Chemokine Receptor Ccr2 Deficiency Reduces Renal Disease and Prolongs Survival in MRL/lpr Lupus-Prone Mice
Guillermo Pérez de Lema*,
Holger Maier*,
Tobias J. Franz,
Maríia Escribese,
Silvia Chilla*,
Stephan Segerer*,
Natalia Camarasa,
Holger Schmid*,
Bernhard Banas*,
Svetoslav Kalaydjiev,
Dirk H. Busch,,
Klaus Pfeffer||,
Francisco Mampaso,
Detlef Schlöndorff* and
Bruno Luckow*
* Klinikum der Universität München, Medizinische Poliklinik - Innenstadt, Munich, Germany; German Mouse Clinic, Institute of Experimental Genetics, GSF - National Research Center for Environment and Health, Munich/Neuherberg, Germany; Department of Pathology, Hospital Ramón y Cajal, Universidad de Alcalá, Madrid, Spain; Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany; and || Institut für Medizinische Mikrobiologie, Universitätsklinikum der Heinrich-Heine-Universität Düsseldorf, Germany
Address correspondence to: Dr. Bruno Luckow, Klinikum der Universität München, Medizinische Poliklinik - Innenstadt, Arbeitsgruppe Klinische Biochemie, Schillerstrasse 42, D-80336 München, Germany. Phone: +49-89-2180-75842; Fax: +49-89-2180-75860; E-mail:bruno.luckow{at}med.uni-muenchen.de
Received for publication May 2, 2005.
Accepted for publication September 22, 2005.
MRL/MpJ-Faslpr/J (MRL/lpr) mice represent a well-establishedmouse model of human systemic lupus erythematosus. MRL/lpr micehomozygous for the spontaneous lymphoproliferation mutation(lpr) are characterized by systemic autoimmunity, massive lymphadenopathyassociated with proliferation of aberrant T cells, splenomegaly,hypergammaglobulinemia, arthritis, and fatal immune complex–mediatedglomerulonephritis. It was reported previously that steady-statemRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2)continuously increase in kidneys of MRL/lpr mice. For examiningthe role of Ccr2 for development and progression of immune complex–mediatedglomerulonephritis, Ccr2-deficient mice were generated and backcrossedonto the MRL/lpr genetic background. Ccr2-deficient MRL/lprmice developed less lymphadenopathy, had less proteinuria, hadreduced lesion scores, and had less infiltration by T cellsand macrophages in the glomerular and tubulointerstitial compartment.Ccr2-deficient MRL/lpr mice survived significantly longer thanMRL/lpr wild-type mice despite similar levels of circulatingimmunoglobulins and comparable immune complex depositions inthe glomeruli of both groups. Anti-dsDNA antibody levels, however,were reduced in the absence of Ccr2. The frequency of CD8+ Tcells in peripheral blood was significantly lower in Ccr2-deficientMRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophageand T cell infiltration in the kidney but also the systemicT cell response in MRL/lpr mice. These data suggest an importantrole for Ccr2 both in the general development of autoimmunityand in the renal involvement of the lupus-like disease. Theseresults identify Ccr2 as an additional possible target for thetreatment of lupus nephritis.
Systemic lupus erythematosus is an autoimmune disease with immunecomplex–mediated glomerulonephritis as a major manifestationand determinant of the disease outcome (1). The MRL/lpr mousecarries a mutation in the apoptosis-related Fas gene, resultingin autoreactive lymphocyte proliferation, and is consideredas a mouse model that closely mimics the human disease withlymphadenopathy; splenomegaly; hypergammaglobulinemia with anti-dsDNAantibodies that lead to tissue deposition; and injury in variousorgans, including lung and kidney (2,3). These pathophysiologicprocesses are mediated by immune complex deposition, complementactivation, and infiltration of inflammatory leukocytes (4,5).The renal leukocyte infiltrates consist mostly of macrophagesand T lymphocytes. These cells are major contributors to thedevelopment of progressive renal insufficiency, ultimately resultingin the death of the MRL/lpr mice.
Numerous studies in both human and experimental murine lupushave provided evidence that chemokines play a considerable roleduring the development and progression of the renal disease.We and others have proposed that deposition of immune complexesand complement activation leads to local generation of chemokinesin the kidney followed by the influx of macrophages and T cells(6). Initially, this may be restricted to the glomerulus withsubsequent expansion to the tubulointerstitial compartment anddevelopment of progressive renal insufficiency (7). This hypothesisis supported by studies with MRL/lpr mice carrying a targeteddeletion of the Ccl2 (murine MCP-1) gene (5) or treated withantagonists for Ccl2 (8,9). In both cases, an attenuation ofthe nephritis has been observed. Furthermore, Ccl2-deficientMRL/lpr mice showed a marked reduction of inflammatory lesions,including in the kidney, leading to prolonged survival (5).The chemokine receptor Ccr2, which represents the only knownreceptor for Ccl2, is considered to play a role not only inthe local infiltration of macrophages and T cells but also inthe development and bias of the overall immune response (10).Surprisingly, Ccr2 and Ccl2 have opposite effects on Th1 versusTh2 polarization. Ccr2-deficient mice show decreased IFN- secretion,i.e., a Th1 defect (11), whereas Ccl2-deficient mice show predominantlya defect of Th2 cytokine secretion (12). Possibly because ofthese differences, conflicting results have been obtained withCcl2- and Ccr2-deficient mice in several disease models (13).These may relate in part to the fact that in addition to Ccl2,other Ccr2 ligands exist (Ccl7/MCP-3, Ccl8/MCP-2, and Ccl12/MCP-5)(13) and that other, yet unidentified, receptor(s) for Ccl2may exist (14).
Although Ccr2-deficient mice have been studied in many diseasemodels (15), data about the role of Ccr2 in lupus nephritisare still missing. This report describes the generation andanalysis of Ccr2-deficient MRL/lpr mice with a main focus onthe development and progression of lupus nephritis.
Animals
MRL/MpJ-Faslpr/J (briefly MRL/lpr) mice were purchased fromThe Jackson Laboratory (Bar Harbor, ME) and subsequently maintainedas a breeding colony in our animal facility. All mice were housedunder specific pathogen free conditions. All animal experimentswere performed in compliance with current guidelines and approvedby local authorities.
Generation of Ccr2-Deficient MRL/lpr Mice
A targeting vector (Figure 1A) was constructed and electroporatedinto E14–1 embryonic stem (ES) cells. A total of 192 EScell clones that were resistant to G418 and Ganciclovir werescreened by Southern blot analysis (Figure 1B). Homologous recombinationwithin the 3'-homology arm was confirmed by long-range PCR (ExpandLong Template PCR System; Roche Applied Science (Penzburg, Germany),forward primer 5'-CAGAAGAACGGCATCAAGGTGAAC-3', reverse primer5'-TGTGGAACAAACTTTATGCTGGGG-3'; Figure 1C). Two positive EScell clones were microinjected into C57BL/6 blastocysts. Resultingchimeras were mated with C57BL/6 wild-type (wt) mice for germlinetransmission. A more detailed description is available uponrequest. The resulting heterozygous Ccr2-deficient mice werebackcrossed for seven generations onto the MRL/lpr backgroundand then intercrossed to obtain Ccr2–/– MRL/lprmice that were used for all experiments. For survival analysis,four additional Ccr2-deficient mice that were backcrossed for10 generations were included. All experiments were performedwith virgin female mice homozygous for the lpr mutation.
Figure 1. Generation and identification of Ccr2-deficient mice. (A) Schematic representation of the targeting strategy. The murine Ccr2 wild-type (wt) locus, the targeting vector and the targeted Ccr2 locus are shown together with selected restriction sites relevant for Southern blot analysis. , 5' and 3' untranslated regions; , coding region of the Ccr2 gene. The homology arms are depicted as thick lines, the floxed neomycin resistance gene (neo) for positive selection as a gray box with black triangles at both ends, the Herpes simplex virus thymidine kinase gene (HSV tk) for negative selection as a hatched box, the enhanced green fluorescence reporter gene (egfp) also as a hatched box, and the SV40 polyadenylation signal (pA) as a cross-hatched box. The wavy line represents prokaryotic sequences in the targeting vector. The sizes of relevant restriction fragments and PCR fragments before and after homologous recombination are depicted. Arrowheads indicate the position of primers used for long-range PCR analysis. (B) Southern blot analysis of wt embryonic stem cells (lane 1) and a successfully targeted ES cell clone (lane 2) using the 5' external probe depicted in A. (C) Long-range PCR to prove homologous recombination within the 3' arm of homology. An 8.3-kb PCR fragment indicates homologous recombination. Lane 1, 1-kb ladder; lane 2, PCR product; lane 3, PCR product digested with HindIII; lane 4, PCR product digested with BamHI. (D) Analysis of peripheral blood leukocytes (PBL) from wt and Ccr2-deficient mice by flow cytometry. PBL were stained with antibodies directed against CD11b and Ccr2 and gated for monocytes.
Genotype Analysis
All mice were genotyped by PCR using genomic DNA that was isolatedfrom tail snips and the following primers: Ccr2 forward 5'-AAAGGAAATGGAAGACAATAATATG-3';Ccr2 reverse 5'-AAAGGCAAACTGTCACTTACTTTAC-3'; EGFP reverse 5'-GTCAGGGTGGTCACGAGG-3';Fas forward 5'-AAAGGTTACAAAAGGTCACCC-3'; Fas reverse 5'-GGTGCAGCCAGAAGCTAG-3';lpr reverse 5'-GTTCCTCTTAGCATCTCTCTGC-3'.
Collection of Blood, Urine, and Tissue Samples and Determinations of Blood Urea Nitrogen, Creatinine, and Urinary Albumin
Blood and spot urine samples were obtained and determinationswere performed as described previously (6,16). After a finalbleeding step, anesthetized mice were killed by cervical dislocation.One kidney was snap-frozen in liquid nitrogen and used exclusivelyfor isolation of total RNA. The other kidney was used for histologicand immunohistologic analyses.
Histopathology
A renal pathologist performed in a blinded manner the histopathologicanalysis as described previously (17). Glomerular lesions weregraded semiquantitatively from 0 to 3+ for hypercellularity,mesangial matrix expansion, and necrosis (0, absence; 1, mild;2, moderate; 3, severe), as well as for the percentage of scleroticglomeruli and presence of crescents (0, <10%; 1, 10 to 25%;2, 25 to 40%; 3, >40%). Tubulointerstitial lesions were alsograded from 0 to 3+ for peritubular and pericapillary mononuclearcell infiltrate, tubular damage, interstitial fibrosis, andvasculitis. Finally, perivascular lymphoproliferative mononuclearcell infiltrate was graded from 0 (absence) to 3+ (maximal intensity).Global glomerular and tubulointerstitial lesion scores werecalculated for each mouse from the sum of all parameters.
Immunofluorescence and Immunohistochemistry
Direct immunofluorescence was performed on 5-µm ether/ethanol-fixedcryostat sections using FITC-conjugated rabbit anti-mouse IgGand goat anti-mouse complement C3 antibodies (16). CD3-, ER-HR3–,Mac-2–, and Ki-67–positive cells were detected using4-µm-thick paraffin sections from renal tissue (17,18).IgG of the corresponding isotype served as negative control.For quantification, positively stained cells were counted eitherwithin 20 glomeruli or within 10 high-power fields (x630) oftubulointerstitial tissue and expressed as cells per glomerularcross-section or high-power field.
RNase Protection Assay
Cytokine, chemokine, and chemokine receptor mRNA expressionwas analyzed in kidney RNA from wt and Ccr2–/– MRL/lprmice using RPA template sets mCK-3, mCK-5b, and mCR-5 (BD Biosciences,Heidelberg, Germany) (19). Integrity of RNA samples was confirmedby agarose gel electrophoresis. For chemokine receptors, 30µg/lane total kidney RNA was used; for cytokines and chemokines,10 µg/lane was used. Bands were quantified by phosphorimaging(Storm 840 PhosphorImager; Molecular Dynamics, Sunnyvale, CA)using ImageQuant software.
Real-Time Reverse Transcriptase–PCR
Spleen IFN- mRNA levels were quantified by real-time RT-PCRas described (20) using a predesigned probe and primer set fromApplied Biosystems (Darmstadt, Germany). The normalized expressionvalues were multiplied by a factor of 105.
Analysis of Peripheral Blood by Flow Cytometry and ELISA
Blood was collected from the retro-orbital plexus of anesthetizedmice and peripheral blood leukocytes were stained after erythrocytelysis with antibodies against CD3 (clone 17A2), CD4 (clone RM4–5),CD8 (clone H35–17.2), CD19 (clone 1D3), CD49b (clone DX5),B220 (clone RA3–6B2), and Gr-1 (clone RB6–8C5; allPharMingen, Heidelberg, Germany). Samples were acquired (25,000leukocytes) using a high-throughput sampler on a FACS Calibur(BD Biosciences, San Jose, CA) and analyzed using FlowJo software(Tree Star, Ashland, OR).
Plasma levels of Ig isotypes were determined by standard sandwichELISA using goat anti-mouse Ig antibodies and alkaline phosphatase(AP) conjugates (SouthernBiotech, Birmingham, AL). Antibodiesagainst double-stranded DNA were detected by indirect ELISAon plates coated with calf thymus DNA (5 µg/ml; Sigma-Aldrich,Taufkirchen, Germany) and AP-conjugated goat anti-mouse polyvalentimmunoglobulins antibody (Sigma-Aldrich). Serum from MRL/lprmice was used in 1:1250 to 1:10,000 dilution as positive controlin autoantibody assays.
MC-21 antibody was used to stain Ccr2 on peripheral blood leukocytes(21). Detection was performed with PE-conjugated polyclonalgoat anti-rat IgG (P64; Biomeda, Foster City, CA). For simultaneousCD11b staining, a FITC-conjugated mAb (clone M1/70, BD Biosciences)was used.
Statistical Analyses
Data are shown as means ± SD and were analyzed eitherby two-tailed unpaired t test (parametric data), Mann-WhitneyU test (nonparametric data), or log-rank test (survival). P< 0.05 was considered significant.
Generation of Ccr2-Deficient MRL/lpr Mice
Ccr2-deficient mice were generated using the targeting strategyoutlined in Figure 1. They were viable, fertile, and indistinguishablefrom their wt littermates. Absence of Ccr2 protein on the cellsurface of blood monocytes was demonstrated by flow cytometry(Figure 1D). Ccr2-deficient mice were backcrossed for sevengenerations onto the inbred strain MRL/lpr and then intercrossedto obtain Ccr2–/– MRL/lpr mice. Because the contributionof the MRL/lpr genetic background in these mice exceeds 99%,wt MRL/lpr mice were used as controls.
Body Weight, Splenomegaly, and Lymphadenopathy
The body, spleen, and right axillary lymph node weights of Ccr2+/+and Ccr2–/– MRL/lpr mice were determined at theage of 14 and 20 wk (Figure 2). There was no difference in bodyweight between wt and Ccr2–/– MRL/lpr mice. In contrast,the spleen weights were significantly reduced in Ccr2–/–MRL/lpr mice at 14 wk but not at 20 wk. Lymph node weights weresignificantly reduced in Ccr2–/– MRL/lpr mice at14 and 20 wk.
Figure 2. Effect of Ccr2 deficiency on body weight, splenomegaly, and lymphadenopathy. The body weight, the spleen weight, and the weight of the right axillary lymph node were determined in 14- and 20-wk-old wt and Ccr2-deficient MRL/lpr mice (body: week 14 wt n = 20, knockout [KO] n = 15; week 20 wt n = 12, KO n = 17; spleen: week 14 wt n = 13, KO n = 15, P = 0.006; week 20 wt n = 12, KO n = 10; lymph node: week 14 wt n = 17, KO n = 15, P = 0.031; week 20 wt n = 12, KO n = 10, P = 0.016).
Time Course of Blood Urea Nitrogen and Albuminuria
No statistically significant differences in blood urea nitrogen(BUN) were observed between wt and Ccr2-deficient MRL/lpr miceat 8, 14, and 20 wk of age (Figure 3). Urinary albumin excretionincreased between weeks 8 and 20 in MRL/lpr wt mice with a markedinteranimal variability and was significantly reduced in Ccr2-deficientMRL/lpr mice at 8 and 14 wk (Figure 3).
Figure 3. Effect of Ccr2 deficiency on blood urea nitrogen (BUN) and proteinuria. BUN levels were measured in serum samples from wt and Ccr2-deficient MRL/lpr mice (week 8 wt n = 8, KO n = 6; week 14 wt n = 14, KO n = 19; week 20 wt n = 13, KO n = 21). Albuminuria was determined by ELISA in urine samples from wt and Ccr2-deficient MRL/lpr mice (week 8 wt n = 20, KO n = 8, P = 0.018; week 14 wt n = 15, KO n = 23, P = 0.0014; week 20 wt n = 14, KO n = 19).
Histopathologic and Immunohistochemical Findings
At the age of 14 wk, Ccr2+/+ MRL/lpr mice showed enlarged hypercellularglomeruli, an increase in mesangial matrix, and a mild peritubularmononuclear cell infiltrate (Figure 4A). In addition, prominentperivascular lymphoid cell accumulations were present at thistime point, which may be related to the lymphoproliferativeprocess of these mice (16,17). At the age of 20 wk, Ccr2+/+MRL/lpr mice showed locally and focally variable signs of intraglomerularnecrosis, sclerosis, and crescent formation and peritubularmononuclear cell infiltration with increased overall tubulardamage and focal tubular atrophy and intratubular protein castformation (Figure 4A). In addition, mild signs of tubulointerstitialfibrosis could be observed. No vasculitis was detected. Theselesions were significantly reduced in Ccr2–/– MRL/lprmice (Figure 4).
Figure 4. Effect of Ccr2 deficiency on glomerular and tubulointerstitial damage. (A) Representative periodic acid-Schiff–stained kidney sections from 14- or 20-wk-old wt and Ccr2-deficient MRL/lpr mice. (B) Glomerular and tubulointerstitial lesion scores for 14- or 20-wk-old wt and Ccr2-deficient MRL/lpr mice (14 wk wt n = 15, KO n = 10; 20 wk wt n = 25, KO n = 14, glomerular scores P = 0.001, tubulointerstitial scores P = 0.006). Magnification, x630 in A.
Immune Complex Deposition
IgG and C3 depositions were analyzed by immunofluorescence withinthe glomeruli of 14- and 20-wk-old Ccr2+/+ and Ccr2–/–MRL/lpr mice. No differences in immune complex and complementC3 depositions were observed between wt and Ccr2-deficient MRL/lprmice (Figure 5A).
Figure 5. Effect of Ccr2 deficiency on immune complex deposition, kidney infiltration, and cellular proliferation. (A) Analysis of C3 and IgG immune complex deposition in glomeruli from 14-wk-old wt and Ccr2-deficient MRL/lpr mice by immunofluorescence. (B) Immunohistochemical analysis of T cell and macrophage infiltration and cellular proliferation in kidneys from 14- and 20-wk-old wt and Ccr2-deficient MRL/lpr mice. The Ki-67 pictures are from 14-wk-old animals; the other ones are from 20-wk-old animals. For illustrative purposes, pictures were chosen showing glomeruli with a higher number of infiltrated macrophages than the mean values obtained from all analyzed glomeruli (see C). (C) Quantitative analysis of infiltration and cellular proliferation in kidneys from wt and Ccr2-deficient MRL/lpr mice at 14 and 20 wk of age (wt n = 4 to 6, KO n = 4 to 6; glomerulus week 20: CD3 P = 0.016, ER-HR3 P = 0.0095; tubulointerstitium week 20: CD3 P = 0.0008, ER-HR3 P = 0.0033; Ki-67 week 14 P = 0.019, week 20 P = 0.020). Magnification, x400 in A.
Kidney T Cell and Macrophage Infiltration
Infiltration of the kidney by T lymphocytes and macrophageswas analyzed by immunohistochemical staining for CD3 and ER-HR3(Figure 5B). At 14 and 20 wk of age, low numbers of T cellsand macrophages were found in the glomerular compartment, whichwere significantly reduced in Ccr2-deficient MRL/lpr mice at20 wk of age (Figure 5C). The number of glomerular macrophageswas also determined by Mac-2 staining, which detects a broaderrange of macrophage subpopulations than ER-HR3 staining (22).As expected, the number of Mac-2+ cells per glomerulus was anorder of magnitude higher for both groups and reduced in Ccr2-deficientmice (week 20 13.4 ± 4.7 versus 9.5 ± 5.0; wtn = 9, KO n = 15; P = 0.034 one-tailed unpaired t test, P =0.068 two-tailed test). In the tubulointerstitial compartment,both CD3+ T cells and ER-HR3+ macrophages were significantlyreduced in Ccr2-deficient MRL/lpr mice at both time points (Figure 5,B and C).
Intraglomerular Cell Proliferation
Intraglomerular cell proliferation was analyzed by immunohistochemicalstaining for Ki-67. A prominent signal was observed mainly inglomeruli of 14-wk-old Ccr2+/+ MRL/lpr mice, consistent witha more acute/proliferative phase of glomerulonephritis (Figure 5B).At 20 wk, fewer Ki-67–positive cells were detectedwithin the glomeruli, and proliferative lesions were replacedby necrosis and sclerosis. Ccr2-deficient MRL/lpr mice had fewerKi-67–positive cells per glomerulus, at both 14 and 20wk of age as compared with age-matched Ccr2+/+ MRL/lpr mice(Figure 5C).
Expression of Proinflammatory Cytokines, Chemokines, and Chemokine Receptors
Multiprobe RPA was used to analyze mRNA expression of selectedcytokines, chemokines, and chemokine receptors in kidneys of14- and 20-wk-old Ccr2+/+ and Ccr2–/– MRL/lpr mice.The results of 20-wk-old mice are shown in Figures 6, 7, and8. Although the expression of all detected genes was somewhatlower in Ccr2–/– mice, a statistically significantreduction was observed only for the cytokines TNF- and IFN-(Figure 6); the chemokine Xcl1 (Figure 7); and the chemokinereceptors Ccr1, Ccr3, and Ccr5 (Figure 8). To examine whetherthe IFN- response was generally lower in Ccr2–/–MRL/lpr mice, we used real-time RT-PCR to quantify IFN- mRNAlevels in spleens from 20-wk-old Ccr2+/+ and Ccr2–/–MRL/lpr mice. No differences were observed between both groups(wt 16.2 ± 7.8, n = 6; KO 15.0 ± 11.6, n = 7;P = 0.833).
Figure 6. Effect of Ccr2 deficiency on renal cytokine expression. Expression of cytokines was analyzed by multiprobe RPA using total kidney RNA from 20-wk-old wt and Ccr2-deficient MRL/lpr mice (wt n = 5, KO n = 5). (A) PhosphorImager scan. (B) Quantitative evaluation (TNF-P = 0.038, IFN-P = 0.039).
Figure 7. Effect of Ccr2 deficiency on renal chemokine expression. Expression of chemokines was analyzed by multiprobe RPA using total kidney RNA from 20-wk-old wt and Ccr2-deficient MRL/lpr mice (wt n = 5, KO n = 5). (A) PhosphorImager scan. (B) Quantitative evaluation (Xcl1 P = 0.038).
Figure 8. Effect of Ccr2 deficiency on renal chemokine receptor expression. Expression of chemokine receptors was analyzed by multiprobe RPA using total kidney RNA from 20-wk-old wt and Ccr2-deficient MRL/lpr mice (wt n = 5, KO n = 5). (A) PhosphorImager scan. (B) Quantitative evaluation (Ccr1 P = 0.012, Ccr3 P = 0.006, Ccr5 P = 0.020).
Circulating Immunoglobulins
Circulating Ig isotype levels and anti-dsDNA antibodies weredetermined in 14- and 20-wk-old wt and Ccr2-deficient MRL/lprmice (Figure 9). We noticed a three- to four-fold decrease inIgG2a between weeks 14 and 20 in all mice. With the exceptionof anti-dsDNA antibodies (Figure 9B), no significant differencesbetween wt and Ccr2-deficient MRL/lpr mice were observed (Figure 9A).
Figure 9. Effect of Ccr2 deficiency on circulating Ig isotype levels. (A) Serum from wt (week 14 n = 7, week 20 n = 6) and Ccr2-deficient MRL/lpr mice (week 14 n = 8, week 20 n = 7) was analyzed by ELISA for Ig isotype levels. (B) Serum from 20-wk-old wt (n = 6) and Ccr2-deficient MRL/lpr mice (n = 7) was analyzed by ELISA for anti-dsDNA antibodies (1:200 P = 0.002, 1:400 P = 0.008).
Immunoscreen of Peripheral Blood
Flow cytometry was used to measure the frequency of selectedleukocyte subpopulations in peripheral blood from 14- and 20-wk-oldCcr2+/+ and Ccr2–/– MRL/lpr mice. Lymphadenopathyin MRL/lpr mice is characterized by a marked accumulation ofCD4– CD8– B220+ T cells (double-negative T cells),which can comprise >90% in older animals. As expected, anincrease of B220+ cells was observed in parallel to an increaseof CD3+ T cells between weeks 14 and 20 (Figure 10). Whereasno differences between wt and Ccr2–/– MRL/lpr micewere observed with respect to the percentage of overall CD3+T cells, B220+ cells, CD19+ B cells, Gr-1+ cells, or DX5+ NKcells at both time points, the frequency of CD4+ T cells wassignificantly lower in Ccr2-deficient MRL/lpr mice at 14 wk(P = 0.007). Furthermore, the percentage of CD8+ T cells wasmarkedly reduced at both 14 (P = 0.00008) and 20 wk (P = 0.00012;Figure 10).
Figure 10. Effect of Ccr2 deficiency on leukocyte subsets in peripheral blood. Peripheral blood from wt (week 14 n = 7, week 20 n = 6) and Ccr2-deficient MRL/lpr mice (week 14 n = 8, week 20 n = 7) was analyzed by flow cytometry using antibodies directed against CD3, CD4, CD8, and B220.
Survival Analysis
Ccr2+/+ MRL/lpr mice showed a median (50%) survival time of25.7 ± 2 wk (95% confidence interval 21.8 to 29.6), whichwas increased to 38.7 ± 10.6 wk (95% confidence interval18.0 to 59.4) in Ccr2–/– MRL/lpr mice. The statisticalanalysis of the survival data revealed a significant prolongationof survival in Ccr2-deficient MRL/lpr mice (Figure 11).
Figure 11. Effect of Ccr2 deficiency on survival. Kaplan-Meier plot showing the survival curves for wt and Ccr2-deficient MRL/lpr mice. One Ccr2-deficient mouse is still alive (August 12, 2005) and 111 wk old.
The various roles of Ccr2 and its ligand Ccl2 have been examinedin numerous experimental studies. Discrepant results have beenobtained concerning the general immune response in Ccl2–/–(12) versus Ccr2–/– mice (23). With regard to infiltrationof T cells and monocytes/macrophages at sites of immune injury,a more uniform picture is emerging. Our results obtained withthe model of systemic lupus in Ccr2-deficient MRL/lpr mice supporta role for this receptor in the development of the systemicautoimmune parameters, as well as in the local leukocyte infiltrates,especially in the kidney, the major target organ in lupus. Consistentwith a role of Ccr2 in the general immune response, Ccr2–/–MRL/lpr mice developed less lymphadenopathy and had a lowerpercentage of CD4+ and CD8+ peripheral T lymphocytes (Figure 10).
In keeping with an important role for Ccr2 in the infiltrationof T cells and macrophages in the kidney, Ccr2-deficient MRL/lprmice showed reduced glomerular and tubulointerstitial CD3+ Tcell and ER-HR3+ and Mac2+ macrophage infiltrates and less glomerularcell proliferation, with a corresponding reduction in proteinuriaand renal disease. This occurred despite unchanged hyperimmunoglobulinemiaand comparable glomerular immune complex and complement deposits.There was, however, a reduction in plasma anti-dsDNA antibodytiters at 20 wk of age. Finally, these changes were associatedwith a prolongation of overall survival of Ccr2–/–MRL/lpr mice as compared with Ccr2+/+ MRL/lpr controls. Theseeffects cannot be attributed to differences in genetic backgroundbetween the Ccr2+/+ and Ccr2–/– MRL/lpr mice, asthe knockout strain had been backcrossed for seven generationsand therefore represents an incipient congenic strain. Our resultssupport roles for Ccr2 in the development of the autoimmunedisease, especially the generation of activated T cells andanti-dsDNA antibody production, and in the development of renalinjury, thereby influencing disease progression and survivalof MRL/lpr mice.
The phenotypes of Ccr2-deficient mice in diverse disease modelshave been summarized recently (15). Depending on the model used,the disease parameters and the outcome in Ccr2–/–mice were worse (24), unchanged (25), or improved (26). Withsome exceptions, autoimmune (27–29) and inflammatory diseasemodels (30) were generally improved in Ccr2–/– mice,whereas most infectious models had a more severe outcome (31,32).Overall, these diverse studies support a role for Ccr2 in leukocyterecruitment to the site of immune injury.
A potential shift of the Th1–Th2 response in Ccr2–/–mice has been discussed, but, depending on the model used, conflictingresults have been obtained (11,32–35). On the basis ofthe lack of difference in the Ig isotypes in Ccr2–/–MRL/lpr mice compared with those in the Ccr2+/+ MRL/lpr mice,our data seem not to support a major shift in Th1–Th2balance in this model. Furthermore, IFN- mRNA levels were unchangedin spleens from wt and Ccr2-deficient mice at 20 wk. We didobserve, however, decreased IFN- mRNA levels in kidneys fromCcr2-deficient mice, which would indicate a defect in Th1 response,as previously reported for Ccr2–/– mice (11). Thereduced IFN- mRNA levels in the kidneys of Ccr2–/–MRL/lpr mice that were observed by us are consistent with theresults of Tesch et al. (5) in Ccl2-deficient MRL/lpr mice andof Hasegawa et al. (8) obtained with a Ccl2 antagonist in MRL/lprmice. IFN- plays a major role in the development of the renaldisease in MRL/lpr mice, as demonstrated by marked improvementof disease parameters in IFN-–deficient MRL/lpr mice (36,37).Infiltrating T cells and macrophages can be the source of theIFN- (38,39). Because there was also less inflammatory infiltratein the kidneys, we cannot differentiate between attributingthe lower renal IFN- to fewer IFN-–producing cells inthe kidney and/or less IFN- produced per infiltrating macrophage.The latter possibility is less likely, however, because Peterset al. (40) could show that the reduced IFN- levels that wereobserved in Ccr2-deficient mice were caused by an in vivo traffickingdefect of IFN-–producing cells and not by diminished production.Furthermore, we observed comparable IFN- mRNA levels in spleensfrom both groups. Similar considerations apply to the lowermRNA levels for TNF. Irrespective of the exact mechanism, thelower IFN- levels in the kidney could contribute to the relativeprotection of the kidney to the development of immune nephritis.
The reduced lymphadenopathy noted in the Ccr2-deficient MRL/lprmice is comparable to data obtained in Ccl2-deficient MRL/lprmice (5). The reason for the attenuation—but not normalization—ofthe size of the lymphatic organs in both the Ccr2–/–and Ccl2–/– MRL/lpr mice remains speculative butpossibly could relate to Ccl2- and Ccr2-mediated cell migrationand sequestration in lymph nodes and spleen (41) or Ccl2-dependentapoptosis of bone marrow precursor cells as reported by Reidet al. (42) in Ccr2–/– mice.
The reduction in size of the lymphatic organs in the Ccr2–/–MRL/lpr mice was associated with a reduction of circulatingCD8+ T cells in the peripheral blood, consistent with the reportedrole of Ccr2 in T cell migration and the enhanced expressionof Ccr2 on activated CD8+ T cells (43). The lower number ofCD8+ T cells that we observed in the blood of Ccr2–/–MRL/lpr mice is consistent with the recent observation of fewerCD62low CD44high CD8+ T cells in the spleen of Ccr2–/–mice that received islet transplants (44). The reduction inCD8+ T cells observed by us in the blood of Ccr2-deficient MRL/lprmice and by Abdi et al. (44) in the spleens of mice that receivedtransplants suggests a regulatory role of Ccr2 in the activationof CD8+ T cells during the immune-mediated disease. A previousreport from our group has shown that in peripheral blood ofBALB/c mice with apoferritin-induced glomerulonephritis, only4% of the CD4+ and 3% of the CD8+ T cells expressed Ccr2, but63 and 30% of these cell types that were isolated from inflamedkidneys were positive for Ccr2 (21). In the context of the MRL/lprmouse, our finding of reduced peripheral CD8+ T cells in Ccr2–/–MRL/lpr mice potentially could point toward a contributory roleof T cells in the development of nephritis (45,46).
Despite the reduction in lymphadenopathy in either the Ccr2–/–(these data) or the Ccl2–/– MRL/lpr mice (5), thepolyclonal hyperimmunoglobulinemia persisted in both models.Thus, the autoreactive B cell expansion of the MRL/lpr mouseseems to be independent of Ccr2 or its ligand. Consistent withthe unchanged circulating antibody levels, the glomerular depositionof Ig and complement were unchanged by Ccr2 deficiency in theMRL/lpr mice. Together, these results add to the growing numberof studies in MRL/lpr mice showing that local immune complexdeposition and complement activation alone seem to be insufficientto induce a full-blown glomerular inflammatory response. Thishas been observed with elimination of the receptor for the IgFcR (47); for the cytokines IFN- (37), IL-12 (48), and CSF-1(49); and for the chemokine Ccl2 (5,8) or the respective receptors,e.g., Ifngr1 (50) or Ccr2 (this report).
The mitigation of the immune-mediated renal disease may be areason for the improved survival. As previously reported byothers and us, glomerular chemokine production follows immunecomplex deposition and precedes the leukocyte infiltration,which in turn precedes the development of proteinuria (5,6).At 20 wk of age, Ccr2–/– MRL/lpr mice showed reducednumbers of infiltrating ER-HR3+ macrophages as well as CD3+T cells in the glomerular and tubulointerstitial compartments.As expected, this was associated with significantly lower histopathologicscores. Similar results have been reported by Hasegawa et al.(8) using a Ccl2 antagonist and by Tesch et al. (5) using Ccl2–/–MRL/lpr mice, with the exception that in the latter study, noreduction in glomerular T cell infiltrate was observed. Thedifference in glomerular T cell infiltrate between the Ccr2–/–and the Ccl2–/– mice could be related to the reducedperipheral CD8+ T cells noted in the Ccr2–/– miceand to the high percentage of Ccr2+ T cells infiltrating kidneyswith immune complex disease as previously reported by us (21).Furthermore, the diminished T cell infiltration may not necessarilyreflect a direct role for Ccr2 in T cell migration but couldalso be a consequence of the reduced macrophage infiltrationleading to less secondary T cell recruitment (46). The reducedCcr1, Ccr3, and Ccr5 mRNA levels in kidneys of Ccr2–/–MRL/lpr mice may simply reflect the decrease in number of infiltratingmacrophages and T cells that express—in addition to Ccr2—Ccr1,Ccr3, or Ccr5 (21). Taken together, the ligand-receptor pairCcl2 and Ccr2 seems to play a significant role in glomerularand in tubulointerstitial macrophage and T cell infiltrationin MRL/lpr lupus nephritis and contribute to its progressiontoward terminal renal insufficiency.
Surprisingly, the reduced renal pathology of the Ccr2–/–MRL/lpr mice at 20 wk of age was not consistently associatedwith a reduction in BUN levels or proteinuria. In contrast,the mild proteinuria at 14 wk of age was significantly reducedin Ccr2-deficient mice, but this did not persist as proteinuriaprogressed. The reasons for this discrepancy between the improvedhistopathology and the unchanged proteinuria remain unclear.
In conclusion, our results in Ccr2-deficient MRL/lpr mice suggesta role for this receptor in the development of lupus-like diseasemanifestations consisting of lymphoproliferation, activationof T cells, generation of anti-dsDNA antibodies, renal diseasewith chemokine and cytokine production, and mononuclear cellinfiltration, resulting in proteinuria and progressive renalinsufficiency. The reduction in the development of the lupus-likedisease in the Ccr2–/– MRL/lpr mice leads to prolongationof life, pointing toward a role for Ccr2 in the developmentand progression in immune complex–mediated renal disease.
Acknowledgments
We thank Dan Draganovic, Ulrike Brandt, Anita Jozak, and SabineSauer for technical assistance and Peter J. Nelson and AntalRot for helpful discussions and comments on the manuscript.
This work was supported by grants from the Deutsche Forschungsgemeinschaftto B.L. (LU612/4-1) and to B.B. and D.S. (BA2137/1-1); fromthe Nationales Genomforschungsnetz to D.H.B. (NGFN PMM-S31T11),and from the Spanish Ministerio de Ciencia y Tecnología(SAF2001-1048-C03-02), FIS-Instituto Salud Carlos III (PI02-0346),and Comunidad de Madrid (08.3/0011.1/2001) to F.M.; and by abilateral exchange program between Spain (HA00-003) and Germany(DAAD 314/AI-e-dr/ia). S.S. was supported by a grant from theElse Kröner-Fresenius-Stiftung. G.P.d.L. was supportedby a postdoctoral fellowship from the Spanish Ministerio deEducación y Cultura (grant EX 97 7230290).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
G.P.d.L. and H.M. share first authorship. D.S. and B.L. sharesenior authorship.
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Top
Abstract
Introduction
secretion, i.e., a Th1 defect (11), whereas Ccl2-deficient mice show predominantly a defect of Th2 cytokine secretion (12). Possibly because of these differences, conflicting results have been obtained with Ccl2- and Ccr2-deficient mice in several disease models (13). These may relate in part to the fact that in addition to Ccl2, other Ccr2 ligands exist (Ccl7/MCP-3, Ccl8/MCP-2, and Ccl12/MCP-5) (13) and that other, yet unidentified, receptor(s) for Ccl2 may exist (14). 
(clone H35–17.2), CD19 (clone 1D3), CD49b (clone DX5), B220 (clone RA3–6B2), and Gr-1 (clone RB6–8C5; all PharMingen, Heidelberg, Germany). Samples were acquired (25,000 leukocytes) using a high-throughput sampler on a FACS Calibur (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). 
and IFN-
, 5' and 3' untranslated regions; 
, coding region of the Ccr2 gene. The homology arms are depicted as thick lines, the floxed neomycin resistance gene (neo) for positive selection as a gray box with black triangles at both ends, the Herpes simplex virus thymidine kinase gene (HSV tk) for negative selection as a hatched box, the enhanced green fluorescence reporter gene (egfp) also as a hatched box, and the SV40 polyadenylation signal (pA) as a cross-hatched box. The wavy line represents prokaryotic sequences in the targeting vector. The sizes of relevant restriction fragments and PCR fragments before and after homologous recombination are depicted. Arrowheads indicate the position of primers used for long-range PCR analysis. (B) Southern blot analysis of wt embryonic stem cells (lane 1) and a successfully targeted ES cell clone (lane 2) using the 5' external probe depicted in A. (C) Long-range PCR to prove homologous recombination within the 3' arm of homology. An 8.3-kb PCR fragment indicates homologous recombination. Lane 1, 1-kb ladder; lane 2, PCR product; lane 3, PCR product digested with HindIII; lane 4, PCR product digested with BamHI. (D) Analysis of peripheral blood leukocytes (PBL) from wt and Ccr2-deficient mice by flow cytometry. PBL were stained with antibodies directed against CD11b and Ccr2 and gated for monocytes.
