CXCL9, but not CXCL10, Promotes CXCR3-Dependent Immune-Mediated Kidney Disease

CXCL9, but not CXCL10, Promotes CXCR3-DependentImmune-Mediated Kidney Disease

Julia Menke,* Geraldine C. Zeller,* Eriya Kikawada,* Terry K. Means,† Xiao R. Huang,‡

Han Y. Lan,‡ Bao Lu,§ Joshua Farber,� Andrew D. Luster,† and Vicki R. Kelley*

*Laboratory of Molecular Autoimmune Disease, Renal Division, Brigham and Women’s Hospital, †Center forImmunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, MassachusettsGeneral Hospital, Harvard Medical School, and §Perlmutter Laboratory, Children’s Hospital and Harvard MedicalSchool, Boston, Massachusetts; ‡Department of Medicine, University of Hong Li Ka Shing Facility of Medicine, HongKong, China; and �Inflammation Biology Section, Laboratory of Molecular Immunology, National Institute of Allergyand Infectious Disease, National Institute of Child Health and Human Development, National Institute of Health,Bethesda, Maryland

ABSTRACTChemokines are instrumental in macrophage- and T cell–dependent diseases. The chemokine CCL2promotes kidney disease in two models of immune-mediated nephritis (MRL-Faslpr mice and thenephrotoxic serum nephritis model), but evidence suggests that multiple chemokines are involved. Foridentification of additional therapeutic targets for immune-mediated nephritis, chemokine ligands andreceptors in CCL2�/� and wild-type (WT) MRL-Faslpr kidneys were profiled. The focus was on intrarenalchemokine ligand/receptor pairs that were highly upregulated downstream of CCL2; the chemokineCXCL10 and its cognate receptor, CXCR3, stood out as potential therapeutic targets. However, renaldisease was not suppressed in CXCL10�/� MRL-Faslpr mice, and CXCL10�/� C57BL/6 mice were notprotected from nephrotoxic serum nephritis compared with WT mice. Because CXCR3 engages with theligand CXCL9, CXCR3�/�, CXCL9�/�, and CXCL10�/� B6 mice were compared with WT mice withnephrotoxic serum nephritis. Kidney disease, measured by loss of renal function and histopathology, wassuppressed in both CXCR3�/� and CXCL9�/� mice but not in CXCL10�/� mice. With nephrotoxic serumnephritis, CXCR3�/� and CXCL9�/� mice had fewer intrarenal activated T cells and activated macro-phages. Both IgG glomerular deposits and antigen-specific IgG in serum were reduced in these mice,suggesting that although CXCR3 and CXCL9 initiate nephritis through cell-mediated events, renalinflammation may be sustained by their regulation of IgG. It is concluded that specific blockade of CXCL9or CXCR3 may be a potential therapeutic target for human immune-mediated kidney diseases.

J Am Soc Nephrol 19: 1177–1189, 2008. doi: 10.1681/ASN.2007111179

Chemokines are instrumental in the recruitment,migration, and effector functions of immune cellsduring inflammation.1– 4 Chemokine ligands en-gaging with their cognate receptors promote the in-flux of leukocytes into the kidney, a hallmark ofnephritis.5,6 Although multiple chemokines sharethe same receptor, they are not necessarily redun-dant.7 Because chemokine ligand/receptors are in-duced during inflammation, these molecules areappealing therapeutic targets for nephritis.

Macrophages (Mø) and T cells in the kidney me-diate inflammation.8 MRL-Faslpr mice and nephro-

toxic serum nephritis (NSN) are two distinct T cell–and Mø-dependent mouse models of immune-me-diated nephritis.9 We previously determined thatCCL2 (monocyte chemoattractant protein-1) is

Received November 8, 2007. Accepted January 9, 2008.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Vicki Rubin Kelley, Harvard Institutes ofMedicine, 4 Blackfan Circle, Boston, MA 02115. Phone: 617-525-5915; Fax: 617-525-5830; E-mail: [email protected]

Copyright � 2008 by the American Society of Nephrology

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pivotal in promoting renal disease in these models. UsingCCL2�/� MRL-Faslpr mice, we established that tubular/inter-stitial and glomerular disease is suppressed.10 By comparison,tubular/interstitial but not glomerular disease is suppressed inCCL2�/� mice during NSN.10,11 In each model, Mø and T cellsare no longer recruited to sites in the interstitium adjacent totubular epithelial cells (TEC), the major source of CCL2 in WTmice with nephritis10,11; however, Mø and T cells remain inperivascular areas lacking CCL2 but rich in CCL5 (RANTES), achemokine capable of inciting local renal inflammation inMRL-Faslpr mice.12 This suggests that multiple chemokinesdictate the tempo and locale of kidney disease.

To identify the chemokines along with CCL2 that are in-strumental in T cell– and Mø-mediated nephritis, we exten-sively profiled kidneys of CCL2�/� and wild-type (WT) MRL-Faslpr mice during the development of lupus nephritis. Weidentified a highly expressed chemokine ligand/receptor pairinstrumental in attracting T cells during inflammation,CXCL10 (IP-10)/CXCR3.13–16 We report that CXCR3 is ex-pressed on intrarenal activated Mø in addition to T cells duringexperimental immune-mediated kidney disease. Furthermore,the expression of CXCR3 on T cells and Mø seems to mediatetheir recruitment into the kidneys expressing CXCL9 duringNSN. Finally, we determined that CXCR3 and one, CXCL9, butnot another, CXCL10, of its ligands promote T cell– and Mø-dependent nephritis. Thus, CXCL9 and CXCR3 are potentialtherapeutic targets for immune-mediated kidney illnesses.

RESULTS

Multiple Chemokine Ligand/Receptors AreUpregulated in MRL-Faslpr Nephritic KidneysTo identify potential therapeutic chemokine ligand/receptortargets in MRL-Faslpr mice, we compared intrarenal chemo-kine ligand/receptor transcripts in mice before (2 mo of age)and after (5 mo of age) onset of nephritis. The majority ofintrarenal chemokine ligands (18 of 23) and chemokine recep-tors (10 of 16) we evaluated increased with advancing nephritis(Supplemental Figure 1). We focused on the groups with thehighest increase in intrarenal transcript expression. Withinthis group of chemokine ligands, we detected an increase inCXCL10 (nine-fold), CXCL9 (45-fold), CXCL11 (10-fold),CXCL13 (133-fold), CCL5 (16-fold), CCL20 (seven-fold), andCX3CL1 (two-fold), and within the group of chemokine re-ceptors, we detected an increase in CXCR3 (nine-fold),CXCR4 (six-fold), CXCR5 (42-fold), CCR2 (10-fold), andCX3CR1 (six-fold; Supplemental Figure 1). Of note, the che-mokine ligand/receptor transcript levels in the MRL-Faslpr andB6 kidneys at 2 mo of age were similar.

Identifying Chemokine Ligand/Receptors Other thanCCL2 that Are Expressed in Nephritic MRL-Faslpr KidneysCCL2 promotes lupus nephritis in MRL-Faslpr mice.10 Toidentify chemokines other than CLL2 that are central to MRL-

Faslpr nephritis, we compared chemokine ligand/receptor tran-scripts in CCL2�/� and WT MRL-Faslpr kidneys. Most(�80%) chemokine ligand/receptor transcripts that were up-regulated in WT MRL-Faslpr nephritic kidneys were sup-pressed in CCL2�/� MRL-Faslpr kidneys (Supplemental Figure2). One possible interpretation is that within the hierarchicalpattern of chemokine ligand/receptor expression regulatingimmune responses, CCL2 is proximal in the chemokine cas-cade leading to nephritis in MRL-Faslpr mice; therefore, ourgoal was to focus on the chemokine ligand/receptors thatmaybe expressed “downstream” of CCL2 in MRL-Faslpr kid-neys during nephritis.

Mø and TEC Are Sources of Intrarenal CXCL10Expression during Lupus Nephritis in MRL-Faslpr MiceCXCL10 engaging with its receptor CXCR3 is a potent T cellchemoattractant.15 Because CXCL10 is among the most highlyupregulated chemokines and may be downstream of CCL2, weexplored the role of CXCL10 in MRL-Faslpr mice. Alveolar Møexpress CXCL10,17 and TEC are a rich source of multiple che-mokines in MRL-Faslpr mice10; therefore, Mø and TEC wereprime candidates in our attempt to identify sources ofCXCL10. We detected CXCL10 in TEC (Figure 1A), leukocytes(Figure 1A, inset), and endothelial cells (data not shown) inMRL-Faslpr mice with nephritis. CXCL10�/� MRL-Faslpr

kidneys served as negative controls. The frequency ofCXCL10� Mø increased (two-fold) in MRL-Faslpr kidneysfrom 3 to 6 mo of age with advancing nephritis as deter-mined by FACS analysis (Figure 1A). By comparison, thefrequency of CXCL10� T cells (CD4�, CD8�) in MRL-Faslpr kidneys, before and during nephritis, was minimal(approximately 2%; data not shown). Similarly, the expres-sion of CXCL10 in primary TEC derived from MRL-Faslpr

mice increased (more than nine-fold) as determined by re-al-time PCR (Figure 1A). Of note, CXCL10� Mø and TEC inMRL-Faslpr kidneys were similar to B6 kidneys (3 mo ofage). Thus, Mø and TEC are intrarenal sources of CXCL10during nephritis in MRL-Faslpr mice.

Frequency of CXCR3� T Cells and Mø Is Increased inMRL-Faslpr Nephritic KidneysBecause there is an increase in the frequency of intrarenalCXCL10� Mø and TEC, we probed for the expression ofCXCR3. The frequency of CD4�, CD8�, and B220� (uniquedouble-negative) T cells and Mø (CD68�) expressing CXCR3in the kidneys of MRL-Faslpr mice increased with nephritis (6mo of age) as compared with non-nephritic MRL-Faslpr and B6mice (2 mo of age; Figure 1B). The increased frequency ofCXCR3� T cells and Mø was not limited to the kidney; theseleukocytes increased in their spleens (p � 0.05; data notshown). Taken together, an increase in intrarenal and extrare-nal CXCR3� T cell subsets and Mø is associated with a rise inintrarenal CXCL10 in nephritic MRL-Faslpr mice.


1178 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008

Nephritis, Systemic Disease, and Survival Are notAltered in CXCL10�/� MRL-Faslpr MiceTo determine whether CXCL10 is central to lupus nephritisand the systemic illness, we compared CXCL10�/� and WTMRL-Faslpr strains (6 mo of age). The severity of glomerular,interstitial, and perivascular pathology was similar inCXCL10�/� and WT MRL-Faslpr mice (Figure 1C). Similarly,we did not detect a difference in the number (CD68�, CD4�,CD8�, B220� cells) and activation stages (CD69�, IFN-��;data not shown) of infiltrating leukocytes and renal function(Figure 1C). Furthermore, we did not detect a difference insurvival in CXCL10�/� MRL-Faslpr mice (data not shown).Thus, despite enhanced intrarenal expression of CXCL10 andCXCR3 during nephritis, CXCL10 is not central to disease inMRL-Faslpr mice.

CXCR3 and Its Ligands, CXCL9, CXCL10, and CXCL11,Are Upregulated during NSNOur results suggest that CXCL10 is not central to lupus nephri-tis; however, it is possible that CXCR3 engaging with otherligands (CXCL9, CXCL11) promotes T cell– and Mø-medi-ated renal diseases. To explore this possibility, we selected arapidly induced T cell– and Mø-dependent nephritis, NSN.Using this model, we evaluated the expression of CXCR3 andits ligands. Intrarenal CXCR3, CXCL10, CXCL9, and CXCL11transcripts increased during NSN (Figure 2A). Thus, as in theMRL-Faslpr nephritic kidneys, intrarenal CXCR3 and its li-gands are upregulated during NSN.

To identify the leukocyte populations expressing CXCR3and its ligands, we probed CD68� and T cells (CD4�, CD8�)for CXCR3 expression. Of note, a similar percentage of intra-

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Figure 1. Despite an increase in intrarenal Mø and TEC expressing CXCL10, and CXCR3 expressing T cells and Mø in MRL-Faslpr

nephritic kidneys, CXCL10�/� MRL-Faslpr mice are not protected from renal disease. (A) CXCL10 expression in the kidney was examinedby immunostaining, flow cytometry (Mø), and real-time PCR (TEC). CXCL10�/� mice served as negative control. (B) CXCR3 expressionwas assessed on CD4�, CD8�, B220� T cells and Mø (CD68�) in kidneys of MRL-Faslpr mice by flow cytometry. B6 mice at 2 mo of ageserved as normal controls. (C) Renal function, pathology, and leukocytic infiltrates are similar in CXCL10�/� MRL-Faslpr mice ascompared with MRL-Faslpr mice. Data are means � SEM.

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J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1179

renal CD68� and CD11b� cells expressedCXCR3; therefore, we refer to theseCD68� cells as Mø (data not shown). Thefrequency of intrarenal CD4�, CD8� Tcells, and Mø expressing CXCR3 in-creased during NSN compared with un-treated mice (Figure 2B). We verifiedCXCR3 antibody (Ab) specificity usingCXCR3�/� mice with NSN. Although it iswidely appreciated that CXCR3 is ex-pressed on activated T cells, reports ofCXCR3� Mø are limited.15,18 To evaluatefurther CXCR3� Mø, we determined thatthere is an increased frequency ofCXCR3� bone marrow macrophages(BMMø), (F4/80�; Figure 2C) andCD11b� (data not shown) after stimula-tion with LPS (Figure 2C) and IFN-�(data not shown) in WT mice. The speci-ficity of CXCR3 Ab was verified by usingCXCR3�/� BMMø and an isotype con-trol. Because activated Mø induce apo-ptosis of renal parenchymal cells,11 weevaluated the frequency of activated Møduring NSN. Intrarenal CXCR3�-acti-vated Mø increased during NSN as com-pared with untreated mice (Figure 2D).Thus, intrarenal CXCR3�-activated Møand T cells are upregulated during NSN.

We next determined the intrarenal localeof the ligands (CXCL9, CXCL10) binding toCXCR3 during NSN. Because there is afunctional loss of CXCL11 protein as a re-sult of a missense mutation at 36 bp (G/A)and a single-nucleotide deletion mutationat 39 bp (C/�) in the gene for CXCL11 in B6mice (Genbank sequence accession no.NT_109320; A.D.L. et al., unpublished ob-servations), we evaluated CXCL9 andCXCL10. CXCL9 is expressed on the major-ity of TEC and absent on CXCL9�/� kid-neys during NSN and in untreated mice(Figure 2E). In addition, a small populationof leukocytes express CXCL9 (Figure 2E).These intrarenal CXCL9� leukocytes thatincreased during NSN are CD4� andCD68� cells (Figure 2E). Similarly,CXCL10 is expressed primarily on TEC andto a lesser extent on leukocytes and is absentin CXCL10�/� control kidneys during NSNand in untreated mice (Figure 2E). The in-crease in intrarenal CXCL10� leukocytesduring NSN is in CD4�, CD8�, and CD68�

cells (Figure 2E). Although the frequency ofthese intrarenal CXCL10� leukocytes is

Figure 2. T cells and activated Mø increase during NSN, whereas TEC are a primarysource of CXCL9 and CXCL10 during NSN (day 14). (A) Expression of CXCR3 and itsligands CXCL10, CXCL9, and CXCL11 during NSN were evaluated by real-time PCR. (B)The frequency of intrarenal T cells (CD4� and CD8�) and Mø expressing CXCR3. Noterepresentative FACS plots of CXCR3 expression on T cells and Mø in WT and controlCXCR3�/� mice during NSN. (C) The frequency of cultured BMMø expressing CXCR3rises in response to stimulation with LPS. To verify staining specificity, we analyzedCXCR3�/� BMMø stimulated with LPS and B6 BMMø stimulated with LPS and stainedwith an isotype control. These data are representative of three samples and twoadditional experiments that used isotype controls alone. (D) Intrarenal activated Møincreased during NSN. We determined the total number of intrarenal activated (CD86�)CXCR3� Mø by multiplying the frequency by the total number of Mø. (E) CXCL9 andCXCL10 are mainly expressed by TEC during NSN (n � 4/group). Intrarenal CXCL9�

CD4� and CD68� cells and CXCL10� CD4�, CD8�, and CD68� cells increase duringNSN. We analyzed 0.5 to 1 � 106 cells (B, D, and E) and 2.5 � 105 cells (C) by FACSanalysis. Data are means � SEM.



somewhat higher than in CXCL9� cells, it was substantially(�2�) lower than in CXCR3� cells (Figure 2, B and E). Thus,intrarenal CXCR3 is primarily expressed on activated T cells and

Mø, whereas the ligands CXCL9 and CXCL10 are largely gener-ated by TEC during NSN. Of note, CXCL9 and CXCL10 expres-sion and CXCR3� leukocytes are rarely detected in glomeruli

Figure 3. CXCR3�/� and CXCL9�/� but not CXCL10�/� mice are protected from loss of renal function and renal pathology duringNSN (day 14). (A) The rise in proteinuria, blood urea nitrogen (BUN; day 14), and serum creatinine (day 14) was suppressed in CXCR3�/�

and CXCL9�/� but not CXCL10�/� mice as compared with WT mice during NSN. Data are means � SEM; n � 4 to 9/group. (B) Tubularand glomerular pathology is reduced in CXCR3�/� and CXCL9�/� mice as compared with WT mice. In contrast, tubular and glomerularpathology is similar in CXCL10�/� mice and WT mice during NSN. Representative photomicrographs are shown. Data are means �SEM; n � 7 to 9/group. Magnification, �40.



during NSN (data not shown). This pattern of enhanced CXCR3and its cognate ligand expression during NSN is consistent withour findings in MRL-Faslpr kidneys.

CXCR3 and CXCL9 but not CXCL10 Promote NSNTo determine whether CXCR3 regulates intrarenal T cell– andMø-mediated renal disease via ligands other than CXCL10, we

compared CXCR3�/�, CXCL9�/�, and CXCL10�/� mice withWT mice during NSN. Because CXCR3�/�, CXCL9�/�, andCXCL10�/� mouse strains do not express a functional CXCL11,for simplicity, we refer to each strain as the single unique “knock-out.” CXCR3�/� mice were protected from the loss of renal func-tion (Figure 3A). By comparison, renal function in CXCL10�/�

mice was similar to WT mice during NSN (Figure 3A). Consistent

Figure 4. Intrarenal activated T cells and activated Mø are suppressed in CXCR3�/� and CXCL9�/� mice as compared with WT miceduring NSN (day 14). (A) The number of CD4� and CD8� T cells and Mø detected by immunostaining is diminished within glomeruli(intra-, periglomerular) and in the interstitium in CXCR3�/� and CXCL9�/� mice as compared with WT mice during NSN. (B) Wedetected a suppression of activated (CD69�) CD4� and CD8� T cells and activated (CD86�) Mø in CXCR3�/� (top) and CXCL9�/�

(bottom) mice as compared with WT mice during NSN. We analyzed 0.5 to 1.0 � 106 cells by FACS (n � 6 to 9/group). (C) We evaluatedkidneys from CXCR3�/� and WT mice for the presence of activated Mø (CD68�, TNF-��) during NSN using immunofluorescence.Representative photomicrographs illustrate CD68� cells expressing TNF-�� (circled) in the CXCR3�/� compared with WT kidneys. Dataare means � SEM; n � 4/group.



with preserving renal function, renal pathology in CXCR3�/� butnot in CXCL10�/� mice was reduced (Figure 3B). Tubular andglomerular pathology were blunted in CXCR3�/� but not inCXCL10�/� mice. Thus, CXCR3 but not CXCL10 promotesNSN, suggesting that another CXCR3 ligand is instrumental inimmune-mediated nephritis.

To determine whether CXCL9, another ligand for CXCR3,promotes immune-mediated nephritis, we compared

CXCL9�/� and WT mice during NSN. Renal function (pro-teinuria, blood urea nitrogen, and serum creatinine) was im-proved in CXCL9�/� mice (Figure 3A). Similarly, renal pa-thology (glomerular and tubular) was suppressed inCXCL9�/� mice (Figure 3B, Supplemental Figure 3). Notably,renal function (Figure 3A) and pathology (data not shown) inCXCR3�/� and CXCL9�/� mice did not return to baseline(untreated mice). This suggests that CXCR3 and one, CXCL9,

Anti-Sheep /Rabbit IgG ELISA

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but not another, CXCL10, of its ligands promote immune-mediated nephritis.

Intrarenal Activated T Cells and Mø Are Decreased inCXCR3�/� and CXCL9�/� Mice during NSNBecause CXCR3 is expressed on intrarenal T cells (CD4�,CD8�) and Mø during NSN, we hypothesized these intrarenalleukocytes decrease in CXCR3�/� as compared with WT mice.Fewer CD4�, CD8�, and Mø were detected in the glomeruli(intra-, periglomerular) and interstitium in CXCR3�/� mice(Figure 4A). A similar decrease in CD4� T cells and Mø oc-curred in the CXCL9�/� kidneys, whereas we did not detect areduction in these leukocytic populations in CXCL10�/� mice(data not shown).

CXCR3 is expressed on activated T cells19 and Mø; there-fore, we compared these intrarenal leukocytes in CXCR3�/�

mice with WT mice during NSN. We detected a decreasedfrequency of activated (CD69�) CD4� and CD8� T cells andactivated (CD86�) Mø in CXCR3�/� mice during NSN (Fig-ure 4B). In support of this decrease in activated Mø, the fre-quency of TNF-�� Mø was diminished (Figure 4C, circled,immunofluorescence). Similarly, CXCL9�/� mice had a re-duced frequency of activated T cells and Mø (Figure 4B). Thus,CXCR3 engaging with CXCL9 fosters an intrarenal accumula-tion of activated CD4�, CD8� T cells and Mø during NSN.

Serum Antigen-Specific IgG Isotypes and GlomerularIgG Deposits Are Suppressed in CXCR3�/� andCXCL9�/� Mice during NSNElevated Ig and Ig class switching are hallmarks of autoimmunedisease20,21; therefore, we determined whether the attenuated se-verity of kidney disease in CXCR3�/� and CXCL9�/� mice wasrelated to a reduction in circulating levels of antigen-specific IgGisotypes during NSN. For this purpose, we measured the anti-sheep and -rabbit IgG isotypes in the sera of CXCR3�/� andCXCL9�/� mice, respectively, in comparison with WT mice dur-ing NSN. We detected a suppression of IgG and IgG1 and notIgG2b at multiple titers (2.0 to 8.0 � 103) in CXCR3�/� andCXCL9�/� mice (Figure 5A). Because serum isotypes were atten-uated in CXCR3�/� and CXL9�/� mice, we hypothesized that thereduced renal pathology resulted from decreased deposition ofIgG within glomeruli of CXCR3�/� and CXCL9�/� mice. Wedetected a reduction in IgG deposits at multiple dilutions (3.0 to7.5 � 103) in CXCR3�/� and CXCL9�/� glomeruli (Figure 5B).Contrastingly, circulating anti-sheep IgG isotypes and glomerularIgG deposits in CXCL10�/� did not differ from WT mice. Takentogether, this suggests CXCR3 engaging with CXCL9 may pro-mote renal disease by increasing systemic IgG and IgG depositswithin glomeruli.

DISCUSSION

Chemokines are tempting therapeutic targets for a wide rangeof diseases, because they are specifically upregulated during

inflammation. Mø and T cells are instrumental in mediatingrenal inflammation; thus, chemokines that regulate Mø and Tcells within the kidney are potential therapeutic targets for ne-phritis. Therefore, we extensively profiled MRL-Faslpr kidneysbefore and during Mø- and T cell– dependent lupus nephritis.Although previous reports claimed that a few chemokine li-gands (four of nine) and chemokine receptors (three of six) areupregulated in MRL-Faslpr kidneys, we now report that themajority of chemokine ligands (18 of 23) and chemokine re-ceptors (10 of 16) that we profiled were upregulated in MRL-Faslpr mice with lupus nephritis.6,22 This difference in our find-ings may be related to the larger panel of chemokine ligand/receptors in our experiments and the detection system(ribonuclease protection assay22 versus real-time PCR, ourstudy). We previously established that CCL2 is required topromote Mø- and T cell– dependent lupus nephritis in MRL-Faslpr mice.10 We now report that the rise in the panel of che-mokine ligand/receptors that increase in MRL-Faslpr mice issuppressed in CCL2�/� MRL-Faslpr mice. This may suggestthat CCL2 is a “proximal master switch” in the chemokinecascade; however, increased expression of a specific chemokineduring disease does not necessarily indicate that it promotesinflammation; it may not have an impact or even thwart in-flammation. Therefore, we investigated the impact of CXCR3and its ligands, CXCL9 and CXCL10, during Mø and T cellimmune–mediated nephritis (NSN). Of note, we could notanalyze the impact of CXCL11 during NSN because the proteinfor this remaining CXCR3 ligand is not expressed in B6 mice.We now report that CXCR3 and CXCL9 but not CXCL10 arecentral to Mø- and T cell– dependent kidney disease.

The impact of CXCL10 in autoimmune and kidney diseaseis controversial. Whereas Ab blockade of CXCL10 in experi-mental insulin-dependent diabetes23 and adjuvant arthritis24

abrogates disease, deleting CXCL10 does not diminish experi-mental autoimmune encephalomyelitis, although it lowersdisease threshold.25 Thus, the consequences of CXCL10 engag-ing with CXCR3 in autoimmune diseases are complex and yetnot fully understood. Furthermore, the impact of CXCL10 onglomerular and tubulointerstitial renal disease is unclear. Ininduced forms of renal injury, puromycin aminonucleoside–and anti-nephrin Ab–induced nephropathy,26 Ab blockade ofCXCL10 exacerbated proteinuria and podocyte injury. Con-versely, Ab blockade of CXCL10 in a model of renal endothelialinjury reduced interstitial T cell infiltration and improved re-nal function; however, pathology was not evaluated.27 We nowreport that despite the abundant protein expression of intrarenalCXCL10 on TEC and leukocyte infiltrates, CXCL10 is not instru-mental in two distinct models of immune-mediated renal disease,MRL-Faslpr mice and NSN. This indicates that CXCL10 alone isnot central to immune-mediated kidney disease.

Because it is possible that other ligands alone or togetherwith CXCL10 promote immune-mediated kidney disease, wecompared CXCR3�/� and CXCL9�/� mice during NSN withWT mice. CXCR3 and CXCL9 but not CXCL10 are central toimmune-mediated kidney disease. This is the first report indi-



cating that CXCL9�/� mice are protected from nephritis. Ourfindings are in agreement with recent studies reporting thatCXCR3�/� mice develop less severe nephritis than WT miceduring NSN.28 In fact, CXCR3 has been shown to incite non-immune-induced renal inflammation because CXCR3 orches-trates the recruitment of T cells instrumental in mediating re-nal ischemic injury.16 Thus, CXCR3 may be a potentialtherapeutic target for a broad array of kidney diseases. Ourfindings are intriguing; although CXCL9 and CXCL10 proteinexpression is similar in locale and intensity during NSN, thefunctional impact of these two CXCR3 ligands differ. This dif-ferential impact on nephritis may be related to distinct bio-availability of CXCL9 and CXCL10. The preferential contribu-tion of one CXCR3 ligand to disease pathology has been seen inother inflammatory models of disease, such as CXCL10 inmouse hepatitis virus and Dengue virus encephalitis29,30 andCXCL9 in herpes simplex virus-1 ocular infection and cyto-megalovirus infection.31,32 Regardless of the exact mecha-nisms, our findings indicate that selective blockade of CXCL9and CXCR3 is a potential therapeutic strategy to combat im-mune-mediated renal diseases.

Although it is well appreciated that activated T cells expressCXCR3, we now report that activated and resting Mø expressCXCR3 during kidney disease. The frequency of intrarenalCD68� cells along with CD4� and CD8� T cells expressingCXCR3 increased during NSN. This rise in CXCR3� Mø in thekidney is consistent with studies reporting that a low percentage ofCXCR3� monocytes in the normal circulation increases in pa-tients with rheumatoid arthritis.33 Furthermore, we determinedthat whereas some primary cultured BMMø express CXCR3, thefrequency increases after IFN-� stimulation and is even more ro-bust after LPS stimulation. This suggests that whereas low num-bers of resting Mø express CXCR3, the increase in CXCR3� Mø isa reflection of a rise in activated Mø during renal inflammation.Consistent with this concept, the total number of intrarenal acti-vated CXCR3� Mø increases during NSN as compared with un-treated mice. Furthermore, CXCR3 is responsible for recruitingthese activated Mø, because there is a decrease in intrarenal acti-vated Mø, along with activated CD4� and CD8� T cells, inCXCR3�/� during NSN. This suggests that activated Mø bearingCXCR3 are central to inducing renal injury, because activated Mørelease mediators that initiate TEC apoptosis.11 Thus, we providethe first evidence that CXCR3 is instrumental in recruiting acti-vated Mø, as well as T cells, into the kidney, that are responsible forinciting renal injury.

Humoral and cell-driven immune mechanisms mediate kid-ney disease.20 We investigated whether the suppression in renaldisease in the CXCR3�/� and CXCL9�/� mice during NSN wasrelated to a decrease in pathogenic Ab within the kidney. We de-tected a reduction in total serum antigen-specific IgG and IgG1Ab in CXCR3�/� and CXCL9�/� mice during NSN. Our findingis reminiscent of reduced Ab against a bacterial pathogen (Fran-cisella tularensis) in CXCL9�/� mice34; however, our data are notin agreement with the recent study of CXCR3�/� mice, indicatingthat there is no difference in total mouse anti-sheep IgG or anti-

sheep IgG1 during NSN (day 8).28 This difference may be relatedto the assay methods, the day of analysis, or other variables relatedto inducing NSN. Nevertheless, our data support the concept thatCXCR3 and CXCL9 enhance Ab production during immune re-sponses. The regulation of serum IgG and glomerular deposits byCXCR3 and CXCL9 remains unclear. We speculate that distinctpatterns of CXCR3�CD4� T cells and CXCL9 expression in ger-minal centers during inflammation and/or the expression ofCXCR3 on a subset of memory B cells and plasma cells may reg-ulate serum IgG and, in turn, glomerular deposits.35,36 Notably,the decline in antigen-specific IgG in CXCR3�/� and CXCL9�/�

serum correlates with diminished IgG deposits within glomeruli;therefore, suppressed renal pathology and improved renal func-tion may, in part, result from a reduced intrarenal deposition ofpathogenic Ab in CXCR3�/� and CXCL9�/� mice during NSN.In addition, we did not detect CXCL9 and CXCL10 expressionand CXCR3� leukocytes in glomeruli of WT mice duringNSN. Thus, the reduction in proteinuria in CXCR3�/� andCXCL9�/� mice during the later stages of NSN may be re-lated to the suppression of humoral immune responses.This is consistent with findings indicating that although thegeneration of autologous murine IgG against the nephro-toxic Ab is not essential for disease initiation, it may con-tribute to sustaining renal dysfunction.37 This may indicatethat although CXCR3 and CXCL9 promote the initiation ofNSN via cell-mediated events, their regulation of the pro-duction and deposition of pathogenic Ab may be responsi-ble for sustaining immune-mediated renal disease.

In conclusion, CXCR3 and one, CXCL9, but not another,CXCL10, of its ligands mediate activated T cells and Mø re-cruitment into the kidney and enhance Ab deposition in glo-meruli that may, in turn, promote immune-mediated kidneydisease. This suggests that blocking CXCR3 and CXCL9 is apotential therapeutic target for human immune-mediated kid-ney diseases.

CONCISE METHODS

MiceWe purchased MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) and C57BL/6

(B6) from the Jackson Laboratory (Bar Harbor, ME). Mice were

housed and bred in our pathogen-free animal facility. CXCL9 null

(�/�), CXCL10�/�, CXCR3�/� (B6 background), and CCL2�/�

(MRL-Faslpr background) mice were generated as described previous-

ly.4,10,34,38 We generated CXCL10�/� MRL-Faslpr mice using a back-

cross-intercross scheme. MRL-Faslpr mice were mated with

CXCL10�/� (B6 background) mice to yield heterozygous F1 off-

spring. We intercrossed F1 mice and screened the progeny for dis-

rupted and intact CXCL10 and the Faslpr mutation in tail genomic

DNA. The DNA was assessed by PCR using oligonucleotide primers

that recognized the normal CXCL10 gene: Sense (5�-TCC CTC CCG

TAA CCA CAC AGT AAA T-3�) and antisense (5�-GCG GAT AGA

CTC TGC TTT CAC TTT GG-3�) and Neo gene sense (5�-TGG ATG

TGG AAT GTG TGC GAG-3�) and antisense (5-TTT CAC TTT GG-



3�). Gel analysis of the PCR products identified the CXCL10 and Neo

gene fragments at 342 and 640 bp, respectively. The Faslpr mutation

was identified as previously reported.39 After five generations of back-

cross matings (N5), we analyzed and compared CXCL10�/� MRL-

Faslpr mice with age- and gender-matched WT MRL-Faslpr litter-

mates. The number of mice analyzed is specified in each figure. We

compared these strains at the N5 generation, because we previously

established that there are sufficient MRL-Faslpr background genes to

result in consistent phenotypic changes characteristic of MRL-Faslpr

mice.40 Use of mice in our study was reviewed and approved by the

Standing Committee on Animals in the Harvard Medical School in

adherence to the National Institutes of Health Guide for the Care and

Use of Laboratory Animals.

Inducing NSNWe prepared nephrotoxic serum by immunizing sheep with a partic-

ulate fraction of mouse glomerular basement membrane from B6

mice kidneys, as described previously.41 To induce NSN, we primed

CXCL10�/�, CXCR3�/�, and WT male mice (6 wk of age) by inject-

ing 0.5 mg of sheep IgG in Freund’s complete adjuvant (Sigma Chem-

ical Co., St. Louis, MO) subcutaneously in each flank. We challenged

these mice 7 d later with an intravenous injection of nephrotoxic

serum (15 �l/g body wt). We used nephrotoxic serum from rabbits to

immunize CXCL9�/� and WT mice as described previously.42 Mice

were killed 14 d after challenge and analyzed. The number of mice in

each group is specified within each figure.

Intrarenal Chemokine and Chemokine ReceptorTranscriptsWe analyzed the expression of chemokines (CXCL10, CXCL9,

CXCL11, CXCL12, CCL13, CCL5, CCL28, CCL22, CCL24, CCL1,

CCL17, CX3CL1, CCL25, CCL2, CCL8, CCL7, CCL3, CXCL2,

CCL28�, CCL4, CCL28�, CCL27, CCL21, CXCL16, and CXCL1) and

chemokine receptors (CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,

CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,

CCR10, and CX3CR1) in MRL-Faslpr mice by using real-time, two-

step, quantitative PCR, as described previously.43 Primers were de-

signed, as previously reported.44 To detect CXCL10 expression during

NSN, we used primers designed by Applied Biosystems (Foster City,

CA). To detect CXCL9, CXCL11, and CXCR3 expression during

NSN, we used the following primers: glyceraldehyde-3-phosphate de-

hydrogenase sense 5�-CAT GGC CTC CAA GGA GTA AG-3� and

antisense 5�-CCT AGG CCC CTC CTG TTA TT-3�; CXCL9 sense

5�-TCC TTT TGG GCA TCA TCT TC-3� and antisense 5�-TTC CCC

CTC TTT TGC TTT TT-3�; CXCL11 sense 5�-AGT AAC GGC TGC

GAC AAA GT-3� and antisense 5�-GCA TGT TCC AAG ACA GCA

GA-3�; and CXCR3 sense 5�-TGA GAC AAC TGA GGC CTC CTA-3�

and antisense 5�-TCT TGC TCC CCA GTT GAT G-3� (Invitrogen,

Carlsbad CA). We evaluated expression as previously reported.45

Isolation of TEC and BMMøWe isolated and cultured TEC from B6 and MRL-Faslpr mice as pre-

viously reported,46 and we isolated and cultured BMMø from B6 mice

as described previously.47

Flow CytometryWe prepared and stained single-cell suspensions from kidneys,

spleens, or primary cultured TEC/BMMø as described previously.48

We collected 0.5 to 1.0 � 106 total kidney or spleen cells and 0.5 to

1.0 � 105 of cultured cells using a FACSCalibur (Becton Dickinson,

San Jose, CA) and analyzed data using Flowjo software (Tree Star,

Palo Alto, CA).

AntibodiesWe used the following Ab from eBioscience (San Diego, CA) for FACS

analysis: FITC-conjugated anti-CD4 (L3T4), anti-CD8 (53-6.7), anti-

B220 (RA3-6B2), and anti-CD45.2 (104); PE-conjugated anti-CD4,

anti-CD45.2, anti-CD69 (H1.2F3), and anti-CD86 (GL1); PE-Cy5–

conjugated anti-CD8 and anti–TCR-� chain (H57-597); and allophy-

cocyanin-conjugated anti-CD4, anti-CD45.2, and anti-F4/80 (BM8).

We used FITC- and allophycocyanin-conjugated anti-CD68 Ab

(FA11; Serotec, Oxford, UK). We used purified rabbit anti-mouse

CXCR3 Ab (Zymed, San Francisco, CA), purified goat anti-mouse

CXCL9 Ab (R&D Systems, Minneapolis, MN), and rabbit anti-mouse

CXCL10 Ab (PeproTech, Rocky Hill, NJ). The following isotype-spe-

cific Ab were used for controls: Rat-IgG2a (BR2a), rat-IgG2b (KLH/

G2b-1-2), and rabbit-IgG (eBioscience). As secondary PE- or allophy-

cocyanin-conjugated Ab, we used goat anti-rabbit (Jackson

ImmunoResearch Laboratories; West Grove, PA) and biotin-conju-

gated rabbit anti-goat Ab (Vector Laboratories, Burlingame, CA). To

detect biotin-conjugated secondary Ab, we used streptavidin PE or

allophycocyanin (Jackson ImmunoResearch Laboratories). We iden-

tified renal proximal tubules by binding of fluorescein-conjugated

lotus lectin (Vector Laboratories).49,50

HistopathologyWe fixed kidneys in 10% formalin and prepared and stained paraffin

sections with the periodic acid Schiff reagent. Slides were coded before

grading the renal pathology.

MRL-Faslpr Mice.We evaluated renal (glomerular, tubular, intersti-

tial, and perivascular) pathology on a scale of 0 (normal) to 3 (severe)

as described previously.45

NSN. We evaluated CXCL9�/�, CXCL10�/�, CXCR3�/�, and

B6 kidneys for glomerular and tubular damage as described previ-

ously.11

Gross PathologyWe evaluated skin lesions monthly from 2 to 6 mo of age using a

scoring system previously described.10

ImmunohistochemistryWe stained cryostat-cut kidney sections for the presence of Mø with

anti-CD68 Ab (FA-11; Serotec) and for T cells with anti-CD4 Ab

(RM4-5), anti-CD8 Ab (53-6.7), and anti-B220 (RA3– 6B2) rat anti-

mouse Ab (Pharmingen, San Diego, CA) according to a previously

described immunoperoxidase method.51 To evaluate CXCL9 and

CXCL10 expression in the kidney, we stained frozen sections with

goat anti-mouse CXCL9 and rabbit anti-mouse CXCL10. The follow-



ing isotype-specific Ab were used for controls: Rat-IgG2a (R35–95),

rat IgG2b (R35-38), rabbit IgG (eBioscience), and goat IgG (Southern

Biotechnology, Birmingham, AL). The secondary Ab for immuno-

staining was biotin-conjugated rabbit anti-rat IgG (Vector Laborato-

ries). The immunostaining was analyzed by counting for the presence

of CD68�, CD4�, CD8�, and B220� cells within and surrounding

glomeruli and in the interstitium in 10 randomly selected high-power

fields. Of note, we have determined that the B220� cells in the kidney

of MRL-Faslpr mice are the unique double-negative (CD4�CD8�

double-negative) T cells and are not B cells.52

To determine the number of activated Mø in the kidney during

NSN, we fixed frozen kidney sections in paraformaldehyde, stained

them with rat anti-mouse TNF-�–PE (MP6-XT22; eBioscience) and

anti–CD68-FITC (FA-11; Serotec), and analyzed these sections using

a fluorescence microscope. The frequency (%) of activated Mø was

assessed by enumerating the number of CD68�TNF-�� cells within

the total number of Mø (CD68�) in five high-power fields.

Renal FunctionWe assessed urine proteins semiquantitatively by dipstick analysis as

described previously.45 We measured blood urea nitrogen levels using

a colorimetric analysis kit (Infinity; Thermo Electron, Melbourne,

Australia) and serum creatinine using the creatinine reagent kit and

Creatinine Analyzer 2 (Beckman Coulter, Galway, Ireland) according

to the manufacturer’s instructions.

SurvivalWe compared survival of the CXCL10�/� MRL-Faslpr and WT MRL-

Faslpr mice using similar numbers of males and females from birth to

10 mo of age.

Serum Anti-Sheep and Anti-Rabbit IgG IsotypesWe measured the levels of mouse anti-sheep IgG Ab (Sigma Chemical

Co.) and anti-rabbit IgG (Jackson ImmunoResearch Laboratories) by

ELISA using sera collected at day 14 during NSN as described previ-

ously.53 We detected bound mouse anti-sheep/anti-rabbit IgG, IgG1,

and IgG2b using peroxidase-conjugated rabbit anti-mouse IgG Ab

(1:5000; Southern Biotechnology).

IgG Deposits within the KidneyWe evaluated IgG deposits within renal glomeruli as described previ-

ously.54 We evaluated stained sections by scoring 20 glomeruli as ei-

ther positive or negative and graded the amount (severity) of deposits

in 20 positive glomeruli per specimen on a scale of 0 to 3 using mul-

tiple dilutions (1:3000, 1:4500, 1:6000, and 1:7500) of the detection

Ab (fluorescein-conjugated goat anti-mouse IgG; MP Biomedicals,

Aurora, OH).40

Statistical AnalysesThe data are means � SEM and were analyzed by GraphPad Prism 4.0

(GraphPad, San Diego, CA). We used the nonparametric Mann-

Whitney U test to determine differences among groups. Survival

curves were compared and analyzed using the log-rank test.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants DK

52369 (V.R.K.), DK 56848 (V.R.K.), DK 36149 (V.R.K.), KO1 AR

051367 (T.K.M.), and RO1 CA 069212 (A.D.L.); by Deutsche For-

schungsgemeinschaft grants ME-3194/1-1 (J.M.) and ZE-711/1

(G.Z.); and the Research Grants Council of Hong Kong HKU 7592/06

(H.Y.L.).

We thank Dr. Craig Gerard for providing CXCR3�/� mice, Dr.

Tanya Mayadas for the rabbit anti– glomerular basement membrane

anti-serum, and Dr. Julie Lucas for editorial assistance.

DISCLOSURESNone.

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