Kidney International, Vol. 56 (1999), pp. 612–620

Prevention of crescentic glomerulonephritis byimmunoneutralization of the fractalkine receptor CX3CR1Rapid Communication

LILI FENG, SHIZHONG CHEN, GABRIELA E. GARCIA, YIYANG XIA, MIKE A. SIANI, PAULO BOTTI,CURTIS B. WILSON, JEFFREY K. HARRISON, and KEVIN B. BACON

Department of Immunology, The Scripps Research Institute, La Jolla; Gryphon Sciences, South San Francisco, and NeurocrineBiosciences Inc., San Diego, California; and Department of Pharmacology and Therapeutics, University of Florida,Gainesville, Florida, USA

Prevention of crescentic glomerulonephritis by immunoneu- of leukocytes from the blood stream into tissues requirestralization of the fractalkine receptor CX3CR1. both the mechanisms for the leukocytes to slow down

Background. Fractalkine is a newly identified T-cell and and adhere to the vascular endothelium and the displaymonocyte/macrophage (Mφ) chemokine with a transmembraneof recruiting signals at sufficient levels at the vicinity ofdomain and is a cell-surface protein on activated endothelium.the intended exit site. The exit of leukocytes would beIt can mediate adhesion of cells expressing the fractalkine recep-

tor CX3CR1. These unique features make fractalkine well suited more difficult in the fast flow of the arterial system. In-for leukocyte recruitment in tissues with high blood flow as in creasing evidence indicates that chemokines are the majorthe renal glomerulus. recruiting molecules of leukocyte emigration [4–6], andMethods. Fractalkine expression in glomeruli and response

it is believed that these small soluble chemoattractantof isolated glomerular inflammatory cells to fractalkine weremolecules depend on their low-affinity binding to proteo-studied in the Wistar-Kyoto (WKY) crescentic glomerulone-

phritis model. Antibody was used to confirm the proinflamma- glycans on the endothelial surface to counter the dis-tory role of fractalkine. persal force of blood flow and to maintain a local concen-

Results. Fractalkine was markedly induced in the endothe- tration at the vessel wall [7–10].lium of nephritic rat glomeruli, and inflammatory leukocytesThe recent identification and cloning of fractalkineinfiltrating the glomeruli expressed increased levels of CX3CR1.

[11, 12] introduced a new member with unique featuresAnti-CX3CR1 antibody treatment dramatically blocked leuko-cyte infiltration in the glomeruli, prevented crescent formation, to the chemokine superfamily. Fractalkine has a noveland improved renal function. CX3C motif in the chemotactic domain. It is mainly ex-

Conclusions. Fractalkine plays a central role in leukocyte pressed by endothelial cells and not by hematopoietictrafficking at the endothelium in the high-flow glomerular cir-and inflammatory cells. Most importantly, it has a trans-cuit and, in turn, implicates CX3CR1 as a prime drug target formembrane domain and can be expressed in activatedtherapeutic intervention of endothelium-related inflammatory

diseases. endothelium as a cell-surface protein. Membrane-boundfractalkine can directly mediate adhesion of cells express-ing the fractalkine receptor CX3CR1, including T cells

Leukocytes are distributed throughout the body via and macrophages (Mφ) [13, 14]. Based on these features,the vascular network. Upon receiving appropriate signals, fractalkine may be especially important for leukocyteleukocytes are cued to exit at specific sites, frequently in recruitment in tissues with a high blood flow rate, forthe slow flow of the venular compartment, to patrol the example, the glomeruli, where the low-affinity bindingsolid tissues (immune surveillance), or to mount reac- of other chemokines to proteoglycans may not withstandtions against pathogens (inflammation) [1–3]. The exit the high shear environment [15, 16].

Infiltration of CD81 T cells and Mφ is the major mech-anism for injury in the aggressive, crescentic glomerulo-Key words: chemokine, inflammation, glomerulus, endothelium, leuko-

cyte trafficking, blood flow. nephritis (GN) of the Wistar-Kyoto (WKY) rat inducedby administration of antiglomerular basement membrane(GBM) antibody (Ab) [17]. Various means to inhibit in-Received for publication April 27, 1999

Accepted for publication May 17, 1999 flux of these two cell types modulate the disease to vary-ing degrees [18–20]. Recently, the use of vMIPII, a viral 1999 by the International Society of Nephrology

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protein that blocks a number of chemokine receptors, Immunofluorescence stainingwas shown to be modulatory in the WKY model [21]. Wistar-Kyoto rats were sacrificed on day 3 after theAmong the chemokine receptors influenced by vMIPII anti-GBM GN induction, and the renal tissue samplesis the fractalkine receptor CX3CR1. To investigate the were snap-frozen in Tissue-Tek (Miles Laboratories Inc.,role of fractalkine directly in this model, an Ab to the Elkhart, IN, USA) at 2708C. For fractalkine staining, 5CX3CR1 was prepared and compared with Ab specific mm kidney sections were reacted with horseradish perox-for the CC chemokine receptor CCR5 in the WKY idase (HRP)-conjugated antifractalkine Ab and detectedmodel. Anti-CX3CR1 was found to be even more effec- with FITC-labeled anti-HRP Ab (Bethyl Laboratories,tive than vMIPII in reducing the glomerular disease, Inc., Montgomery, TX, USA). For GBM staining, kidneysuggesting the pivotal role of fractalkine in this model. sections were reacted with FITC-labeled swine antirabbitThe striking reduction in CD81 and Mφ suggests that the IgG Ab (DAKO Corp., Carpinteria, CA, USA). Fluores-membrane-bound nature of fractalkine is of particular cent images of the stained slides were captured in aimportance in the influx of inflammatory cells in the digital camera and merged to the phase-contrast micro-glomerular circuit. In addition, other chemokines that scopic background.may bind via heparin binding sites could contribute tosubsequent accumulation and activation of inflammatory Stable transfectant cells of rat CX3CR1 cDNAcells, especially as the influx of cells likely alters glomeru- cDNA containing the open reading frame of CX3CR1lar hemodynamics and probably slows flow in segments [28] was cloned into mammalian expression vectorof the glomerular capillary. pcDNA3. HEK 293 cells were transfected with 10 mg of

plasmid DNA using LipofectAMINE reagent (Life Tech-nologies, Inc., Gaithersburg, MD, USA). G418-resistantMETHODScells expressing CX3CR1 were characterized by flow cy-Production of antibodiestometry analysis.

A polymerase chain reaction (PCR)-amplified cDNAfragment encoding amino acids 2 to 22 of rat CCR5 Preparation of inflammatory glomerular leukocytesprotein was cloned in-frame into the E. coli expression and normal peritoneal Mφ, steady-state binding assay,vector pETM1 [22]. The plasmid was transformed into and chemotaxis assayE. coli host strain BL21(DE3) for the expression and

Inflammatory leukocytes were isolated from the ne-purification of His-tagged recombinant protein. The pu-phritic glomeruli of WKY rats three days after anti-GBMrified recombinant protein was injected into a rabbit toGN induction, following the method of Cook, Smith,raise Ab. A similar approach was used to generate rabbitand Cattell [29]. Peritoneal Mφ were collected from nor-Ab against rat fractalkine as described previously [23].mal rats by washing the peritoneal cavity twice with 50ml of phosphate-buffered saline. These cells, togetherInduction, treatment, and analysis of antiglomerularwith the stable transfectants of CX3CR1, were used inbasement membrane glomerulonephritisboth steady-state binding and chemotaxis assays, whichAnti-GBM GN was induced at day 0 in WKY rats (10were performed as previously described [30].to 12 weeks old) by intravenous injection of anti-GBM

Ab [24] at a dose of 25 ml/100 g body weight. For Ab Flow cytometry analysistreatment, the induction of anti-GBM GN was followed

Inflammatory leukocytes isolated from the nephriticby daily intravenous injection of 0.5 ml of different Absglomeruli of WKY rats three days after anti-GBM GNup to eight days. Blood samples and urine excretionsinduction were used in two-color flow cytometry analysis.(24-hr period) were collected on days 0, 3, 5, 7, and 9.Cells (5 3 10 5) were suspended in Hank’s balanced saltProteinuria was assayed by the sulfosalicylic method [25].solution (HBSS)/4% fetal calf serum (FCS) and were incu-Urine and blood creatinine were determined using abated with affinity-purified anti-CX3CR1 Ab or an unre-creatinine diagnostic kit (Sigma, St. Louis, MO, USA),lated Ab at the final concentration of 6 mg/ml for 20 minutesfollowing the manufacturer’s instructions.on ice. The cells were washed, incubated with a 1:50

RNA isolation and RNase protection assay dilution of a PE-labeled donkey antirabbit IgG F(ab9)2

(Jackson ImmunoResearch Laboratories, Inc., WestAt different time points after the induction of anti-GBMGrove, PA, USA) for 20 minutes, and washed again.GN, the rats were sacrificed and glomeruli were preparedThese cells were next stained with FITC-labeled, cell-as previously described [25]. Total RNA was isolated fromspecific Abs. For the identification of T cells, the cellsglomeruli using a one-step method [26]. Five microgramswere reacted with FITC-labeled mouse antirat CD3of total RNA from each sample were used for chemokinemonoclonal antibody (mAb; Pharmingen, San Diego,mRNA expression analysis by RNase protection assay,

following a previously described protocol [27]. CA, USA). For identification of macrophages, the cells

Feng et al: Fractalkine in GN614

were fixed in freshly prepared HBSS/2% paraformalde-hyde for one hour on ice, washed, and permeabilizedwith 0.1% Saponin (Sigma) in staining buffer for onehour on ice. The permeabilized cells were reacted withFITC-labeled anti-ED1 mAb (Biosource International,Camarillo, CA, USA) or FITC-labeled anti-CD45RAMAb OX-33 (Pharmingen) as a control. Stained cellswere analyzed on a FACScan station (Becton DickinsonImmunocytometry Systems, San Jose, CA, USA). Cellswere gated according to their forward and side scatter,and 10,000 gated events were collected for analysis.

HistopathologyKidney tissue samples were fixed in 10% neutralized

buffered formalin (NBF) or methanol-Carnoy fixativesolution and were embedded in paraffin. For light mi-croscopy examination, 5 mm paraffin sections of NBF-fixed tissues were stained with periodic acid-Schiff (PAS)reagent. For identification of infiltrating leukocytes, 5 mmparaffin sections of the methanol-Carnoy–fixed tissueswere immunohistochemically stained for CD81 andED11 cells, as previously described [30].

RESULTSIncreased fractalkine expression in the endothelium ofnephritic glomeruli

Induction of anti-GBM GN in WKY rats led to a pro-found increase in the expression of fractalkine mRNA(Fig. 1A). The induction of fractalkine mRNA was prom-inent on days 3 and 5 after the Ab injection, persisted

Fig. 1. Characterization of chemokine and chemokine receptor expres-through day 7, and started to subside by day 9. Whension in the glomeruli of Wistar-Kyoto (WKY) rats. (A) mRNA expres-the glomeruli were fractionated by an enzyme digestionsion of fractalkine. Antiglomerular basement membrane (GBM) glo-

method [29], it was found that fractalkine was expressed merulonephritis (GN) was induced in WKY rats on day 0. On days 0,3, 5, 7, and 9, the rats were sacrificed, and glomerular RNA samplesexclusively in glomerular resident cells. Immunofluores-were prepared for analysis. Probes contain polylinker regions and arecence staining of fractalkine in the nephritic kidneylonger than the protected bands. Rat ribosomal L32 gene was used asshowed a nonlinear pattern typical of glomerular endothe- a housekeeping gene. Each lane represents one rat sample. (B, facing

lium, in contrast to the linear staining of anti-GBM Ab page, top panels) Immunofluorescence staining of fractalkine (left) andGBM (right) in the kidneys of WKY rats with anti-GBM GN. Thethat outlined the GBM (Fig. 1B). Fractalkine staining wasgreen fluorescent staining concentrated in the glomeruli is evident overonly detected in nephritic glomeruli, but not in normal the purple phase contrast microscopic background. The arrow in the

control kidneys (data not shown). Fractalkine-absorbed left panel denotes a small vessel positively stained for fractalkine. (C)mRNA expression of chemokine receptors CX3CR1, CCR2, and CCR5.Ab and preimmune serum did not stain nephritic glomer-Each lane represents one rat sample. (D, p. 616) Flow cytometry analysisuli, indicating that the fractalkine staining was specific. of CX3CR1 expression in ED11 Mφ and CD31 T cells isolated fromnephritic glomeruli. Two-dimensional dot plots showing glomerular

Increased CX3CR1 expression in glomerular leukocytes stained with (A) an unrelated Ab and anti-CD3 Ab; (B)anti-CX3CR1 Ab and anti-CD3 Ab; (C) an unrelated Ab and anti-inflammatory leukocytes and responseCD45RA Ab (as isotype control); and (D) anti-CX3CR1 Ab and anti-of these cells to fractalkineED1 Ab.

Concordant with the increased expression of fractal-kine, the expression of CX3CR1 mRNA was also in-creased in nephritic glomeruli (Fig. 1C). The time-course

matory leukocytes isolated from nephritic glomeruliof CX3CR1 mRNA expression mirrored that of its ligand.(Fig. 1D). Two-color flow cytometry analysis further lo-As a comparison, the mRNA expression of several CCcalized CX3CR1 expression to CD31 T cells and ED11chemokine receptors was also examined. CCR2 andMφ. CX3CR1 expressed in these cells was characterizedCCR5 were induced between days 3 and 7 (Fig. 1C),in functional studies. In steady-state binding assays, ne-but not CCR1 and CCR4 (not shown). Flow cytometry

analysis showed that CX3CR1 was expressed in inflam- phritic leukocytes showed a specific binding of 125I-labeled

Fig. 3. Photomicrographs (3400 magnification) of the glomeruli of antiglomerular basement membrane (GBM) glomerulonephritis (GN) in WKYrats treated with normal rabbit serum (NRS) or rabbit antibodies (Abs) against CX3CR1 (anti-CX3CR1) or CCR5 (anti-CCR5). Kidney sectionswere immunohistochemically stained for CD81 cells or ED11 Mφ or were stained with periodic acid-Schiff (PAS) reagent. Sections shown weresampled on day 3 (for CD8 staining) or day 5 (for ED1 and PAS staining) after anti-GBM Ab injection.

Feng et al: Fractalkine in GN616

Fig. 1. See legend on p. 614.

fractalkine similar to that of HEK 293 cells transfected we investigated whether normal peritoneal Mφ wouldrespond to fractalkine. Normal peritoneal Mφ showedwith CX3CR1 cDNA (Fig. 2A). The binding was inhib-little fractalkine binding (data not shown), and althoughited by anti-CX3CR1 Ab (Fig. 2B), but not by anti-CCR5responding well to RANTES (not shown), it did notAb (data not shown). In chemotaxis assays, the glomeru-respond to fractalkine in the chemotaxis assay (Fig. 2C).lar infiltrates displayed a robust chemotactic responseThese results suggest that CX3CR1 receptor regulationto fractalkine (Fig. 2C). The chemotaxis response wasmay be an important step in fractalkine recruitment andinhibited by anti-CX3CR1 Ab, but not by anti-CCR5 Abactivation of leukocytes.(Fig. 2D). As a control, glomerular leukocyte chemotaxis

response to RANTES was inhibited by anti-CCR5 Ab, Prevention of crescent glomerulonephritis by anti-CX3CR1 antibody treatmentbut not by anti-CX3CR1 Ab (Fig. 2D). Because a basal

level of CX3CR1 is expressed in normal mononuclear Because the anti-CX3CR1 Ab was capable of blockingglomerular leukocyte chemotaxis in vitro, the effect ofcells [13] and normal resident Mφ (our unpublished data),

Feng et al: Fractalkine in GN 617

Fig. 2. Leukocyte binding of and chemotaxis to fractalkine. (A) Com-petitive binding assay of 125I fractalkine with activated leukocytes fromnephritic glomeruli (left panel) and HEK 293 cells transfected withCX3CR1 (right panel). (B) Anti-CX3CR1 antibody (Ab) inhibition of125I fractalkine binding by inflammatory glomerular leukocytes andCX3CR1 transfectants. (C) Fractalkine-induced chemotaxis of inflam-matory glomerular leukocytes (j) and normal peritoneal Mφ (h). (D)Effects of anti-CX3CR1 and anti-CCR5 Abs on chemotaxis responsesof inflammatory glomerular leukocytes to fractalkine (upper panel) andRANTES (lower panel).

this Ab on the progress of anti-GBM GN was tested in treatment attenuated glomerular leukocyte infiltration inthe experimental group, with significant reduction invivo. The induction of GN in WKY rats was followed

by treatment with daily injections of anti-CX3CR1 Ab both the ED11 Mφ and CD81 cells (Fig. 3). The blockingeffect was specific; CX3CR1-adsorbed anti-CX3CR1 Abor normal rabbit serum as a control. In the control group,

a prominent accumulation of CD81 cells and ED11 Mφ failed to show any reduction in leukocyte infiltration(data not shown). As a comparison, anti-GBM GN inin the glomeruli was observed (Fig. 3). Anti-CX3CR1 Ab

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Fig. 4. Effect of various antibody (Ab) treat-ments [normal rabbit serum (NRS; j); anti-CX3CR1 Ab (h); anti-CCR5 Ab ( ) on theantiglomerular basement membrane (GBM)glomerulonephritis (GN) in Wistar-Kyoto(WKY) rats. (A) Quantitation of CD81 andED11 infiltrates per glomerulus. Kidney sec-tions stained as shown in Figure 3 were exam-ined and counted for positively stained cells.Data represent 100 glomeruli counted fromthree rat (N 5 3) kidney sections and areexpressed as mean 6 sem. The asterisk de-notes significant difference compared withNRS-treated group (P , 0.01, Student’s t-test). (B) Twenty-four–hour urinary proteinexcretion. Results were sampled from six ratsper group (N 5 6) and were expressed asmean 6 sem. The asterisk denotes a significantdifference compared with NRS-treated group(P , 0.01, Student’s t-test).

WKY rats was also treated with anti-CCR5 Ab. Anti- with the proteinuria level in different groups of rats (datanot shown).CCR5 Ab treatment led to a moderate reduction in leu-

kocyte infiltration (Fig. 3). The quantitation of glomeru-lar leukocyte infiltration in different groups of rats is DISCUSSIONshown in Figure 4A. The effects of various Ab treatments

The results establish a central role for fractalkine andon leukocyte infiltration were also reflected in the overallits receptor, CX3CR1, in the pathogenesis of crescentickidney histology (Fig. 3). The control group of rats dis-GN in the WKY rat. In vitro studies have indicated thatplayed severe glomerular hypercellularity and crescentfractalkine can be induced as a membrane protein information in the kidneys. In stark contrast, the anti-activated endothelial cells and can mediate both chemo-CX3CR1 Ab-treated group had minimal pathologicaltaxis and adhesion of cells bearing CX3CR1 [11, 13].

damage in the kidneys; crescentic formation, a majorBased on these in vitro studies, it has been suggested that

characteristic of this model, was virtually abolished by fractalkine may be specialized for directing leukocytethe anti-CX3CR1 Ab treatment. As expected from their trafficking at the endothelium and that, in turn, fractal-effects on glomerular leukocyte infiltration, the anti- kine/CX3CR1 interactions could be of critical importanceCCR5 Ab treatment showed a moderate beneficial effect at the interface between the leukocyte cell surface andover the control group. Finally, urine protein excretions the endothelium, especially in high-flow situations suchand serum creatinine levels were monitored in the con- as the renal glomeruli [15, 16].trol and treatment groups of rats as indications of renal The observation that activated glomerular leukocytesfunction. Normal renal function was largely maintained show a greater response to fractalkine than normal peri-in anti-GBM GN WKY rats treated with anti-CX3CR1 toneal Mφ suggests that the regulation of CX3CR1 ex-Ab. Urinary protein of the anti-CX3CR1 Ab-treated pression contributes in an important way in the controlgroup was remarkably low, being less than 15% of that of the fractalkine/CX3CR1 interaction. It was reportedin the control group at all time points (P , 0.01; Fig. that the fractalkine/CX3CR1 interaction has a hybrid4B). Anti-CCR5 Ab treatment also reduced proteinuria, function of adhesion and chemotaxis and suggested thatalthough not as effectively as the anti-CX3CR1 Ab treat- the fractalkine/CX3CR1-mediated adhesion was inde-

pendent of the fractalkine/CX3CR1 signaling for chemo-ment (Fig. 4B). The serum creatinine level correlated

Feng et al: Fractalkine in GN 619

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sion injury, and allograft rejection. and lymphocyte function-associated antigen-1 prevent glomerularinjury in rat experimental crescentic glomerulonephritis. J Immu-nol 150:1074–1083, 1993ACKNOWLEDGMENTS

19. Nishikawa K, Guo YJ, Miyasaka M, Tamatani T, Collins AB,Sy MS, McCluskey RT, Andres G: Antibodies to intercellularThis work was supported in part by the National Institutes of Healthadhesion molecule 1/lymphocyte function-associated antigen 1 pre-Grants R29 DK-49832 (Feng), AR-40770 (Feng), DK20043 (Wilson),vent crescent formation in rat autoimmune glomerulonephritis.5T32AI07233 (Chen), and Novartis Special Funding Project-1223J Exp Med 177:667–677, 1993(Feng). Dr. Xia was the recipient of a fellowship from the National

20. Fujinaka H, Yamamoto T, Takeya M, Feng L, Kawasaki K,Kidney Foundation of Southern California. This is publication no.Yaoita E, Kondo D, Wilson CB, Uchiyama M, Kihara I: Suppres-11792-IMM from the Department of Immunology, The Scripps Re-sion of anti-glomerular basement membrane nephritis by adminis-search Institute, La Jolla, CA, USA. We thank Ms. Carolyn Douglastration of anti-monocyte chemoattractant protein-1 antibody infor her expert technical assistance.WKY rats. J Am Soc Nephrol 8:1174–1178, 1997

21. Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, ThompsonReprint requests to Lili Feng, M.D., Department of ImmunologyDA, Siani MA, Yamamoto T, Harrison JK, Feng L: In vivo(IMM5), The Scripps Research Institute, 10550 North Torrey Pinesinhibition of CC and CX3C chemokine-induced leukocyte infiltra-Road, La Jolla, California 92037, USA.tion and attenuation of glomerulonephritis in Wistar-Kyoto (WKY)E-mail: llfimm@scripps.edurats by vMIP-II. J Exp Med 188:193–198, 1998

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