UP FRONT MATTERSBasic Research

Decoy Receptor 3 Ameliorates an Autoimmune Crescentic Glomerulonephritis Model in Mice

Shuk-Man Ka, Huey-Kang Sytwu, Deh-Ming Chang, Shie-Liang Hsieh, Pei-Yi Tsai and Ann Chen
JASN September 2007, 18 (9) 2473-2485; DOI: https://doi.org/10.1681/ASN.2006111242

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 μg/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.

Systemic lupus erythematosus (SLE) is a typical autoimmune disease involving multiple organs. Although its cause is not entirely clear, the basic abnormality seems to be a failure of mechanisms that maintain self-tolerance, resulting in the production of diverse autoantibodies, especially antinuclear antibodies,1 T cell abnormalities,2,3 and apoptosis.4–6 In patients and mouse models, pathogenic T cells recognize self-antigens and drive B cell hyperactivity, confirming their central role in the pathogenesis of SLE.3,7–9 Lupus nephritis is a major renal injury in SLE and is characterized by immune complex deposition in the glomerular and renal tubular and peritubular capillary basement membranes.10 Clinically, autoimmune crescentic glomerulonephritis (ACGN) is an extremely progressive form of lupus nephritis and is classified as a type of crescentic glomerulonephritis11,12 in which widespread glomerular crescents, consisting of a mixture of parietal epithelial cells, monocytes/macrophages, and lymphocytes, are formed.13,14 We have shown that a murine chronic graft-versus-host disease, induced in C57BL/6 × DBA/2J F1 hybrid mice by giving DBA/2J donor lymphocytes, can progress to ACGN, with extensive, characteristic glomerular crescent formation (up to 80% of glomeruli examined), sclerosis, and intense interstitial inflammation.15 Although recovery of renal functions in patients with crescentic glomerulonephritis can occur after early intensive plasma exchange and/or treatment with steroids and cytotoxic agents, patients eventually require long-term dialysis or transplantation.11

Decoy receptor 3 (DCR3) lacks the transmembrane domain of the conventional TNF receptor and is a secreted protein.16,17 DCR3 can interact with Fas ligand (FasL),16 LIGHT (homologous to lymphotoxins, shows inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes),18 and TNF-like molecule 1A.19 Binding of DCR3 to LIGHT also downregulates the alloresponsiveness of T cells.17,20 By helping tumor cells avoid immune attack through lymphocyte infiltration and FasL/LIGHT-mediated apoptosis, increased DCR3 expression might benefit their growth.16,18 DCR3 exerts another regulatory function by directly modulating the differentiation and function of macrophages and dendritic cells.21,22

Recently, Sung et al.23 reported that transgenic human DCR3 (hDCR3) protects mice from autoimmune and cyclophosphamide-induced diabetes in a dosage-dependent manner and significantly reduces the severity of insulitis in an autoimmune diabetes model. In addition, in vivo administration of hDCR3 ameliorates allograft rejection.17 The effects of hDCR3 on immune regulation should be explored for its possible therapeutic use in controlling undesirable immune responses. Clinically, DCR3 gene expression has been detected in peripheral blood mononuclear leukocytes derived from patients with SLE,24 although its clinical significance remains to be determined.

In this study, we tested the hypothesis that in vivo overexpression of hDCR3 would prevent crescentic formation of an experimental ACGN. Using hydrodynamics-based gene delivery, we demonstrated that hDCR3 gene therapy is an effective therapeutic approach in the ACGN model. Regulation of both T cell function and apoptosis in lymphoid organs and in the kidney seems to be the major factor responsible for its favorable effects in this autoimmune kidney disease model.

RESULTS

Serum Levels of hDCR3 Protein in Treated ACGN Mice

Based on the hDCR3 expression (Figure 1, methods as described in Concise Methods), we delivered the hDCR3 plasmid to the mice at 14-d intervals. After administrations of hDCR3 plasmid or empty vector, ELISA tests showed high serum levels of hDCR3 protein in the hDCR3-treated ACGN mice at 3 wk (Figure 2A) and 9 wk (Figure 2B), respectively, after ACGN induction but not in the empty vector–treated ACGN mice or normal control mice.

Figure 1.
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 ± SEM for groups of 10 mice. The arrow indicates the time of gene delivery with hDCR3 or empty vector. (B) hDCR3 protein expression in the liver of normal control mice on day 2 after a single injection of hDCR3 plasmid or empty vector. Magnification, ×400.

Figure 2.
Figure 2.

Serum levels of hDCR3 in treated autoimmune crescentic glomerulonephritis (ACGN) mice. Serum levels at week 3 (A) and week 9 (B) in ACGN mice after administration of hDCR3 or empty vector. Data are means ± SEM for groups of 10 mice. #Not detectable.

DCR3 Suppresses T/B Cell Activities

Inhibition of T/B Cell Activation.

Abnormal T and B cell cooperation can cause a graft-versus-host reaction,9,25 the basic mechanism for the induction of the ACGN model. DCR3 has been reported to inhibit T cell proliferation and lymphokine secretion.17,19,21 We tested whether hDCR3 could ameliorate the development of the ACGN model by negatively regulating T cell and/or B cell activation by performing flow cytometry of splenocytes.

As shown in Figure 3, the percentage of CD3+CD69+ cells (activated T cells) in the spleen was significantly increased in empty vector–treated ACGN mice at 3 wk (Figure 3, A and C) or 9 wk (Figure 3, B and D) after ACGN induction, compared with normal control mice (both P < 0.005). However, this effect was significantly suppressed by administration of hDCR3 plasmid to ACGN mice compared with empty vector–treated ACGN mice (P < 0.05 at 3 wk; P < 0.005 at 9 wk). Moreover, hDCR3-treated ACGN mice showed a significantly lower percentage of CD4+CD69+ cells (activated T helper cells) than that of empty vector–treated ACGN mice at week 3 (4.0 32 ± 1.0 versus 8.1 ± 1.5%; P < 0.05) and week 9 (9.5 ± 0.5 versus 18.2 ± 2.9%; P < 0.05), respectively. Conversely, the percentage of CD19+CD69+ cells (activated B cells) was significantly decreased in hDCR3-treated ACGN mice to the levels seen in normal control mice at week 9 (P < 0.005; Figure 3, B and D), although there was no significant difference in the percentage of CD19+CD69+ 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.

Figure 3.
Figure 3.

Flow cytometry for T and B cells in the spleen. Immunofluorescence dot-plot pattern of the CD69 activation marker on CD3+ 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 ± SEM for groups of 10 mice. *P < 0.05; ***P < 0.005; NS, no significant difference.

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 further measured the production of intracellular IL-2, IFN-γ, 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-γ, or IL-4 between the empty vector–treated ACGN and hDCR3-treated ACGN mice at 3 wk, although the percentage of IFN-γ was significantly increased in empty vector–treated ACGN or hDCR3-treated ACGN mice, compared with normal control mice (P < 0.005). However, the percentage of T cells expressing IL-2, IFN-γ, or IL-4 was significantly inhibited by the administration of hDCR3 plasmid to ACGN mice at 9 wk, compared with empty vector–treated ACGN mice (each P < 0.05; Figure 4B).

Figure 4.
Figure 4.

Flow cytometry for intracellular cytokine staining in the spleen. The percentage of T cells expressing IL-2, IFN-γ, or IL-4 from spleen. (A) Week 3. (B) Week 9. Each bar represents the means ± SEM for groups of 10 mice. *P < 0.05; **P < 0.01; ***P < 0.005; NS, no significant difference.

Blocking of T Cell Proliferation.

As shown in Figure 5, compared with empty vector–treated ACGN mice, hDCR3-treated ACGN mice showed significant suppression of spleen T cell proliferation to the level seen in normal control mice at 3 and 9 wk (both P < 0.05).

Figure 5.
Figure 5.

T cell proliferation assay in the spleen. Thymidine incorporation by anti–CD3 antibody–stimulated spleen T cells from normal control mice and ACGN mice that were administered an injection of empty vector or hDCR3 plasmid at week 3 (A) and week 9 (B). Data are means ± SEM for groups of 10 mice. *P < 0.05; ***P < 0.005.

DCR3 Decreases Serum Levels of Proinflammatory Cytokines

Monocyte chemoattractant protein-1 (MCP-1),27,28 IL-4,29,30 and IFN-γ31,32 have been implicated in the pathogenesis of SLE. To determine whether hDCR3 affected systemic proinflammatory 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 < 0.05), although there was no significant difference in the levels of IL-4 or IFN-γ 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 < 0.05) in hDCR3-treated ACGN mice, the levels being similar to those in normal control mice (Figure 6, D through F).

Figure 6.
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-γ. (C and F) IL-4 measured by ELISA. Data are means ± SEM for groups of 10 mice. *P < 0.05; **P < 0.01; ***P < 0.005; NS, no significant difference.

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 improved 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 hDCR3-treated ACGN mice, as compared with empty vector–treated ACGN mice at week 9, but significantly lower serum levels of blood urea nitrogen (BUN; P < 0.005; Figure 7B) and creatinine (Cr; P < 0.005; Figure 7C) were seen in the hDCR3-treated mice, levels being similar to those in normal control mice. There was no significant difference in the levels of BUN or creatinine between the hDCR3-treated and empty vector–treated ACGN mice at week 3.

Figure 7.
Figure 7.

Proteinuria and renal function. (A) Time-course studies of proteinuria. The arrows indicate the time of gene delivery. (B) Serum blood urea nitrogen (BUN) levels. (C) Serum creatinine levels. Data are means ± SEM for groups of 10 mice. ***P < 0.005; NS, no significant difference.

DCR3 Prevents Glomerular Crescent Formation, Glomerulosclerosis, and Renal Interstitial Inflammation.

Empty vector–treated ACGN mice showed extensive crescent formation, glomerulosclerosis, and interstitial (mainly periglomerular) mononuclear leukocyte infiltration at week 9 (Figure 8, E and H), all of which were substantially inhibited by hDCR3 administration (P < 0.005; Figure 8, F and H). There were no significant histopathologic changes in the empty vector–treated ACGN or hDCR3-treated ACGN mice compared with normal control mice at week 3 (Figure 8, A through C and G).

Figure 8.
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 ± SEM for groups of 10 mice. ***P < 0.005. #Not detectable. Magnification, ×400.

DCR3 Reduces Serum Levels of Autoantibodies and Glomerular Immune Deposits.

Because autoantibodies play a major pathogenic role in the development of SLE,1,9 we measured serum levels of anti–double-stranded DNA (anti-dsDNA) antibody in sera obtained at week 3 and week 9, respectively, by ELISA. As shown in Figure 9A, the autoantibody levels were significantly lower in the hDCR3-treated ACGN mice (P < 0.01) than in empty vector–treated ACGN mice at 9 wk, although there was no significant difference in autoantibody levels between empty vector–treated ACGN and hDCR3-treated ACGN mice at 3 wk. In addition, at week 9, the glomerular deposits of IgG (Figure 9B) seen in empty vector–treated ACGN mice were significantly reduced in hDCR3-treated ACGN mice (P < 0.005; Figure 9C). There was no significant difference in glomerular IgG deposits between empty vector–treated ACGN and hDCR3-treated ACGN mice at 3 wk (Figure 9, B and C), although both empty vector–treated and hDCR3-treated mice showed higher levels of glomerular IgG deposits than normal control mice (both P < 0.05).

Figure 9.
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 ± SEM for groups of 10 mice. *P < 0.05; **P < 0.01; ***P < 0.005; NS, no significant difference. Magnification, ×400.

DCR3 Inhibits Apoptosis in the Spleen and Kidney

Apoptosis is a tightly regulated process of programmed cell death, and its abnormal regulation is responsible for the pathogenesis of both SLE and lupus nephritis.4–6,33 Because DCR3 has been shown to modulate apoptosis in some tumor cells and cultured lymphocytes,16,18 we tested whether hDCR3 overexpression could inhibit apoptosis in vivo in the spleen or kidney in ACGN mice.

With the use of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, significant suppression of apoptosis was seen in the spleen of hDCR3-treated ACGN mice compared with empty vector–treated ACGN mice (P < 0.01; Figure 10, J through L, and V) at week 9. Again, in the kidney, only a few apoptotic figures were identified in hDCR3-treated ACGN mice (Figure 10O) at 9 wk, although empty vector–treated ACGN mice showed prominent apoptosis that was widespread in the glomerulus and renal tubular compartment, respectively (both P < 0.01; Figure 10, M, N, and W). Besides, earlier at week 3, suppression of apoptosis in the spleen was noted in hDCR3-treated ACGN mice, compared with empty vector–treated ACGN mice (Figure 10, A through C, and S), although there was no statistical significance. At this stage, there was no detectable apoptosis in empty vector–treated ACGN or hDCR3-treated ACGN mice (Figure 10, D through F and T).

Figure 10.
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 ± SEM for groups of 10 mice. *P < 0.05; **P < 0.01. #Not detectable; NS, no significant difference. Magnification, ×400.

Considering that TUNEL is unlikely to be able to differentiate apoptosis from necrosis, we performed immunohistochemistry (IHC) with anti–single-stranded DNA (anti-ssDNA) monoclonal antibody (mAb) in kidney tissues.34 Again, hDCR3-treated ACGN mice showed a significant reduction of apoptosis compared with empty vector–treated ACGN mice (Figure 10, P through R, and X) at week 9, although only a few apoptotic figures were noted at week 3 in both hDCR3-treated ACGN and empty vector–treated ACGN mice (Figure 10, G through I, and U).

DCR3 Prevents T Cell and Macrophage Infiltration of the Kidney

To gain further insights into the mechanisms by which hDCR3 prevented glomerular crescent formation and relevant renal injury in the ACGN model, we next assessed whether renal infiltration of mononuclear leukocytes was altered by hDCR3 gene therapy. Infiltration of T cells35,36 and/or monocytes/macrophages14,36 plays an important role in the formation of the glomerular crescents invariably 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 macrophage-dominated advanced and fibrocellular crescents. In this study, at week 9, IHC showed diffuse infiltration of CD3+ T cells, CD4+ T cells (Figure 11H), and F4/80 macrophages (Figure 11K) in the periglomerular region of the renal interstitium in empty vector–treated ACGN mice and that this was markedly reduced in hDCR3-treated ACGN mice (each P < 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).

Figure 11.
Figure 11.

Renal infiltration of T cells and macrophages. (A through F) Week 3 (G through L) Week 9. (A through C and G through I) CD4 T helper cells. (D through F and J through L) F4/80 monocytes/macrophages. (A, D, G, and J) Normal control mice (B, E, H, and K) Empty vector–treated ACGN mice (C, F, I, and L) hDCR3-treated ACGN mice. (M and N) Infiltrating cell score in the periglomeruli. Data are means ± SEM for groups of 10 mice. ***P < 0.005; NS, no significant difference. Magnification, ×400 each.

DCR3 Suppresses Fibrosis-Related Gene Expression in the Kidney

As a subcategory of crescentic glomerulonephritis, ACGN tends to evolve rapidly to diffuse glomerulosclerosis and interstitial fibrosis.15 We therefore evaluated the effects of hDCR3 gene therapy on renal fibrosis in the ACGN model, focusing on α-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 < 0.01) and that hDCR3 plasmid administration was associated with significant suppression of the expression of both mRNA (P < 0.01), their mRNA levels in hDCR3-treated ACGN not being significantly different from those in normal control mice. Again, although greatly enhanced expression of the proteins encoded by these fibrosis-related genes was detected in empty vector–treated ACGN mice compared with normal control mice, this was significantly reduced in hDCR3-treated ACGN mice (Figure 12, B and C).

Figure 12.
Figure 12.

Renal fibrosis-related gene expression at week 9. (A) Real-time PCR for α-smooth muscle actin and collagen IV mRNA levels. Each bar represents the means ± SEM for groups of 10 mice. **P < 0.01; ***P < 0.005. (B) α-Smooth muscle actin protein expression by IHC. (C) Collagen IV protein expression by IHC. Magnification, ×400.

DISCUSSION

This study is the first to report that in an experimental ACGN mouse model, showing clinical immunologic and pathologic features similar to those of patients with lupus nephritis, overexpression of hDCR3 by hydrodynamics-based gene delivery protects the animals from developing glomerular crescents, renal interstitial inflammation, and glomerulosclerosis. The favorable effects of hDCR3 gene therapy on the ACGN model may be due to one or more of the following three mechanisms: (1) Inhibition of T cell activation/proliferation and B cell activation, (2) protection against apoptosis in lymphoid organs (e.g., spleen) and kidney, and (3) suppression of mononuclear leukocyte infiltration of the kidney.

Regarding the first proposed mechanism, it is generally accepted that abnormal T cell function plays an important role in SLE.2,3,7,8 Pathogenic alloreactive25 and host37 T cells have been demonstrated to mediate the development of chronic graft-versus-host disease in mice, featuring SLE-like syndrome and renal lesions of various degrees.38 In this regard, DCR3 might downregulate alloresponsiveness of T cell function17,20 and lymphokine production17,19,21 systemically, which might, in turn, prevent the evolution of ACGN. In this study, we administered the hDCR3 plasmid by hydrodynamic-based gene delivery to ACGN mice. Our data showed that at early stage (week 3) of the experiment, hDCR3 significantly inhibited T cell activation and reduced T cell proliferation in the hDCR3-treated mice, although its potential suppressive effects on autoantibody and glomerular immune deposits were not seen. At 9 wk, in addition to significant suppression of T cell activation and proliferation, hDCR3-treated mice demonstrated a great inhibition on B cell activation, autoantibody production, and glomerular immune deposition. The data suggested that the earlier suppressive effect on T cells than that on B cells by hDCR3 administration could be operated in this experimental model of ACGN. However, T cells exert their effector function partly through the production and release of cytokines.26 In this study, hDCR3 administration significantly inhibited the percentage of T cells expressing IL-2, IFN-γ, and IL-4 in the spleen at 9 wk of the experiment. It should be noted that IL-429,30 and IFN-γ31,32 contribute to the pathogenesis of human lupus nephritis. hDCR3 administration also greatly depressed the sera levels of both IL-4 and IFN-γ at week 9. This could in part explain the favorable effects of hDCR3 on the condition of the kidney. On the whole, our data support the idea that blockade of T cell activity could be the key mechanism by which hDCR3 prevents renal injury.

The second possible mechanism is that hDCR3 prevents apoptosis in lymphoid organs (e.g., spleen) and kidney. Dysregulation of apoptosis and the clearance of apoptotic products have been implicated in the pathogenesis of SLE.4,5,33,39 Xue et al.40 showed that acceleration of lymphocytic apoptosis plays a crucial role in immune pathogenic injury. High apoptotic rates are also identified in severe active glomerular lesions in patients with lupus nephritis.41 The Fas pathway of apoptosis has been shown to be involved in the process of immune tolerance by deletion of unwanted autoreactive T cells and B cells.40,42 DCR3 can bind to FasL and inhibit FasL-induced apoptosis.16 We found that hDCR3 overexpression significantly inhibited splenic apoptosis at week 9, which has been shown to contribute to the development of lupus nephritis.43 Consistent with these findings, Seery et al.44 proposed that the use of an apoptosis inhibitor might be beneficial in the treatment of patients. Our data showed that blocking of apoptosis in both the glomerular and tubulointerstitial compartments of the kidney was associated with lower histopathologic severity of renal damage. Inhibition of apoptosis in lymphoid tissues (as represented by the spleen) and in the kidney is probably involved in the beneficial effects of DCR3 on ACGN.

Finally, as regards the third proposed mechanism, deletion of T cells and/or macrophages attenuates crescentic glomerulonephritis,12,14,36 suggesting an essential role for mononuclear leukocytes in the pathogenesis of crescents. The intraglomerular cellular structures of crescents are formed partly by proliferation of the parietal epithelial cells and partly by mononuclear infiltrates.11,13 hDCR3 administration resulted in (1) persistent reduction of serum MCP-1 levels starting early at week 3 and (2) almost total absence of periglomerular and interstitial mononuclear leukocyte infiltration in the kidney, reducing the formation of crescents in the glomeruli. Consistent with our data, Roth et al.45 showed that DCR3 decreases CD4+ T cell and macrophage infiltration in a rat gliosarcoma model. We believe that this effect might serve as a crucial and direct mechanism operating locally in the kidney for the beneficial effect of hDCR3 on ACGN.

Our data show that administration of an hDCR3 plasmid, given by hydrodynamics-based gene delivery, prevents the development of ACGN. We provide evidence that blocking of T cell activation/proliferation and B cell activation could play a role in the mode of action of hDCR3. Suppression of apoptosis in lymphoid organs and the kidney and inhibition of mononuclear leukocyte infiltration of the kidney are also highly implicated in its effects.

CONCISE METHODS

Induction of ACGN in the Model System

ACGN was induced in 7- to 8-wk-old female (C57BL/6 × DBA/2J) F1 hybrid mice by injection of DBA/2J donor lymphocytes, as described previously.15 Briefly, a cell suspension containing a mixture of donor cells from the thymus, spleen, and lymph nodes (neck, axillary, and inguinal regions) was injected intravenously three times at 3- to 4-d intervals. All mice were killed at week 3 or week 9 after disease induction. Spleen, renal cortical tissue, blood, and urine samples were collected and stored appropriately until analysis. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Plasmid hDCR3

hDCR3 cDNA (sequence data available from GenBank/EMBL/DDBJ under accession no. AF104419) was produced as described previously.22 Briefly, the hDCR3 gene was isolated by the reverse transcriptase–PCR using the forward primer 5′-CAAGGACCATGAGGGCGCTG-3′ and reverse primer 5′-GTGCACAGGGAGGAAGCGC-3′. The amplified product was cloned using the pGEM-T Easy Vector System (Promega, Madison, WI), and then the gene was subcloned into the pCMV vector (Clontech, Palo Alto, CA) to obtain the pCMV-hDCR3 expression construct. The plasmids were prepared using EndoFree plasmid kits (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

Hydrodynamics-Based Gene Delivery

To characterize the expression pattern and determine the interval at which hDCR3 plasmids were to be administered to the animals, we first injected a single dose of 100 μg/10 g body wt hDCR3 plasmid (diluted in 1.6 ml of normal saline) into normal (C57BL/6 × DBA/2J) F1 hybrid mice (n = 10) by hydrodynamics-based gene delivery through tail vein as described previously.46 Mice that received empty 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 expression 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 decided to deliver the hDCR3 plasmid at 14-d intervals in the subsequent therapeutic study. The first dose of hDCR3 plasmid was given on the day when the mice received the first shot of lymphocytes 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 disease 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 determined using a Pierce BCA protein assay kit (Perbio 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 paraffin. Sections (4 μm) were stained with hematoxylin and eosin. One hundred glomeruli were examined by light microscopy at the magnification of ×400 for the slide with at least two renal tissue sections each. Scoring of the severity of renal lesions was performed as described previously.47 The proportion (percentage) was calculated for the following three major components: Crescent formation, glomerular sclerosis, and periglomerular inflammation.

Immunofluorescence, IHC, and Detection of Apoptosis

For immunofluorescence (IF), frozen renal tissues were cut, air-dried, fixed in acetone for 5 min at room temperature, and incubated with FITC-conjugated goat anti-mouse IgG (Cappel, Durham, NC). Scoring of staining intensity was performed as described previously.47

For IHC, methyl Carnoy solution or formalin-fixed, paraffin-embedded tissue sections (4 μm) were stained with biotin-labeled mouse anti–α-smooth muscle actin antibodies (Neomarkers, Fremont, CA), goat anti–collagen IV (Southern Biotech, Birmingham, AL), or rat anti-F4/80 (monocytes/macrophages; Serotec, Raleigh, NC) antibodies. For detection of hDCR3 protein in the liver and the kidney, frozen sections of the tissues were fixed in acetone for 5 min and incubated with biotin-conjugated anti-hDCR3 antibody (Anawrahta, Taipei) as described previously.23 To detect pan-T cells and T helper cells, renal tissues were fixed in periodate-lysine paraformaldehyde as described previously,48 then incubated with biotin-conjugated anti-mouse CD3 (pan-T cell; Serotec) or CD4 (T helper cell; BioLegend, San Diego, CA) antibodies. Scoring of staining intensity was performed as described previously.15

For the detection of apoptosis, both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and IHC with anti-ssDNA were used. For TUNEL, formalin-fixed tissue sections were stained with ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Chemicon, Temecula, CA) according to the manufacturer’s instructions. For IHC with anti-ssDNA, formalin-fixed tissue sections were incubated with proteinase K (DAKO, Carpinteria, CA) for 20 min and then were incubated in 50% (vol/vol) formamide in distilled water at 56°C for 20 min, followed by the incubation with mouse anti-ssDNA mAb (Chemicon) at 4°C overnight, as described previously.34 For scoring, 50 randomly selected glomeruli were examined, and 20 randomly selected fields of renal tubules in the cortical area were examined by light microscopy at the magnification of ×400, as described previously.49

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/streptomycin (all from Life Technologies, Invitrogen, Carlsbad, CA). The cells were stained with either surface markers for T or B cell activation or intracellular 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 surface 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 × 106 cells in 500 μl/well) in the presence or absence of 20 ng/ml phorbol nyristate acetate, 1 μM ionomycin, and 4 μM monensin (all from Sigma, St. Louis, MO) for 6 h, as described previously.50 The cells were stained with FITC-conjugated anti-mouse CD3 (BD Biosciences) for 1 h at 4°C, fixed in 1% paraformaldehyde (Sigma), and resuspended in 100 μl of permeabilization buffer (0.1% saponin, 1% BSA, and 0.1% sodium azide; all from Sigma). Cytokine staining was stained with phycoerythrin-conjugated anti-mouse IL-2, IFN-γ, or IL-4 antibodies (all from BD Biosciences) for 1 h at 4°C, with FACSCalibur (BD Biosciences) as described previously.

T Cell Proliferation Analysis

Splenocytes from the mice were prepared as described previously, then were cultured in triplicate in wells (5 × 105 cells in 200 μl/well) in 96-well flat-bottom microtiter plates previously coated overnight at 4°C with 0.25 μg/ml anti-mouse CD3 (145–2C11) antibodies (BD Biosciences). After 48 h, the cultures were pulsed with 1 μCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) and harvested 16 h later, and the incorporated 3H-methyl thymidine was measured using a TopCount (Packard, PerkinElmer, Boston, MA) as described previously.23

Real-Time PCR Assay

RNA was extracted from the renal cortex using TriZOL reagents (Invitrogen). For first-strand cDNA synthesis, 1.5 μg of RNA was used in a single-round reverse transcriptase reaction. The reaction mixture consisted of 0.9 μl of Oligo (dT) 12 to 18 primer, 1.0 mM dNTP, 1× first-strand buffer, 0.4 mM dithiothreitol, 80 U of RNaseout recombinant ribonuclease inhibitor, and 300 U of Superscript II RNase H (Invitrogen) in a total volume of 25 μl. Real-time PCR was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). All of the probes and primers were Assays-on-Demand Gene expression products (Applied Biosystems). Real-time PCR reactions were performed using 10 μl of cDNA, 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μl of the specific probe/primer mixture in a total volume of 25 μl. The thermal cycler conditions were 1 × 2 min at 50°C, 1 × 10 min at 95°C, and 40 cycles of denaturation (15 s at 95°C) and combined annealing/extension (1 min at 60°C). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as the internal standard.

ELISA

Serum levels of hDCR3 were measured using an hDCR3 ELISA kit (Anawrahta), according to the manufacturer’s instructions. Serum levels of MCP-1, IFN-γ, and IL-4 were measured using commercial ELISA kits (BD Biosciences) according to the manufacturer’s instructions. Anti-dsDNA antibody was measured using an anti-mouse dsDNA ELISA kit (Alpha Diagnostic, San Antonio, TX). In all ELISA, the absorbance at 450 nm was measured using an ELISA plate reader (Bio-Tek, Winooski, VT).

Statistical Analysis

Values are presented as the means ± SEM. Comparison between two groups was performed using t test. P < 0.05 was taken as a statistical difference.

DISCLOSURES

None.

Acknowledgments

This study was supported by grants from Tri-Service General Hospital (TSGH-C92-4-S02) and Ministry of Economy (95-EC-17-A-20-S1-028), Taiwan, Republic of China.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

References

  1. ↵
    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 227
  2. ↵
    Hoffman RW: T cells in the pathogenesis of systemic lupus erythematosus. Clin Immunol 113 : 4 –13, 2004
    OpenUrlCrossRefPubMed
  3. ↵
    Nagy G, Koncz A, Perl A: T- and B-cell abnormalities in systemic lupus erythematosus. Crit Rev Immunol 25 : 123 –140, 2005
    OpenUrlCrossRefPubMed
  4. ↵
    Mevorach D: Systemic lupus erythematosus and apoptosis: A question of balance. Clin Rev Allergy Immunol 25 : 49 –60, 2003
    OpenUrlCrossRefPubMed
  5. ↵
    Gaipl US, Kuhn A, Sheriff A, Munoz LE, Franz S, Voll RE, Kalden JR, Herrmann M: Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun 9 : 173 –187, 2006
    OpenUrlPubMed
  6. ↵
    Liu CC, Navratil JS, Sabatine JM, Ahearn JM: Apoptosis, complement and systemic lupus erythematosus: A mechanistic view. Curr Dir Autoimmun 7 : 49 –86, 2004
    OpenUrlPubMed
  7. ↵
    Burlingame RW, Rubin RL, Balderas RS, Theofilopoulos AN: Genesis and evolution of antichromatin autoantibodies in murine lupus implicates T-dependent immunization with self antigen. J Clin Invest 91 : 1687 –1696, 1993
    OpenUrlCrossRefPubMed
  8. ↵
    Desai-Mehta A, Mao C, Rajagopalan S, Robinson T, Datta SK: Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus. J Clin Invest 95 : 531 –541, 1995
    OpenUrlCrossRefPubMed
  9. ↵
    Gleichmann E, Van Elven EH, Van der Veen JP: A systemic lupus erythematosus (SLE)-like disease in mice induced by abnormal T-B cell cooperation. Preferential formation of autoantibodies characteristic of SLE. Eur J Immunol 12 : 152 –159, 1982
    OpenUrlCrossRefPubMed
  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
  11. ↵
    Alpers C: The Kidney. In: Robbins and Cotran Pathologic Basis of Disease, 7th Ed., edited by Kumar V, Abbas AK, Fausto N, Philadelphia, Saunders, 2005 , pp 976 –978
  12. ↵
    Lan HY, Nikolic-Paterson DJ, Mu W, Atkins RC: Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis. Clin Exp Immunol 110 : 233 –240, 1997
    OpenUrlCrossRefPubMed
  13. ↵
    Lan HY, Nikolic-Paterson DJ, Atkins RC: Involvement of activated periglomerular leukocytes in the rupture of Bowman’s capsule and glomerular crescent progression in experimental glomerulonephritis. Lab Invest 67 : 743 –751, 1992
    OpenUrlPubMed
  14. ↵
    Isome M, Fujinaka H, Adhikary LP, Kovalenko P, El-Shemi AG, Yoshida Y, Yaoita E, Takeishi T, Takeya M, Naito M, Suzuki H, Yamamoto T: Important role for macrophages in induction of crescentic anti-GBM glomerulonephritis in WKY rats. Nephrol Dial Transplant 19 : 2997 –3004, 2004
    OpenUrlCrossRefPubMed
  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 –298, 2006
    OpenUrlCrossRefPubMed
  16. ↵
    Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, Huang A, Donahue CJ, Sherwood SW, Baldwin DT, Godowski PJ, Wood WI, Gurney AL, Hillan KJ, Cohen RL, Goddard AD, Botstein D, Ashkenazi A: Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396 : 699 –703, 1998
    OpenUrlCrossRefPubMed
  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
    OpenUrlCrossRefPubMed
  18. ↵
    Yu KY, Kwon B, Ni J, Zhai Y, Ebner R, Kwon BS: A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem 274 : 13733 –13736, 1999
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Migone TS, Zhang J, Luo X, Zhuang L, Chen C, Hu B, Hong JS, Perry JW, Chen SF, Zhou JX, Cho YH, Ullrich S, Kanakaraj P, Carrell J, Boyd E, Olsen HS, Hu G, Pukac L, Liu D, Ni J, Kim S, Gentz R, Feng P, Moore PA, Ruben SM, Wei P: TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity 16 : 479 –492, 2002
    OpenUrlCrossRefPubMed
  20. ↵
    Shi G, Wu Y, Zhang J, Wu J: Death decoy receptor TR6/DcR3 inhibits T cell chemotaxis in vitro and in vivo. J Immunol 171 : 3407 –3414, 2003
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Chang YC, Hsu TL, Lin HH, Chio CC, Chiu AW, Chen NJ, Lin CH, Hsieh SL: Modulation of macrophage differentiation and activation by decoy receptor 3. J Leukoc Biol 75 : 486 –494, 2004
    OpenUrlCrossRefPubMed
  22. ↵
    Hsu TL, Chang YC, Chen SJ, Liu YJ, Chiu AW, Chio CC, Chen L, Hsieh SL: Modulation of dendritic cell differentiation and maturation by decoy receptor 3. J Immunol 168 : 4846 –4853, 2002
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Sung HH, Juang JH, Lin YC, Kuo CH, Hung JT, Chen A, Chang DM, Chang SY, Hsieh SL, Sytwu HK: Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice. J Exp Med 199 : 1143 –1151, 2004
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Otsuki T, Tomokuni A, Sakaguchi H, Aikoh T, Matsuki T, Isozaki Y, Hyodoh F, Ueki H, Kusaka M, Kita S, Ueki A: Over-expression of the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) derived from silicosis patients. Clin Exp Immunol 119 : 323 –327, 2000
    OpenUrlCrossRefPubMed
  25. ↵
    Van Elven EH, Rolink AG, Veen FV, Gleichmann E: Capacity of genetically different T lymphocytes to induce lethal graft-versus-host disease correlates with their capacity to generate suppression but not with their capacity to generate anti-F1 killer cells. A non-H-2 locus determines the inability to induce lethal graft-versus-host disease. J Exp Med 153 : 1474 –1488, 1981
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Goldsby RA, Kindt TJ, Osborne BA, Kuby J: Cytokines. In: Immunology, 5th Ed., edited by Goldsby RA, New York, W.H. Freeman, 2003 , pp 276 –281
  27. ↵
    Aguilar F, Gonzalez-Escribano MF, Sanchez-Roman J, Nunez-Roldan A: MCP-1 promoter polymorphism in Spanish patients with systemic lupus erythematosus. Tissue Antigens 58 : 335 –338, 2001
    OpenUrlCrossRefPubMed
  28. ↵
    Wada T, Furuichi K, Segawa-Takaeda C, Shimizu M, Sakai N, Takeda SI, Takasawa K, Kida H, Kobayashi KI, Mukaida N, Ohmoto Y, Matsushima K, Yokoyama H: MIP-1alpha and MCP-1 contribute to crescents and interstitial lesions in human crescentic glomerulonephritis. Kidney Int 56 : 995 –1003, 1999
    OpenUrlCrossRefPubMed
  29. ↵
    Wong CK, Ho CY, Li EK, Lam CW: Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 9 : 589 –593, 2000
    OpenUrlCrossRefPubMed
  30. ↵
    Umland SP, Razac S, Nahrebne DK, Seymour BW: Effects of in vivo administration of interferon (IFN)-gamma, anti-IFN-gamma, or anti-interleukin-4 monoclonal antibodies in chronic autoimmune graft-versus-host disease. Clin Immunol Immunopathol 63 : 66 –73, 1992
    OpenUrlCrossRefPubMed
  31. ↵
    Masutani K, Akahoshi M, Tsuruya K, Tokumoto M, Ninomiya T, Kohsaka T, Fukuda K, Kanai H, Nakashima H, Otsuka T, Hirakata H: Predominance of Th1 immune response in diffuse proliferative lupus nephritis. Arthritis Rheum 44 : 2097 –2106, 2001
    OpenUrlCrossRefPubMed
  32. ↵
    Uhm WS, Na K, Song GW, Jung SS, Lee T, Park MH, Yoo DH: Cytokine balance in kidney tissue from lupus nephritis patients. Rheumatology (Oxford) 42 : 935 –938, 2003
    OpenUrlCrossRefPubMed
  33. ↵
    Licht R, Dieker JW, Jacobs CW, Tax WJ, Berden JH: Decreased phagocytosis of apoptotic cells in diseased SLE mice. J Autoimmun 22 : 139 –145, 2004
    OpenUrlCrossRefPubMed
  34. ↵
    Frankfurt OS, Robb JA, Sugarbaker EV, Villa L: Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res 226 : 387 –397, 1996
    OpenUrlCrossRefPubMed
  35. Huang XR, Tipping PG, Apostolopoulos J, Oettinger C, D’Souza M, Milton G, Holdsworth SR: Mechanisms of T cell-induced glomerular injury in anti-glomerular basement membrane (GBM) glomerulonephritis in rats. Clin Exp Immunol 109 : 134 –142, 1997
    OpenUrlCrossRefPubMed
  36. ↵
    Lenda DM, Stanley ER, Kelley VR: Negative role of colony-stimulating factor-1 in macrophage, T cell, and B cell mediated autoimmune disease in MRL-Fas(lpr) mice. J Immunol 173 : 4744 –4754, 2004
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Choudhury A, Maldonado MA, Cohen PL, Eisenberg RA: The role of host CD4 T cells in the pathogenesis of the chronic graft-versus-host model of systemic lupus erythematosus. J Immunol 174 : 7600 –7609, 2005
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Lang TJ, Nguyen P, Papadimitriou JC, Via CS: Increased severity of murine lupus in female mice is due to enhanced expansion of pathogenic T cells. J Immunol 171 : 5795 –5801, 2003
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Berden JH, Grootscholten C, Jurgen WC, van der Vlag J: Lupus nephritis: A nucleosome waste disposal defect? J Nephrol 15[Suppl 6] : S1 –S10, 2002
    OpenUrl
  40. ↵
    Xue C, Lan-Lan W, Bei C, Jie C, Wei-Hua F: Abnormal Fas/FasL and caspase-3-mediated apoptotic signaling pathways of T lymphocyte subset in patients with systemic lupus erythematosus. Cell Immunol 239 : 121 –128, 2006
    OpenUrlCrossRefPubMed
  41. ↵
    Jeruc J, Vizjak A, Rozman B, Ferluga D: Immunohistochemical expression of activated caspase-3 as a marker of apoptosis in glomeruli of human lupus nephritis. Am J Kidney Dis 48 : 410 –418, 2006
    OpenUrlCrossRefPubMed
  42. ↵
    Kamradt T, Mitchison NA: Tolerance and autoimmunity. N Engl J Med 344 : 655 –664, 2001
    OpenUrlCrossRefPubMed
  43. ↵
    Kalled SL, Cutler AH, Burkly LC: Apoptosis and altered dendritic cell homeostasis in lupus nephritis are limited by anti-CD154 treatment. J Immunol 167 : 1740 –1747, 2001
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Seery JP, Cattell V, Watt FM: Cutting edge: Amelioration of kidney disease in a transgenic mouse model of lupus nephritis by administration of the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(beta-o-methyl)-fluoromethylketone. J Immunol 167 : 2452 –2455, 2001
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Roth W, Isenmann S, Nakamura M, Platten M, Wick W, Kleihues P, Bahr M, Ohgaki H, Ashkenazi A, Weller M: Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 61 : 2759 –2765, 2001
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Liu F, Song Y, Liu D: Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6 : 1258 –1266, 1999
    OpenUrlCrossRefPubMed
  47. ↵
    Chen A, Sheu LF, Ho YS, Lin YF, Chou WY, Chou TC, Lee WH: Experimental focal segmental glomerulosclerosis in mice. Nephron 78 : 440 –452, 1998
    OpenUrlCrossRefPubMed
  48. ↵
    Chao TK, Rifai A, Ka SM, Yang SM, Shui HA, Lin YF, Sytwu HK, Lee WH, Kung JT, Chen A: The endogenous immune response modulates the course of IgA-immune complex mediated nephropathy. Kidney Int 70 : 283 –297, 2006
    OpenUrlCrossRefPubMed
  49. ↵
    Cheng CW, Rifai A, Ka SM, Shui HA, Lin YF, Lee WH, Chen A: Calcium-binding proteins annexin A2 and S100A6 are sensors of tubular injury and recovery in acute renal failure. Kidney Int 68 : 2694 –2703, 2005
    OpenUrlCrossRefPubMed
  50. ↵
    Assenmacher M, Schmitz J, Radbruch A: Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells. Eur J Immunol 24 : 1097 –1101, 1994
    OpenUrlCrossRefPubMed
View Abstract
Back to top

In this issue

View Selected Citations (0)
Print
Download PDF
Email Article
Citation Tools
Request Permissions
Decoy Receptor 3 Ameliorates an Autoimmune Crescentic Glomerulonephritis Model in Mice
Shuk-Man Ka, Huey-Kang Sytwu, Deh-Ming Chang, Shie-Liang Hsieh, Pei-Yi Tsai, Ann Chen
JASN Sep 2007, 18 (9) 2473-2485; DOI: 10.1681/ASN.2006111242
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section