BASIC RESEARCH www.jasn.org

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

Julia Menke,* Geraldine C. Zeller,* Eriya Kikawada,* Terry K. Means,† Xiao R. Huang,‡ ʈ 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 for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, and §Perlmutter Laboratory, Children’s Hospital and Harvard Medical School, Boston, Massachusetts; ‡Department of Medicine, University of Hong Li Ka Shing Facility of Medicine, Hong ʈ Kong, China; and Inflammation Biology Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Disease, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, Maryland

ABSTRACT are instrumental in - and –dependent diseases. The CCL2 promotes kidney disease in two models of immune-mediated nephritis (MRL-Faslpr mice and the nephrotoxic serum nephritis model), but evidence suggests that multiple chemokines are involved. For identification of additional therapeutic targets for immune-mediated nephritis, chemokine ligands and Ϫ Ϫ receptors in CCL2 / and wild-type (WT) MRL-Faslpr kidneys were profiled. The focus was on intrarenal chemokine ligand/ pairs that were highly upregulated downstream of CCL2; the chemokine CXCL10 and its cognate receptor, CXCR3, stood out as potential therapeutic targets. However, renal Ϫ Ϫ Ϫ Ϫ disease was not suppressed in CXCL10 / MRL-Faslpr mice, and CXCL10 / C57BL/6 mice were not protected from nephrotoxic serum nephritis compared with WT mice. Because CXCR3 engages with the Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ligand CXCL9, CXCR3 / , CXCL9 / , and CXCL10 / B6 mice were compared with WT mice with nephrotoxic serum nephritis. Kidney disease, measured by loss of renal function and histopathology, was Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ suppressed in both CXCR3 / and CXCL9 / mice but not in CXCL10 / mice. With nephrotoxic serum Ϫ Ϫ Ϫ Ϫ nephritis, 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, renal inflammation may be sustained by their regulation of IgG. It is concluded that specific blockade of CXCL9 or 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, toxic serum nephritis (NSN) are two distinct T cell– migration, and effector functions of immune cells and Mø-dependent mouse models of immune-me- during inflammation.1–4 Chemokine ligands en- diated nephritis.9 We previously determined that gaging with their cognate receptors promote the in- CCL2 ( chemoattractant -1) is flux of leukocytes into the kidney, a hallmark of nephritis.5,6 Although multiple chemokines share Received November 8, 2007. Accepted January 9, 2008. the same receptor, they are not necessarily redun- Published online ahead of print. Publication date available at dant.7 Because chemokine ligand/receptors are in- www.jasn.org. duced during inflammation, these molecules are Correspondence: Dr. Vicki Rubin Kelley, Harvard Institutes of appealing therapeutic targets for nephritis. Medicine, 4 Blackfan Circle, Boston, MA 02115. Phone: 617-525- (Mø) and T cells in the kidney me- 5915; Fax: 617-525-5830; E-mail: [email protected] lpr diate inflammation.8 MRL-Fas mice and nephro- Copyright ᮊ 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 1177–1189, 2008 ISSN : 1046-6673/1906-1177 1177 BASIC RESEARCH www.jasn.org pivotal in promoting renal disease in these models. Using Faslpr nephritis, we compared chemokine ligand/receptor tran- CCL2Ϫ/Ϫ MRL-Faslpr mice, we established that tubular/inter- scripts in CCL2Ϫ/Ϫ and WT MRL-Faslpr kidneys. Most stitial and glomerular disease is suppressed.10 By comparison, (Ͼ80%) chemokine ligand/receptor transcripts that were up- tubular/interstitial but not glomerular disease is suppressed in regulated in WT MRL-Faslpr nephritic kidneys were sup- Ϫ Ϫ Ϫ Ϫ CCL2 / mice during NSN.10,11 In each model, Mø and T cells pressed in CCL2 / MRL-Faslpr kidneys (Supplemental Figure are no longer recruited to sites in the interstitium adjacent to 2). One possible interpretation is that within the hierarchical tubular epithelial cells (TEC), the major source of CCL2 in WT pattern of chemokine ligand/receptor expression regulating mice with nephritis10,11; however, Mø and T cells remain in immune responses, CCL2 is proximal in the chemokine cas- perivascular areas lacking CCL2 but rich in CCL5 (RANTES), a cade leading to nephritis in MRL-Faslpr mice; therefore, our chemokine capable of inciting local renal inflammation in goal was to focus on the chemokine ligand/receptors that MRL-Faslpr mice.12 This suggests that multiple chemokines maybe expressed “downstream” of CCL2 in MRL-Faslpr kid- dictate the tempo and locale of kidney disease. neys during nephritis. To identify the chemokines along with CCL2 that are in- strumental in T cell– and Mø-mediated nephritis, we exten- Ϫ Ϫ sively profiled kidneys of CCL2 / and wild-type (WT) MRL- Mø and TEC Are Sources of Intrarenal CXCL10 Faslpr mice during the development of lupus nephritis. We Expression during Lupus Nephritis in MRL-Faslpr Mice identified a highly expressed chemokine ligand/receptor pair CXCL10 engaging with its receptor CXCR3 is a potent T cell instrumental in attracting T cells during inflammation, chemoattractant.15 Because CXCL10 is among the most highly CXCL10 (IP-10)/CXCR3.13–16 We report that CXCR3 is ex- upregulated chemokines and may be downstream of CCL2, we pressed on intrarenal activated Mø in addition to T cells during explored the role of CXCL10 in MRL-Faslpr mice. Alveolar Mø experimental immune-mediated kidney disease. Furthermore, express CXCL10,17 and TEC are a rich source of multiple che- the expression of CXCR3 on T cells and Mø seems to mediate mokines in MRL-Faslpr mice10; therefore, Mø and TEC were their recruitment into the kidneys expressing CXCL9 during prime candidates in our attempt to identify sources of NSN. Finally, we determined that CXCR3 and one, CXCL9, but CXCL10. We detected CXCL10 in TEC (Figure 1A), leukocytes not another, CXCL10, of its ligands promote T cell– and Mø- (Figure 1A, inset), and endothelial cells (data not shown) in dependent nephritis. Thus, CXCL9 and CXCR3 are potential MRL-Faslpr mice with nephritis. CXCL10Ϫ/Ϫ MRL-Faslpr therapeutic targets for immune-mediated kidney illnesses. kidneys served as negative controls. The frequency of CXCL10ϩ Mø increased (two-fold) in MRL-Faslpr kidneys from 3 to 6 mo of age with advancing nephritis as deter- RESULTS mined by FACS analysis (Figure 1A). By comparison, the frequency of CXCL10ϩ T cells (CD4ϩ, CD8ϩ)inMRL- Multiple Chemokine Ligand/Receptors Are Faslpr kidneys, before and during nephritis, was minimal Upregulated in MRL-Faslpr Nephritic Kidneys (approximately 2%; data not shown). Similarly, the expres- To identify potential therapeutic chemokine ligand/receptor sion of CXCL10 in primary TEC derived from MRL-Faslpr targets in MRL-Faslpr mice, we compared intrarenal chemo- mice increased (more than nine-fold) as determined by re- kine ligand/receptor transcripts in mice before (2 mo of age) al-time PCR (Figure 1A). Of note, CXCL10ϩ Mø and TEC in and after (5 mo of age) onset of nephritis. The majority of MRL-Faslpr kidneys were similar to B6 kidneys (3 mo of intrarenal chemokine ligands (18 of 23) and chemokine recep- age). Thus, Mø and TEC are intrarenal sources of CXCL10 tors (10 of 16) we evaluated increased with advancing nephritis during nephritis in MRL-Faslpr mice. (Supplemental Figure 1). We focused on the groups with the highest increase in intrarenal transcript expression. Within this group of chemokine ligands, we detected an increase in Frequency of CXCR3؉ T Cells and Mø Is Increased in CXCL10 (nine-fold), CXCL9 (45-fold), CXCL11 (10-fold), MRL-Faslpr Nephritic Kidneys CXCL13 (133-fold), CCL5 (16-fold), CCL20 (seven-fold), and Because there is an increase in the frequency of intrarenal CX3CL1 (two-fold), and within the group of chemokine re- CXCL10ϩ Mø and TEC, we probed for the expression of ceptors, we detected an increase in CXCR3 (nine-fold), CXCR3. The frequency of CD4ϩ, CD8ϩ, and B220ϩ (unique CXCR4 (six-fold), CXCR5 (42-fold), CCR2 (10-fold), and double-negative) T cells and Mø (CD68ϩ) expressing CXCR3 lpr CX3CR1 (six-fold; Supplemental Figure 1). Of note, the che- in the kidneys of MRL-Fas mice increased with nephritis (6 mokine ligand/receptor transcript levels in the MRL-Faslpr and mo of age) as compared with non-nephritic MRL-Faslpr and B6 B6 kidneys at 2 mo of age were similar. mice (2 mo of age; Figure 1B). The increased frequency of CXCR3ϩ T cells and Mø was not limited to the kidney; these Identifying Chemokine Ligand/Receptors Other than leukocytes increased in their spleens (p Ͻ 0.05; data not CCL2 that Are Expressed in Nephritic MRL-Faslpr Kidneys shown). Taken together, an increase in intrarenal and extrare- ϩ CCL2 promotes lupus nephritis in MRL-Faslpr mice.10 To nal CXCR3 T cell subsets and Mø is associated with a rise in identify chemokines other than CLL2 that are central to MRL- intrarenal CXCL10 in nephritic MRL-Faslpr mice.

1178 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH

A CXCL10 Expression (MRL-Fas lpr)

5mo5mo 2mo2mo CXCL10-/-CXCL10 -/-

CXCL10+ Intra-renal- Mø (FACS) CXCL10+ TEC (RTQ-PCR)- B CXCR3 + Intra- -renal Cells ** Strain Age(mo) n 10 * 10 MRL- Faslpr 6 5 * 30 MRL- Faslpr 2 5 n=3-4-- * B6 2 5 Mø p<0.05 + *p<0.01 20 *p<0.05 / GAPDH

** + * 5 5 10 * Frequency CXCL10 Frequency * CXCL10 0 0 cellsCXCR3+ (%) 0 Age (mo) 6 3 3 Age (mo) 6 3 3 CD4 CD8 B220 CD68

C Kidney/Systemic Disease (CXCL10-/--- MRL -Fas lpr ) Kidney MRLFaslpr n 4 Pathology CXCL10-/--/--- 11 15 Glomerular Leukocyte Subsets Renal function CXCL10+/+ 9 40 BUN

10 20 2

Score 0 5 3 Proteinuria

Cells/ glomerulus 2 Score mg/dl 0 0 1 glomerular interstitial perivascular CD4 CD8 B220 CD 68 0

Figure 1. Despite an increase in intrarenal Mø and TEC expressing CXCL10, and CXCR3 expressing T cells and Mø in MRL-Faslpr Ϫ Ϫ nephritic kidneys, CXCL10 / MRL-Faslpr mice are not protected from renal disease. (A) CXCL10 expression in the kidney was examined Ϫ Ϫ by immunostaining, flow cytometry (Mø), and real-time PCR (TEC). CXCL10 / mice served as negative control. (B) CXCR3 expression ϩ ϩ ϩ ϩ was 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 age Ϫ Ϫ served as normal controls. (C) Renal function, pathology, and leukocytic infiltrates are similar in CXCL10 / MRL-Faslpr mice as compared with MRL-Faslpr mice. Data are means Ϯ SEM.

Nephritis, Systemic Disease, and Survival Are not CXCR3 and Its Ligands, CXCL9, CXCL10, and CXCL11, Altered in CXCL10؊/؊ MRL-Faslpr Mice Are Upregulated during NSN To determine whether CXCL10 is central to lupus nephritis Our results suggest that CXCL10 is not central to lupus nephri- and the systemic illness, we compared CXCL10Ϫ/Ϫ and WT tis; however, it is possible that CXCR3 engaging with other MRL-Faslpr strains (6 mo of age). The severity of glomerular, ligands (CXCL9, CXCL11) promotes T cell– and Mø-medi- interstitial, and perivascular pathology was similar in ated renal diseases. To explore this possibility, we selected a CXCL10Ϫ/Ϫ and WT MRL-Faslpr mice (Figure 1C). Similarly, rapidly induced T cell– and Mø-dependent nephritis, NSN. we did not detect a difference in the number (CD68ϩ, CD4ϩ, Using this model, we evaluated the expression of CXCR3 and CD8ϩ, B220ϩ cells) and activation stages (CD69ϩ, IFN-␥ϩ; its ligands. Intrarenal CXCR3, CXCL10, CXCL9, and CXCL11 data not shown) of infiltrating leukocytes and renal function transcripts increased during NSN (Figure 2A). Thus, as in the (Figure 1C). Furthermore, we did not detect a difference in MRL-Faslpr nephritic kidneys, intrarenal CXCR3 and its li- survival in CXCL10Ϫ/Ϫ MRL-Faslpr mice (data not shown). gands are upregulated during NSN. Thus, despite enhanced intrarenal expression of CXCL10 and To identify the leukocyte populations expressing CXCR3 CXCR3 during nephritis, CXCL10 is not central to disease in and its ligands, we probed CD68ϩ and T cells (CD4ϩ, CD8ϩ) MRL-Faslpr mice. for CXCR3 expression. Of note, a similar percentage of intra-

J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1179 BASIC RESEARCH www.jasn.org

renal CD68ϩ and CD11bϩ cells expressed CXCR3; therefore, we refer to these CD68ϩ cells as Mø (data not shown). The frequency of intrarenal CD4ϩ, CD8ϩ T cells, and Mø expressing CXCR3 in- creased during NSN compared with un- treated mice (Figure 2B). We verified CXCR3 (Ab) specificity using CXCR3Ϫ/Ϫ mice with NSN. Although it is widely appreciated that CXCR3 is ex- pressed on activated T cells, reports of ϩ CXCR3 Mø are limited.15,18 To evaluate further CXCR3ϩ Mø, we determined that there is an increased frequency of CXCR3ϩ macrophages (BMMø), (F4/80ϩ; Figure 2C) and CD11bϩ (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 using CXCR3Ϫ/Ϫ BMMø and an isotype con- trol. Because activated Mø induce apo- ptosis of renal parenchymal cells,11 we evaluated the frequency of activated Mø during NSN. Intrarenal CXCR3ϩ-acti- vated Mø increased during NSN as com- pared with untreated mice (Figure 2D). Thus, intrarenal CXCR3ϩ-activated Mø and T cells are upregulated during NSN. We next determined the intrarenal locale of the ligands (CXCL9, CXCL10) binding to CXCR3 during NSN. Because there is a functional loss of CXCL11 protein as a re- sult of a missense mutation at 36 bp (G/A) and a single-nucleotide deletion mutation at 39 bp (C/Ϫ) in the for CXCL11 in B6 mice (Genbank sequence accession no. NT_109320; A.D.L. et al., unpublished ob- servations), we evaluated CXCL9 and CXCL10. CXCL9 is expressed on the major- ity of TEC and absent on CXCL9Ϫ/Ϫ kid- Figure 2. T cells and activated Mø increase during NSN, whereas TEC are a primary neys during NSN and in untreated mice source of CXCL9 and CXCL10 during NSN (day 14). (A) Expression of CXCR3 and its (Figure 2E). In addition, a small population ligands CXCL10, CXCL9, and CXCL11 during NSN were evaluated by real-time PCR. (B) ϩ ϩ of leukocytes express CXCL9 (Figure 2E). The frequency of intrarenal T cells (CD4 and CD8 ) and Mø expressing CXCR3. Note These intrarenal CXCL9ϩ leukocytes that representative FACS plots of CXCR3 expression on T cells and Mø in WT and control ϩ Ϫ/Ϫ increased during NSN are CD4 and CXCR3 mice during NSN. (C) The frequency of cultured BMMø expressing CXCR3 ϩ rises in response to stimulation with LPS. To verify staining specificity, we analyzed CD68 cells (Figure 2E). Similarly, Ϫ Ϫ CXCR3 / BMMø stimulated with LPS and B6 BMMø stimulated with LPS and stained CXCL10 is expressed primarily on TEC and with an isotype control. These data are representative of three samples and two to a lesser extent on leukocytes and is absent Ϫ/Ϫ additional experiments that used isotype controls alone. (D) Intrarenal activated Mø in CXCL10 control kidneys during NSN ϩ increased during NSN. We determined the total number of intrarenal activated (CD86 ) and in untreated mice (Figure 2E). The in- ϩ ϩ CXCR3 Mø by multiplying the frequency by the total number of Mø. (E) CXCL9 and crease in intrarenal CXCL10 leukocytes ϩ CXCL10 are mainly expressed by TEC during NSN (n ϭ 4/group). Intrarenal CXCL9 during NSN is in CD4ϩ, CD8ϩ, and CD68ϩ ϩ ϩ ϩ ϩ ϩ ϩ CD4 and CD68 cells and CXCL10 CD4 , CD8 , and CD68 cells increase during cells (Figure 2E). Although the frequency of ϫ 6 ϫ 5 ϩ NSN. We analyzed 0.5 to 1 10 cells (B, D, and E) and 2.5 10 cells (C) by FACS these intrarenal CXCL10 leukocytes is analysis. Data are means Ϯ SEM.

1180 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH

Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Figure 3. CXCR3 / and CXCL9 / but not CXCL10 / mice are protected from loss of renal function and renal pathology during Ϫ Ϫ NSN (day 14). (A) The rise in proteinuria, blood urea nitrogen (BUN; day 14), and serum creatinine (day 14) was suppressed in CXCR3 / Ϫ Ϫ Ϫ Ϫ and CXCL9 / but not CXCL10 / mice as compared with WT mice during NSN. Data are means Ϯ SEM; n ϭ 4 to 9/group. (B) Tubular Ϫ Ϫ Ϫ Ϫ and glomerular pathology is reduced in CXCR3 / and CXCL9 / mice as compared with WT mice. In contrast, tubular and glomerular Ϫ Ϫ pathology is similar in CXCL10 / mice and WT mice during NSN. Representative photomicrographs are shown. Data are means Ϯ SEM; n ϭ 7 to 9/group. Magnification, ϫ40. somewhat higher than in CXCL9ϩ cells, it was substantially Mø, whereas the ligands CXCL9 and CXCL10 are largely gener- (Ͼ2ϫ) lower than in CXCR3ϩ cells (Figure 2, B and E). Thus, ated by TEC during NSN. Of note, CXCL9 and CXCL10 expres- intrarenal CXCR3 is primarily expressed on activated T cells and sion and CXCR3ϩ leukocytes are rarely detected in glomeruli

J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1181 BASIC RESEARCH www.jasn.org

Ϫ Ϫ Ϫ Ϫ Figure 4. Intrarenal activated T cells and activated Mø are suppressed in CXCR3 / and CXCL9 / mice as compared with WT mice ϩ ϩ during NSN (day 14). (A) The number of CD4 and CD8 T cells and Mø detected by immunostaining is diminished within glomeruli Ϫ Ϫ Ϫ Ϫ (intra-, periglomerular) and in the interstitium in CXCR3 / and CXCL9 / mice as compared with WT mice during NSN. (B) We ϩ ϩ ϩ ϩ Ϫ Ϫ Ϫ Ϫ detected 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 ϫ 106 cells by FACS (n ϭ 6 to 9/group). (C) We evaluated Ϫ Ϫ ϩ ϩ kidneys from CXCR3 / and WT mice for the presence of activated Mø (CD68 , TNF-␣ ) during NSN using immunofluorescence. ϩ ϩ Ϫ Ϫ Representative photomicrographs illustrate CD68 cells expressing TNF-␣ (circled) in the CXCR3 / compared with WT kidneys. Data are means Ϯ SEM; n ϭ 4/group. during NSN (data not shown). This pattern of enhanced CXCR3 compared CXCR3Ϫ/Ϫ, CXCL9Ϫ/Ϫ, and CXCL10Ϫ/Ϫ mice with and its cognate ligand expression during NSN is consistent with WT mice during NSN. Because CXCR3Ϫ/Ϫ, CXCL9Ϫ/Ϫ, and our findings in MRL-Faslpr kidneys. CXCL10Ϫ/Ϫ mouse strains do not express a functional CXCL11, for simplicity, we refer to each strain as the single unique “knock- Ϫ Ϫ CXCR3 and CXCL9 but not CXCL10 Promote NSN out.” CXCR3 / mice were protected from the loss of renal func- To determine whether CXCR3 regulates intrarenal T cell– and tion (Figure 3A). By comparison, renal function in CXCL10Ϫ/Ϫ Mø-mediated renal disease via ligands other than CXCL10, we mice was similar to WT mice during NSN (Figure 3A). Consistent

1182 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH

Anti-Sheep # /Rabbit + IgG ELISA

Total IgG IgG1 IgG2b B6 n 0.8 1.2 0.6 * * CXCR3-/- 6 WT 6 * * * ** #CXCR3-/- 0.4 0.6 0.3 **

* B6 n * CXCL9-/- 0.8 1.0 1.0 7 * * WT 8 +CXCL9-/- *

0.4 * 0.5 0.5 Absorbance (450nm) Absorbance

B6 n *p<0.05 0.2 0.4 0.2 CXCL10-/- 6 **p<0.01 WT 6 #CXCL10-/- 0.1 0.2 0.1

0.0 0.0 0.0 248 4816 0.25 0.5 1.0 Serum dilutions (1:10 3)

B Intra-renal IgG deposits

3 B6 n CXCR3-/- 4 * WT 4 *p<0.05 CXCR3-/- 2 * *

1

B6 n CXCL9-/- 4 2 * WT 4

CXCL9-/- Intensity) * 1 * Score ( Score

3 B6 n CXCL10-/- 3 WT 3 CXCL10-/- 2

1

3.0 4.5 6.0 7.5 Ab dilutions (1:10 3)

Ϫ Ϫ Ϫ Ϫ Figure 5. Decreased serum anti-sheep/rabbit IgG isotype Ab and IgG deposits in CXCR3 / and CXCL9 / mice. We detected a Ϫ Ϫ decrease in serum total anti-sheep IgG and IgG1 (n ϭ 6/group) and glomerular IgG deposits (n ϭ 4/group) in CXCR3 / compared with WT mice. Similarly, we detected a reduction in serum total anti-rabbit IgG and IgG1 (n ϭ 7 to 8/group) and in glomerular IgG deposits Ϫ Ϫ (n ϭ 4/group) in CXCL9 / compared with WT mice. In contrast, serum anti-sheep IgG isotypes (n ϭ 6/group) and IgG glomerular Ϫ Ϫ deposits (n ϭ 4/group) in CXCL10 / mice did not differ from WT mice. Data are means Ϯ SEM. with preserving renal function, renal pathology in CXCR3Ϫ/Ϫ but CXCL9Ϫ/Ϫ and WT mice during NSN. Renal function (pro- not in CXCL10Ϫ/Ϫ mice was reduced (Figure 3B). Tubular and teinuria, blood urea nitrogen, and serum creatinine) was im- glomerular pathology were blunted in CXCR3Ϫ/Ϫ but not in proved in CXCL9Ϫ/Ϫ mice (Figure 3A). Similarly, renal pa- CXCL10Ϫ/Ϫ mice. Thus, CXCR3 but not CXCL10 promotes thology (glomerular and tubular) was suppressed in NSN, suggesting that another CXCR3 ligand is instrumental in CXCL9Ϫ/Ϫ mice (Figure 3B, Supplemental Figure 3). Notably, immune-mediated nephritis. renal function (Figure 3A) and pathology (data not shown) in To determine whether CXCL9, another ligand for CXCR3, CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ mice did not return to baseline promotes immune-mediated nephritis, we compared (untreated mice). This suggests that CXCR3 and one, CXCL9,

J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1183 BASIC RESEARCH www.jasn.org but not another, CXCL10, of its ligands promote immune- inflammation. Mø and T cells are instrumental in mediating mediated nephritis. renal inflammation; thus, chemokines that regulate Mø and T cells within the kidney are potential therapeutic targets for ne- Intrarenal Activated T Cells and Mø Are Decreased in phritis. Therefore, we extensively profiled MRL-Faslpr kidneys .CXCR3؊/؊ and CXCL9؊/؊ Mice during NSN before and during Mø- and T cell–dependent lupus nephritis Because CXCR3 is expressed on intrarenal T cells (CD4ϩ, Although previous reports claimed that a few chemokine li- CD8ϩ) and Mø during NSN, we hypothesized these intrarenal gands (four of nine) and chemokine receptors (three of six) are leukocytes decrease in CXCR3Ϫ/Ϫ as compared with WT mice. upregulated in MRL-Faslpr kidneys, we now report that the Fewer CD4ϩ, CD8ϩ, and Mø were detected in the glomeruli majority of chemokine ligands (18 of 23) and chemokine re- (intra-, periglomerular) and interstitium in CXCR3Ϫ/Ϫ mice ceptors (10 of 16) that we profiled were upregulated in MRL- ϩ (Figure 4A). A similar decrease in CD4 T cells and Mø oc- Faslpr mice with lupus nephritis.6,22 This difference in our find- curred in the CXCL9Ϫ/Ϫ kidneys, whereas we did not detect a ings may be related to the larger panel of chemokine ligand/ reduction in these leukocytic populations in CXCL10Ϫ/Ϫ mice receptors in our experiments and the detection system (data not shown). (ribonuclease protection assay22 versus real-time PCR, our CXCR3 is expressed on activated T cells19 and Mø; there- study). We previously established that CCL2 is required to fore, we compared these intrarenal leukocytes in CXCR3Ϫ/Ϫ promote Mø- and T cell–dependent lupus nephritis in MRL- mice with WT mice during NSN. We detected a decreased Faslpr mice.10 We now report that the rise in the panel of che- frequency of activated (CD69ϩ) CD4ϩ and CD8ϩ T cells and mokine ligand/receptors that increase in MRL-Faslpr mice is activated (CD86ϩ) Mø in CXCR3Ϫ/Ϫ mice during NSN (Fig- suppressed in CCL2Ϫ/Ϫ MRL-Faslpr mice. This may suggest ure 4B). In support of this decrease in activated Mø, the fre- that CCL2 is a “proximal master switch” in the chemokine quency of TNF-␣ϩ Mø was diminished (Figure 4C, circled, cascade; however, increased expression of a specific chemokine immunofluorescence). Similarly, CXCL9Ϫ/Ϫ mice had a re- during disease does not necessarily indicate that it promotes duced frequency of activated T cells and Mø (Figure 4B). Thus, inflammation; it may not have an impact or even thwart in- CXCR3 engaging with CXCL9 fosters an intrarenal accumula- flammation. Therefore, we investigated the impact of CXCR3 tion of activated CD4ϩ, CD8ϩ T cells and Mø during NSN. and its ligands, CXCL9 and CXCL10, during Mø and T cell immune–mediated nephritis (NSN). Of note, we could not Serum Antigen-Specific IgG Isotypes and Glomerular analyze the impact of CXCL11 during NSN because the protein .IgG Deposits Are Suppressed in CXCR3؊/؊ and for this remaining CXCR3 ligand is not expressed in B6 mice CXCL9؊/؊ Mice during NSN We now report that CXCR3 and CXCL9 but not CXCL10 are Elevated Ig and Ig class switching are hallmarks of autoimmune central to Mø- and T cell–dependent kidney disease. disease20,21; therefore, we determined whether the attenuated se- The impact of CXCL10 in autoimmune and kidney disease verity of kidney disease in CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ mice was is controversial. Whereas Ab blockade of CXCL10 in experi- related to a reduction in circulating levels of antigen-specific IgG mental insulin-dependent diabetes23 and adjuvant arthritis24 isotypes during NSN. For this purpose, we measured the anti- abrogates disease, deleting CXCL10 does not diminish experi- sheep and -rabbit IgG isotypes in the sera of CXCR3Ϫ/Ϫ and mental autoimmune encephalomyelitis, although it lowers Ϫ Ϫ CXCL9 / mice, respectively, in comparison with WT mice dur- disease threshold.25 Thus, the consequences of CXCL10 engag- ing NSN. We detected a suppression of IgG and IgG1 and not ing with CXCR3 in autoimmune diseases are complex and yet IgG2b at multiple titers (2.0 to 8.0 ϫ 103) in CXCR3Ϫ/Ϫ and not fully understood. Furthermore, the impact of CXCL10 on CXCL9Ϫ/Ϫ mice (Figure 5A). Because serum isotypes were atten- glomerular and tubulointerstitial renal disease is unclear. In uated in CXCR3Ϫ/Ϫ and CXL9Ϫ/Ϫ mice, we hypothesized that the induced forms of renal injury, puromycin aminonucleoside– reduced renal pathology resulted from decreased deposition of and anti-nephrin Ab–induced nephropathy,26 Ab blockade of IgG within glomeruli of CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ mice. We CXCL10 exacerbated proteinuria and podocyte injury. Con- detected a reduction in IgG deposits at multiple dilutions (3.0 to versely, Ab blockade of CXCL10 in a model of renal endothelial 7.5 ϫ 103) in CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ glomeruli (Figure 5B). injury reduced interstitial T cell infiltration and improved re- Contrastingly, circulating anti-sheep IgG isotypes and glomerular nal function; however, pathology was not evaluated.27 We now IgG deposits in CXCL10Ϫ/Ϫ did not differ from WT mice. Taken report that despite the abundant protein expression of intrarenal together, this suggests CXCR3 engaging with CXCL9 may pro- CXCL10 on TEC and leukocyte infiltrates, CXCL10 is not instru- mote renal disease by increasing systemic IgG and IgG deposits mental in two distinct models of immune-mediated renal disease, within glomeruli. MRL-Faslpr mice and NSN. This indicates that CXCL10 alone is not central to immune-mediated kidney disease. Because it is possible that other ligands alone or together DISCUSSION with CXCL10 promote immune-mediated kidney disease, we compared CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ mice during NSN with Chemokines are tempting therapeutic targets for a wide range WT mice. CXCR3 and CXCL9 but not CXCL10 are central to of diseases, because they are specifically upregulated during immune-mediated kidney disease. This is the first report indi-

1184 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH

Ϫ Ϫ cating that CXCL9 / mice are protected from nephritis. Our sheep IgG1 during NSN (day 8).28 This difference may be related findings are in agreement with recent studies reporting that to the assay methods, the day of analysis, or other variables related CXCR3Ϫ/Ϫ mice develop less severe nephritis than WT mice to inducing NSN. Nevertheless, our data support the concept that during NSN.28 In fact, CXCR3 has been shown to incite non- CXCR3 and CXCL9 enhance Ab production during immune re- immune-induced renal inflammation because CXCR3 orches- sponses. The regulation of serum IgG and glomerular deposits by trates the recruitment of T cells instrumental in mediating re- CXCR3 and CXCL9 remains unclear. We speculate that distinct ϩ ϩ nal ischemic injury.16 Thus, CXCR3 may be a potential patterns of CXCR3 CD4 T cells and CXCL9 expression in ger- therapeutic target for a broad array of kidney diseases. Our minal centers during inflammation and/or the expression of findings are intriguing; although CXCL9 and CXCL10 protein CXCR3 on a subset of memory B cells and plasma cells may reg- expression is similar in locale and intensity during NSN, the ulate serum IgG and, in turn, glomerular deposits.35,36 Notably, functional impact of these two CXCR3 ligands differ. This dif- the decline in antigen-specific IgG in CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ ferential impact on nephritis may be related to distinct bio- serum correlates with diminished IgG deposits within glomeruli; availability of CXCL9 and CXCL10. The preferential contribu- therefore, suppressed renal pathology and improved renal func- tion of one CXCR3 ligand to disease pathology has been seen in tion may, in part, result from a reduced intrarenal deposition of other inflammatory models of disease, such as CXCL10 in pathogenic Ab in CXCR3Ϫ/Ϫ and CXCL9Ϫ/Ϫ mice during NSN. mouse hepatitis virus and Dengue virus encephalitis29,30 and In addition, we did not detect CXCL9 and CXCL10 expression CXCL9 in herpes simplex virus-1 ocular infection and cyto- and CXCR3ϩ leukocytes in glomeruli of WT mice during Ϫ Ϫ megalovirus infection.31,32 Regardless of the exact mecha- NSN. Thus, the reduction in proteinuria in CXCR3 / and nisms, our findings indicate that selective blockade of CXCL9 CXCL9Ϫ/Ϫ mice during the later stages of NSN may be re- and CXCR3 is a potential therapeutic strategy to combat im- lated to the suppression of humoral immune responses. mune-mediated renal diseases. This is consistent with findings indicating that although the Although it is well appreciated that activated T cells express generation of autologous murine IgG against the nephro- CXCR3, we now report that activated and resting Mø express toxic Ab is not essential for disease initiation, it may con- CXCR3 during kidney disease. The frequency of intrarenal tribute to sustaining renal dysfunction.37 This may indicate CD68ϩ cells along with CD4ϩ and CD8ϩ T cells expressing that although CXCR3 and CXCL9 promote the initiation of CXCR3 increased during NSN. This rise in CXCR3ϩ Mø in the NSN via cell-mediated events, their regulation of the pro- kidney is consistent with studies reporting that a low percentage of duction and deposition of pathogenic Ab may be responsi- CXCR3ϩ in the normal circulation increases in pa- ble for sustaining immune-mediated renal disease. tients with rheumatoid arthritis.33 Furthermore, we determined In conclusion, CXCR3 and one, CXCL9, but not another, that whereas some primary cultured BMMø express CXCR3, the CXCL10, of its ligands mediate activated T cells and Mø re- frequency increases after IFN-␥ stimulation and is even more ro- cruitment into the kidney and enhance Ab deposition in glo- bust after LPS stimulation. This suggests that whereas low num- meruli that may, in turn, promote immune-mediated kidney bers of resting Mø express CXCR3, the increase in CXCR3ϩ Mø is disease. This suggests that blocking CXCR3 and CXCL9 is a a reflection of a rise in activated Mø during renal inflammation. potential therapeutic target for human immune-mediated kid- Consistent with this concept, the total number of intrarenal acti- ney diseases. vated CXCR3ϩ Mø increases during NSN as compared with un- treated mice. Furthermore, CXCR3 is responsible for recruiting these activated Mø, because there is a decrease in intrarenal acti- CONCISE METHODS vated Mø, along with activated CD4ϩ and CD8ϩ T cells, in Ϫ Ϫ CXCR3 / during NSN. This suggests that activated Mø bearing Mice CXCR3 are central to inducing renal injury, because activated Mø We purchased MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) and C57BL/6 release mediators that initiate TEC .11 Thus, we provide (B6) from the Jackson Laboratory (Bar Harbor, ME). Mice were the first evidence that CXCR3 is instrumental in recruiting acti- housed and bred in our pathogen-free animal facility. CXCL9 null vated Mø, as well as T cells, into the kidney, that are responsible for (Ϫ/Ϫ), CXCL10Ϫ/Ϫ, CXCR3Ϫ/Ϫ (B6 background), and CCL2Ϫ/Ϫ inciting renal injury. (MRL-Faslpr background) mice were generated as described previous- Humoral and cell-driven immune mechanisms mediate kid- ly.4,10,34,38 We generated CXCL10Ϫ/Ϫ MRL-Faslpr mice using a back- ney disease.20 We investigated whether the suppression in renal cross-intercross scheme. MRL-Faslpr mice were mated with Ϫ Ϫ Ϫ Ϫ disease in the CXCR3 / and CXCL9 / mice during NSN was CXCL10Ϫ/Ϫ (B6 background) mice to yield heterozygous F1 off- related to a decrease in pathogenic Ab within the kidney. We de- spring. We intercrossed F1 mice and screened the progeny for dis- tected a reduction in total serum antigen-specific IgG and IgG1 rupted and intact CXCL10 and the Faslpr mutation in tail genomic Ϫ Ϫ Ϫ Ϫ Ab in CXCR3 / and CXCL9 / mice during NSN. Our finding DNA. The DNA was assessed by PCR using oligonucleotide primers is reminiscent of reduced Ab against a bacterial pathogen (Fran- that recognized the normal CXCL10 gene: Sense (5Ј-TCC CTC CCG Ϫ Ϫ cisella tularensis) in CXCL9 / mice34; however, our data are not TAA CCA CAC AGT AAA T-3Ј) and antisense (5Ј-GCG GAT AGA Ϫ Ϫ in agreement with the recent study of CXCR3 / mice, indicating CTC TGC TTT CAC TTT GG-3Ј) and Neo gene sense (5Ј-TGG ATG that there is no difference in total mouse anti-sheep IgG or anti- TGG AAT GTG TGC GAG-3Ј) and antisense (5-TTT CAC TTT GG-

J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1185 BASIC RESEARCH www.jasn.org

3Ј). Gel analysis of the PCR products identified the CXCL10 and Neo Flow Cytometry lpr gene fragments at 342 and 640 bp, respectively. The Fas mutation We prepared and stained single-cell suspensions from kidneys, was identified as previously reported.39 After five generations of back- spleens, or primary cultured TEC/BMMø as described previously.48 Ϫ/Ϫ cross matings (N5), we analyzed and compared CXCL10 MRL- We collected 0.5 to 1.0 ϫ 106 total kidney or spleen cells and 0.5 to lpr lpr Fas mice with age- and gender-matched WT MRL-Fas litter- 1.0 ϫ 105 of cultured cells using a FACSCalibur (Becton Dickinson, mates. The number of mice analyzed is specified in each figure. We San Jose, CA) and analyzed data using Flowjo software (Tree Star, compared these strains at the N5 generation, because we previously Palo Alto, CA). established that there are sufficient MRL-Faslpr background to result in consistent phenotypic changes characteristic of MRL-Faslpr mice.40 Use of mice in our study was reviewed and approved by the We used the following Ab from eBioscience (San Diego, CA) for FACS Standing Committee on Animals in the Harvard Medical School in analysis: FITC-conjugated anti-CD4 (L3T4), anti-CD8 (53-6.7), anti- adherence to the National Institutes of Health Guide for the Care and B220 (RA3-6B2), and anti-CD45.2 (104); PE-conjugated anti-CD4, Use of Laboratory Animals. anti-CD45.2, anti-CD69 (H1.2F3), and anti-CD86 (GL1); PE-Cy5– conjugated anti-CD8 and anti–TCR-␤ chain (H57-597); and allophy- Inducing NSN cocyanin-conjugated anti-CD4, anti-CD45.2, and anti-F4/80 (BM8). We prepared nephrotoxic serum by immunizing sheep with a partic- We used FITC- and allophycocyanin-conjugated anti-CD68 Ab ulate fraction of mouse glomerular basement membrane from B6 (FA11; Serotec, Oxford, UK). We used purified rabbit anti-mouse mice kidneys, as described previously.41 To induce NSN, we primed CXCR3 Ab (Zymed, San Francisco, CA), purified goat anti-mouse CXCL10Ϫ/Ϫ, CXCR3Ϫ/Ϫ, and WT male mice (6 wk of age) by inject- CXCL9 Ab (R&D Systems, Minneapolis, MN), and rabbit anti-mouse ing 0.5 mg of sheep IgG in Freund’s complete adjuvant (Sigma Chem- CXCL10 Ab (PeproTech, Rocky Hill, NJ). The following isotype-spe- ical Co., St. Louis, MO) subcutaneously in each flank. We challenged cific Ab were used for controls: Rat-IgG2a (BR2a), rat-IgG2b (KLH/ these mice 7 d later with an intravenous injection of nephrotoxic G2b-1-2), and rabbit-IgG (eBioscience). As secondary PE- or allophy- serum (15 ␮l/g body wt). We used nephrotoxic serum from rabbits to cocyanin-conjugated Ab, we used goat anti-rabbit (Jackson immunize CXCL9Ϫ/Ϫ and WT mice as described previously.42 Mice ImmunoResearch Laboratories; West Grove, PA) and biotin-conju- were killed 14 d after challenge and analyzed. The number of mice in gated rabbit anti-goat Ab (Vector Laboratories, Burlingame, CA). To each group is specified within each figure. 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 Intrarenal Chemokine and lotus lectin (Vector Laboratories).49,50 Transcripts We analyzed the expression of chemokines (CXCL10, CXCL9, Histopathology CXCL11, CXCL12, CCL13, CCL5, CCL28, CCL22, CCL24, CCL1, We fixed kidneys in 10% formalin and prepared and stained paraffin CCL17, CX3CL1, CCL25, CCL2, CCL8, CCL7, CCL3, CXCL2, sections with the periodic acid Schiff reagent. Slides were coded before CCL28␣, CCL4, CCL28␤, CCL27, CCL21, CXCL16, and CXCL1) and grading the renal pathology. chemokine receptors (CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, MRL-Faslpr Mice.We evaluated renal (glomerular, tubular, intersti- CCR10, and CX CR1) in MRL-Faslpr mice by using real-time, two- 3 tial, and perivascular) pathology on a scale of 0 (normal) to 3 (severe) step, quantitative PCR, as described previously.43 Primers were de- as described previously.45 signed, as previously reported.44 To detect CXCL10 expression during NSN, we used primers designed by Applied Biosystems (Foster City, NSN. We evaluated CXCL9Ϫ/Ϫ, CXCL10Ϫ/Ϫ, CXCR3Ϫ/Ϫ, and CA). To detect CXCL9, CXCL11, and CXCR3 expression during B6 kidneys for glomerular and tubular damage as described previ- NSN, we used the following primers: glyceraldehyde-3-phosphate de- ously.11 hydrogenase sense 5Ј-CAT GGC CTC CAA GGA GTA AG-3Ј and Ј Ј antisense 5 -CCT AGG CCC CTC CTG TTA TT-3 ; CXCL9 sense Gross Pathology 5Ј-TCC TTT TGG GCA TCA TCT TC-3Ј and antisense 5Ј-TTC CCC We evaluated skin lesions monthly from 2 to 6 mo of age using a CTC TTT TGC TTT TT-3Ј; CXCL11 sense 5Ј-AGT AAC GGC TGC scoring system previously described.10 GAC AAA GT-3Ј and antisense 5Ј-GCA TGT TCC AAG ACA GCA Ј Ј Ј GA-3 ; and CXCR3 sense 5 -TGA GAC AAC TGA GGC CTC CTA-3 Immunohistochemistry and antisense 5Ј-TCT TGC TCC CCA GTT GAT G-3Ј (Invitrogen, We stained cryostat-cut kidney sections for the presence of Mø with Carlsbad CA). We evaluated expression as previously reported.45 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- Isolation of TEC and BMMø mouse Ab (Pharmingen, San Diego, CA) according to a previously We isolated and cultured TEC from B6 and MRL-Faslpr mice as pre- described immunoperoxidase method.51 To evaluate CXCL9 and viously reported,46 and we isolated and cultured BMMø from B6 mice CXCL10 expression in the kidney, we stained frozen sections with as described previously.47 goat anti-mouse CXCL9 and rabbit anti-mouse CXCL10. The follow-

1186 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH ing isotype-specific Ab were used for controls: Rat-IgG2a (R35–95), ACKNOWLEDGMENTS rat IgG2b (R35-38), rabbit IgG (eBioscience), and goat IgG (Southern Biotechnology, Birmingham, AL). The secondary Ab for immuno- This work was supported by National Institutes of Health grants DK staining was biotin-conjugated rabbit anti-rat IgG (Vector Laborato- 52369 (V.R.K.), DK 56848 (V.R.K.), DK 36149 (V.R.K.), KO1 AR ries). The immunostaining was analyzed by counting for the presence 051367 (T.K.M.), and RO1 CA 069212 (A.D.L.); by Deutsche For- ϩ ϩ ϩ ϩ of CD68 , CD4 , CD8 , and B220 cells within and surrounding schungsgemeinschaft grants ME-3194/1-1 (J.M.) and ZE-711/1 glomeruli and in the interstitium in 10 randomly selected high-power (G.Z.); and the Research Grants Council of Hong Kong HKU 7592/06 ϩ fields. Of note, we have determined that the B220 cells in the kidney (H.Y.L.). lpr Ϫ Ϫ of MRL-Fas mice are the unique double-negative (CD4 CD8 We thank Dr. Craig Gerard for providing CXCR3Ϫ/Ϫ mice, Dr. double-negative) T cells and are not B cells.52 Tanya Mayadas for the rabbit anti–glomerular basement membrane To determine the number of activated Mø in the kidney during anti-serum, and Dr. Julie Lucas for editorial assistance. 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 DISCLOSURES a fluorescence microscope. The frequency (%) of activated Mø was None. assessed by enumerating the number of CD68ϩTNF-␣ϩ cells within the total number of Mø (CD68ϩ) in five high-power fields. REFERENCES Renal Function We assessed urine semiquantitatively by dipstick analysis as 1. Sallusto F, Mackay CR, Lanzavecchia A: The role of chemokine recep- tors in primary, effector, and memory immune responses. Annu Rev described previously.45 We measured blood urea nitrogen levels using Immunol 18: 593–620, 2000 a colorimetric analysis kit (Infinity; Thermo Electron, Melbourne, 2. Rossi D, Zlotnik A: The biology of chemokines and their receptors. Australia) and serum creatinine using the creatinine reagent kit and Annu Rev Immunol 18: 217–242, 2000 Creatinine Analyzer 2 (Beckman Coulter, Galway, Ireland) according 3. Segerer S, Cui Y, Hudkins KL, Goodpaster T, Eitner F, Mack M, to the manufacturer’s instructions. Schlondorff D, Alpers CE: Expression of the chemokine monocyte chemoattractant protein-1 and its receptor chemokine receptor 2 in human crescentic glomerulonephritis. J Am Soc Nephrol 11: 2231– Survival 2242, 2000 We compared survival of the CXCL10Ϫ/Ϫ MRL-Faslpr and WT MRL- 4. Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD: lpr IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice re- Fas mice using similar numbers of males and females from birth to veal a role for IP-10 in effector T cell generation and trafficking. 10 mo of age. J Immunol 168: 3195–3204, 2002 5. Anders HJ, Vielhauer V, Schlondorff D: Chemokines and chemokine receptors are involved in the resolution or progression of renal dis- Serum Anti-Sheep and Anti-Rabbit IgG Isotypes ease. Kidney Int 63: 401–415, 2003 We measured the levels of mouse anti-sheep IgG Ab (Sigma Chemical 6. Vielhauer V, Anders HJ, Schlondorff D: Chemokines and chemokine Co.) and anti-rabbit IgG (Jackson ImmunoResearch Laboratories) by receptors as therapeutic targets in lupus nephritis. Semin Nephrol 27: ELISA using sera collected at day 14 during NSN as described previ- 81–97, 2007 7. Premack BA, Schall TJ: Chemokine receptors: Gateways to inflamma- ously.53 We detected bound mouse anti-sheep/anti-rabbit IgG, IgG1, tion and infection. Nat Med 2: 1174–1178, 1996 and IgG2b using peroxidase-conjugated rabbit anti-mouse IgG Ab 8. Hooke DH, Gee DC, Atkins RC: Leukocyte analysis using monoclonal (1:5000; Southern Biotechnology). antibodies in human glomerulonephritis. Kidney Int 31: 964–972, 1987 9. Kelley VE, Roths JB: Interaction of mutant lpr gene with background IgG Deposits within the Kidney strain influences renal disease. Clin Immunol Immunopathol 37: 220– We evaluated IgG deposits within renal glomeruli as described previ- 229, 1985 ously.54 We evaluated stained sections by scoring 20 glomeruli as ei- 10. Tesch GH, Maifert S, Schwarting A, Rollins BJ, Kelley VR: Monocyte ther positive or negative and graded the amount (severity) of deposits chemoattractant protein 1-dependent leukocytic infiltrates are re- sponsible for autoimmune disease in MRL-Fas(lpr) mice. J Exp Med in 20 positive glomeruli per specimen on a scale of 0 to 3 using mul- 190: 1813–1824, 1999 tiple dilutions (1:3000, 1:4500, 1:6000, and 1:7500) of the detection 11. Tesch GH, Schwarting A, Kinoshita K, Lan HY, Rollins BJ, Kelley VR: Ab (fluorescein-conjugated goat anti-mouse IgG; MP Biomedicals, Monocyte chemoattractant protein-1 promotes macrophage-medi- Aurora, OH).40 ated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis. J Clin Invest 103: 73–80, 1999 12. Moore KJ, Wada T, Barbee SD, Kelley VR: Gene transfer of RANTES Statistical Analyses elicits autoimmune renal injury in MRL-Fas(lpr) mice. Kidney Int 53: The data are means Ϯ SEM and were analyzed by GraphPad Prism 4.0 1631–1641, 1998 13. Luster AD, Ravetch JV: Biochemical characterization of a gamma (GraphPad, San Diego, CA). We used the nonparametric Mann- -inducible (IP-10). J Exp Med 166: 1084–1097, Whitney U test to determine differences among groups. Survival 1987 curves were compared and analyzed using the log-rank test. 14. Xie JH, Nomura N, Lu M, Chen SL, Koch GE, Weng Y, Rosa R, Di Salvo

J Am Soc Nephrol 19: 1177–1189, 2008 CXCL9, not CXCL10, Promotes Nephritis 1187 BASIC RESEARCH www.jasn.org

J, Mudgett J, Peterson LB, Wicker LS, DeMartino JA: Antibody-medi- Luster A, Liao F: Both CXCR3 and CXCL10/IFN-inducible protein 10 ated blockade of the CXCR3 chemokine receptor results in diminished are required for resistance to primary infection by dengue virus. J Im- recruitment of T helper 1 cells into sites of inflammation. J Leukoc Biol munol 177: 1855–1863, 2006 73: 771–780, 2003 31. Wuest T, Farber J, Luster A, Carr DJ: CD4ϩ T into the 15. Liu L, Callahan MK, Huang D, Ransohoff RM: Chemokine receptor cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice CXCR3: An unexpected enigma. Curr Top Dev Biol 68: 149–181, 2005 following herpes simplex virus type 1 infection. Cell Immunol 243: 16. Fiorina P, Ansari MJ, Jurewicz M, Barry M, Ricchiuti V, Smith RN, Shea 83–89, 2006 S, Means TK, Auchincloss H Jr, Luster AD, Sayegh MH, Abdi R: Role of 32. Salazar-Mather TP, Hamilton TA, Biron CA: A chemokine-to-cytokine- CXC chemokine receptor 3 pathway in renal ischemic injury. JAmSoc to-chemokine cascade critical in antiviral defense. J Clin Invest 105: Nephrol 17: 716–723, 2006 985–993, 2000 17. Shiozawa F, Kasama T, Yajima N, Odai T, Isozaki T, Matsunawa M, 33. Katschke KJ Jr, Rottman JB, Ruth JH, Qin S, Wu L, LaRosa G, Ponath Yoda Y, Negishi M, Ide H, Adachi M: Enhanced expression of inter- P, Park CC, Pope RM, Koch AE: Differential expression of chemokine feron-inducible protein 10 associated with Th1 profiles of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue receptor in autoimmune pulmonary inflammation of MRL/lpr mice. monocytes/macrophages in rheumatoid arthritis. Arthritis Rheum 44: Arthritis Res Ther 6: R78–R86, 2004 1022–1032, 2001 18. Rappert A, Bechmann I, Pivneva T, Mahlo J, Biber K, Nolte C, Kovac 34. Park MK, Amichay D, Love P, Wick E, Liao F, Grinberg A, Rabin RL, AD, Gerard C, Boddeke HW, Nitsch R, Kettenmann H: CXCR3-depen- Zhang HH, Gebeyehu S, Wright TM, Iwasaki A, Weng Y, DeMartino dent microglial recruitment is essential for dendrite loss after brain JA, Elkins KL, Farber JM: The CXC chemokine murine lesion. J Neurosci 24: 8500–8509, 2004 induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, 19. Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher M, targets including activated B cells, and supports anti- Koch AE, Moser B, Mackay CR: The chemokine receptors CXCR3 and body responses to a bacterial pathogen in vivo. J Immunol 169: CCR5 mark subsets of T cells associated with certain inflammatory 1433–1443, 2002 reactions. J Clin Invest 101: 746–754, 1998 35. Rabin RL, Alston MA, Sircus JC, Knollmann-Ritschel B, Moratz C, Ngo 20. Dean EG, Wilson GR, Li M, Edgtton KL, O’Sullivan KM, Hudson BG, D, Farber JM: CXCR3 is induced early on the pathway of CD4ϩ T cell Holdsworth SR, Kitching AR: Experimental autoimmune Goodpas- differentiation and bridges central and peripheral functions. J Immu- ture’s disease: A pathogenetic role for both effector cells and anti- nol 171: 2812–2824, 2003 body in injury. Kidney Int 67: 566–575, 2005 36. Muehlinghaus G, Cigliano L, Huehn S, Peddinghaus A, Leyendeckers 21. Izui S, Fossati-Jimack L, da Silveira SA, Moll T: Isotype-dependent patho- H, Hauser AE, Hiepe F, Radbruch A, Arce S, Manz RA: Regulation of genicity of autoantibodies: Analysis in experimental autoimmune hemo- CXCR3 and CXCR4 expression during terminal differentiation of mem- lytic anemia. Springer Semin Immunopathol 23: 433–445, 2001 ory B cells into plasma cells. Blood 105: 3965–3971, 2005 22. Perez de Lema G, Maier H, Nieto E, Vielhauer V, Luckow B, Mampaso 37. Rosenkranz AR, Knight S, Sethi S, Alexander SI, Cotran RS, Mayadas F, Schlondorff D: Chemokine expression precedes inflammatory cell TN: Regulatory interactions of alphabeta and gammadelta T cells in infiltration and chemokine receptor and cytokine expression during glomerulonephritis. Kidney Int 58: 1055–1066, 2000 the initiation of murine lupus nephritis. J Am Soc Nephrol 12: 1369– 38. Hancock WW, Gao W, Csizmadia V, Faia KL, Shemmeri N, Luster AD: 1382, 2001 Donor-derived IP-10 initiates development of acute allograft rejection. 23. Christen U, McGavern DB, Luster AD, von Herrath MG, Oldstone MB: J Exp Med 193: 975–980, 2001 Among CXCR3 chemokines, IFN-gamma-inducible protein of 10 kDa 39. Wu J, Zhou T, He J, Mountz JD: Autoimmune disease in mice due to (CXC chemokine ligand (CXCL) 10) but not monokine induced by integration of an endogenous retrovirus in an apoptosis gene. J Exp IFN-gamma (CXCL9) imprints a pattern for the subsequent develop- Med 178: 461–468, 1993 ment of autoimmune disease. J Immunol 171: 6838–6845, 2003 40. Kikawada E, Lenda DM, Kelley VR: IL-12 deficiency in MRL-Fas(lpr) 24. Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N: mice delays nephritis and intrarenal IFN-gamma expression, and di- Targeting the function of IFN-gamma-inducible protein 10 suppresses minishes systemic pathology. J Immunol 170: 3915–3925, 2003 ongoing adjuvant arthritis. J Immunol 169: 2685–2693, 2002 41. Tipping PG, Cornthwaite LJ, Holdsworth SR: Beta 2 integrin indepen- 25. Klein RS, Izikson L, Means T, Gibson HD, Lin E, Sobel RA, Weiner HL, dent recruitment and injury in anti-GBM glomerulonephritis Luster AD: IFN-inducible protein 10/CXC chemokine ligand 10-inde- in rabbits. Immunol Cell Biol 72: 471–479, 1994 pendent induction of experimental autoimmune encephalomyelitis. 42. Vielhauer V, Stavrakis G, Mayadas TN: Renal cell-expressed TNF re- J Immunol 172: 550–559, 2004 ceptor 2, not receptor 1, is essential for the development of glomer- 26. Han GD, Suzuki K, Koike H, Yoneyama H, Narumi S, Shimizu F, ulonephritis. J Clin Invest 115: 1199–1209, 2005 Kawachi H: IFN-inducible protein-10 plays a pivotal role in maintaining 43. Abdi R, Means TK, Ito T, Smith RN, Najafian N, Jurewicz M, Tchipach- slit-diaphragm function by regulating podocyte cell-cycle balance. vili V, Charo I, Auchincloss H Jr, Sayegh MH, Luster AD: Differential J Am Soc Nephrol 17: 442–453, 2006 role of CCR2 in islet and heart allograft rejection: Tissue specificity of 27. Panzer U, Steinmetz OM, Reinking RR, Meyer TN, Fehr S, Schneider A, chemokine/chemokine receptor function in vivo. J Immunol 172: 767– Zahner G, Wolf G, Helmchen U, Schaerli P, Stahl RA, Thaiss F: Com- 775, 2004 partment-specific expression and function of the chemokine IP-10/ 44. Means TK, Hayashi F, Smith KD, Aderem A, Luster AD: The Toll-like CXCL10 in a model of renal endothelial microvascular injury. JAmSoc receptor 5 stimulus bacterial flagellin induces maturation and chemokine Nephrol 17: 454–464, 2006 production in human dendritic cells. J Immunol 170: 5165–5175, 2003 28. Panzer U, Steinmetz OM, Paust HJ, Meyer-Schwesinger C, Peters A, 45. Lenda DM, Stanley ER, Kelley VR: Negative role of colony-stimulating Turner JE, Zahner G, Heymann F, Kurts C, Hopfer H, Helmchen U, factor-1 in macrophage, T cell, and mediated autoimmune Haag F, Schneider A, Stahl RA: Chemokine receptor CXCR3 mediates disease in MRL-Fas(lpr) mice. J Immunol 173: 4744–4754, 2004 T cell recruitment and tissue injury in nephrotoxic nephritis in mice. 46. Wuthrich RP, Glimcher LH, Yui MA, Jevnikar AM, Dumas SE, Kelley VE: J Am Soc Nephrol 18: 2071–2084, 2007 MHC class II, antigen presentation and in renal 29. Liu MT, Keirstead HS, Lane TE: Neutralization of the chemokine tubular epithelial cells. Kidney Int 37: 783–792, 1990 CXCL10 reduces inflammatory cell invasion and demyelination and 47. Chow FY, Nikolic-Paterson DJ, Atkins RC, Tesch GH: Macrophages in improves neurological function in a viral model of multiple sclerosis. streptozotocin-induced diabetic nephropathy: Potential role in renal J Immunol 167: 4091–4097, 2001 fibrosis. Nephrol Dial Transplant 19: 2987–2996, 2004 30. Hsieh MF, Lai SL, Chen JP, Sung JM, Lin YL, Wu-Hsieh BA, Gerard C, 48. Lenda D, Kikawada E, Stanley ES, Kelley VR: Reduced macrophage

1188 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1177–1189, 2008 www.jasn.org BASIC RESEARCH

recruitment, proliferation, and activation in colony-stimulating factor- destruction of autoimmune kidney disease in MRL- Fas(lpr) mice. 1-deficient mice results in decreased tubular apoptosis during renal J Immunol 161: 494–503, 1998 inflammation. J Immunol 170: 3254–3262, 2003 53. Topham PS, Csizmadia V, Soler D, Hines D, Gerard CJ, Salant DJ, 49. Faraggiana T, Malchiodi F, Prado A, Churg J: Lectin-peroxidase con- Hancock WW: Lack of chemokine receptor CCR1 enhances Th1 re- jugate reactivity in normal human kidney. J Histochem Cytochem 30: sponses and glomerular injury during nephrotoxic nephritis. J Clin 451–458, 1982 Invest 104: 1549–1557, 1999 50. Swinford AE, Bernstein J, Toriello HV, Higgins JV: Renal tubular dys- 54. Kinoshita K, Tesch G, Schwarting A, Maron R, Sharpe AH, Kelley VR: genesis: Delayed onset of oligohydramnios. Am J Med Genet 32: Costimulation by B7–1 and B7–2 is required for autoimmune disease 127–132, 1989 in MRL-Faslpr mice. J Immunol 164: 6046–6056, 2000 51. Moore KJ, Naito T, Martin C, Kelley VR: Enhanced response of mac- rophages to CSF-1 in autoimmune mice: A gene transfer strategy. J Immunol 157: 433–440, 1996 52. Schwarting A, Wada T, Kinoshita K, Tesch G, Kelley VR: IFN-gamma Supplemental information for this article is available online at http://www. receptor signaling is essential for the initiation, acceleration, and jasn.org/.

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