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Decoy Receptor 3 Ameliorates an Autoimmune Crescentic Glomerulonephritis Model in Mice

ʈ Shuk-Man Ka,* Huey-Kang Sytwu,†‡ Deh-Ming Chang,§ Shie-Liang Hsieh, Pei-Yi Tsai,† and Ann Chen*

*Department of Pathology, §Division of Rheumatology/Immunology & Allergy, Department of Medicine, Tri-Service General Hospital, †Graduate Institute of Medical Sciences, ‡Department of Microbiology and Immunology, National ʈ Defense Medical Center, and Institute and Department of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China

ABSTRACT Autoimmune crescentic glomerulonephritis (ACGN) is a variant of crescentic glomerulonephritis. The outcome of treatment of crescentic glomerulonephritis is poor. Binding of decoy receptor 3 (DCR3) to its ligand is capable of downregulating the alloresponsiveness of T cells. DCR3 has also been shown to benefit an experimental autoimmune model of diabetes. This study tested the hypothesis that a potential immune regulator, DCR3, could prevent the evolution of ACGN. With the use of an established ACGN model in mice, mice were treated with 100 ␮g/10 g body wt human DCR3 by hydrodynamics- based gene delivery at 14-d intervals. The results showed that the gene therapy resulted in (1) suppression of T and B cell activation and T cell proliferation; (2) a reduction in serum levels of proinflammatory cytokines; (3) improvement of proteinuria and renal dysfunction; (4) prevention of glomerular crescent formation, renal interstitial inflammation, and glomerulosclerosis; (5) a reduction in serum levels of autoantibodies and glomerular immune deposits; (6) inhibition of apoptosis in the spleen and kidney; (7) prevention of T cell and macrophage infiltration of the kidney; and (8) suppression of fibrosis-related gene expression in the kidney compared with empty vector–treated (disease control) ACGN mice. On the basis of these findings, it is proposed that human DCR3 exerts its preventive and protective effects on ACGN through modulation of T cell activation/proliferation, B cell activation, protection against apoptosis, and suppression of mononuclear leukocyte infiltration in the kidney.

J Am Soc Nephrol 18: 2473–2485, 2007. doi: 10.1681/ASN.2006111242

Systemic lupus erythematosus (SLE) is a typical au- (ACGN) is an extremely progressive form of lupus toimmune disease involving multiple organs. Al- nephritis and is classified as a type of crescentic though its cause is not entirely clear, the basic ab- glomerulonephritis11,12 in which widespread normality seems to be a failure of mechanisms that glomerular crescents, consisting of a mixture of maintain self-tolerance, resulting in the production parietal epithelial cells, monocytes/macrophages, of diverse autoantibodies, especially antinuclear an- tibodies,1 T cell abnormalities,2,3 and apoptosis.4–6 In patients and mouse models, pathogenic T cells Received November 15, 2006. Accepted May 24, 2007. recognize self-antigens and drive B cell hyperactiv- Published online ahead of print. Publication date available at ity, confirming their central role in the pathogenesis www.jasn.org. 3,7–9 of SLE. Lupus nephritis is a major renal injury Correspondence: Dr. Ann Chen, Department of Pathology, Tri- in SLE and is characterized by immune complex Service General Hospital, National Defense Medical Center, No. deposition in the glomerular and renal tubular and 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC, Phone: ϩ886-2-8792-7008; Fax: ϩ886-2-8792-7009; E-mail: doc31717@ 10 peritubular capillary basement membranes. Clin- ndmctsgh.edu.tw ically, autoimmune crescentic glomerulonephritis Copyright © 2007 by the American Society of Nephrology

J Am Soc Nephrol 18: 2473–2485, 2007 ISSN : 1046-6673/1809-2473 2473 BASIC RESEARCH www.jasn.org and lymphocytes, are formed.13,14 We have shown that a ner and significantly reduces the severity of insulitis in an au- murine chronic graft-versus-host disease, induced in toimmune diabetes model. In addition, in vivo administration C57BL/6 ϫ DBA/2J F1 hybrid mice by giving DBA/2J donor of hDCR3 ameliorates allograft rejection.17 The effects of lymphocytes, can progress to ACGN, with extensive, char- hDCR3 on immune regulation should be explored for its pos- acteristic glomerular crescent formation (up to 80% of glo- sible therapeutic use in controlling undesirable immune re- meruli examined), sclerosis, and intense interstitial inflam- sponses. Clinically, DCR3 gene expression has been detected in mation.15 Although recovery of renal functions in patients peripheral blood mononuclear leukocytes derived from pa- with crescentic glomerulonephritis can occur after early in- tients with SLE,24 although its clinical significance remains to tensive plasma exchange and/or treatment with steroids and be determined. cytotoxic agents, patients eventually require long-term di- In this study, we tested the hypothesis that in vivo over- alysis or transplantation.11 expression of hDCR3 would prevent crescentic formation of Decoy receptor 3 (DCR3) lacks the transmembrane do- an experimental ACGN. Using hydrodynamics-based gene main of the conventional TNF receptor and is a secreted delivery, we demonstrated that hDCR3 gene therapy is an protein.16,17 DCR3 can interact with Fas ligand (FasL),16 effective therapeutic approach in the ACGN model. Regu- LIGHT (homologous to lymphotoxins, shows inducible ex- lation of both T cell function and apoptosis in lymphoid pression and competes with HSV glycoprotein D for herpes organs and in the kidney seems to be the major factor re- virus entry mediator, a receptor expressed by T lympho- sponsible for its favorable effects in this autoimmune kid- cytes),18 and TNF-like molecule 1A.19 Binding of DCR3 to ney disease model. LIGHT also downregulates the alloresponsiveness of T cells.17,20 By helping tumor cells avoid immune attack through lymphocyte infiltration and FasL/LIGHT-medi- RESULTS ated apoptosis, increased DCR3 expression might benefit their growth.16,18 DCR3 exerts another regulatory function Serum Levels of hDCR3 Protein in Treated ACGN by directly modulating the differentiation and function of Mice macrophages and dendritic cells.21,22 Based on the hDCR3 expression (Figure 1, methods as de- Recently, Sung et al.23 reported that transgenic human scribed in Concise Methods), we delivered the hDCR3 plasmid DCR3 (hDCR3) protects mice from autoimmune and cyclo- to the mice at 14-d intervals. After administrations of hDCR3 phosphamide-induced diabetes in a dosage-dependent man- plasmid or empty vector, ELISA tests showed high serum levels

Figure 1. Serum levels and hepatic expression of human decoy receptor 3 (hDCR3) protein. (A) Serum hDCR3 levels in normal control mice after a single injection of hDCR3 plasmid or empty vector measured by ELISA. Data are means Ϯ SEM for groups of Figure 2. Serum levels of hDCR3 in treated autoimmune crescen- 10 mice. The arrow indicates the time of gene delivery with tic glomerulonephritis (ACGN) mice. Serum levels at week 3 (A) hDCR3 or empty vector. (B) hDCR3 protein expression in the liver and week 9 (B) in ACGN mice after administration of hDCR3 or of normal control mice on day 2 after a single injection of hDCR3 empty vector. Data are means Ϯ SEM for groups of 10 mice. #Not plasmid or empty vector. Magnification, ϫ400. detectable.

2474 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2473–2485, 2007 www.jasn.org BASIC RESEARCH of hDCR3 protein in the hDCR3-treated ACGN mice at 3 wk empty vector–treated ACGN mice at 3 wk (Figure 3, A and C) (Figure 2A) and 9 wk (Figure 2B), respectively, after ACGN or 9 wk (Figure 3, B and D) after ACGN induction, compared induction but not in the empty vector–treated ACGN mice or with normal control mice (both P Ͻ 0.005). However, this normal control mice. effect was significantly suppressed by administration of hDCR3 plasmid to ACGN mice compared with empty vec- DCR3 Suppresses T/B Cell Activities tor–treated ACGN mice (P Ͻ 0.05 at 3 wk; P Ͻ 0.005 at 9 Inhibition of T/B Cell Activation. wk). Moreover, hDCR3-treated ACGN mice showed a sig- ϩ ϩ Abnormal T and B cell cooperation can cause a graft-versus- nificantly lower percentage of CD4 CD69 cells (activated host reaction,9,25 the basic mechanism for the induction of the T helper cells) than that of empty vector–treated ACGN ACGN model. DCR3 has been reported to inhibit T cell pro- mice at week 3 (4.0 32 Ϯ 1.0 versus 8.1 Ϯ 1.5%; P Ͻ 0.05) and liferation and lymphokine secretion.17,19,21 We tested whether week 9 (9.5 Ϯ 0.5 versus 18.2 Ϯ 2.9%; P Ͻ 0.05), respec- ϩ ϩ hDCR3 could ameliorate the development of the ACGN model tively. Conversely, the percentage of CD19 CD69 cells by negatively regulating T cell and/or B cell activation by per- (activated B cells) was significantly decreased in hDCR3- forming flow cytometry of splenocytes. treated ACGN mice to the levels seen in normal control As shown in Figure 3, the percentage of CD3ϩCD69ϩ cells mice at week 9 (P Ͻ 0.005; Figure 3, B and D), although (activated T cells) in the spleen was significantly increased in there was no significant difference in the percentage of

ϩ Figure 3. Flow cytometry for T and B cells in the spleen. Immunofluorescence dot-plot pattern of the CD69 activation marker on CD3 ϩ ϩ ϩ ϩ ϩ T cells or CD19 B cells. (A and C) Week 3. (B and D) Week 9. (C and D) Percentage of CD3 CD69 T cells and CD19 CD69 . Each bar represents the means Ϯ SEM for groups of 10 mice. *P Ͻ 0.05; ***P Ͻ 0.005; NS, no significant difference.

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CD19ϩCD69ϩ cells between empty vector–treated ACGN and hDCR3-treated ACGN mice (Figure 3, A and C) at week 3 compared with empty vector–treated ACGN mice. Activation of T cell can lead to cytokine production, which in turn may assist in recruitment of other cells to the site of inflammation or mediate tissue damage.26 Therefore, we fur- ther measured the production of intracellular IL-2, IFN-␥,or IL-4 of T lymphocytes in the spleen by flow cytometry. As shown in Figure 4A, there was no significant difference in the percentage of T cells expressing IL-2, IFN-␥, or IL-4 between the empty vector–treated ACGN and hDCR3-treated ACGN mice at 3 wk, although the percentage of IFN-␥ was signifi- cantly increased in empty vector–treated ACGN or hDCR3- treated ACGN mice, compared with normal control mice (P Ͻ 0.005). However, the percentage of T cells expressing IL-2, IFN-␥, or IL-4 was significantly inhibited by the administra- tion of hDCR3 plasmid to ACGN mice at 9 wk, compared with empty vector–treated ACGN mice (each P Ͻ 0.05; Figure 4B).

Blocking of T Cell Proliferation. As shown in Figure 5, compared with empty vector–treated ACGN mice, hDCR3-treated ACGN mice showed significant Figure 5. T cell proliferation assay in the spleen. Thymidine suppression of spleen T cell proliferation to the level seen in Ͻ incorporation by anti–CD3 antibody–stimulated spleen T cells normal control mice at 3 and 9 wk (both P 0.05). from normal control mice and ACGN mice that were administered an injection of empty vector or hDCR3 plasmid at week 3 (A) and DCR3 Decreases Serum Levels of Proinflammatory week 9 (B). Data are means Ϯ SEM for groups of 10 mice. *P Ͻ Cytokines 0.05; ***P Ͻ 0.005. Monocyte chemoattractant protein-1 (MCP-1),27,28 IL-4,29,30 and IFN-␥31,32 have been implicated in the pathogenesis of SLE. To determine whether hDCR3 affected systemic proin- flammatory cytokine production, we measured serum levels of these proteins in the mice. At week 3, hDCR3-treated ACGN mice showed significantly lower levels of MCP-1 than empty vector–treated ACGN mice (P Ͻ 0.05), although there was no significant difference in the levels of IL-4 or IFN-␥ between hDCR3-treated ACGN and empty vector–treated ACGN mice (Figure 6). At week 9, however, compared with empty vector– treated ACGN mice, significantly lower serum levels of these cytokines were seen (each P Ͻ 0.05) in hDCR3-treated ACGN mice, the levels being similar to those in normal control mice (Figure 6, D through F).

DCR3 Interferes with ACGN Development DCR3 Mitigates Proteinuria and Abnormal Renal Function. As shown in Figure 7A, empty vector–treated ACGN mice developed proteinuria that started at week 4 and plateau between weeks 7 and 9. This clinical sign was greatly im- proved in the hDCR3-treated ACGN mice as early as week 4, returning to the normal range at weeks 7 to 9. Likewise, a dramatic improvement in renal function was noted in the Figure 4. Flow cytometry for intracellular cytokine staining in the hDCR3-treated ACGN mice, as compared with empty vec- spleen. The percentage of T cells expressing IL-2, IFN-␥, or IL-4 tor–treated ACGN mice at week 9, but significantly lower Ͻ from spleen. (A) Week 3. (B) Week 9. Each bar represents the serum levels of blood urea nitrogen (BUN; P 0.005; Figure means Ϯ SEM for groups of 10 mice. *P Ͻ 0.05; **P Ͻ 0.01; 7B) and creatinine (Cr; P Ͻ 0.005; Figure 7C) were seen in ***P Ͻ 0.005; NS, no significant difference. the hDCR3-treated mice, levels being similar to those in

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Figure 6. Serum levels of proinflammatory cytokines. (A through C) Week 3. (D through F) Week 9. (A and D) Monocyte chemoattractant protein 1 (MCP-1). (B and E) IFN-␥. (C and F) IL-4 measured by ELISA. Data are means Ϯ SEM for groups of 10 mice. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.005; NS, no significant difference. normal control mice. There was no significant difference in treated ACGN mice at 3 wk (Figure 9, B and C), although both the levels of BUN or creatinine between the hDCR3-treated empty vector–treated and hDCR3-treated mice showed higher and empty vector–treated ACGN mice at week 3. levels of glomerular IgG deposits than normal control mice (both P Ͻ 0.05). DCR3 Prevents Glomerular Crescent Formation, Glomeruloscle- rosis, and Renal Interstitial Inflammation. DCR3 Inhibits Apoptosis in the Spleen and Kidney Empty vector–treated ACGN mice showed extensive cres- Apoptosis is a tightly regulated process of programmed cell cent formation, glomerulosclerosis, and interstitial (mainly death, and its abnormal regulation is responsible for the patho- periglomerular) mononuclear leukocyte infiltration at week genesis of both SLE and lupus nephritis.4–6,33 Because DCR3 9 (Figure 8, E and H), all of which were substantially inhib- has been shown to modulate apoptosis in some tumor cells and ited by hDCR3 administration (P Ͻ 0.005; Figure 8, F and cultured lymphocytes,16,18 we tested whether hDCR3 overex- H). There were no significant histopathologic changes in pression could inhibit apoptosis in vivo in the spleen or kidney the empty vector–treated ACGN or hDCR3-treated ACGN in ACGN mice. mice compared with normal control mice at week 3 (Figure With the use of the terminal deoxynucleotidyl transferase- 8, A through C and G). mediated dUTP nick-end labeling (TUNEL) assay, significant suppression of apoptosis was seen in the spleen of hDCR3- DCR3 Reduces Serum Levels of Autoantibodies and Glomerular treated ACGN mice compared with empty vector–treated Immune Deposits. ACGN mice (P Ͻ 0.01; Figure 10, J through L, and V) at week Because autoantibodies play a major pathogenic role in the 9. Again, in the kidney, only a few apoptotic figures were iden- development of SLE,1,9 we measured serum levels of anti–dou- tified in hDCR3-treated ACGN mice (Figure 10O) at 9 wk, ble-stranded DNA (anti-dsDNA) antibody in sera obtained at although empty vector–treated ACGN mice showed promi- week 3 and week 9, respectively, by ELISA. As shown in Figure nent apoptosis that was widespread in the glomerulus and re- 9A, the autoantibody levels were significantly lower in the nal tubular compartment, respectively (both P Ͻ 0.01; Figure hDCR3-treated ACGN mice (P Ͻ 0.01) than in empty vector– 10, M, N, and W). Besides, earlier at week 3, suppression of treated ACGN mice at 9 wk, although there was no significant apoptosis in the spleen was noted in hDCR3-treated ACGN difference in autoantibody levels between empty vector– mice, compared with empty vector–treated ACGN mice (Fig- treated ACGN and hDCR3-treated ACGN mice at 3 wk. In ure 10, A through C, and S), although there was no statistical addition, at week 9, the glomerular deposits of IgG (Figure 9B) significance. At this stage, there was no detectable apoptosis in seen in empty vector–treated ACGN mice were significantly empty vector–treated ACGN or hDCR3-treated ACGN mice reduced in hDCR3-treated ACGN mice (P Ͻ 0.005; Figure (Figure 10, D through F and T). 9C). There was no significant difference in glomerular IgG Considering that TUNEL is unlikely to be able to differ- deposits between empty vector–treated ACGN and hDCR3- entiate apoptosis from necrosis, we performed immunohis-

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present in crescentic glomerulonephritis, including ACGN, and Lan et al.12 proposed that macrophage accumulation within crescents plays an important role in the progression of epithelial-dominated early cellular crescents to macroph- age-dominated advanced and fibrocellular crescents. In this study, at week 9, IHC showed diffuse infiltration of CD3ϩ T cells, CD4ϩ T cells (Figure 11H), and F4/80 macrophages (Figure 11K) in the periglomerular region of the renal in- terstitium in empty vector–treated ACGN mice and that this was markedly reduced in hDCR3-treated ACGN mice (each P Ͻ 0.005; Figure 11, G, I, J, L, and N). At week 3, only a few or no such mononuclear cells were noted in either empty vector–treated ACGN or hDCR3-treated ACGN (Figure 11, A through F, and M).

DCR3 Suppresses Fibrosis-Related Gene Expression in the Kidney As a subcategory of crescentic glomerulonephritis, ACGN tends to evolve rapidly to diffuse glomerulosclerosis and inter- stitial fibrosis.15 We therefore evaluated the effects of hDCR3 gene therapy on renal fibrosis in the ACGN model, focusing on ␣-smooth muscle actin and collagen IV. As shown in Figure 12A, real-time PCR demonstrated marked renal expression of both mRNA in empty vector–treated ACGN mice compared with normal controls (P Ͻ 0.01) and that hDCR3 plasmid administration was associated with significant suppression of the expression of both mRNA (P Ͻ 0.01), their mRNA levels in hDCR3-treated ACGN not being significantly different from those in normal control mice. Again, although greatly en- hanced expression of the proteins encoded by these fibrosis- related genes was detected in empty vector–treated ACGN Figure 7. Proteinuria and renal function. (A) Time-course studies mice compared with normal control mice, this was signifi- of proteinuria. The arrows indicate the time of gene delivery. (B) cantly reduced in hDCR3-treated ACGN mice (Figure 12, B Serum blood urea nitrogen (BUN) levels. (C) Serum creatinine and C). levels. Data are means Ϯ SEM for groups of 10 mice. ***P Ͻ 0.005; NS, no significant difference. tochemistry (IHC) with anti–single-stranded DNA (anti- DISCUSSION ssDNA) monoclonal antibody (mAb) in kidney tissues.34 Again, hDCR3-treated ACGN mice showed a significant re- This study is the first to report that in an experimental ACGN duction of apoptosis compared with empty vector–treated mouse model, showing clinical immunologic and pathologic ACGN mice (Figure 10, P through R, and X) at week 9, features similar to those of patients with lupus nephritis, over- although only a few apoptotic figures were noted at week 3 expression of hDCR3 by hydrodynamics-based gene delivery in both hDCR3-treated ACGN and empty vector–treated protects the animals from developing glomerular crescents, ACGN mice (Figure 10, G through I, and U). renal interstitial inflammation, and glomerulosclerosis. The favorable effects of hDCR3 gene therapy on the ACGN model DCR3 Prevents T Cell and Macrophage Infiltration of may be due to one or more of the following three mechanisms: the Kidney (1) Inhibition of T cell activation/proliferation and B cell acti- To gain further insights into the mechanisms by which vation, (2) protection against apoptosis in lymphoid organs hDCR3 prevented glomerular crescent formation and rele- (e.g., spleen) and kidney, and (3) suppression of mononuclear vant renal injury in the ACGN model, we next assessed leukocyte infiltration of the kidney. whether renal infiltration of mononuclear leukocytes was Regarding the first proposed mechanism, it is generally ac- altered by hDCR3 gene therapy. Infiltration of T cells35,36 cepted that abnormal T cell function plays an important role in and/or monocytes/macrophages14,36 plays an important SLE.2,3,7,8 Pathogenic alloreactive25 and host37 T cells have been role in the formation of the glomerular crescents invariably demonstrated to mediate the development of chronic graft-

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Figure 8. Renal pathology. (A through C) Week 3. (D through F) Week 9. (A and D) Normal control mice. (B and E) Empty vector–treated ACGN mice. (C and F) hDCR3-treated ACGN mice. (G and H) Renal lesion scores. Data are means Ϯ SEM for groups of 10 mice. ***P Ͻ 0.005. #Not detectable. Magnification, ϫ400. versus-host disease in mice, featuring SLE-like syndrome and percentage of T cells expressing IL-2, IFN-␥, and IL-4 in the renal lesions of various degrees.38 In this regard, DCR3 might spleen at 9 wk of the experiment. It should be noted that IL- downregulate alloresponsiveness of T cell function17,20 and 429,30 and IFN-␥31,32 contribute to the pathogenesis of human lymphokine production17,19,21 systemically, which might, in lupus nephritis. hDCR3 administration also greatly depressed turn, prevent the evolution of ACGN. In this study, we admin- the sera levels of both IL-4 and IFN-␥ at week 9. This could in istered the hDCR3 plasmid by hydrodynamic-based gene de- part explain the favorable effects of hDCR3 on the condition of livery to ACGN mice. Our data showed that at early stage the kidney. On the whole, our data support the idea that block- (week 3) of the experiment, hDCR3 significantly inhibited T ade of T cell activity could be the key mechanism by which cell activation and reduced T cell proliferation in the hDCR3- hDCR3 prevents renal injury. treated mice, although its potential suppressive effects on au- The second possible mechanism is that hDCR3 prevents toantibody and glomerular immune deposits were not seen. At apoptosis in lymphoid organs (e.g., spleen) and kidney. Dys- 9 wk, in addition to significant suppression of T cell activation regulation of apoptosis and the clearance of apoptotic prod- and proliferation, hDCR3-treated mice demonstrated a great ucts have been implicated in the pathogenesis of SLE.4,5,33,39 inhibition on B cell activation, autoantibody production, and Xue et al.40 showed that acceleration of lymphocytic apoptosis glomerular immune deposition. The data suggested that the plays a crucial role in immune pathogenic injury. High apo- earlier suppressive effect on T cells than that on B cells by ptotic rates are also identified in severe active glomerular le- hDCR3 administration could be operated in this experimental sions in patients with lupus nephritis.41 The Fas pathway of model of ACGN. However, T cells exert their effector function apoptosis has been shown to be involved in the process of partly through the production and release of cytokines.26 In immune tolerance by deletion of unwanted autoreactive T cells this study, hDCR3 administration significantly inhibited the and B cells.40,42 DCR3 can bind to FasL and inhibit FasL-in-

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Figure 9. Serum levels of autoantibodies and glomerular immune deposits. (A) Anti-double-stranded DNA (anti-dsDNA) levels by ELISA at week 3 and week 9. (B) Immunohistochemistry (IHC) showing glomerular IgG deposition. (C) Staining intensity score. Data are means Ϯ SEM for groups of 10 mice. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.005; NS, no significant difference. Magnification, ϫ400. duced apoptosis.16 We found that hDCR3 overexpression sig- cell activation/proliferation and B cell activation could play a nificantly inhibited splenic apoptosis at week 9, which has been role in the mode of action of hDCR3. Suppression of apoptosis shown to contribute to the development of lupus nephritis.43 in lymphoid organs and the kidney and inhibition of mononu- Consistent with these findings, Seery et al.44 proposed that the clear leukocyte infiltration of the kidney are also highly impli- use of an apoptosis inhibitor might be beneficial in the treat- cated in its effects. ment of patients. Our data showed that blocking of apoptosis in both the glomerular and tubulointerstitial compartments of CONCISE METHODS the kidney was associated with lower histopathologic severity of renal damage. Inhibition of apoptosis in lymphoid tissues Induction of ACGN in the Model System (as represented by the spleen) and in the kidney is probably ACGN was induced in 7- to 8-wk-old female (C57BL/6 ϫ DBA/2J) F1 involved in the beneficial effects of DCR3 on ACGN. hybrid mice by injection of DBA/2J donor lymphocytes, as described Finally, as regards the third proposed mechanism, deletion previously.15 Briefly, a cell suspension containing a mixture of donor of T cells and/or macrophages attenuates crescentic glomeru- cells from the thymus, spleen, and lymph nodes (neck, axillary, and lonephritis,12,14,36 suggesting an essential role for mononuclear inguinal regions) was injected intravenously three times at 3- to 4-d leukocytes in the pathogenesis of crescents. The intraglomeru- intervals. All mice were killed at week 3 or week 9 after disease induc- lar cellular structures of crescents are formed partly by prolif- tion. Spleen, renal cortical tissue, blood, and urine samples were col- eration of the parietal epithelial cells and partly by mononu- lected and stored appropriately until analysis. All animal experiments clear infiltrates.11,13 hDCR3 administration resulted in (1) were performed with the approval of the Institutional Animal Care persistent reduction of serum MCP-1 levels starting early at and Use Committee of the National Defense Medical Center, Taiwan, week 3 and (2) almost total absence of periglomerular and and were consistent with the National Institutes of Health Guide for interstitial mononuclear leukocyte infiltration in the kidney, the Care and Use of Laboratory Animals. reducing the formation of crescents in the glomeruli. Consis- tent with our data, Roth et al.45 showed that DCR3 decreases Plasmid hDCR3 ϩ CD4 T cell and macrophage infiltration in a rat gliosarcoma hDCR3 cDNA (sequence data available from GenBank/EMBL/ model. We believe that this effect might serve as a crucial and DDBJ under accession no. AF104419) was produced as described direct mechanism operating locally in the kidney for the ben- previously.22 Briefly, the hDCR3 gene was isolated by the reverse eficial effect of hDCR3 on ACGN. transcriptase–PCR using the forward primer 5Ј-CAAGGAC Our data show that administration of an hDCR3 plasmid, CATGAGGGCGCTG-3Ј and reverse primer 5Ј-GTGCACAGG- given by hydrodynamics-based gene delivery, prevents the de- GAGGAAGCGC-3Ј. The amplified product was cloned using the velopment of ACGN. We provide evidence that blocking of T pGEM-T Easy Vector System (Promega, Madison, WI), and then

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Figure 10. Detection of apoptosis. (A through I) Week 3. (J through R) Week 9. (A through F and J through O) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). (G through I and P through R) Anti–single-stranded DNA (anti-ssDNA) mAb. (A through C and J through L) Spleen. (D through I and M) Kidney. (A, D, G, J, M, and P) Normal control mice. (B, E, H, K, N, and Q) Empty vector–treated ACGN mice. (C, F, I, L, O, and R) hDCR3-treated ACGN mice. The arrow indicates the positively stained cell. (S and V) Scoring of positive cells in spleen. (T, U, W, and X) Scoring of positive cells in kidney. Data are means Ϯ SEM for groups of 10 mice. *P Ͻ 0.05; **P Ͻ 0.01. #Not detectable; NS, no significant difference. Magnification, ϫ400.

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vector (pCMV) served as controls. The animals were then killed at days 0, 1, 2, 3, 7, 14, and 28 after plasmid administration, and serum and liver tissues were stored appropriately until analysis. As shown in Figure 1A, serum hDCR3 levels started to increase on day 1, peaked on day 2, gradually declined to day 14, and returned to baseline levels by day 28, as demonstrated by ELISA. By IHC, the liver showed strong expres- sion of hDCR3 (Figure 1B), although there was no signal of staining in the kidney. No staining was seen in the liver (Figure 1B) or kidney at any time point in mice that were given empty vector. On the basis of the hDCR3 expression, we de- cided to deliver the hDCR3 plasmid at 14-d in- tervals in the subsequent therapeutic study. The first dose of hDCR3 plasmid was given on the day when the mice received the first shot of lym- phocytes for the induction of the ACGN model and the second after all of the injections for ACGN induction had been given. ACGN mice that were given empty vector were used as dis- ease controls.

Clinical and Pathologic Evaluation Collection and assay of blood and urine samples were performed as described previously.47 Urine samples were collected in metabolic cages weekly, and urinary levels of protein were deter- mined using a Pierce BCA protein assay kit (Per- bio Science, Etten-Leur, Netherlands); serum samples were collected at weeks 3 and week 9, respectively, to measure serum levels of BUN and Cr. For histopathology, the tissues were fixed in 10% buffered formalin and embedded in paraf- ␮ Figure 11. Renal infiltration of T cells and macrophages. (A through F) Week 3 (G fin. Sections (4 m) were stained with hema- through L) Week 9. (A through C and G through I) CD4 T helper cells. (D through F and toxylin and eosin. One hundred glomeruli were J through L) F4/80 monocytes/macrophages. (A, D, G, and J) Normal control mice (B, examined by light microscopy at the magnifica- E, H, and K) Empty vector–treated ACGN mice (C, F, I, and L) hDCR3-treated ACGN tion of ϫ400 for the slide with at least two renal mice. (M and N) Infiltrating cell score in the periglomeruli. Data are means Ϯ SEM for tissue sections each. Scoring of the severity of groups of 10 mice. ***P Ͻ 0.005; NS, no significant difference. Magnification, ϫ400 renal lesions was performed as described previ- each. ously.47 The proportion (percentage) was calcu- lated for the following three major components: the gene was subcloned into the pCMV vector (Clontech, Palo Crescent formation, glomerular sclerosis, and periglomerular inflam- Alto, CA) to obtain the pCMV-hDCR3 expression construct. The mation. plasmids were prepared using EndoFree plasmid kits (Qiagen, Va- lencia, CA) according to the manufacturer’s instructions. Immunofluorescence, IHC, and Detection of Apoptosis Hydrodynamics-Based Gene Delivery For immunofluorescence (IF), frozen renal tissues were cut, air-dried, To characterize the expression pattern and determine the interval at fixed in acetone for 5 min at room temperature, and incubated with which hDCR3 plasmids were to be administered to the animals, we FITC-conjugated goat anti-mouse IgG (Cappel, Durham, NC). Scor- first injected a single dose of 100 ␮g/10 g body wt hDCR3 plasmid ing of staining intensity was performed as described previously.47 (diluted in 1.6 ml of normal saline) into normal (C57BL/6 ϫ DBA/2J) For IHC, methyl Carnoy solution or formalin-fixed, paraffin-em- F1 hybrid mice (n ϭ 10) by hydrodynamics-based gene delivery bedded tissue sections (4 ␮m) were stained with biotin-labeled mouse through tail vein as described previously.46 Mice that received empty anti–␣-smooth muscle actin antibodies (Neomarkers, Fremont, CA),

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Flow Cytometry Splenocytes from the mice were treated with Tris-buffered ammonium chloride to eliminate erythrocytes; washed; and resuspended in RPMI 1640 supplemented with 10% FCS, HEPES buffer, L-glutamine, and penicillin/streptomy- cin (all from Life Technologies, Invitrogen, Carlsbad, CA). The cells were stained with either surface markers for T or B cell activation or in- tracellular cytokines produced by T cells. FITC- conjugated anti-mouse CD3 (17A2), CD4 (G11.5), or CD19 (B cell, 1D3) antibodies and phycoerythrin-conjugated anti-mouse CD69 (H1.2F3) antibodies (all from BD Biosciences, San Jose, CA) were used for the analysis of sur- face markers for T or B cell activation with FACSCalibur (BD Biosciences), as described previously.23 For intracellular cytokine staining, the cells were cultured in 24-well flat-bottom microtiter plates (1 ϫ 106 cells in 500 ␮l/well) in the presence or absence of 20 ng/ml phorbol nyristate acetate, 1 ␮M ionomycin, and 4 ␮M monensin (all from Sigma, St. Louis, MO) for 6 h, as described previously.50 The cells were Figure 12. Renal fibrosis-related gene expression at week 9. (A) Real-time PCR for stained with FITC-conjugated anti-mouse CD3 ␣-smooth muscle actin and collagen IV mRNA levels. Each bar represents the means Ϯ (BD Biosciences) for1hat4°C, fixed in 1% Ͻ Ͻ ␣ SEM for groups of 10 mice. **P 0.01; ***P 0.005. (B) -Smooth muscle actin paraformaldehyde (Sigma), and resuspended in protein expression by IHC. (C) Collagen IV protein expression by IHC. Magnification, 100 ␮l of permeabilization buffer (0.1% sapo- ϫ400. nin, 1% BSA, and 0.1% sodium azide; all from Sigma). Cytokine staining was stained with phy- goat anti–collagen IV (Southern Biotech, Birmingham, AL), or rat coerythrin-conjugated anti-mouse IL-2, IFN-␥, or IL-4 antibodies anti-F4/80 (monocytes/macrophages; Serotec, Raleigh, NC) antibod- (all from BD Biosciences) for1hat4°C, with FACSCalibur (BD Bio- ies. For detection of hDCR3 protein in the liver and the kidney, frozen sciences) as described previously. sections of the tissues were fixed in acetone for 5 min and incubated with biotin-conjugated anti-hDCR3 antibody (Anawrahta, Taipei) as T Cell Proliferation Analysis described previously.23 To detect pan-T cells and T helper cells, renal Splenocytes from the mice were prepared as described previously, tissues were fixed in periodate-lysine paraformaldehyde as described then were cultured in triplicate in wells (5 ϫ 105 cells in 200 ␮l/well) previously,48 then incubated with biotin-conjugated anti-mouse CD3 in 96-well flat-bottom microtiter plates previously coated overnight (pan-T cell; Serotec) or CD4 (T helper cell; BioLegend, San Diego, at 4°C with 0.25 ␮g/ml anti-mouse CD3 (145–2C11) antibodies (BD CA) antibodies. Scoring of staining intensity was performed as de- Biosciences). After 48 h, the cultures were pulsed with 1 ␮Ci of 3H- scribed previously.15 methyl thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) For the detection of apoptosis, both terminal deoxynucleotidyl and harvested 16 h later, and the incorporated 3H-methyl thymidine transferase-mediated dUTP nick-end labeling (TUNEL) and IHC was measured using a TopCount (Packard, PerkinElmer, Boston, with anti-ssDNA were used. For TUNEL, formalin-fixed tissue sec- MA) as described previously.23 tions were stained with ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Chemicon, Temecula, CA) according to the manufac- Real-Time PCR Assay turer’s instructions. For IHC with anti-ssDNA, formalin-fixed tissue RNA was extracted from the renal cortex using TriZOL reagents (In- sections were incubated with proteinase K (DAKO, Carpinteria, CA) vitrogen). For first-strand cDNA synthesis, 1.5 ␮g of RNA was used in for 20 min and then were incubated in 50% (vol/vol) formamide in a single-round reverse transcriptase reaction. The reaction mixture distilled water at 56°C for 20 min, followed by the incubation with consisted of 0.9 ␮l of Oligo (dT) 12 to 18 primer, 1.0 mM dNTP, 1ϫ mouse anti-ssDNA mAb (Chemicon) at 4°C overnight, as described first-strand buffer, 0.4 mM dithiothreitol, 80 U of RNaseout recom- previously.34 For scoring, 50 randomly selected glomeruli were exam- binant ribonuclease inhibitor, and 300 U of Superscript II RNase H ined, and 20 randomly selected fields of renal tubules in the cortical (Invitrogen) in a total volume of 25 ␮l. Real-time PCR was performed area were examined by light microscopy at the magnification of ϫ400, on an ABI Prism 7700 Sequence Detection System (Applied Biosys- as described previously.49 tems, Foster City, CA). All of the probes and primers were Assays-on-

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Demand Gene expression products (Applied Biosystems). Real-time 8. Desai-Mehta A, Mao C, Rajagopalan S, Robinson T, Datta SK: Struc- PCR reactions were performed using 10 ␮l of cDNA, 12.5 ␮l of Taq- ture and specificity of T cell receptors expressed by potentially patho- Man Universal PCR Master Mix (Applied Biosystems), and 1.25 ␮lof genic anti-DNA autoantibody-inducing T cells in human lupus. J Clin ␮ Invest 95: 531–541, 1995 the specific probe/primer mixture in a total volume of 25 l. The 9. Gleichmann E, Van Elven EH, Van der Veen JP: A systemic lupus thermal cycler conditions were 1 ϫ 2 min at 50°C, 1 ϫ 10 min at 95°C, erythematosus (SLE)-like disease in mice induced by abnormal T-B cell and 40 cycles of denaturation (15 s at 95°C) and combined annealing/ cooperation. Preferential formation of autoantibodies characteristic of extension (1 min at 60°C). The housekeeping gene glyceraldehyde-3- SLE. Eur J Immunol 12: 152–159, 1982 phosphate dehydrogenase was used as the internal standard. 10. Abbas AK: Disease of immunity. In: Robbins and Cotran Pathologic Basis of Disease, 7th Ed., edited by Kumar V, Abbas AK, Fausto N, Philadelphia, Saunders, 2005, pp 231 ELISA 11. Alpers C: The Kidney. In: Robbins and Cotran Pathologic Basis of Serum levels of hDCR3 were measured using an hDCR3 ELISA kit Disease, 7th Ed., edited by Kumar V, Abbas AK, Fausto N, Philadel- (Anawrahta), according to the manufacturer’s instructions. Serum phia, Saunders, 2005, pp 976–978 levels of MCP-1, IFN-␥, and IL-4 were measured using commer- 12. Lan HY, Nikolic-Paterson DJ, Mu W, Atkins RC: Local macrophage cial ELISA kits (BD Biosciences) according to the manufacturer’s proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis. instructions. Anti-dsDNA antibody was measured using an anti- Clin Exp Immunol 110: 233–240, 1997 mouse dsDNA ELISA kit (Alpha Diagnostic, San Antonio, TX). In 13. Lan HY, Nikolic-Paterson DJ, Atkins RC: Involvement of activated all ELISA, the absorbance at 450 nm was measured using an ELISA periglomerular leukocytes in the rupture of Bowman’s capsule and plate reader (Bio-Tek, Winooski, VT). glomerular crescent progression in experimental glomerulonephritis. Lab Invest 67: 743–751, 1992 14. Isome M, Fujinaka H, Adhikary LP, Kovalenko P, El-Shemi AG, Yoshida Statistical Analysis Y, Yaoita E, Takeishi T, Takeya M, Naito M, Suzuki H, Yamamoto T: Values are presented as the means Ϯ SEM. Comparison between two Important role for macrophages in induction of crescentic anti-GBM groups was performed using t test. P Ͻ 0.05 was taken as a statistical glomerulonephritis in WKY rats. Nephrol Dial Transplant 19: 2997– difference. 3004, 2004 15. Ka SM, Rifai A, Chen JH, Cheng CW, Shui HA, Lee HS, Lin YF, Hsu LF, Chen A: Glomerular crescent-related biomarkers in a murine model of chronic graft versus host disease. Nephrol Dial Transplant 21: 288– ACKNOWLEDGMENTS 298, 2006 16. Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, Huang A, Donahue CJ, Sherwood SW, Baldwin DT, Godowski PJ, This study was supported by grants from Tri-Service General Hospital Wood WI, Gurney AL, Hillan KJ, Cohen RL, Goddard AD, Botstein D, (TSGH-C92-4-S02) and Ministry of Economy (95-EC-17-A-20-S1- Ashkenazi A: Genomic amplification of a decoy receptor for Fas ligand 028), Taiwan, Republic of China. in lung and colon cancer. Nature 396: 699–703, 1998 17. Zhang J, Salcedo TW, Wan X, Ullrich S, Hu B, Gregorio T, Feng P, Qi S, Chen H, Cho YH, Li Y, Moore PA, Wu J: Modulation of T-cell responses to alloantigens by TR6/DcR3. J Clin Invest 107: 1459–1468, 2001 DISCLOSURES 18. 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