocytes from the Bone Marrow
Monocyte emigration from bone marrow during bacterial infectionwas shown to involve chemokine receptor CCR2,21 but the roleof CCR2 in the context of autoimmunity remains hypothetical.To verify the effect of CCL2 blockade on monocyte migrationwith Spiegelmer treatment, we performed a single-dose studyof the anti-CCL2 Spiegelmer (50 mg/kg), control Spiegelmer (Co,50 mg/kg), and 5% glucose (control). Anti-CCL2 Spiegelmer significantlyreduced the percentage of the circulating CCR2-positive cells6 h after injection, compared with the vehicle-treated group.By contrast, the control Spiegelmer had no effect (Figure 4A).Furthermore, we examined the CCR2-positive monocyte populationin peripheral blood and bone marrow in 24-wk-old MRLlpr/lprmice that had been treated with anti-CCL2 Spiegelmer or salinefrom weeks 14 to 24 of age. Anti-CCL2 Spiegelmer increased CCR2-positivecells from 13 to 26% in the bone marrow and reduced this populationfrom 26 to 11% in the peripheral blood, respectively (Figure 4B).These data support a novel role for CCL2 for the evasion ofCCR2-positive cells from the bone marrow during autoimmune diseaseof MRLlpr/lpr mice.
Figure 4. Flow cytometry for CCR2 in peripheral blood and bone marrow of MRLlpr/lpr mice. (A) Effect of single dose of anti-CCL2 Spiegelmer (50 mg/kg), Spiegelmer (Co, 50 mg/kg), and 5% glucose on circulating CCR2+ cells. Data are mean percentage of CCR2+F4/80+ cells ± SEM in blood 6 h after injection. (B) To study the effects of anti-CCL2 Spiegelmer on leukocyte evasion from the bone marrow, we performed flow cytometry for CCR2 on bone marrow and peripheral blood in 24-wk-old vehicle- or anti–CCL2 Spiegelmer–treated MRLlpr/lpr mice. Data are mean percentage of CCR2+ cells ± SEM in either bone marrow or peripheral blood in five mice of each group.
Anti-CCL2 Spiegelmer Improves Survival and Kidney Disease of MRLlpr/lpr Mice
Female MRLlpr/lpr mice develop and subsequently die from proliferativeimmune complex glomerulonephritis with striking similaritiesto diffuse proliferative lupus nephritis in humans. We treatedMRLlpr/lpr mice with anti-CCL2 Spiegelmers, nonfunctional Co-Spiegelmers(Co-/Co-PEG), or vehicle from weeks 14 to 24 of age, thus usinga therapeutic dosage regimen. In fact, at 14 wk of age, MRLlpr/lprmice revealed proliferative glomerulonephritis with an activityindex of 4.1 ± 1.1 and a chronicity index of 0.1 ±0.2 (data not shown). At this age, major abnormalities of thetubulointerstitial compartment could not be detected. After10 wk of treatment, vehicle-, Co-, or Co-PEG–treated MRLlpr/lprmice showed diffuse proliferative glomerulonephritis characterizedby glomerular macrophage infiltration and a mixed periglomerularand interstitial inflammatory cell infiltrate consisting ofglomerular and interstitial Mac2-positive macrophages and interstitialCD3-positive lymphocytes (Figure 5A, Table 1). Anti-CCL2 Spiegelmerimproved the activity and chronicity index of lupus nephritisas well as the aforementioned markers of renal inflammation(Table 1). Non-PEGylated anti-CCL2 Spiegelmer was less effectiveon the chronicity index and interstitial macrophage and T cellcounts (Table 1). Advanced chronic kidney disease was furtherillustrated by tubular atrophy and confluent areas of interstitialfibrosis in vehicle- and Co-Spiegelmer-treated mice (Figure 5A).We used morphometry to quantify these changes and found thatPEGylated and non-PEGylated anti-CCL2 Spiegelmers reduced interstitialvolume, tubular cell damage, and tubular dilation, all beingmarkers of the severity and prognosis of chronic kidney disease(Figure 5B). These histomorphologic markers of lupus nephritiswere associated with significant mortality and marked albuminuriaat 24 wk of age (Figure 6A, Table 1). In fact, PEGylated butnot non-PEGylated anti-CCL2 Spiegelmers improved the parameter50% mortality. Thus, anti-CCL2 Spiegelmers treatment reducedthe number of renal macrophage and T cell infiltrates, improvedthe symptoms of lupus nephritis, and increased (renal) survivalof MRLlpr/lpr mice. To study whether treatment with PEGylatedor non-PEGylated anti-CCL2 Spiegelmer affects intrarenal inflammationin MRLlpr/lpr mice, we performed real-time reverse transcriptase–PCRfor the proinflammatory chemokines CCL2 and CCL5 which werepreviously shown to be progressively upregulated in kidneysof MRLlpr/lpr mice during progression of renal disease.22 Treatmentwith both anti-CCL2 Spiegelmers reduced renal expression ofCCL2 and CCL5 mRNA compared with vehicle-treated controls (Figure 6B).
Figure 5. Renal histopathology in MRLlpr/lpr mice. (A) Renal sections of 24-wk-old MRLlpr/lpr mice were stained with periodic acid-Schiff (PAS) and antibodies for Mac2 (macrophages) and CD3 (T cells) as indicated. Images are representative for seven to 12 mice in each group. (B) Morphometry was performed on silver-stained sections of mice from all groups as described in the Concise Methods section. The interstitial volume index, tubular cell damage index, and tubular dilation index were calculated as percentage of high-power field and are expressed as means ± SEM. *P < 0.05 versus respective control Spiegelmer. Magnifications: x100 in PAS and PAS inserts and CD3; x400 in Mac2.
Figure 6. Survival and renal chemokine expression of MRLlpr/lpr mice with experimental lupus. (A) Survival of mice of the various treatment groups was calculated by Kaplan-Meier analysis. (B) Renal mRNA expression for the CC-chemokines CCL2 and CCL5 was determined by real-time reverse transcriptase–PCR using total renal RNA pooled from five mice of each group. RNA levels for each group of mice are expressed per respective 18s rRNA expression. Co, control Spiegelmer.
Anti-CCL2 Spiegelmers Reduce Extrarenal Autoimmune Tissue Injury in MRLlpr/lpr Mice
Skin and lungs are also commonly affected from autoimmune tissueinjury in MRLlpr/lpr mice. In vehicle-treated mice, autoimmunelung disease was characterized by moderate peribronchiolar andperivascular inflammatory cell infiltrates, and skin lesionswere observed in 60% of mice (Figure 7, Table 1). Compared withvehicle- and Co-Spiegelmer-treated MRLlpr/lpr mice, administrationsof both anti-CCL2 Spiegelmers reduced peribronchial inflammationand skin disease (Figure 7, Table 1). Hence, the effects ofCCL2-specific Spiegelmers are not limited to lupus nephritisbut extend to other manifestations of autoimmune tissue injuryin MRLlpr/lpr mice.
Figure 7. Extrarenal autoimmune tissue injury in MRLlpr/lpr mice. MRLlpr/lpr mice of all groups were regularly checked for cutaneous lupus manifestations, which typically occur at the facial or neck area and were less common in anti–mCCL2 Spiegelmer–treated mice (A). At 24 wk of age, lung tissue was prepared from MRLlpr/lpr mice of all groups and scored semiquantitatively as described in the Concise Methods section. Treatment with anti-CCL2 Spiegelmers reduced peribronchiolar inflammation in MRLlpr/lpr mice. Images are representative of seven to 11 mice in each group. Magnification: x100.
Anti-CCL2 Spiegelmers and the Lymphoproliferative Syndrome and dsDNA Autoantibodies in MRLlpr/lpr Mice
Female MRLlpr/lpr mice are known to develop a lymphoproliferativesyndrome characterized by massive splenomegaly and bulks ofcervical, axillary, inguinal, and mesenterial lymph nodes. Bothanti-CCL2 Spiegelmers had no effect on the weight of spleensand lymph nodes in MRLlpr/lpr mice (Table 2). Autoimmunity inMRLlpr/lpr mice is characterized by the production of autoantibodiesagainst multiple nuclear antigens including dsDNA. In 24-wk-oldMRLlpr/lpr mice, serum dsDNA IgG, IgG1, IgG2a, and IgG2b autoantibodieswere present at high levels. Both anti-CCL2 Spiegelmers bothhad no effect on either of these DNA autoantibodies (Table 2).Thus, anti-CCL2 Spiegelmers do not affect lymphoproliferationand anti-dsDNA IgG production in MRLlpr/lpr mice.
Aptamers can neutralize biologic functions of target moleculesconceptually similar to antibodies. Unlike aptamers, Spiegelmer-basedoligonucleotides are nuclease resistant and thus biostable withoutfurther modifications,11 which makes them very well suited forin vivo applications. We generated the Spiegelmer mNOX-E36,which binds with high affinity to murine CCL2 and blocks itsfunction in vitro at low nanomolar concentrations. The PEGylatedform of mNOX-E36 displayed higher plasma levels compared withthe non-PEGylated mNOX-E36, but progressive renal failure inMRLlpr/lpr mice did not affect their plasma levels.
Synthetic oligonucleotides may induce IFN- in dendritic cells17,18through innate pattern recognition receptors in cytosolic orendosomal compartments such as Toll-like receptor-7, retinoicacid–inducible protein-1, or melanoma differentiation–associatedgene-5.16–19,23 IFN- is an important mediator of autoimmunity.24,25The induction of IFN- would hamper the use of mNOX-36 in lupusnephritis. However, Spiegelmers did not induce IFN- productionin dendritic cells, because the l-enantiomeric configurationof Spiegelmers is probably not recognized by RNA-specific patternrecognition receptors.
Evidence for a pathogenic role of the CCL2/CCR2 axis in lupus-associatedtissue injury already exists. CCL2- and CCR2-deficient MRLlpr/lprmice are protected from kidney, lung, and skin pathology.5,6Thus, CCL2 and CCR2 both are crucial for the recruitment ofcytokine-producing leukocytes to peripheral tissues. Our datademonstrate that even nonprophylactic late-onset treatment withanti-CCL2 Spiegelmer at 14 wk of age—a point at whichautoimmune tissue injury is already established5,6—iseffective to the same extent as CCL2 or CCR2 deficiency, althoughsuch knockout animals lack CCL2/CCR2 from birth.
The consistency of the data from CCL/CCR2 deficiency and therapeuticCCL2 blockade may relate to the predominant role of the CCL2/CCR2axis during the late leukocyte recruitment–dependent autoimmunetissue injury rather than the early development of autoimmunityin MRLlpr/lpr mice. In fact, both CCL2-deficient and anti-CCL2Spiegelmer–treated MRLlpr/lpr mice did not affect thelymphoproliferative syndrome, DNA autoantibodies, and renalimmune complex deposition, all being early disease markers.6Our findings are consistent with other studies that initiatedblockade of the CCL2/CCR2 axis after onset of autoimmunity.Hasegawa et al.7 injected 5 x 106 cells that were transfectedwith an NH2-truncated CCL2 analog subcutaneously into 12-wk-oldfemale MRLlpr/lpr mice which was associated with an improvedpathology at 20 wk. However, the cell transfer approach causedlocal tumors of considerable size.7 Similar results were obtainedby Shimizu et al.8 who injected 7ND, a NH2-terminal deletionmutant of the Ccl2 gene, into skeletal muscles of 16-wk-oldfemale MRLlpr/lpr mice. The caveat for the use of this genetherapy–like approach in humans is that CCL2 antagonismcannot be abandoned once initiated.
Ccl2 blockade is thought to target CCL2-dependent leukocyteadhesion to activated endothelia and transendothelial migrationof leukocytes at sites of inflammation.3 A recent study foundthat CCR2 is also required for the evasion of monocytes fromthe bone marrow into the intravascular compartment during bacterialinfection.21 Our finding that CCL2 blockade has a similar effecton monocyte homing to the bone marrow during systemic autoimmunityextends this findings two-fold: First, monocyte homing to thebone marrow involves CCL2, possibly acting through CCR2, andsecond, this mechanism seems to apply to systemic inflammationcaused by infection and autoimmunity.
The Spiegelmer-based inhibition of CCL2/monocyte-chemoattractantprotein-1 offers a new and promising way to treat lupus nephritis.The compound mNOX-E36 not only shows efficacy in MRLlpr/lprmice but also demonstrates its suitability in terms of pharmacokineticprofile as well as its absence of immunostimulatory adverseeffects.
Peptides and Nucleic Acids
Recombinant mCCL2 was obtained from R&D Systems (Wiesbaden,Germany). C-terminally biotinylated murine all-d-CCL2 (1 to76; QPDAVNAPLT CCYSFTSKMI PMSRLESYKR ITSSRCPKEA VVFVTKLKRE VCADPKKEWVQTYIKNLDRN QMRSEP) was custom synthesized by Bachem (Bubendorf,Switzerland). Oligonucleotides were synthesized at NOXXON byusing standard phosphoramidite chemistry. l-Phosporamiditeswere from ChemGenes Corp. (Wilmington, MA). The DNA librarycontaining 40 internal random positions had the sequence 5'-GAATTCTAATACGACTCACT ATAGGAAGAG ATGGCGAC-N40-GCCATTCGAA CCGTG-3' and wasamplified with the forward primer 5'-GAATTCTAAT ACGACTCACT ATAGGAAGAGATGGCGAC-3' and the reverse primer 5'-CACGGTTCGAATGGC-3'. Forin vivo application, the mCCL2-binding Spiegelmer mNOX-E36 (5'-GGCGACAUUGGUUGGGCAUG AGGCGAGGCC CUUUGAUGAA UCCGCGGCCA-3') and the nonfunctionalcontrol Spiegelmer PoC (5'-UAAGGAAACU CGGUCUGAUG CGGUAGCGCUGUGCAGAGCU-3') were used nonmodified or modified with 40-kDPEG at the 3' and 5' terminus, respectively. The PoC Spiegelmeris representative for most Spiegelmers identified at NOXXONto date, and its size completely matches the human-specificanti-CCL2 Spiegelmer NOX-E36, which is intended for clinicaldevelopment. In vivo doses of anti-CCL2 Spiegelmer in mg/kgapply to the combined total weight of the oligonucleotide partplus the coupled PEG moiety.
Spiegelmer In Vitro Selection and Truncation
With the use of a library of approximately 6 x 1014 RNA molecules,Spiegelmers to murine d-CCL2 were identified after 11 roundsof in vitro selection.13 The best binding RNA sequence was truncatedto give the final candidate mNOX-E36: 5'-GGCGACAUUGGUUGGGCAU-GAGGCGAGGCCCUUUGAUGAAUCCGCGGCCA-3'.
Determination of Binding Affinities and Inhibition of mCCL2-Induced Chemotaxis In Vitro
The affinity of Spiegelmers to murine d-CCL2 was measured ina pull-down assay format at 37°C using a constant amountof radioactively labeled Spiegelmer and a dilution series ofbiotinylated d-CCL2.13 The percentage of binding was plottedagainst the concentration of biotinylated murine d-CCL2, anddissociation constants were calculated assuming a 1:1 stoichiometry(GRAFIT; Erithacus Software, Surrey, UK).
The inhibitory activity of mNOX-E36 on CCR2 activation in culturedcells was determined by measurement of mCCL2-stimulated chemotaxisusing THP-1 human acute monocytic leukemia cells (DSMZ, Braunschweig,Germany) that express the CCL2 receptor CCL2. The migrationof THP-1 cells was stimulated with 0.5 nM mCCL2 (R&D Systems)in the presence of increasing mNOX-E36 concentrations. Overnight-grownTHP-1 cells (3 x 105) were transferred to each insert of a transwellplate (polycarbonate membrane, pore size 5 µm; Corning3421, Corning Life Sciences, Corning, NY) and placed into theprefilled lower compartments of the transwell plate that contained600 µl of stimulation solution each (0.5 nM mCCL2 in HBSS,0.1% BSA, 20 mM HEPES, and varying amounts of mNOX-E36). Thenumber of migrated cells was determined using resazurin stainingand fluorescence measurement. IC50 values were determined graphicallyby setting the value for the samples without mNOX-E36 (mCCL2only) to 100%, calculating the values for the samples with mNOX-E36as percentage of this, and plotting against the mNOX-E36 concentrations.
Immunostimulatory Effects of Spiegelmers In Vitro
pDC were generated by incubation with Ftl-3 ligand from bonemarrow–derived dendritic cells of MRLlpr/lpr mice andcultured as described.23 pDC were incubated with 10 µgof various types of synthetic RNA in 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP; Roche, Mannheim, Germany; i.e., d- and l-enantiomericphosphorothioates RNA40, RNA41, and RNA42, respectively17),as well as various concentrations of the Spiegelmers in DOTAPfor 24 h. Supernatants were analyzed for IFN- by ELISA (PBLBiomedical Labs, Piscataway, NJ).
Animals and Experimental Protocol
Seven-week-old female MRLlpr/lpr mice were obtained from HarlanWinkelmann (Borchen, Germany) and kept under normal housingconditions in a 12-h light and dark cycle. Water and standardchow (Ssniff, Soest, Germany) were available ad libitum. Atan age of 14 wk, 10 mice were killed for determination of theextent of renal injury at the time of starting the intervention.Additional groups of 12 mice each received subcutaneous injectionsof Spiegelmers of 4 ml/kg in 5% glucose as vehicle three timesper week: mNOX-E36, 25 mg/kg; mNOX-E36–3'PEG, 50 mg/kg;nonfunctional control Spiegelmer PoC, 25 mg/kg; PoC-PEG, 50mg/kg; or vehicle. The plasma levels of mNOX-E36 and mNOX-E36–3'PEGwere determined weekly 3 or 24 h after injection as describedpreviously.26 All experiments were approved by the local governmentauthorities.
Evaluation of Systemic Lupus
Skin lesions were recorded by a semiquantitative score.27 Urinealbumin/creatinine ratios and serum dsDNA autoantibody IgG isotypetiters were determined as described previously.23 Paraffin sections(5 µm) for silver and periodic acid-Schiff stains wereprepared following routine protocols.28 The severity of therenal lesions was graded using the indices for activity andchronicity as described for human lupus nephritis,29 and morphometryof renal interstitial injury was conducted as described previously.28The severity of the peribronchial inflammation was graded semiquantitativelyfrom 0 to 4. Immunostaining was performed as described previously.28The following primary antibodies were used: Rat anti-Mac2 (macrophages;Cederlane; Hornby, ON, Canada; 1:50), anti-mouse CD3 (1:100,clone 500A2; BD Biosciences), anti-mouse IgG1 (1:100, M32015;Caltag Laboratories, Burlingame, CA), and anti-mouse IgG2a (1:100,M32215; Caltag). Glomerular cells were counted in 15 corticalglomeruli per section. Glomerular Ig deposits were scored from0 to 3 on 15 glomerular sections.
RNA Preparation and Real-Time Quantitative (TaqMan) Reverse Transcriptase–PCR
Renal tissue from each mouse was snap-frozen in liquid nitrogenand stored at –80°C. From each mouse, total renalRNA preparation and reverse transcription were performed asdescribed previously.28 Primers and probes were from PE Biosystems(Weiterstadt, Germany). Oligonucleotide primer (300 nM) andprobes (100 nM) were used as listed in Table 3.
Table 3. Primers and probes used for real-time RT-PCR
Flow Cytometry
Flow cytometry was performed using a FACSCalibur machine andthe previously characterized MC21 anti-mCCR2 antibody.30 A biotinylatedanti-rat IgG antibody (BD Biosciences) was used for detection.A rat IgG2b (BD Biosciences) was used as isotype control.
Statistical Analyses
Data were expressed as means ± SEM. Comparison betweengroups were performed using univariate ANOVA (post hoc Bonferronicorrection was used for multiple comparisons) and t test. P< 0.05 indicated statistical significance.
W.P., D.E., N.S., K.B., and S.K. are employees of NOXXON PharmaAG. H.J.A. received consultancy fees from NOXXON Pharma AG.
Acknowledgments
This work was supported by NOXXON Pharma AG.
Parts of this project were prepared as a doctoral thesis atthe Faculty of Medicine, University of Munich, by O.K.
The expert technical assistance of Dan Draganovic, Jana Mandelbaum,and Ewa Radomska is gratefully acknowledged. We thank ChristianLange and Hilke Hansen for Spiegelmer quantification in plasma,the NOXXON chemistry group for providing the oligonucleotides,and the biology group for carrying out the cell culture assays.The authors are grateful to Dr. Bruno Luckow for the generousgift of dsDNA.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
production in pDC (Figure 2). Furthermore, IFN-
