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Chemokine and Chemokine Receptor Expression duringInitiation and Resolution of Immune ComplexGlomerulonephritis

HANS-JOACHIM ANDERS,* VOLKER VIELHAUER,* MATTHIAS KRETZLER,*CLEMENS D. COHEN,* STEPHAN SEGERER,* BRUNO LUCKOW,*LARS WELLER,* HERMANN-JOSEF GRONE,† and DETLEF SCHLONDORFF**Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Munich; and†Division ofExperimental Pathology, German Cancer Research Center, Heidelberg, Germany.

Abstract. Chemokines participate in leukocyte infiltration,which plays a major role in glomerular injury during immunecomplex glomerulonephritis (IC-GN). Because target cell ex-pression of chemokine receptors (CCR) is thought to mediateleukocyte migration, the expression pattern of chemokines andCCR in a model of IC-GN was examined. The transient courseand predominant glomerular pathology of this model allowsthe examination of both the induction and resolution phases ofIC-GN. GN was induced in mice by daily apoferritin injectionfor 2 wk. Urine samples and kidneys were obtained at 1, 2, and4 wk. Albuminuria was noted at 2 wk, but resolved after 4 wk.This was associated with glomerular IC deposits and mesangialproliferation. Glomerular macrophage infiltration was promi-nent at 1 and 2 wk, which resolved at 4 wk. Expression ofmonocyte chemoattractant protein-1 (MCP-1) and RANTESmRNA was upregulated at week 1 and decreased to control

levels at weeks 2 and 4. The expression was localized toglomeruli by in situ hybridization and immunohistochemistry.The mRNA of CCR1, CCR2, and CCR5 but not CCR3 orCCR4 were upregulated at week 1 and decreased at weeks 2and 4. Expression of CCR5 was located to the glomerulus byin situ hybridization and quantitative reverse transcription-PCR of isolated glomeruli. In summary, in a model of transientIC-GN, MCP-1 and RANTES and their receptors CCR1,CCR2, and CCR5 are expressed early and are already down-regulated at the peak of proteinuria and leukocyte infiltration.Resolution of glomerulonephritis is associated with a return tobaseline of chemokine and CCR expression. Therefore, it isconcluded that glomerular MCP-1 and RANTES productiondirects circulating leukocytes that express CCR1, CCR2, andCCR5 into the glomerulus. After initiating GN, MCP-1 andRANTES and their receptors are readily downregulated.

The infiltration of leukocytes into the glomerulus is a mainstayof inflammatory glomerular damage in proliferative glomeru-lonephritis (GN) (1). Attraction of circulating leukocytes ininflammatory kidney diseases has been proposed to be causedby chemokines (2). The chemokines, an expanding family ofsmall chemotactic cytokines, are classified into the C, CC,CXC, and CX3C subfamilies (where X represents any inter-vening amino acid residue between the first two cysteines inthe conserved amino acid sequence). In the kidney, many celltypes, including mesangial, endothelial, tubular epithelial, andinterstitial, have the ability to secrete chemokines in responseto stimulation with proinflammatory cytokines and immunecomplexes (2). Important members of the CC subfamily are thechemokines RANTES, monocyte chemoattractant protein-1(MCP-1), macrophage inflammatory protein-1a (MIP-1a), andMIP-1b. Upregulation of these chemokines has been reported

in rat anti-Thy1.1 and rat and mouse nephrotoxic serum ne-phritis (NSN) as well as in murine models of lupus nephritis(3–6). Functional roles for the chemokines are suggested bythe observation that neutralizing antibodies against MCP-1reduced monocyte influx and proteinuria in rat NSN (6) andabrogated crescent formation in murine NSN (7). In MCP-1–deficient mice with NSN, a marked reduction of concomitanttubulointerstitial damage was observed (8). Furthermore,MCP-1-deficient MRL-Faslpr mice were protected from pro-gressive renal injury by reduced leukocyte recruitment (9).Treatment with met-RANTES, a RANTES antagonist, de-creased infiltrating leukocytes in rat renal transplant rejection(10). An antagonist blocking multiple chemokine receptors(CCR), vMIP-2, decreased interstitial leukocyte infiltration andproteinuria in rat NSN (11). This indicates that chemokines andCCR antagonists may provide a new therapeutic approach forinflammatory renal diseases. The biologic effects of CC che-mokines on their target cells are mediated through a group ofCCR, called CCR1-10 (12). Despite that many chemokines canbind to several CCRin vitro, the selective expression of CCRby leukocyte subsets seems to be important in the regulation ofinduction, maintenance, and abrogation of the inflammatorycell infiltrate in vivo. In contrast to the substantial amount ofdata on chemokine expression, studies on CCR expression in

Received March 14, 2000. Accepted August 12, 2000.Correspondence to Prof. Dr. Detlef Schlo¨ndorff, Medizinische Poliklinik derLMU, Pettenkoferstrasse 8a, 80336 Munich, Germany. Phone:1149-89-51603500; Fax:1149-89-51604439; E-mail: [email protected]

1046-6673/1205-0919Journal of the American Society of NephrologyCopyright © 2001 by the American Society of Nephrology

J Am Soc Nephrol 12: 919–931, 2001

renal disease are still sparse. In human renal biopsies, CCR5was localized to interstitial mononuclear cells, predominantlyT cells, in various kidney diseases (13). In human crescenticGN, CCR2 mRNA expression was found in crescentic lesionson interstitial leukocytes (14). CXCR4 was found on infiltrat-ing leukocytes during human renal allograft rejection (15).Furthermore, CCR1, CCR2, and CCR5 mRNA were upregu-lated in cortical isolates from mice with NSN with crescenticGN and a strong periglomerular leukocyte accumulation(5,16).

NSN is the most widely used experimental model of immunecomplex GN (IC-GN). NSN is characterized by a necrotizingand crescentic GN in mice, accompanied by a strong periglo-merular and interstitial leukocytic infiltrate resembling humanrapidly progressive GN‘ (5,7,17). Therefore, murine NSN can-not be considered as a model of isolated GN. Consistent withthe prominent tubulointerstitial injury, a predominantly tubularexpression of MCP-1 was observed in wild-type mice withNSN (8). Concordantly, mice that were deficient of MCP-1were protected from tubulointerstitial injury, in correlationwith a decreased number of interstitial macrophages, but notfrom glomerular damage (8). A model of an isolated andreversible IC-GN resembling human diseases such as postin-fectious GN, IgA nephropathy, cryoglobulin-related GN typeII, or early lupus nephritis has not been studied.

We therefore established a murine model of reversibleIC-GN by daily injection of apoferritin for 2 wk, which al-lowed us to study the expression of chemokines and theirreceptors during both the induction and resolution phases ofIC-GN. Horse apoferritin-induced GN (HAF-GN) was charac-terized by mesangial IC deposition, diffuse mesangial prolif-eration, a glomerular influx of macrophages, and proteinuriawithout significant tubulointerstitial injury. We observed thatthe maximum mRNA expression of MCP-1 and RANTES andtheir receptors CCR1, CCR2, and CCR5 occurs early in theinitiation phase of GN and before the onset of proteinuria.CCR5 was localized to glomeruli only. At the peak of protein-uria, the expression of both chemokines and CCR decreaseddespite persistent glomerular leukocyte infiltration. Resolutionof GN with normalization of the glomerular leukocyte countand the proteinuria was associated with return to control ofchemokine and CCR expression. These data point toward arole of MCP-1 and RANTES for the initiation of glomerularleukocyte influx, which then results in proteinuria.

Materials and MethodsAnimals

Female inbred Balb/c mice (body weight, 18 to 21 g) were obtainedfrom Charles River (Sulzfeld, Germany) and were kept in macrolonetype III cages under a 12-h light/dark cycle. Water and standard chow(Sniff, Soest, Germany) were availablead libitum. For each timepoint, mice and controls that received apoferritin injections were keptin a single cage. Mice were killed by cervical dislocation. All exper-imental protocols were approved by the Animal Care and EthicsCommittee of the Bavarian government.

Induction and Evaluation of IC-GNGN was induced by daily intraperitoneal injections of 4 mg of HAF

in 80 ml of 0.1 M sodium chloride (Sigma-Aldrich Chemicals, Stein-heim, Germany) for 14 consecutive days (18). Controls receivedinjections of 80ml of 0.1 M sodium chloride.

Body weight was determined at the beginning of the study and atweekly intervals. Blood samples were collected from each animal 1wk before the beginning and at the end of the study by bleeding fromthe retro-orbital venous plexus under general anesthesia with inhaledether. After centrifugation, all serum samples were stored at280°Cuntil analyzed. Spot urine samples were collected from each animalinitially and at the end of the study. For the determination of urineprotein/creatinine ratio (Up/Ucr [mg/mg]), serum creatinine and ureanitrogen concentrations were determined using a Hitachi 717 Auto-analyzer (Roche, Mannheim, Germany), and urine albumin concen-tration was measured using a mouse albumin enzyme-linked immu-nosorbent assay (ELISA) kit (Albuwell M, Exocell Inc., Philadelphia,PA).

The immune response to the injected antigen was assessed by mea-suring anti-HAF titers based on a previously described ELISA method(19). In brief, 96-well polystyrene microtiter plates (Greiner, Munich,Germany) were coated at 4°C overnight with 0.1 mg/ml HAF in phos-phate-buffered saline (PBS; 0.15 M NaCl, 0.01 M NaH2PO4 [pH 7.4]).After washing the plates with 0.05% Tween-20 (Sigma-Aldrich, Deisen-hofen, Germany) in PBS, nonspecific binding of protein to the wells wasblocked by 1 h of incubation with 1% bovine serum albumin at roomtemperature followed by three washes with 0.05% Tween-20 in PBS.Serial dilutions of serum samples were added in duplicate for 1 h atroomtemperature. After three washes with 0.05% Tween-20 in PBS, a 1:2000dilution of horseradish-peroxidase anti-mouse Ig antibody (P260; Dako,Hamburg, Germany) was added for 1 h. Peroxidase was developed with2,2-amino-bis(3-ethylbenzothiazoline)-6-sulfonate substrate (BoehringerMannheim, Manheim, Germany), and the optical density was measuredat 450 nm applying a microtiter plate reader (Molecular Devices Corp.,Munich, Germany). Serum samples of HAF-immunized mice were com-pared with nonimmunized mice. Pooled serum from three mice that wereimmunized three times at 2-wk intervals by intramuscular injections with0.05 mg of HAF in TiterMax Gold Adjuvans (Sigma-Aldrich) served aspositive controls in the ELISA.

Morphologic EvaluationFrom each mouse, the left kidney was used for histologic assess-

ment. Each kidney was divided horizontally into two halves by amidline cut. For quantitative analysis, 2-mm-thick slices cut from thehorizontal cut surface were used. Every fifth of 15 subsequent slices,chosen by systematic uniformly random sampling, were stained andanalyzed. Intraglomerular cells were counted in at least 30 corticalglomeruli per section, selected by uniformly random sampling, fromeach animal and were analyzed. Subcapsular glomeruli (up to 50mm)were not evaluated to avoid stain-related edge artifacts. Glomeruliwere assessed only when more than 10 capillary loops were present toexclude evaluation of pole cuts. Only cells within the glomerular tuftwere counted. Cells were considered periglomerular when localizedadjacent to the outside of Bowmann’s capsule. Interstitial leukocyteswere counted in 20 high-power fields, randomly chosen out of 40cortical high-power fields per animal. Positive cells were counted perhigh-power field, omitting positive cells in glomerular fields.

Light and Electron Microscopy. The lower half of the kidneywas fixed in 4% buffered formalin, processed, and embedded inparaffin. Two-mm-thick sections were cut and stained with periodicacid-Schiff reagent. A small piece of cortical tissue from the lower

920 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 919–931, 2001

kidney pole was fixed in glutaraldehyde and embedded in araldite forelectron microscopic analysis. Slices that were cut with an ultramic-rotome were stained with osmiumtetroxide and lead citrate. Electronmicroscopy was performed with a transmission electron microscope(Philips, Stuttgart, Germany).

Immunofluorescence. The upper kidney half was snap-frozen inliquid nitrogen, vacuum sealed, and stored at280°C. Two-mm-thicksections from the cut surface of the upper kidney half were analyzed.Glomerular IgG and HAF deposits were detected by a fluorescein-conjugated goat anti-mouse IgG antibody (1:250; Dianova, Hamburg,Germany), and an anti-horse ferritin antibody (1:100; Jackson Immu-noresearch Laboratories, West Grove, PA), respectively. A nuclearcounterstain with propidium-iodide (Sigma-Aldrich) was performedto localize glomeruli in controls. Thirty cortical glomeruli were as-sessed from each section. Glomeruli were within 50mm from thecapsule to avoid edge artifacts. Only positive glomerular signals wereassessed by a semiquantitative score as follows: 0, background signal;1, low signal intensity;11, moderate signal intensity;111, strongsignal intensity.

Immunohistochemistry. Acetone-fixed frozen sections wereair-dried and incubated in 3% hydrogen peroxidase to block endog-enous peroxidases. Biotin was blocked applying the Vector BlockingKit (Vector Laboratories, Burlingame, CA). Slices were washed inPBS and incubated with the primary antibody for 1 h at room tem-perature. The following rat and rabbit antibodies were used as primaryantibodies: anti-mCD45 (leukocytes; Pharmingen, San Diego, CA;1:200), anti-F4/80 (macrophages; Serotec, Oxford, UK; 1:50), anti-Mac-3 (macrophages; Pharmingen; 1:200), anti-CD3 (lymphocytes;Pharmingen; 1:100), anti Ki-67 (cell proliferation; Dianova; rabbit,1:100), and anti-murine RANTES (Peprotech, Rocky Hill, NJ; 1:50).Anti-mouse MCP-1 antibodies were raised in rabbits using the peptideYIKNLDRNQMRSEPTC (murine MCP-1 amino acids 86 to 100 withan additional C at the carboxy terminus for coupling). The peptide wassynthesized, conjugated to keyhole limpet hemocyanin as a carriermolecule, and used to immunize two rabbits followed by two boosterinjections given in 14-d intervals and two injections in 28-d intervals.The specificity of the antiserum was demonstrated by a dot blot assay.Serial dilutions (1mg, 100 ng, 10 ng, and 1 ng) of the peptide used forimmunization and an unrelated peptide were spotted onto a nitrocel-lulose membrane and incubated with 1:1000 dilutions of the preim-mune and immune serum. Bound antibodies were detected using acommercial enhanced chemiluminescence kit (Amersham, Freiburg,Germany) according to the manufacturer’s instructions (data notshown). For immunohistochemistry, a dilution of 1:50 was used.Rabbit preimmune serum served as a negative control. Signals of allprimary antibodies were detected with a commercial mouse link andlabel kit following the instructions of the supplier (Biogenex Super-Sensitive, San Ramon, CA). 3-Amino-9-ethylcarbazole substrate wasused for signal development. All slices except for Ki-67 and Mac-3staining were counterstained with hemalaun.

In Situ HybridizationRadiolabeled riboprobes containing mRANTES, mMCP-1,

mCCR2, or mCCR5 cDNA fragments, flanked by RNA promotorsequences for SP6 and T7 RNA polymerases were used. The 321-bpmRANTES fragment represents positions 124 to 444 of the nucleotidesequence (GenBank accession number S37648). The 161-bp mMCP-1fragment corresponds to nucleotides 299 to 459 (GenBank accessionnumber J04467). The 153-bp mCCR2 fragment represents positions1738 to 1888 of the nucleotide sequence (GenBank accession numberU47035). The 220-bp mCCR5 fragment corresponds to nucleotides

1384 to 1604 (GenBank accession number D83648). Recombinantplasmids were used to transformEscherichia coliXL1-Blue. PlasmidDNA was prepared by alkaline lysis and purified over a cesiumchloride gradient. The subcloned inserts were sequenced by thedideoxy-chain-termination method using a Big Dye Terminator CycleSequencing Kit (PE Applied Biosystems, Weiterstadt, Germany) toconfirm sequence identity. The constructs were linearized and used astemplates forin vitro transcription by T7 and SP6 RNA polymerases(Boehringer Mannheim) generating antisense and sense probes, re-spectively. The probes were labeled witha35S-UTP (1250 Ci/mmol;NEN, Cologne, Germany) to a specific activity of 8.33 108 cpm/mg.Isotopic in situ hybridization with a35S-labeled RNA probes wasperformed according to modified standard protocols (20). Seven-mmfrozen sections were treated with 0.2 N HCl at room temperature for20 min for protein denaturation. Slides were rinsed in water andsubmerged for 30 min in prewarmed 23 SSC at 70°C. After a shortrinse in distilled water, slides were incubated with 5mg/ml proteinaseK in 50 mM Tris-HCl (pH 7.4) and 5 mM ethylenediaminetetraacetate(EDTA) for 10 min at room temperature. Sections were treated withglycine (0.2% in PBS) for 5 min and postfixed in 4% paraformalde-hyde for 10 min at room temperature, followed by two washes in PBS.Sections were acetylated with 0.25% (vol/vol) acetic anhydride in 0.1M triethanolamine-HCl (pH 8.0) for 10 min, to avoid nonspecificbinding of the probe. After the slides were rinsed twice in PBS, theywere prehybridized in 50% formamide, 0.3 M NaCl, 10 mM sodiumphosphate buffer (10 mM NaH2PO4, 10 mM Na2HPO4 [pH 6.8]), 10mM Tris-HCl, 5 mM EDTA (pH 7.4), 10% dextran sulfate, 25 mMDTT, 1 3 Denhardt’s, and 1.25 mg/ml tRNA at 52°C for 2 h. Afterprehybridization, slides were washed once in PBS and dehydrated ingraded ethanol concentrations. The35S-labeled antisense and senseRNA transcripts served as hybridization probe and control, respec-tively. Sections were hybridized at 52°C overnight with 12ml ofprehybridization solution containing labeled RNA probes with anactivity of 1.03 106 cpm per section, leading to a probe concentrationof 0.3 ng/ml per kb (mRANTES probe) or 0.6 ng/ml per kb (mMCP-1probe). After hybridization, four initial washes with 43 SSC, 10 mMDTT (15 min, room temperature) were followed by a washing pro-cedure with 50% formamide, 13 SSC, 10 mM DTT at 50°C for 60min. Sections were then incubated in a mixture of RNase A (40mg/ml) and RNase T1 (50 U/ml) in RNase buffer (0.5 M NaCl, 10mM Tris-HCl, 1 mM EDTA [pH 7.4]) at 37°C for 30 min. They werewashed three times in RNase buffer, followed by two high-stringencywashes in 0.13 SSC, 10 mMb-mercaptoethanol (60°C, 15 min).Finally, slides were dehydrated in a graded series of ethanol contain-ing 0.3 M ammonium acetate, 10 mMb-mercaptoethanol. For auto-radiography, sections were dipped in Ilford K2 nuclear researchemulsion diluted 1:1 with distilled water at 42°C and exposed for 2 wkat 4°C in a dry chamber. After development, tissue was counterstainedwith Harris hematoxylin and eosin.

Real-Time Quantitative Reverse Transcription-PCR onMicrodissected Renal Tissue

Glomeruli and tubulointerstitial specimen were microdissectedmanually and transferred to liquid nitrogen. Reverse transcriptionfollowed a protocol previously established for single glomerularpodocytes (21). In brief, microdissected samples underwent randomprimed reverse transcription for 1 h at42°C using a modified Moloneymurine leukemia virus reverse transcriptase (Superscript; Life Tech-nologies, Karlsruhe, Germany). Real-time reverse transcription-PCR(RT-PCR) was performed on a TaqMan ABI 7700 Sequence Detec-tion System (PE Biosystems) using a heat-activated TaqDNA poly-

J Am Soc Nephrol 12: 919–931, 2001 Chemokines and CCR in GN 921

merase (Amplitaq Gold, PE Biosystems). Thermal cycler conditionscontained holds at 50°C for 2 min and 95°C for 10 min followed by40 cycles of 95°C for 15 s and 60°C for 60 s. Messenger RNAexpression for each signal was calculated following theDCt procedure(22). Glyceraldehyde phosphate dehydrogenase (GAPDH) was usedas reference gene. The amplification efficiency of target and referencewas shown to be similar, with a slope of log input amount toDCt ,0.1, i.e., 0.0057 for CCR5versusGAPDH. Controls consisting ofddH2O were negative for target and housekeeper. The followingoligonucleotide primers (300 nM) and probes (100 nM) were used:Murine CCR 5 (gb D 83648; bp 1065 to 1191): sense, 5'-CAAGA-CAATCCTGATCGTGCAA-3', antisense, 5'-TCCTACTCCCAAG-CTGCATAGAA-3'; internal fluorescence-labeled probe (FAM):5'-TCTATACCCGATCCACAGGAGAACATGAAGTTT-3'; CCR5specificity of primers and probe were tested on CCR plasmids; murineGAPDH (gb M32599; bp 730 to 836): sense, 5'-CATGGCCTTC-CGTGTTCCTA-3', antisense, 5'-ATGCCTGCTTCACCACCTTCT-3'; internal fluorescence labeled probe (VIC): 5'-CCCAATGTGTC-CGTCGTGGATCTGA-3'. Primers and probes were obtained fromPE Biosystems.

RNA Preparation and RNase Protection Assay fromKidneys

From each animal, the right kidney was snap-frozen in liquidnitrogen and stored at280°C. Total RNA was prepared using themethod of Chomczynski and Sacci (23). The final product was airdried, dissolved in dielaidoylphosphatedylcholine-treated water, andstored at280°C.

Multiprobe template sets for mouse CC chemokines (mCK-5) andmouse CCR (mCR-5) for use in RNase protection assays were ob-tained from Pharmingen. Antisense riboprobes were prepared byinvitro transcription with T7 RNA polymerase (Promega, Madison, WI)and the incorporation of [32P]UTP at 37°C. Tenmg of total kidneyRNA was used to analyze the expression pattern of CC chemokines,and 50mg of RNA was used for analysis of CCR. Efficacy of RNasedigestion was ensured by a yeast t-RNA sample in every assay. RNAsamples were hybridized with 33 105 counts of each [32P] UTP-labeled riboprobe for 14 h at 56°C. Free probe and other single-stranded RNA was digested with RNase A (Sigma) and T1 (Boehr-inger Mannheim) at 30°C for 30 min. RNases were digested withproteinase K (Promega). After phenol-chloroform extraction and so-dium acetate-ethanol precipitation, the samples were electrophoresedthrough a denaturing 6% polyacrylamide gel. Gels were dried andexposed on phosphor screens of a Storm 840 PhosphorImager (Mo-lecular Dynamics, Sunnyvale, CA). Bands were quantified using theImageQuant software (Molecular Dynamics). Values are expressedrelative to the GAPDH mRNA level in each lane after backgroundsubtraction.

Statistical AnalysisData were expressed as means6 SEM and analyzed byt test for

unpaired data. Statistical significance was defined asP , 0.05.

ResultsInduction of GN

Albuminuria was increased significantly after 2 wk in micethat received HAF injections compared with controls (Up/Ucr,140 6 20 versus17 6 10; P , 0.01) but resolved aftercessation of antigen injection (Figure 1). Body weight, serumcreatinine, and blood urea nitrogen levels remained unchanged

Figure 1. Course in mice that received horse apoferritin (HAF)injections (□) and controls (m). (A) Albuminuria as detected bymouse albumin enzyme-linked immunosorbent assay in spot urinesamples, expressed as Ualb/Ucreatinine(mg/mg). (B) Number of intra-glomerular CD45-positive cells per glomerulus. (C) Levels of CC-chemokine mRNA related to respective glyceraldehyde phosphatedehydrogenase (GAPDH) mRNA as detected by RNase protectionassay;m, RANTES;‚, monocyte chemoattractant protein-1 (MCP-1); Œ, macrophage inflammatory protein-1b (MIP-1b). (D) Levels ofCC-chemokine receptor (CCR) mRNA related to respective GAPDHmRNA as detected by RNase protection assay;□, CCR5;Œ, CCR2;e, CCR1. Values are means6 SEM from four controls and 6 micethat received HAF injections at each time point for proteinuria andCD45-positive staining. RNA values were derived from three animalsat each time point. *,P , 0.05versuswk 0.


from prestudy values for mice that received HAF and salineinjections at all time points. Anti-HAF titers were alreadyelevated after 1 wk and increased further until week 4,i.e.,after cessation of antigen injection in all mice that receivedHAF injections (Table 1).

HistopathologyLight microscopy revealed mild mesangial hypercellularity

at week 1 (Figure 2). After 2 wk, diffuse mesangial expansion,with increase of matrix and mesangial hypercellularity, wasobserved in all animals that received HAF injections. Four wkafter cessation of antigen injection, the histologic changes hadreturned to control levels. Immunohistochemical staining forthe marker of cell proliferation Ki-67 revealed increased mes-angial proliferation at weeks 1 and 2, which resolved at week4 (Figure 3, Table 1). No endocapillary necrosis or crescentswere noted. No overt tubulointerstitial lesions were noted atany time point. Kidneys of mice that received saline injectionsshowed normal histology.

Mesangial deposits containing HAF and IgG were noted inmice that received HAF injections at all time points but not incontrols (Figure 2). Semiquantitative assessment showed in-creasing mesangial HAF and IgG deposits from 1 to 2 wk andsubsequent decrease after 4 wk (Table 1). Ultrastructurallyelectron-dense deposits were located in the mesangium andwithin the mesangial glomerular basement membrane (GBM)but not at the capillary loop GBM (Figure 4). Fusion of

podocyte foot processes was not observed, which may berelated to the moderate degree of proteinuria.

Leukocytic Cell InfiltrateIn mice that received HAF injections, infiltrating CD45-

positive leukocytes were found within the glomerular tuft,periglomerulus, and cortical interstitium (Figure 3). However,there was a much stronger increase of the glomerular CD45-positive leukocyte cell count at week 2 compared with theinterstitial compartment (which includes a much larger areacompared with a single glomerular profile) (Table 1). Thenumber of glomerular CD45-positive leukocytes and macro-phages increased from week 1 to week 2 but had resolved atweek 4 (Table 1). Glomerular Mac-3–positive macrophageswere increased at week 2 (3.86 0.9 versus0.3 6 0.2 incontrols;P , 0.01; see Table 1, Figure 3), but CD3-positivelymphocytes were absent (data not shown). Because neutro-phils and T cells were absent in glomeruli of mice that receivedHAF injections, the discrepancy of the CD45-positive andMac-3–positive glomerular cell counts should be due to thefact that Mac-3 stains only a particular activation state of themonocyte-macrophage lineage.

Chemokine ExpressionRNase Protection Assays. Total RNA was isolated from

kidneys of 12 mice that received HAF injections and 6 controlmice and analyzed by RNase protection assay. MCP-1 and

Table 1. Serum, urinary, and histologic findings in HAF-induced glomerulonephritisa

Week 1 Week 2 Week 4

HAF Controls HAF Controls HAF Controls

Functional parametersUalb/Ucr (mg/mg) 206 8 246 10 1406 20b 176 10 106 10 106 1Scr (mg/dl) 0.46 0.1 0.36 0.1 0.46 0.1 0.36 0.1 0.56 0.1 0.36 0.1SBUN (mg/dl) 336 5.6 266 7.8 316 3.3 256 6.8 296 7.5 246 3.6body weight (g) 196 1.7 196 1.6 196 1.6 196 3.0 296 7.5 246 3.6

Humoral responseanti-HAF IgG 526 16b 4 6 2 15366 255b 4 6 2 30726 565b 4 6 0glomerular IgG deposits 1/11 2 11 2 1/11 2glomerular HAF deposits 11/111 2 11/111 2 1/11 2

Cellular responseKi-671 (cells/glomerulus) 2.46 0.8b 0.56 0.3 5.66 2.5b 0.46 0.3 0.56 0.2 0.26 0.1leukocytes

CD451 intraglomerular 5.76 0.7b 0.56 0.1 7.76 0.7b 0.66 0.1 1.16 0.3 0.56 0.1CD451 periglomerular 5.56 0.9b 1.56 0.1 7.06 1.1b 1.56 0.2 2.36 0.4b 1.26 0.1CD451 interstitial 8.46 1.2b 3.76 1.4 11.36 1b 4.56 1.1 4.86 0.8 4.06 0.7MAC31

intraglomerular2.06 0.7b 0.46 0.1 3.86 0.9b 0.36 0.2 0.46 0.2 0.36 0.1

F4/801 periglomerular 2.76 1.0b 0.56 0.1 5.76 0.5b 0.56 0.2 2.36 0.3b 0.46 0.1F4/801 interstitial 3.96 1.5b 0.86 0.1 7.66 0.4b 0.66 0.1 2.66 0.7b 0.46 0.1

a Intra- and periglomerular cells were counted per glomerulus, interstitial cells were counted per high-power field (see Materials andMethods section). HAF, horse apoferritin; Ualb/Ucr, urine albumin/urine creatinine ratio; Scr, serum creatinine; SBUN, serum blood ureanitrogen.1, low signal intensity;11, moderate signal intensity;111, strong signal intensity.

b P , 0.01versuscontrols.


RANTES mRNA expression was increased after 1 wk ofantigen injection compared with saline injection (Figure 5A).Surprisingly, MCP-1 and RANTES mRNA expression haddecreased toward control values at week 2, the time point ofstrong glomerular leukocyte infiltration and proteinuria (Figure1). At week 4, when proteinuria and cell infiltrate had resolved,MCP-1 and RANTES mRNA expression were at control lev-els. Expression of eotaxin, MIP-1a, MIP-1b, MIP-2, T-cellactivation gene-3, and interferon-inducible protein-10 were notincreased when related to GAPDH levels at any time point.

In Situ Hybridization. To localize the sites of chemo-kine expression, we performedin situ hybridization onfrozen kidney sections with antisense probes for RANTESand MCP-1, the two upregulated chemokines. For bothMCP-1 and RANTES, a positive staining was found onlyafter 1 wk in mice that received HAF injections (Figure 6).Positive cells were detected exclusively within but not out-side the glomeruli. Hybridization with sense probes wasnegative as were tissues from controls that received salineinjections.

Immunohistochemistry. Consistent with thein situ hy-bridization data, staining with antibodies against MCP-1 andRANTES revealed positive staining of only intraglomerularcells in mice that received HAF injections at week 1 (Figure 6).MCP-1 and RANTES were not detected at later time points orin glomeruli of mice that received saline injections. The loca-tion of positive cells was similar to that found by the respectivein situ hybridization. Immunohistologic staining with rabbitpreimmune serum was negative.

CCR ExpressionRNase Protection Assays. RNase protection assays with

RNA from kidneys of mice that received saline injectionsrevealed no signals for any of the CCR tested (CCR1 to CCR5)despite strong signals for GAPDH and L32 (Figure 5B). Inkidneys of mice that received HAF injections, elevated levelswere observed for CCR1, CCR2, and CCR5 at 1 and 2 wk. Nosignals for CCR3 and CCR4 were detectable. Expression ofCCR1 was elevated threefold at week 1, decreased to twofoldat week 2, and had returned to the low levels of the controls

Figure 2.Histopathology of HAF-glomerulonephritis (GN) at week 2. (A and B) Diffuse mesangioproliferative GN in kidneys of mice thatreceived HAF injections and normal kidney in controls. (C and D) Mesangial HAF deposits in experimental mice, absent in controls. (E andF) Mesangial IgG deposits in experimental mice, not found in controls. Magnification,31000 (periodic acid-Schiff in A and B; FITC–anti-HAFin C and D; FITC–anti-mouse IgG in E and F).


that received saline injections at week 4 (Figure 1). Levels ofmRNA for CCR2 were elevated threefold at week 1 anddecreased at weeks 2 and 4 to twofold and 1.5-fold of controls,respectively. Similarly, mRNA of CCR5 was elevated fourfoldat week 1 and decreased to twofold of controls.

Real-Time Quantitative RT-PCR. To localize better theCCR expression, RT-PCR of microdissected glomeruli andtubulointerstitial specimens andin situ hybridization were per-formed. The glomerular mRNA expression of CCR5 was four-fold higher in mice that received HAF injections comparedwith that of controls that received saline injections (P , 0.01;Figure 7). Comparison of tubulointerstitial specimens betweenmice that received HAF and saline injections revealed nodifference in CCR5 expression. The signal for CCR2 was tooweak for quantitative analysis. RT-PCR was not conducted forCCR1.

In Situ Hybridization. Hybridization with an antisenseprobe for CCR5 revealed a positive staining for CCR5 in micethat received HAF injections but not saline injections at week

1 or hybridization with sense probes (Figure 8). Only in glo-meruli were single cells positive for CCR5. No CCR5-positivecells could be detected in the tubulointerstitial space, confirm-ing the results obtained by RT-PCR. Also in agreement withthe RT-PCR data, no consistent signal for CCR2 could beobtained byin situ hybridization.

DiscussionThe present data show that in a model of murine IC-GN with

glomerular macrophage accumulation and mesangial cell pro-liferation but without significant tubulointerstitial injury, theexpression of chemokines and CCR is restricted to the glomer-ulus. The generation of MCP-1 and RANTES and the expres-sion of their respective receptors CCR1, CCR2, and CCR5precedes proteinuria and the maximum of leukocyte infiltrationand is already downregulated during active and resolved IC-GN. These results argue for an important role of chemokinesand CCR in the early initiation phase of glomerular leukocyteinfiltration and for a lesser role during the phase with persistent

Figure 3. Histopathology of HAF-GN at week 2. Glomerular CD45-positive cell infiltrate in HAF-GN (A), absent in control mice (B).Accumulation of glomerular Mac-3–positive macrophages in mice that received HAF injections (C, arrows) but not in mice that received salineinjections (D). Glomerular Ki-67–positive cells indicate proliferative GN (E, arrows) compared with controls that received saline injections (F).Magnifications:31000 in A and B;3400 in C, D, E, and F.


leukocyte infiltration and proteinuria at the height of thedisease.

HAF-Induced GN Is a Reversible, Proliferative DiseaseRestricted to the Glomerulus

A 14-d course of HAF injection into Balb/c mice resulted inIC-GN with glomerular macrophage infiltration, mesangialproliferation, proteinuria, and the absence of major tubuloin-terstitial inflammation or damage. Glomerular changes of thisIC-GN model were induced by deposition of immune com-plexes consisting of the injected antigen and autologous Ig atthe paramesangial GBM and the mesangium. When the antigenadministration was stopped, the proliferative changes, leuko-cyte infiltration, and proteinuria resolved within a 2-wk inter-val and the normal structure of the glomerular tuft was re-stored. This resolution occurred despite persistently elevatedanti-HAF serum antibody titers and only slightly reduced glo-merular IC deposits, as judged by immunofluorescence. Thus,to generate a glomerular inflammatory response, a continuousde novointraglomerular IC deposition seems to be required.

This feature of IC trailing the resolution of the proliferative GNis also reminiscent of some human IC-GN, such as postinfec-tious GN or IgA nephropathy (24). Thus, the histology ofHAF-GN bears close similarities to human IC-GN, such aspostinfectious GN, lupus nephritis class IIb, cryoglobulin-re-lated GN type II, and other forms of mesangioproliferativeIC-GN. The course of initiation and resolution resembles inparticular postinfectious GN or IgA nephropathy. Furthermore,HAF-GN shows almost exclusively glomerular lesions withvery little interstitial cell infiltration or damage. This allows thestudy of the role of mediators for predominant glomerularlesions during the initiation and resolution phases of glomer-ular IC nephritis without major interference by tubulointersti-tial disease.

Early MCP-1 and RANTES Expression IsDownregulated already at the Height of GN

We used the course of HAF-induced GN to study chemokineand CCR expression during evolution and resolution of aglomerular leukocytic infiltrate. The finding of MCP-1 and

Figure 4.Electron microscopy of mice that received HAF injections (A) and controls that received saline injections (B) at 2 wk. In mice thatreceived HAF injections (A), a widened mesangium with mesangial hypercellularity and an increase of mesangial matrix is noted comparedwith controls (B). Subepithelial and subendothelial immune complex deposits cause an increase of basement membrane diameter in mice thatreceived HAF injections (C, large arrow) compared with controls that received saline injections (D). No podocyte foot process effacement isobserved in mice that received HAF injections (C, small arrows). Magnifications:35000 in A and B;315,000 in C and D.


RANTES expression early in the course of GN is in concor-dance with results reported by previous studies of murine NSNas well as in human GN (5,8,25–27). Surprisingly, we foundthat glomerular MCP-1 and RANTES expression is alreadycompletely downregulated at the peak of glomerular leukocyteinfiltration and the onset of proteinuria. This argues for down-modulation of local chemokine generation despite persistent ICdeposition and against a self-maintaining amplification loop ofchemokine production by infiltrating leukocytes. To the con-trary, locally generated “antagonists” might inhibit further che-mokine expression. Potential candidates for a counterregula-tive local suppression of chemokine production are,e.g.,transforming growth factor-b1, nitric oxide, prostaglandin E1,and other yet unknown factors (28–32).

Furthermore, we could demonstrate that in evolving GN,MCP-1 and RANTES are expressed by some but not all glo-merular cells. As judged from thein situ hybridization andimmunohistology, these could be endothelial cells, mesangialcells, and infiltrating monocytes but not podocytes. BecauseTeschet al. (8) observed tubular and interstitial but not glo-merular MCP-1 expression in a model of predominantly tubu-lointerstitial leukocyte infiltration, the present data indicate

that depending on the disease model, MCP-1 expression islocalized to the predominant site of inflammation.

In view of the many chemokines known, the limited reper-tory of CC chemokines that are found to be upregulated duringin vivo studies is striking. Predominantly, MCP-1 and RAN-TES among many others examined have been reported invarious models and in human renal disease (2). RANTES isknown to attract T cells and monocytesin vitro, as well as inthe spontaneous nephritis of MRL-Fas(1pr) mice, in experimen-tal murine NSN, and during renal transplant rejection in rats(8,10,33). Interestingly, the marked RANTES expression inHAF-GN was not associated with lymphocyte infiltration. Thisobservation supports the hypothesis that the monotonous pat-tern of chemokine expression unfolds its selectivity locally,perhaps by selective CCR and adhesion molecule expressionon restricted target cells (2).

CCR Expression also Precedes the Peak of HAF-GNActivity

Chemokines attract leukocytes by binding and activatingspecific CCR on subclasses of leukocytes. Therefore, it wasinteresting to find that indeed CCR1, CCR2, and CCR5 mRNA

Figure 5. (A) RNase protection assay for the detection of chemokine mRNA in HAF-GN. Total kidney RNA of six mice that received HAFinjections and four mice that received saline injections was extracted after 1, 2, and 4 wk. Chemokine expression was analyzed by a commercialmultiprobe RNase protection assay using32P-labeled templates specific for RANTES, IP-10, MIP-1a, MIP-1b, MIP-2, MCP-1, and TCA-3.The unprotected probe is shown on the left, and the protected fragments are indicated on the right. The signals for MCP-1 and RANTES wereincreased at week 1 as compared with respective controls. Because of the conditions of the multiprobe RNase protection assay, the MCP-1signal appears in multiple bands. (B) RNase protection assay for the detection of CCR mRNA in HAF-GN. CCR expression was analyzed bya commercial multiprobe RNase protection assay using templates specific for CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5. Expression ofCCR1, CCR2, and CCR5 is increased at week 1 and declines to almost normal values at week 4. Signals for CCR3 and CCR4 are absent.


expression was upregulated together with their respective li-gands MCP-1 and RANTES early in the course of GN. InHAF-GN, CCR2 and CCR5 mRNA expression originates fromglomeruli, most likely from their presence on infiltrating mac-rophages by analysis of isolated glomeruli by RT-PCR andinsituhybridization of renal sections. The peak of chemokine andCCR levels preceded the maximum leukocyte infiltration dur-ing the course of disease. At 2 wk, when MCP-1 and RANTES

mRNA levels had returned to normal, CCR mRNA levelsremained elevated, consistent with the glomerular leuko-cytic cell infiltrate. We and others recently described asimilar upregulation of CCR1, CCR2, and CCR5 in corticaland glomerular preparations of NSN (5,16). In various hu-man renal diseases, CCR5 is found only on infiltratingleukocytes, mostly T cells and monocyte/macrophages (13–15). With the exception of CXCR3, CCR have not been

Figure 6.Expression of MCP-1 protein in single cells (arrow) within the glomerular tuft was found only in HAF mice after 1 wk (A) comparedwith controls (B). A similar expression pattern was noticed for RANTES protein (arrow) in HAF-induced GN (C) but not in control animalsthat received saline injections at week 1 (D).In situ hybridization for mRNA of MCP-1 in mice that received HAF injections (E) and mice thatreceived saline injections (F). Note that positive cellular signals are found only within the glomerular tuft of mice that received HAF injectionsat week 1 in a focal intraglomerular distribution.In situ hybridization for mRNA of RANTES with a similar pattern as for MCP-1 in mice thatreceived HAF injections (G) but not in controls (H) at the same time point. Magnification,31000.


found on intrinsic renal cells, in both normal and diseasedhuman kidneys (34).

CCR1 (receptor for RANTES and MIP-1a) is expressed onneutrophils, monocytes, and activated T cells (35). As recentlyshown by us (36), it can also be induced in human mesangialcells upon stimulation with interferon-g. Therefore, duringearly HAF-GN, mesangial cells might contribute to the in-crease of CCR1 mRNA expression. Once activated, CCR1 israpidly downregulated on effector cells, which is supported byour finding that CCR1 mRNA expression precedes the maxi-mum of leukocyte infiltration in GN (37). CCR1 may also playa role in regulating the immune response in addition to itschemoattractive properties. For example, a recent report noteda worsening of NSN in CCR1-deficient mice in comparison towild-type mice (16). This was related to an enhanced Th1

response and a counterregulatory role of CCR1. Unfortunately,we could not localize further the CCR expression to specificcell types in HAF-GN. Therefore, future work will have toidentify these cells and their role in IC-GN.

CCR2, the only known receptor for murine MCP-1, is ex-pressed on monocytes, macrophages, activated T cells, andactivated endothelial cells (38,39). Apart from mediating che-moattractive effects of MCP-1 on circulating leukocytes,CCR2-positive cells seem to enhance directly Th1-type im-mune responses,e.g., crescentic GN, by increased expressionof interferon-g (40). In murine crescentic GN, CCR2 tran-scripts have been detected in glomerular isolates by RT-PCR.Also in NSN, CCR2-deficient mice exhibit reduced infiltrationof macrophages and proteinuria despite aggravated histopatho-logic damage (5,41). Declining CCR2 mRNA expression dur-ing the maximum of leukocyte infiltration in HAF-GN may bea consequence of receptor downregulation during the differen-tiation of monocytes to tissue macrophages, a known feedbackmechanism in the regulation of the chemotactic response ofmonocytes/macrophages (42). Unfortunately, the signal forCCR2 was too weak to allow further localization by RT-PCRor in situ hybridization in HAF-GN.

CCR5 (receptor for MIP-1a, MIP-1b, and RANTES) can beexpressed on monocytes, activated T cells, and natural killercells. In human allograft rejection as well as in a variety ofother glomerular and tubulointerstitial diseases, CCR5 wasdetected only on infiltrating cells, predominantly T cells andmacrophages, but not on intrinsic renal cells (13,14). In thismodel of IC-GN with predominant glomerular macrophageinfiltration, the expression of CCR5 was also localized to theglomerulus by both RT-PCR andin situ hybridization. Asshown in Figure 8, only single cells within a glomerular sectionare positive. This argues in favor of the CCR5-positive cells’being infiltrating leukocytes, as positivity of intrinsic glomer-ular cells, e.g., endothelial, mesangial, or epithelial cells,should have resulted in multiple signals per glomerular section.Unfortunately, this issue cannot be resolved at present becauseof a lack of appropriate reagents. Of special interest is thedecrease of CCR5 expression during active IC-GN despitepersistent leukocyte infiltration. Most likely, this relates toreceptor downregulation on the respective target cells afterbinding of the chemokine ligand (43). Furthermore, additionalhumoral factors such as prostaglandin E2 may contribute torapid CCR5 downregulation (44). When antigen injection andhencede novoimmune complex deposition are stopped, che-mokine and CCR expression decreased further within 14 d.This occurred in parallel with the resolution of mesangialproliferation, leukocyte infiltration, and proteinuria. Presum-ably, the lack of further chemokine secretion as well as CCRdownregulation results in decreased chemotaxis and adherenceof leukocytes. The resolution of glomerular proliferation maybe a result of cell emigration and/or cellular apoptosis (45).

In summary, we describe a murine model of transient IC-GN, which is characterized by mesangial proliferation andglomerular macrophage infiltration in the absence of majortubulointerstitial disease. During the course of IC-GN, theearly glomerular expression of MCP-1 and RANTES precedes

Figure 7.Expression of mRNA for CCR5 in microdissected nephronsegments of mice that received HAF injections and controls thatreceived saline injections. One wk after induction of nephritis,nephron segments of two control and two experimental animals weremicrodissected manually. Four glomeruli and four tubulointerstitialspecimens per animal were analyzed. mRNA expression was deter-mined by real-time quantitative reverse transcription-PCR. Expressionwas normalized to the housekeeper GAPDH and is shown as ratio tocontrol animals. One percent of the total cDNA was analyzed intriplicate; comparative controls without reverse transcription werenegative for CCR5. Relative quantification was performed followingthe DDCt method. Here, the relative quantification is determined bythe difference in threshold cycles (Ct) of target and reference genes(5DCts) in experimental and control groups, respectively. Datashown are means, error bars represent values calculated by the SD ofthe DCts. The mRNA expression of CCR5 is fourfold higher inglomeruli of mice that received HAF injections compared with con-trols that received saline injections (P , 0.01). No difference wasfound for tubulointerstitial specimens from HAF and control animals.


proteinuria and is already downregulated at the maximum ofglomerular leukocyte infiltration. Their respective receptorsCCR1, CCR2, and CCR5 are also upregulated early togetherwith the leukocyte infiltration. Resolution of IC-GN is associ-ated with a decline of chemokine and CCR mRNA levels.These results suggest a role for MCP-1 and RANTES as initialchemotactic signals attracting CCR1-, CCR2-, and CCR5-positive leukocytes. Overexpression of MCP-1 and RANTESseems not to be necessary for the persistence of leukocyteinfiltration and the resulting proteinuria at the height of glo-merular injury.

AcknowledgmentsThe authors thank Y. Linde and E. Go¨ckel-Krzikalla for their

technical assistance. The data have been reported previously in ab-stract form (J Am Soc Nephrol10: A2652, 1999). The work wassupported by grants from the German Human Genome Project and theElse-Kroener-Fresenius Foundation (M.K.), from the University ofMunich (H.J.A.), and from the Deutsche Forschungsgemeinschaft(B.L.).

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