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Glomerulonephritis in MiceCCR5 Deficiency Aggravates Crescentic
Stahl and Ulf PanzerWenzel, Christian Kurts, Hans-Willi Mittrücker, Rolf A. K.Udo Helmchen, Susanne Fehr, Richard Horuk, Ulrich
Heymann,Anett Peters, Catherine Meyer-Schwesinger, Felix Jan-Eric Turner, Hans-Joachim Paust, Oliver M. Steinmetz,
http://www.jimmunol.org/content/181/9/65462008; 181:6546-6556; ;J Immunol
Referenceshttp://www.jimmunol.org/content/181/9/6546.full#ref-list-1
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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,
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CCR5 Deficiency Aggravates Crescentic Glomerulonephritisin Mice1
Jan-Eric Turner,* Hans-Joachim Paust,* Oliver M. Steinmetz,* Anett Peters,*Catherine Meyer-Schwesinger,* Felix Heymann,† Udo Helmchen,‡ Susanne Fehr,§
Richard Horuk,¶ Ulrich Wenzel,* Christian Kurts,‡ Hans-Willi Mittrucker,�
Rolf A. K. Stahl,* and Ulf Panzer2*
The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important rolein T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation,we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5�/� mice. Induction of ne-phritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5(15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokineexpression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renalfunction. Nephritic CCR5�/� mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3(14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renalT cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5�/� mice showed an increased renalTh1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade inCCR5�/� mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicatingthat increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5�/� mice depended on functionalCCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment. The Journal of Immunology, 2008, 181: 6546–6556.
T he infiltration of leukocytes into the kidney is a hallmarkof human and experimental crescentic glomerulonephri-tis. In particular, monocytes and effector T cells of the
Th1 type are thought to play a central role in immune-mediatedtissue damage, which ultimately leads to progressive loss of kidneyfunction (1).
The molecular family of chemokines and their receptors areamong the main regulators of directional leukocyte trafficking un-der homeostatic and inflammatory conditions. According to theposition of two cysteine residues near the N terminus, chemokinesare classified into four subfamilies named C, CC, CXC, and CX3Cchemokines (2). By detecting chemokine concentration gradientsvia corresponding chemokine receptors, inflammatory cells areguided toward the focus of inflammation, where, in the case of
autoimmune disease, they exert their fatal effector functions (3).The specific composition of the inflammatory infiltrate is achievedby differential expression patterns of chemokine receptors on leu-kocyte subsets (4, 5).
The chemokine receptor CCR5 is predominantly expressed onTh1-polarized T cells, monocytes, and NK cells (6, 7). Recruitment ofCCR5-positive T cells and monocytes is regarded as a typical featurein a variety of human autoimmune diseases, including rheumatoidarthritis, multiple sclerosis, and glomerulonephritis (5, 8–10). In hu-man crescentic anti-neutrophil cytoplasmatic Ab-associated glomer-ulonephritis, anti-glomerular basement membrane glomerulonephri-tis, and lupus nephritis, up-regulated renal expression of the CCR5ligands CCL3, CCL4, and CCL5 has been demonstrated (11), andrenal CCR5-positive infiltrates are a common feature of lupus nephri-tis and IgA nephropathy (12). Furthermore, application of the modi-fied CCL5 analogues Met-RANTES and aminooxypentane-RANTESwith mainly antagonistic effects on CCR5, CCR1, and CCR3 led to areduction in leukocyte infiltration and improvement of the diseaseoutcome in various experimental models of glomerulonephritis (13,14). In contrast, aggravation of glomerular damage was recently re-ported in a model of immune complex-mediated glomerulonephritisin Met-RANTES- and aminooxypentane-RANTES-treated animals(15). No experimental data are yet available on selective CCR5 block-ade in experimental models of glomerulonephritis. Likewise, it re-mains unclear how CCR5 signaling contributes to cell-mediated renaltissue damage. The expression of CCR5 on monocytes and effectorTh1 cells is presumed to play a central role in the recruitment of thesecells into inflamed renal tissue, and thus in cell-mediated kidney in-jury. Therefore, blockade of CCR5 might represent a new therapeuticapproach for the treatment of renal autoimmune disease. This is of
*III. Medizinische Klinik, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Ger-many; †Institute for Molecular Medicine and Experimental Immunology, Universita-tsklinikum Bonn, Bonn, Germany; ‡Institut fur Pathologie, UniversitatsklinikumHamburg-Eppendorf, Hamburg, Germany; §Servicegruppe Morphologie, Zentrum furMolekulare Neurobiologie, Hamburg, Germany; ¶Department of Immunology, Ber-lex Biosciences, Richmond, CA 94804; and �Institut fur Immunologie, Universita-tsklinikum Hamburg-Eppendorf, Hamburg, Germany
Received for publication January 30, 2008. Accepted for publication August 21, 2008.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (PA754/6-3) and by a fellowship of the German National Academic Foundation to F.H.2 Address correspondence and reprint requests to Dr. Ulf Panzer, Universitatsklini-kum Hamburg-Eppendorf III. Medizinische Klinik, Martinistr. 52, 20246 HamburgGermany. E-mail address: [email protected]
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
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particular interest because a pharmacologic antagonist for humanCCR5 has recently become available and has been tested in the clin-ical setting of HIV therapy (16).
Nephrotoxic serum nephritis (NTN)3 in mice is a well-charac-terized experimental model of human crescentic glomerulonephri-tis (17). Induction of NTN by injection of a polyclonal nephrotoxicsheep Ab initially results in glomerular complement activation andrenal infiltration of neutrophils (18). This heterologous phase(�day 4) is followed by an autologous phase of adaptive immuneresponse directed against the planted glomerular sheep Abs, lead-ing to Th1 cell- and monocyte-mediated kidney injury (19–21).
The aim of the present study was to determine the contributionof CCR5 signaling to the formation of inflammatory infiltrates dur-ing the course of crescentic glomerulonephritis. We hypothesizedthat selective blockade of CCR5 might be sufficient to reduce renalT cell and monocyte recruitment. To address this question, weinduced NTN in wild-type (WT) and CCR5�/� mice.
Materials and MethodsAnimals
CCR5�/� mice (C57BL/6 background; 129P2-Ccr5tm1Kuz/J) were pur-chased from The Jackson Laboratory, and CCR5�/� genotype was con-firmed by PCR analysis in each animal. Knockout mice underwent embryotransfer to meet the general standards of our institution. Age-matchedC57BL/6 WT controls (10–12 wk old) also derived from the strain bred inour animal facility. All animals were raised in specific pathogen-free con-ditions. Animal experiments were performed according to national andinstitutional animal care and ethical guidelines, and were approved by localcommittees.
Animal experiments
NTN was induced in 10- to 12-wk-old male C57BL/6 CCR5�/� andC57BL/6 WT mice by i.p. injection of 2.5 mg of nephrotoxic sheep serumper gram of mouse body weight, as described (22). Controls were injectedi.p. with an equal amount of nonspecific sheep IgG. Altogether, nephritiswas induced in 33 WT and 17 CCR5�/� animals. In experiments withCCR1 blockade, mice received s.c. injections of the CCR1-specific antag-onist BX471 from day 3–10 after induction of NTN. Mice were dosed with50 mg/kg BX471 in 50–60 �l vehicle at 8-h intervals. Control mice re-ceived an equal amount of the vehicle 2-hydroxypropyl-�-cyclodextrin(cyclodextrin; Sigma-Aldrich) (23). Solution of BX471 in 40% cyclodex-trin was prepared, as previously described (24).
Functional studies
For urine sample collection, mice were housed in metabolic cages for 6 h.Albuminuria was determined by standard ELISA analysis (mice-albuminkit; Bethyl Laboratories). Blood samples for blood urea nitrogen (BUN)measurement and assessment of systemic Ab response were obtained at thetime of sacrifice. Urinary creatinine and BUN were measured by standardlaboratory methods.
Real-time RT-PCR analysis
Total RNA of renal cortex was prepared according to standard laboratorymethods. Real-time PCR was performed for 40 cycles (initial denaturation:95°C, 10 min; denaturation: 95°C, 15 s; primer annealing and elongation:60°C, 1 min) with 1.5 �l of cDNA samples in the presence of 2.5 �l (0.9�M) of specific murine primers (primer sequences are available upon re-quest) and 12.5 �l of 2� Platinum SYBR Green qPCR Supermix (Invitro-gen) in an AbiPrism Sequence Detection System 7000 (Applied Biosys-tems). All samples were run in duplicate and normalized to 18S rRNA toaccount for small RNA and cDNA variability (25).
In situ hybridization
In situ hybridization procedures were performed, as described previously(26). In brief, CCL5 and CCR5 cRNA probes were prepared by in vitrotranscription of subcloned cDNA. The CCL5 probe corresponds to nt 117–386 of cDNA sequence NM_013653. The CCR5 probe corresponds to nt
736–975 of cDNA sequence NM_009917. Antisense hybridization probeand sense control were labeled with [35S]UTP (20 �Ci/ml; Amersham),and free nucleotides were separated with a Sephadex G-50 column (quick-spin columns; Roche). In situ hybridization was performed on 12-�m cryo-sections of renal tissue using 5 ng of the 35S-labeled antisense and senseRNA probes, respectively. After exposing sections overnight to KodakBiomax MR x-ray films, they were treated with Kodak NTB-3 nucleartrack emulsion and exposed for 3 wk, followed by development in KodakD19 and fixation with Kodak Unifix. Finally, sections were stained withMayer�s Hemalaun.
Morphological examinations
Light microscopy and immunohistochemistry were performed by routineprocedures. Crescent formation and glomerular sclerosis (deposition of Pe-riodic acid Schiff (PAS)-positive material) were assessed in 50 glomeruliper mouse in a blinded fashion in PAS-stained paraffin sections. As a mea-sure for tubulointersitial injury, interstitial area was estimated by pointcounting three independent areas of renal cortex per mouse in low mag-nification fields (�200), as previously described (26). Paraffin-embeddedsections (2 �m) were either stained with an Ab directed against the pan-Tcell marker CD3 (A0452; DakoCytomation), the monocyte-specific mark-ers F4/80 (BM8; BMA Biomedicals) and MAC-2 (M3/38; Cedarlane Lab-oratories), C3 (Cappel Laboratories; Organon Teknika), or sheep IgG andmouse IgG (both Jackson ImmunoResearch Laboratories). Tissue sectionswere developed with the Vectastain ABC-AP kit (Vector Laboratories).MAC-2- and CD3-positive cells in 50 glomerular cross-sections (gcs) andF4/80- and CD3-positive cells in 30 tubulointerstitial high power fields(hpf) per kidney were counted by light microscopy in a blinded fashion.For electron microscopy, kidney tissue was fixed in 4% paraformaldehydein 0.1 M cacodylate buffer, embedded in araldite, cut with an Ultracut Eultramicrotome (Reichert Jung), and stained with uranyl acetate and leadcitrate. Specimens were examined with a Zeiss EM 109 electronmicroscope.
Ag-specific humoral immune response
Mouse anti-sheep IgG Ab titers were measured by ELISA using sera col-lected 10 days after induction of the nephritis, as described recently (27).In brief, ELISA microtiter plates were coated with 100 �l of 100 �g/mlsheep IgG (Sigma-Aldrich) in carbonate-bicarbonate buffer overnight at4°C. After blocking with 1% BSA in TBS (Sigma-Aldrich), the plates wereincubated with serial dilutions of mouse serum (1/100 to 1/12,500) for 1 hat room temperature. Bound mouse IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Biozol) at 1/1000, tetramethylbenzidineperoxidase substrate, and absorbance readings (at 450 nm) on a spectro-photometer. Lack of cross-reactivity of the secondary Ab with sheep IgGwas demonstrated by omitting the primary Ab. Ig isotypes (IgG1, IgG2a,and IgG2b) were measured using the ELISA technique already described.The bound mouse Ig isotypes were detected using peroxidase-conjugatedrabbit anti-mouse IgG1, IgG2a, and IgG2b Abs (Zymed Laboratories-In-vitrogen) at a dilution of 1/1000.
Renal leukocyte isolation
Previously described methods for renal cell isolation from murine kidneyswere used (22). In brief, kidneys were finely minced and digested for 45min at 37°C with 0.4 mg/ml collagenase D (Roche) and 0.01 mg/ml DNaseI in DMEM (Roche) supplemented with 10% heat-inactivated FCS (In-vitrogen). Cell suspensions were sequentially filtered through 70- and40-�m nylon meshes and washed with HBSS without Ca2� and Mg2�
(Invitrogen). Single-cell suspensions were separated using Percoll densitygradient (70 and 40%) centrifugation (28). The leukocyte-enriched cellsuspension was aspirated from the Percoll interface. Viability of the cellswas assessed by trypan blue staining before flow cytometry.
Splenocyte isolation
Single-cell suspension of splenocytes was prepared according to standardlaboratory procedures. In brief, spleens were minced and sequentiallypassed through 70- and 40-�m nylon meshes. After lysis of erythrocyteswith ammonium chloride, cells were washed several times with HBSSwithout Ca2� and Mg2� and resuspended in RPMI 1640 with 10% FCS(Invitrogen).
Flow cytometry
For T cell differentiation, isolated cells were stained for 25 min at 4°C withfluorochrome-labeled Abs specific for CD3 (allophycocyanin; 17A2; R&DSystems) and CD4 (PE; GK1.5; Miltenyi Biotec). Before Ab incubation,unspecific staining was blocked with normal mouse serum (Sigma-Aldrich).
3 Abbreviations used in this paper: NTN, nephrotoxic serum nephritis; BUN, bloodurea nitrogen; gcs, glomerular cross-section; hpf, high power field; PAS, Periodic acidSchiff; WT, wild type.
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Staining of intracellular IFN-� was performed, as recently described by Kornet al. (29). In brief, splenocytes or isolated renal leukocytes were activatedby incubation at 37°C, 5% CO2 for 5 h with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (1 �g/ml; Calbiochem-Merck) in RPMI 1640 with10% FCS. After 30 min of incubation, brefeldin A (10 �g/ml; Sigma-Aldrich) was added. After several washing steps and staining of cell sur-face markers, cells were incubated for 20 min at 4°C in Cytofix/Cytoperm(BD Biosciences) to permeabilize cell membranes. Then, intracellularIFN-� was stained using a rat anti-mouse IFN-� Ab (FITC; XMG1.2; BDBiosciences) (28). Experiments were performed on a BD BiosciencesFACSCalibur system using the CellQuest Professional software.
Statistical analysis
Results are expressed as mean � SD. Differences between individual ex-perimental groups were compared by Kruskal-Wallis test with post hocanalysis by Mann-Whitney U test. For survival analysis, Kaplan-Meyerplot with log-rank test was used. Experiments that did not yield enoughindependent data for statistical analysis due to the experimental setup wererepeated at least three times.
ResultsRenal phenotype of CCR5�/� mice
To examine whether CCR5 deficiency leads to structural or func-tional renal defects, we analyzed 40-wk-old C57BL/6 CCR5�/�
and C57BL/6 WT mice with respect to histological and functional
alterations (Fig. 1). Gross morphology in PAS-stained kidney sec-tions (Fig. 1A) and fine structure of the glomerular filtration barrier(Fig. 1B) were well preserved in CCR5�/� mice. Furthermore,functional analysis revealed no differences in BUN and urinaryalbumin excretion between WT and CCR5�/� mice under homeo-static conditions (Fig. 1C).
Induction of experimental glomerulonephritis in WT andCCR5�/� mice
NTN in C57BL/6 WT and C57BL/6 CCR5�/� mice was inducedby i.p. injection of nephrotoxic sheep serum. Specific glomerularbinding and linear deposition patterns of the polyclonal nephro-toxic Ab were demonstrated by immunohistochemical staining ofsheep IgG on renal tissue 48 h after serum injection (Fig. 2). In-tensity and distribution of sheep IgG staining did not differ be-tween WT and CCR5�/� mice. A critical step in the induction ofthe heterologous phase of NTN is the binding and activation ofcomplement (18). To evaluate whether this initial inflammatoryinsult is altered in CCR5�/� mice, we performed immunohisto-chemistry for complement factor C3 in the heterologous phase ofNTN at 48 h (Fig. 2). Nephritic kidneys of both WT and CCR5�/�
mice showed intensive glomerular and tubulointerstitial binding ofC3, demonstrating unchanged and effective induction of NTN inCCR5�/� mice.
Expression of chemokines and chemokine receptors duringnephrotoxic nephritis
To gain further insight into the role of CCR5 and its ligandsCCL3, CCL4, and CCL5 in the initiation and maintenance of
FIGURE 1. Renal phenotype of CCR5�/� mice. A, Representative pho-tographs of PAS-stained kidney sections of 40-wk-old WT and CCR5�/�
mice (magnification �400). B, Representative electron microscopy of kid-ney tissue from 40-wk-old WT and CCR5�/� mice shows no structuraldifference of the glomerular filtration barrier (original magnification�7000; BM, basal membrane). C, BUN levels (left) and albumin to cre-atinine ratio (right) of 40-wk-old WT (n � 4) and CCR5�/� mice (n �5–6). Symbols represent individual data points, and the horizontal linesindicate mean values.
FIGURE 2. Induction of experimental glomerulonephritis. Representa-tive photographs of kidney sections of WT and CCR5�/� mice 48 h afterinduction of NTN (n � 5 per group) stained immunohistochemically forsheep IgG (magnification �200) and complement factor C3 (magnification�400). Higher magnification reveals linear glomerular binding pattern ofsheep-IgG (inset, oil immersion �1000).
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cell-mediated tissue damage, we performed real-time RT-PCRanalysis on renal cortex of healthy and nephritic WT andCCR5�/� mice (Fig. 3).
Basal renal chemokine expression levels were low and did notdiffer significantly between healthy WT and CCR5�/� mice. Like-wise, no early up-regulation of CCR5 ligands could be detected inthe heterologous phase of nephritis at 48 h (data not shown).
However, mRNA expression of all CCR5 ligands showed sig-nificant up-regulation in the autologous phase at day 10 when com-pared with nonnephritic controls (nephritic WT and CCR5�/� vsnonnephritic controls, p � 0.01; Fig. 3A). Interestingly, at this timepoint, renal CCL3 (WT, 4.9-fold; CCR5�/�, 14.1-fold; p � 0.01)and CCL5 expression (WT, 15-fold; CCR5�/�, 61.6-fold; p �0.01) was significantly higher in nephritic CCR5�/� mice whencompared with nephritic WT mice. CCL4 expression was not dif-ferentially regulated in nephritic WT or CCR5�/� mice (WT, 3.4-fold; CCR5�/�, 5.5-fold; Fig. 3A).
To address the question as to whether increased induction ofchemokines in nephritic CCR5�/� mice is specific to CCR5 li-gands, we analyzed mRNA levels of the CCR5-independent che-mokine CCL2. The monocyte-attracting chemokine CCL2 was al-ready slightly up-regulated in the heterologous phase at 48 h (WT,5.1-fold; CCR5�/�, 3.9-fold; p � 0.01 vs nonnephritic controls)
and showed strong induction in the autologous phase at day 10.However, in contrast to CCL3 and CCL5, no differential expres-sion of CCL2 could be demonstrated between nephritic WT andCCR5�/� mice (WT, 54.2-fold; CCR5�/�, 59.7-fold; Fig. 3A).
Induction of nephritis also resulted in significant up-regulationof CCR5 mRNA during the autologous phase in WT mice com-pared with nonnephritic controls (6.7-fold; p � 0.01 vs nonne-phritic controls; Fig. 3A). No CCR5 mRNA was detectable inCCR5�/� mice.
Analysis of spleen chemokine expression revealed that in-creased production of CCL3 and CCL5 in nephritic CCR5�/�
mice was specific to the kidney. Total spleen mRNA levels ofCCL2, CCL3, CCL4, and CCL5 were not differentially regulatedbetween nephritic WT and CCR5�/� mice. A comparison showedthat they were similar to mRNA levels of nonnephritic controlsimmunized with nonspecific sheep IgG (data not shown).
Localization of CCR5 and its ligand CCL5 in nephrotoxicnephritis
To identify the renal compartments expressing the most abun-dantly induced CCR5 ligand CCL5 and the CCR5 receptor, weperformed in situ hybridization experiments (Fig. 3B). mRNAexpression of CCL5 in both nephritic WT and CCR5�/� mice
FIGURE 3. Chemokine and chemokinereceptor expression. A, Real-time RT-PCRanalysis of renal chemokine and chemokinereceptor mRNA expression at day 10 ofNTN. mRNA levels of nephritic WT mice(n � 11) and nephritic CCR5�/� mice (n �7) are expressed as x-fold of nonnephriticWT controls (n � 13). Symbols represent in-dividual data points, and the horizontal linesindicate mean values (��, p � 0.01). B, Rep-resentative photographs of in situ hybridiza-tions with specific cRNA probes for CCL5and CCR5 at day 10 of NTN (magnification�400). Nephritic mice show CCL5 andCCR5 signals in the periglomerular infil-trates (arrows), and single CCL5-positivecells in the glomerular tuft (arrowheads) andin dilated tubules (inset, �200). SpecificCCR5 signal is absent in CCR5�/� mice.
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at day 10 was predominantly detected in periglomerular andtubulointerstitial infiltrates. Sporadically, strong expression wasalso detectable in epithelial cells of markedly dilated tubules,whereas intraglomerular CCL5 signals were apparently re-stricted to cellular crescents and single infiltrating cells in theglomerular tuft. Arterioles and peritubular capillaries werelargely negative for CCL5 mRNA expression. No differentialCCL5 expression pattern was found in nephritic WT andCCR5�/� mice. Similar to CCL5, CCR5 mRNA was mainlyexpressed in infiltrating cells in periglomerular and tubuloin-terstitial compartments and in glomerular crescents. No CCR5mRNA expression could be found in resident renal cells. CCL5and CCR5 mRNAs were only detected in low levels in nonne-phritic control mice. Nephritic CCR5�/� mice showed no spe-cific expression of CCR5 mRNA above background level.
Increased renal recruitment of monocytes and T cells inCCR5�/� mice
To investigate the effects of CCR5 deficiency on T cell and mono-cyte recruitment, kidney sections were stained immunohistochemi-cally for tubulointerstitial and glomerular T cells (CD3) and mac-rophages (F4/80 and MAC-2, respectively) (Fig. 4A).
No relevant tubulointerstitial infiltration of monocytes and onlya slight increase in T cell numbers were found at 48 h, which is inline with the lack of monocyte- and T cell-attracting chemokineexpression during the heterologous phase of nephritis (data notshown). Up-regulated chemokine expression in the autologousphase at day 10, mainly in the tubulointerstitium and in the peri-glomerular region (as shown by in situ hybridization), was paral-leled by infiltration of T cells (nonnephritic controls, 2.8 � 1.6/hpf; nephritic WT, 12.8 � 10.8/hpf) and macrophages(nonnephritic controls, 1.4 � 1.1/hpf; nephritic WT, 10.8 � 6.8/hpf; Fig. 4B) mainly into these intrarenal compartments.
Quantification of CD3- and F4/80-positive tubulointerstitialcells in nephritic CCR5�/� mice at day 10 revealed a significantincrease in renal infiltration of T cells (WT, 12.8 � 10.8/hpf;CCR5�/�, 26.4 � 7.2/hpf; p � 0.05) and macrophages (WT,10.8 � 6.8/hpf; CCR5�/�, 41.4 � 28.6/hpf; p � 0.01) com-pared with nephritic WT mice (Fig. 4B). Glomerular infiltrationof T cells (nonnephritic controls, 0.16 � 0.23/gcs; nephriticWT, 0.34 � 0.4/gcs; nephritic CCR5�/�, 0.77 � 0.63/gcs) andof MAC-2-positive (nonnephritic controls, 0.62 � 0.2/gcs; ne-phritic WT, 1.79 � 0.84/gcs; nephritic CCR5�/�, 2.24 � 0.41/gcs) monocytes in diseased animals was less pronounced anddid not differ between nephritic CCR5�/� and nephritic WTmice (Fig. 4B).
Aggravated renal tissue damage in CCR5�/� mice
Examination of PAS-stained kidney sections of nephritic mice inthe autologous phase at day 10 showed severe focal glomerularand tubular alterations with complete destruction of regular tissuestructures (Fig. 5A). Glomerular changes included hypercellularityand formation of cellular crescents, capillary aneurysms, and in-traglomerular deposition of PAS-positive material. In addition tomassive leukocyte infiltrates, the tubulointerstitial compartmentshowed tubular dilatation, necrosis and atrophy, and protein castsand tubular protein reuptake due to proteinuria. Glomerular andtubulointerstitial damage was particularly pronounced inCCR5�/� mice. To quantify renal tissue damage, PAS-stained kid-ney sections were evaluated for the presence of crescents, glomer-ular sclerosis (deposition of PAS-positive material), and tubuloin-terstitial injury (Fig. 5B), as described previously (22, 26). Thefrequency of glomerular crescents at day 10 was significantly in-creased in CCR5�/� mice compared with WT controls (nonne-
phritic controls, 0.5 � 1.3%; WT, 13.6 � 8.9%; CCR5�/�, 28.9 �7.8%; p � 0.01). Nephritic kidneys showed a high percentage ofglomerulosclerosis (nonnephritic controls, 0%; nephritic WT,
FIGURE 4. Renal T cell and monocyte recruitment. A, Representativephotographs of kidney sections immunohistochemically stained for CD3 andF4/80 at day 10 of NTN (magnification �200; inset, oil immersion �1000).B, Quantification of tubulointerstitial (left) and glomerular (right) CD3-posi-tive T cells and F4/80- or MAC-2-positive monocytes in nephritic WT mice(n � 11), nephritic CCR5�/� mice (n � 6) at day 10 of NTN, and nonnephriticWT controls (n � 13). Symbols represent individual data points, and the hor-izontal lines indicate mean values (�, p � 0.05; ��, p � 0.01).
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27.3 � 16.6%; p � 0.01; Fig. 5B) and considerable tubulointer-stitial injury, as indicated by a significant increase in interstitialarea (nonnephritic controls, 7.7 � 2.3%; nephritic WT, 16.6 �5.9%; p � 0.01). Interstitial injury appeared pronounced in ne-phritic CCR5�/� mice (CCR5�/�, 21.1 � 8.1%), but was notstatistically significant, whereas glomerulosclerosis was similar inboth groups (CCR5�/�, 31.3 � 9.9%).
CCR5 deficiency deteriorates renal function in nephrotoxicnephritis
After 10 days, mice were sacrificed for assessment of renal func-tion (Fig. 5C). BUN of nephritic mice were significantly elevatedat day 10 compared with nonnephritic controls ( p � 0.01). In theheterologous phase at 48 h, there was no difference in impairmentof renal function between WT and CCR5�/� mice (data notshown). During the T cell-mediated autologous phase at day 10,however, we found a significant increase in BUN in nephriticCCR5�/� mice (50 � 8 mg/dl) compared with nephritic WT mice(42 � 10 mg/dl; p � 0.05; Fig. 5C).
As an indicator of renal tissue damage, urinary albumin excre-tion was monitored (Fig. 5C). WT and CCR5�/� mice showed a
markedly increased albumin to creatinine ratio at day 10 after induc-tion of nephritis (nonnephritic controls, 0.04 � 0.03; WT, 47.9 � 36;CCR5�/�, 28.4 � 16.5; p � 0.01 vs nonnephritic controls). Therewas no statistically significant difference in albumin excretion be-tween nephritic CCR5�/� and nephritic WT mice.
Increased lethality of nephrotoxic nephritis in CCR5�/� mice
Induction of NTN is usually well tolerated in WT mice. They showonly minor behavioral changes, and only a few animals succumbto the disease. In the setting of CCR5 deficiency, however, ne-phritic mice showed more severe clinical impairment. In addition,the lethality rate was significantly increased from day 4 after seruminjection (Fig. 5D). Induction of NTN in CCR5�/� mice was lethalin 30% compared with 3% in WT mice within the first 10 days( p � 0.01; log-rank test).
Two CCR5�/� mice suffering from terminal disease were sac-rificed at days 6 and 7, respectively. Their BUN levels were 381and 219 mg/dl, respectively, clearly demonstrating that loss of re-nal function was the cause of increased lethality in CCR5-deficientmice. Nephritic CCR5�/� mice sacrificed at day 10 thereforerepresented a select population of mice that survived the early
FIGURE 5. Renal tissue damage and impair-ment of renal function. A, Representative photo-graphs of PAS-stained kidney sections of WTand CCR5�/� mice at day 10 of NTN. Nephriticmice show glomerular crescents (arrows) andglomerular sclerosis (arrowheads) (magnifica-tion: upper panel, �200; lower panel, �400). B,Quantification of glomerular (left) and tubuloin-terstitial (right) tissue damage in nephritic WTmice (n � 11), nephritic CCR5�/� mice (n � 6)at day 10 of NTN, and nonnephritic WT controls(n � 13). C, Left, BUN levels of nephritic WTmice (n � 16), nephritic CCR5�/� mice (n � 7)at day 10 of NTN, and nonnephritic WT controls(n � 12). Right, Albuminuria of nephritic WTmice (n � 9), nephritic CCR5�/� mice (n � 7)at day 10 of NTN, and nonnephritic WT controls(n � 13) expressed as urinary albumin to creat-inine ratio. Symbols represent individual datapoints, and the horizontal lines indicate meanvalues (�, p � 0.05). D, Kaplan-Meyer survivalanalysis of WT (n � 33) and CCR5�/� mice(n � 17) with NTN (��, p � 0.01).
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autologous phase of the disease. Compared with nephritic WTmice, the differences observed in renal function are thus probablyeven more pronounced than documented.
Unaltered humoral and cellular systemic immune responses inCCR5�/� mice
To address the question as to whether CCR5 deficiency inducesalterations in Ab production directed against the nephritogenic Ag,we performed immunohistochemistry for mouse IgG on kidneysections 10 days after induction of nephritis (Fig. 6A). The amountof deposition of glomerular mouse IgG and the distribution pat-terns were similar in WT and CCR5�/� kidneys.
For a more precise description of Ag-specific humoral immuneresponses, we analyzed by ELISA the isotype pattern of Ab re-sponse directed against sheep IgG in the serum of nephritic mice(Fig. 6B). There was no significant difference in sheep IgG-specificAb titers of total mouse IgG at day 10 of NTN. Furthermore, theanalysis of IgG isotypes revealed no bias for either Th1-type(IgG2a) or Th2-type (IgG1 and IgG2b) Ab production inCCR5�/� compared with WT mice.
To analyze the systemic cellular Th1 response, we isolated spleencells from CCR5�/� and WT mice at day 10 of NTN (Fig. 6C).Spleen cells were restimulated by incubation with PMA and ionomy-cin. IFN-� production by CD4-positive T cells was assessed by in-tracellular cytokine staining and subsequent FACS analysis. The per-centages of IFN-�-positive T cells in spleens of nephritic CCR5�/�
mice were not different from those of nephritic WT mice. Consistentwith this finding, real-time RT-PCR analysis of spleen mRNA fromnephritic CCR5�/� and WT mice revealed no difference in the ex-pression of the Th1 cytokines IFN-� and TNF-� (data not shown).
Increased renal Th1 response in CCR5�/� mice
Because the local Th1 response is assumed to play a central role inimmune-mediated tissue damage during NTN, we quantified renal
infiltration of IFN-�-producing CD4-positive T cells. Renal leu-kocytes were isolated from CCR5�/� and WT kidneys at day 10 ofNTN (Fig. 7A). Both immunohistochemical quantification andFACS analysis showed an approximate 5-fold increase in T cellrecruitment into nephritic WT kidneys compared with nonne-phritic controls. The numbers of infiltrating CD4-positive T cellsin nephritic CCR5�/� kidneys were even higher. Most impor-tantly, however, infiltration of IFN-�-producing CD4-positive Tcells, as determined by intracellular cytokine staining after re-stimulation with PMA/ionomycin, was increased by �40% in ne-phritic CCR5�/� compared with nephritic WT kidneys. Further-more, renal mRNA expression of the Th1 cytokine TNF-� wasincreased in nephritic CCR5�/� mice compared with WT controls(WT, 6.2-fold; CCR5�/�, 19.2-fold; p � 0.05; Fig. 7B). The ex-pression levels of IFN-� tended to be higher in CCR5�/� kidneys(WT, 2.6-fold; CCR5�/�, 14.1-fold), but failed to reach statisticalsignificance because of considerable variability among the ani-mals. Induction of the anti-inflammatory cytokine IL-10 was notregulated differently in nephritic CCR5�/� or WT mice (WT,30.8-fold; CCR5�/�, 56-fold). Renal mRNA expression of thetranscription factor FoxP3, which is a marker for regulatory Tcells, was also similar in nephritic CCR5�/� and WT mice. Levelsof mRNA for the Th2 cytokine IL-4 were below detection limits innephritic and control animals (data not shown).
CCR1 blockade improves the outcome in nephritic CCR5�/�
mice
Because CCL3 and CCL5 may attract leukocytes by interactionwith their alternate receptor CCR1, increased renal CCL3 andCCL5 levels in nephritic CCR5�/� mice might lead to in-creased T cell and monocyte recruitment via a CCR1-dependentmechanism. To test this hypothesis, we treated nephritic WTand CCR5�/� mice with the CCR1-specific antagonist BX471during the autologous phase of NTN (days 3–10). The elevated
FIGURE 6. Humoral and cellularsystemic immune response. A, Repre-sentative photographs of glomerulifrom kidney sections immunohisto-chemically stained for mouse IgG atday 10 of NTN (magnification �400).B, ELISA for sheep IgG-specific totalmouse IgG (dilution 1/500) and IgGsubtypes (dilution 1/100) at day 10 ofNTN (n � 4 per group) (OD (450):OD at 450 nm). C, RepresentativeFACS analysis of splenocytes for pro-duction of IFN-� at day 10 of NTN.Plots were generated from 500,000 to-tal events that were gated for CD4-positive T cells. Numbers refer to pos-itive events in percentage of gatedevents. All experiments were repeatedfour times.
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renal production of CCL3 and CCL5 in nephritic CCR5�/�
mice was abolished by CCR1 blockade (Fig. 8A). At day 10 ofNTN, renal mRNA expression of CCL5 in BX471-treatedCCR5�/� mice was significantly reduced compared with vehi-cle-treated CCR5�/� mice (nephritic CCR5�/� � Vh, 19-fold;nephritic CCR5�/� � BX471, 7.9-fold; p � 0.01). Expressionof CCL3 was also reduced, though not reaching a statisticallysignificant level (nephritic CCR5�/� � Vh, 37.8-fold; nephriticCCR5�/� � BX471, 10.3-fold). Importantly, expression levelsof CCL3 and CCL5 in BX471-treated CCR5�/� mice were notdifferent from that seen in vehicle- or BX471-treated nephriticWT mice. Compared with previous experiments, up-regulationof CCL5 mRNA expression in nephritic WT mice was slightlyreduced. This difference was caused by a �2-fold increase inbasal CCL5 levels of nonnephritic WT mice treated with thevehicle 2-hydroxypropyl-�-cyclodextrin (cyclodextrin), whichserve as a reference value for calculation of the relative in-crease. Cyclodextrin is widely used to improve water solubilityof lipophilic drugs for pharmacological application in animalsand humans. As it has been described (23) that cyclodextrintreatment can lead to minor histopathological changes in thekidneys, which are reversible and do not impair renal function,this slight up-regulation of CCL5 expression in controls mightbe a side effect of the vehicle treatment.
Double blockade of CCR5 and CCR1 by application ofBX471 to nephritic CCR5�/� mice significantly reduced renalT cell recruitment in comparison with nephritic vehicle-treatedCCR5�/� and WT mice (CD3-positive T cells: nonnephriticcontrols, 4.9 � 3.2/hpf; nephritic WT � Vh, 23.6 � 5.8/hpf; ne-phritic CCR5�/� � Vh, 51.3 � 5.1/hpf; nephritic CCR5�/� �BX471, 11.3 � 4.7/hpf; p � 0.01; Fig. 8B). Furthermore, exces-sive renal monocyte infiltration in nephritic CCR5�/� mice was
inhibited by CCR1 blockade (F4/80-positive monocytes: nonne-phritic controls, 1.2 � 0.4/hpf; nephritic WT � Vh, 20.2 � 12.2/hpf; nephritic CCR5�/� � Vh, 48.9 � 3.4/hpf; nephriticCCR5�/� � BX471, 23.0 � 9.4/hpf; p � 0.01; Fig. 8B). Mostimportantly, double blockade of the two chemokine receptorseffectively protected mice from immune-mediated kidney in-jury, as demonstrated by a reduced number of crescentic glo-meruli in CCR5�/� mice receiving BX471 injections comparedwith nephritic vehicle-treated CCR5�/� and WT mice (nonne-phritic controls, 0.9 � 1.6; nephritic WT � Vh, 20.0 � 3.9/hpf;nephritic CCR5�/� � Vh, 44.4 � 8.1/hpf; nephritic CCR5�/�
� BX471, 13.3 � 6.2/hpf; p � 0.05; Fig. 8C).In line with the previous experiments, the lethality rate was
increased in nephritic vehicle-treated CCR5�/� mice (25%),whereas no mouse deceased in the group of BX471-treatedCCR5�/� animals. This positive selection of surviving vehicle-treated CCR5�/� mice showed an insignificant tendency to in-creased BUN levels (nonnephritic controls, 40.9 � 6.3 mg/dl;nephritic WT � Vh, 60.4 � 18.4 mg/dl; nephritic CCR5�/� �Vh, 67.6 � 1.3 mg/dl; p � 0.073, NS). BUN levels in nephriticBX471-treated CCR5�/�, however, were similar to nephriticvehicle-treated WT mice (nephritic CCR5�/� � BX471,57.1 � 15.3 mg/dl; Fig. 8D). The albumin to creatinine ratiowas also significantly decreased in nephritic BX471-treatedCCR5�/� animals compared with nephritic vehicle-treated WTmice (Fig. 8D).
In nephritic WT mice, CCR1 blockade significantly reduced Tcell infiltration (WT � BX471, 14.3 � 7.3/hpf; p � 0.05 vs ne-phritic WT � Vh), but it did not prevent monocyte infiltration andfailed to improve markers of renal tissue damage and parametersof renal function (Fig. 8, B–D).
FIGURE 7. Renal Th1 immune response. A, Representative FACS analysis of renal leukocytes for production of IFN-� at day 10 of NTN. Plots weregenerated from 200,000 total events, which were gated for CD4-positive T cells. Numbers refer to positive events in percentage of gated events. Allexperiments were repeated four times. B, Real-time RT-PCR analysis of renal cytokine mRNA expression in nephritic WT mice (n � 11), nephriticCCR5�/� mice (n � 7) at day 10 of NTN, and nonnephritic WT controls (n � 13). mRNA levels are expressed as x-fold of nonnephritic WT controls.Symbols represent individual data points, and the horizontal lines indicate mean values (��, p � 0.01).
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DiscussionStudies of the past few years have highlighted the role of chemo-kines and their receptors as main regulators of leukocyte recruit-ment in renal inflammation (30, 31). The chemokine receptorCCR5 is expressed on monocytes and activated Th1 cells, andmight therefore play an important role in directional trafficking ofthese leukocyte subsets (4–7).
Because of the lack of data on the functional role of CCR5 inrenal autoimmune inflammation, we induced a T cell-mediatedmodel of crescentic glomerulonephritis in CCR5-deficient and WTmice. The main finding of our study is that CCR5�/� mice devel-oped an exacerbated form of crescentic glomerulonephritis asso-ciated with significantly increased lethality due to uremia (30 vs3% in WT mice). The high susceptibility of CCR5�/� mice tosevere kidney injury was caused by excessive renal up-regulationof the CCR5 ligands CCL3 and CCL5, resulting in enhancedCCR1-driven monocyte and T cell infiltration with aggravated tis-sue damage and deterioration of renal function.
The excessive up-regulation of CCR5 ligands in CCR5�/� micealong with augmented cellular infiltration and a worse disease out-come has been described in models of cutaneous Arthus reaction(32) and T cell-mediated liver injury (33). In Con A-induced hep-atitis, application of the mainly antagonistic CCL5 analog Met-RANTES to CCR5�/� mice reduced the recruitment of CCR1-positive NK cells to the liver (34). This observation suggestsfacilitated CCR1-dependent hepatic leukocyte infiltration, which isdriven by increased intrahepatic levels of shared CCR5/CCR1 li-gands in the setting of CCR5 deficiency.
To test this hypothesis, we blocked CCR1 signaling in nephriticmice by application of the CCR1-specific small molecule antago-nist BX471 (35). BX471 has been used for treatment in a varietyof disease models, including murine lupus nephritis (36) and uni-lateral ureter obstruction, a model of renal fibrosis (24, 37). In ourexperiments, double blockade of CCR5 and CCR1 almost abol-ished renal T cell recruitment and consistently protected from au-toimmune tissue damage. Furthermore, the CCR1 antagonist pro-tected CCR5�/� mice against deterioration of renal function andlethality. This indicates a predominant role for these two chemo-kine receptors in renal Th1 cell trafficking.
Monocyte infiltration was normalized to levels of nephritic WTmice by treatment of CCR5�/� mice with the CCR1 blocker.However, double receptor blockade did not lead to further inhibi-tion of monocyte infiltration below the level of nephritic WT an-imals, suggesting that other chemokine receptors (e.g., CCR2)compensate for the lack of functional CCR5 and CCR1 in thissetting.
Beneficial effects of selective CCR1 blockade by BX471 treat-ment or ccr1 gene deletion with reduction in tubulointerstitial leu-kocyte infiltration and organ damage have been shown in murinelupus nephritis (36) and renal fibrosis (24, 37). Although BX471treatment significantly reduced renal T cell infiltration in our ex-periments, it did not protect nephritic WT mice against kidneyinjury. The unfavorable results obtained in CCR1�/� mice withNTN (38) support the theory that single blockade of CCR1, likesingle blockade of CCR5, is not a successful strategy in crescenticglomerulonephritis.
The mechanisms underlying excessive up-regulation of CCR5ligands in nephritic CCR5�/� kidneys remain unclear. Interest-ingly, however, CCR1 blockade led to normalization of CCL3 andCCL5 levels. We showed by in situ hybridization that inflamma-tory infiltrates are the main source of CCL5 in NTN. Increasedrenal CCL5 mRNA expression could therefore be secondary toincreased cellular infiltration and decreased to WT levels by re-
ducing the inflammatory infiltrate, as was the case in our CCR1blockade experiments. However, one could also speculate thatCCR5 signaling in chemokine-producing cells is needed to regu-late the expression of its own ligands in terms of a negative feed-back loop. It has been demonstrated that stimulated T cells and NKcells of CCR5�/� mice show increased IFN-� production (34, 39),but no dysregulation of CCR5 ligand production has been reportedto date (39).
Another reason for a worse outcome in nephritic CCR5�/� micemight be an altered systemic or local immune response. Previousexperiments have shown that CCR5�/� mice produce equalamounts of total IgG in response to challenge with a T cell-de-pendent Ag (39, 40). Zhou et al. (39) reported increased IgG iso-type 1 production in CCR5�/� mice, but these results have notbeen confirmed by others (40). In our experiments, total IgG andIgG isotypes directed against sheep IgG, the nephritogenic Ag,
FIGURE 8. CCR1 blockade in nephritic CCR5�/� mice. A, Real-timeRT-PCR analysis of renal CCL5 mRNA expression at day 10 of NTN.mRNA levels of nephritic WT mice treated with vehicle (Vh; n � 7) orBX471 (n � 7) and nephritic CCR5�/� mice treated with vehicle (n � 6)or BX471 (n � 11) are expressed as x-fold of nonnephritic, vehicle-treatedWT controls (n � 7). B, Quantification of tubulointerstitial T cell andmonocyte infiltration by immunohistochemistry for CD3 and F4/80. C,Percentage of crescentic glomeruli in PAS-stained kidney sections. D,BUN (left) and albuminuria (right) of the nephritic mice at day 10 of NTNand nonnephritic control mice. Symbols represent individual data points,and the horizontal lines indicate mean values (group numbers in B–D as inA; �, p � 0.05; ��, p � 0.01).
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were consistently similar in CCR5�/� and WT mice. Furthermore,number and frequency of IFN-�-producing Th1-polarized cells inthe spleen were constant in CCR5�/� mice, arguing against de-fective T cell priming, which has been demonstrated for alloreac-tive CCR5�/� T cells in a model of heart transplantation (40).Despite an unchanged systemic immune response, we found anincreased local Th1 response in CCR5�/� mice, which was re-flected by an elevated number and a higher frequency of renalIFN-�-producing CD4-positive T cells and by increased renal pro-duction of the Th1 prototype cytokine TNF-�. This is consistentwith the finding of an augmented cutaneous delayed-type hyper-sensitivity reaction in CCR5�/� mice (39). The mechanisms un-derlying these observations are still unclear and require furtherstudy.
The role of a human mutation leading to the loss of functionalCCR5 due to a 32-bp deletion in the encoding gene (CCR532) inglomerulonephritis is under debate. Our results of improved renalsurvival of heterozygous CCR532 carriers in IgA nephropathy(41) could not be reproduced by others (42). Large differences instudy populations, endpoints, and time periods studied could ac-count for these different results. It is noteworthy, however, thatbecause of the low frequency of homozygous CCR532 allelecarriers in the Caucasian population (�1%), both studies includedonly low numbers of homozygous patients, and therefore did notallow statistical analysis of this completely CCR5-deficient group.In the control group of patients with intact CCR5, 27% developedend-stage renal disease. In the group of heterozygous CCR532patients who have reduced CCR5 expression on leukocytes, only19% developed end-stage renal disease during followup (41, 42).In striking contrast, however, four of the seven (57%) homozygousand completely CCR5-deficient patients developed end-stage renaldisease (41, 42), indicating that CCR5 deficiency does not protectagainst initiation and progression of IgA nephropathy, but mighteven exacerbate this form of glomerulonephritis. Whether CCR5deficiency in humans is generally disadvantageous in renal auto-immune disease, as shown in this study for mice, cannot be an-swered on the basis of these data. This issue, however, is especiallyimportant because a CCR5-specific antagonist has recently be-come available for HIV therapy (16) and could also represent anew therapeutic option in fatal autoimmune disease. Our presentstudy emphasizes the potential danger that lies in a blockade of asingle chemokine receptor. In view of counterregulatory mecha-nisms and a high degree of redundancy in the chemokine system,we suggest that a therapeutic strategy for human autoimmune dis-ease should include blockade of a group of chemokine receptorsinvolved in the recruitment of pathogenic leukocyte subsets.
DisclosuresThe authors have no financial conflict of interest.
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