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*
* 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 Defense Medical Center, and || Institute and Department of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China
Correspondence: Dr. Ann Chen, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC, Phone: +886-2-8792-7008; Fax: +886-2-8792-7009; E-mail: doc31717{at}ndmctsgh.edu.tw
Received for publication November 15, 2006.
Accepted for publication May 24, 2007.
Autoimmune crescentic glomerulonephritis (ACGN) is a variantof crescentic glomerulonephritis. The outcome of treatment ofcrescentic glomerulonephritis is poor. Binding of decoy receptor3 (DCR3) to its ligand is capable of downregulating the alloresponsivenessof T cells. DCR3 has also been shown to benefit an experimentalautoimmune model of diabetes. This study tested the hypothesisthat a potential immune regulator, DCR3, could prevent the evolutionof ACGN. With the use of an established ACGN model in mice,mice were treated with 100 µg/10 g body wt human DCR3by hydrodynamics-based gene delivery at 14-d intervals. Theresults showed that the gene therapy resulted in (1) suppressionof T and B cell activation and T cell proliferation; (2) a reductionin serum levels of proinflammatory cytokines; (3) improvementof proteinuria and renal dysfunction; (4) prevention of glomerularcrescent formation, renal interstitial inflammation, and glomerulosclerosis;(5) a reduction in serum levels of autoantibodies and glomerularimmune deposits; (6) inhibition of apoptosis in the spleen andkidney; (7) prevention of T cell and macrophage infiltrationof the kidney; and (8) suppression of fibrosis-related geneexpression 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 protectiveeffects on ACGN through modulation of T cell activation/proliferation,B cell activation, protection against apoptosis, and suppressionof mononuclear leukocyte infiltration in the kidney.
Systemic lupus erythematosus (SLE) is a typical autoimmune diseaseinvolving multiple organs. Although its cause is not entirelyclear, the basic abnormality seems to be a failure of mechanismsthat maintain self-tolerance, resulting in the production ofdiverse autoantibodies, especially antinuclear antibodies,1T cell abnormalities,2,3 and apoptosis.4–6 In patientsand mouse models, pathogenic T cells recognize self-antigensand drive B cell hyperactivity, confirming their central rolein the pathogenesis of SLE.3,7–9 Lupus nephritis is amajor renal injury in SLE and is characterized by immune complexdeposition in the glomerular and renal tubular and peritubularcapillary basement membranes.10 Clinically, autoimmune crescenticglomerulonephritis (ACGN) is an extremely progressive form oflupus nephritis and is classified as a type of crescentic glomerulonephritis11,12in which widespread glomerular crescents, consisting of a mixtureof parietal epithelial cells, monocytes/macrophages, and lymphocytes,are formed.13,14 We have shown that a murine chronic graft-versus-hostdisease, induced in C57BL/6 x DBA/2J F1 hybrid mice by givingDBA/2J donor lymphocytes, can progress to ACGN, with extensive,characteristic glomerular crescent formation (up to 80% of glomeruliexamined), sclerosis, and intense interstitial inflammation.15Although recovery of renal functions in patients with crescenticglomerulonephritis can occur after early intensive plasma exchangeand/or treatment with steroids and cytotoxic agents, patientseventually require long-term dialysis or transplantation.11
Decoy receptor 3 (DCR3) lacks the transmembrane domain of theconventional TNF receptor and is a secreted protein.16,17 DCR3can interact with Fas ligand (FasL),16 LIGHT (homologous tolymphotoxins, shows inducible expression and competes with HSVglycoprotein D for herpes virus entry mediator, a receptor expressedby T lymphocytes),18 and TNF-like molecule 1A.19 Binding ofDCR3 to LIGHT also downregulates the alloresponsiveness of Tcells.17,20 By helping tumor cells avoid immune attack throughlymphocyte infiltration and FasL/LIGHT-mediated apoptosis, increasedDCR3 expression might benefit their growth.16,18 DCR3 exertsanother regulatory function by directly modulating the differentiationand 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-induceddiabetes in a dosage-dependent manner and significantly reducesthe severity of insulitis in an autoimmune diabetes model. Inaddition, in vivo administration of hDCR3 ameliorates allograftrejection.17 The effects of hDCR3 on immune regulation shouldbe explored for its possible therapeutic use in controllingundesirable immune responses. Clinically, DCR3 gene expressionhas been detected in peripheral blood mononuclear leukocytesderived from patients with SLE,24 although its clinical significanceremains to be determined.
In this study, we tested the hypothesis that in vivo overexpressionof hDCR3 would prevent crescentic formation of an experimentalACGN. Using hydrodynamics-based gene delivery, we demonstratedthat hDCR3 gene therapy is an effective therapeutic approachin the ACGN model. Regulation of both T cell function and apoptosisin lymphoid organs and in the kidney seems to be the major factorresponsible for its favorable effects in this autoimmune kidneydisease model.
Serum Levels of hDCR3 Protein in Treated ACGN Mice
Based on the hDCR3 expression (Figure 1, methods as describedin Concise Methods), we delivered the hDCR3 plasmid to the miceat 14-d intervals. After administrations of hDCR3 plasmid orempty vector, ELISA tests showed high serum levels of hDCR3protein in the hDCR3-treated ACGN mice at 3 wk (Figure 2A) and9 wk (Figure 2B), respectively, after ACGN induction but notin the empty vector–treated ACGN mice or normal controlmice.
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, x400.
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-hostreaction,9,25 the basic mechanism for the induction of the ACGNmodel. DCR3 has been reported to inhibit T cell proliferationand lymphokine secretion.17,19,21 We tested whether hDCR3 couldameliorate the development of the ACGN model by negatively regulatingT cell and/or B cell activation by performing flow cytometryof splenocytes.
As shown in Figure 3, the percentage of CD3+CD69+ cells (activatedT cells) in the spleen was significantly increased in emptyvector–treated ACGN mice at 3 wk (Figure 3, A and C) or9 wk (Figure 3, B and D) after ACGN induction, compared withnormal control mice (both P < 0.005). However, this effectwas significantly suppressed by administration of hDCR3 plasmidto ACGN mice compared with empty vector–treated ACGN mice(P < 0.05 at 3 wk; P < 0.005 at 9 wk). Moreover, hDCR3-treatedACGN mice showed a significantly lower percentage of CD4+CD69+cells (activated T helper cells) than that of empty vector–treatedACGN 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 percentageof CD19+CD69+ cells (activated B cells) was significantly decreasedin hDCR3-treated ACGN mice to the levels seen in normal controlmice at week 9 (P < 0.005; Figure 3, B and D), although therewas no significant difference in the percentage of CD19+CD69+cells between empty vector–treated ACGN and hDCR3-treatedACGN mice (Figure 3, A and C) at week 3 compared with emptyvector–treated ACGN mice.
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, whichin turn may assist in recruitment of other cells to the siteof inflammation or mediate tissue damage.26 Therefore, we furthermeasured the production of intracellular IL-2, IFN-, or IL-4of T lymphocytes in the spleen by flow cytometry. As shown inFigure 4A, there was no significant difference in the percentageof T cells expressing IL-2, IFN-, or IL-4 between the emptyvector–treated ACGN and hDCR3-treated ACGN mice at 3 wk,although the percentage of IFN- was significantly increasedin empty vector–treated ACGN or hDCR3-treated ACGN mice,compared with normal control mice (P < 0.005). However, thepercentage of T cells expressing IL-2, IFN-, or IL-4 was significantlyinhibited by the administration of hDCR3 plasmid to ACGN miceat 9 wk, compared with empty vector–treated ACGN mice(each P < 0.05; Figure 4B).
Figure 4. Flow cytometry for intracellular cytokine staining in the spleen. The percentage of T cells expressing IL-2, IFN-, 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–treatedACGN mice, hDCR3-treated ACGN mice showed significant suppressionof spleen T cell proliferation to the level seen in normal controlmice at 3 and 9 wk (both P < 0.05).
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-4,29,30and IFN-31,32 have been implicated in the pathogenesis of SLE.To determine whether hDCR3 affected systemic proinflammatorycytokine production, we measured serum levels of these proteinsin the mice. At week 3, hDCR3-treated ACGN mice showed significantlylower levels of MCP-1 than empty vector–treated ACGN mice(P < 0.05), although there was no significant differencein the levels of IL-4 or IFN- between hDCR3-treated ACGN andempty 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 beingsimilar to those in normal control mice (Figure 6, D throughF).
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 ± 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 micedeveloped proteinuria that started at week 4 and plateau betweenweeks 7 and 9. This clinical sign was greatly improved in thehDCR3-treated ACGN mice as early as week 4, returning to thenormal range at weeks 7 to 9. Likewise, a dramatic improvementin renal function was noted in the hDCR3-treated ACGN mice,as compared with empty vector–treated ACGN mice at week9, 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 beingsimilar to those in normal control mice. There was no significantdifference in the levels of BUN or creatinine between the hDCR3-treatedand empty vector–treated ACGN mice at week 3.
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 crescentformation, glomerulosclerosis, and interstitial (mainly periglomerular)mononuclear leukocyte infiltration at week 9 (Figure 8, E andH), all of which were substantially inhibited by hDCR3 administration(P < 0.005; Figure 8, F and H). There were no significanthistopathologic changes in the empty vector–treated ACGNor hDCR3-treated ACGN mice compared with normal control miceat week 3 (Figure 8, A through C and G).
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, x400.
DCR3 Reduces Serum Levels of Autoantibodies and Glomerular Immune Deposits.
Because autoantibodies play a major pathogenic role in the developmentof SLE,1,9 we measured serum levels of anti–double-strandedDNA (anti-dsDNA) antibody in sera obtained at week 3 and week9, respectively, by ELISA. As shown in Figure 9A, the autoantibodylevels were significantly lower in the hDCR3-treated ACGN mice(P < 0.01) than in empty vector–treated ACGN mice at9 wk, although there was no significant difference in autoantibodylevels between empty vector–treated ACGN and hDCR3-treatedACGN mice at 3 wk. In addition, at week 9, the glomerular depositsof IgG (Figure 9B) seen in empty vector–treated ACGN micewere significantly reduced in hDCR3-treated ACGN mice (P <0.005; Figure 9C). There was no significant difference in glomerularIgG deposits between empty vector–treated ACGN and hDCR3-treatedACGN mice at 3 wk (Figure 9, B and C), although both empty vector–treatedand hDCR3-treated mice showed higher levels of glomerular IgGdeposits than normal control mice (both P < 0.05).
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, x400.
DCR3 Inhibits Apoptosis in the Spleen and Kidney
Apoptosis is a tightly regulated process of programmed celldeath, and its abnormal regulation is responsible for the pathogenesisof both SLE and lupus nephritis.4–6,33 Because DCR3 hasbeen shown to modulate apoptosis in some tumor cells and culturedlymphocytes,16,18 we tested whether hDCR3 overexpression couldinhibit apoptosis in vivo in the spleen or kidney in ACGN mice.
With the use of the terminal deoxynucleotidyl transferase-mediateddUTP nick-end labeling (TUNEL) assay, significant suppressionof apoptosis was seen in the spleen of hDCR3-treated ACGN micecompared 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-treatedACGN mice (Figure 10O) at 9 wk, although empty vector–treatedACGN mice showed prominent apoptosis that was widespread inthe glomerulus and renal tubular compartment, respectively (bothP < 0.01; Figure 10, M, N, and W). Besides, earlier at week3, suppression of apoptosis in the spleen was noted in hDCR3-treatedACGN mice, compared with empty vector–treated ACGN mice(Figure 10, A through C, and S), although there was no statisticalsignificance. At this stage, there was no detectable apoptosisin empty vector–treated ACGN or hDCR3-treated ACGN mice(Figure 10, D through F and T).
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, x400.
Considering that TUNEL is unlikely to be able to differentiateapoptosis from necrosis, we performed immunohistochemistry (IHC)with anti–single-stranded DNA (anti-ssDNA) monoclonalantibody (mAb) in kidney tissues.34 Again, hDCR3-treated ACGNmice showed a significant reduction of apoptosis compared withempty vector–treated ACGN mice (Figure 10, P through R,and X) at week 9, although only a few apoptotic figures werenoted at week 3 in both hDCR3-treated ACGN and empty vector–treatedACGN 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 hDCR3prevented glomerular crescent formation and relevant renal injuryin the ACGN model, we next assessed whether renal infiltrationof mononuclear leukocytes was altered by hDCR3 gene therapy.Infiltration of T cells35,36 and/or monocytes/macrophages14,36plays an important role in the formation of the glomerular crescentsinvariably present in crescentic glomerulonephritis, includingACGN, and Lan et al.12 proposed that macrophage accumulationwithin crescents plays an important role in the progressionof epithelial-dominated early cellular crescents to macrophage-dominatedadvanced and fibrocellular crescents. In this study, at week9, IHC showed diffuse infiltration of CD3+ T cells, CD4+ T cells(Figure 11H), and F4/80 macrophages (Figure 11K) in the periglomerularregion of the renal interstitium in empty vector–treatedACGN mice and that this was markedly reduced in hDCR3-treatedACGN 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 notedin either empty vector–treated ACGN or hDCR3-treated ACGN(Figure 11, A through F, and M).
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, x400 each.
DCR3 Suppresses Fibrosis-Related Gene Expression in the Kidney
As a subcategory of crescentic glomerulonephritis, ACGN tendsto evolve rapidly to diffuse glomerulosclerosis and interstitialfibrosis.15 We therefore evaluated the effects of hDCR3 genetherapy on renal fibrosis in the ACGN model, focusing on -smoothmuscle actin and collagen IV. As shown in Figure 12A, real-timePCR demonstrated marked renal expression of both mRNA in emptyvector–treated ACGN mice compared with normal controls(P < 0.01) and that hDCR3 plasmid administration was associatedwith significant suppression of the expression of both mRNA(P < 0.01), their mRNA levels in hDCR3-treated ACGN not beingsignificantly different from those in normal control mice. Again,although greatly enhanced expression of the proteins encodedby these fibrosis-related genes was detected in empty vector–treatedACGN mice compared with normal control mice, this was significantlyreduced in hDCR3-treated ACGN mice (Figure 12, B and C).
Figure 12. Renal fibrosis-related gene expression at week 9. (A) Real-time PCR for -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) -Smooth muscle actin protein expression by IHC. (C) Collagen IV protein expression by IHC. Magnification, x400.
This study is the first to report that in an experimental ACGNmouse model, showing clinical immunologic and pathologic featuressimilar to those of patients with lupus nephritis, overexpressionof hDCR3 by hydrodynamics-based gene delivery protects the animalsfrom developing glomerular crescents, renal interstitial inflammation,and glomerulosclerosis. The favorable effects of hDCR3 genetherapy on the ACGN model may be due to one or more of the followingthree mechanisms: (1) Inhibition of T cell activation/proliferationand B cell activation, (2) protection against apoptosis in lymphoidorgans (e.g., spleen) and kidney, and (3) suppression of mononuclearleukocyte infiltration of the kidney.
Regarding the first proposed mechanism, it is generally acceptedthat abnormal T cell function plays an important role in SLE.2,3,7,8Pathogenic alloreactive25 and host37 T cells have been demonstratedto mediate the development of chronic graft-versus-host diseasein mice, featuring SLE-like syndrome and renal lesions of variousdegrees.38 In this regard, DCR3 might downregulate alloresponsivenessof T cell function17,20 and lymphokine production17,19,21 systemically,which might, in turn, prevent the evolution of ACGN. In thisstudy, we administered the hDCR3 plasmid by hydrodynamic-basedgene delivery to ACGN mice. Our data showed that at early stage(week 3) of the experiment, hDCR3 significantly inhibited Tcell activation and reduced T cell proliferation in the hDCR3-treatedmice, although its potential suppressive effects on autoantibodyand glomerular immune deposits were not seen. At 9 wk, in additionto significant suppression of T cell activation and proliferation,hDCR3-treated mice demonstrated a great inhibition on B cellactivation, autoantibody production, and glomerular immune deposition.The data suggested that the earlier suppressive effect on Tcells than that on B cells by hDCR3 administration could beoperated in this experimental model of ACGN. However, T cellsexert their effector function partly through the productionand release of cytokines.26 In this study, hDCR3 administrationsignificantly inhibited the percentage of T cells expressingIL-2, IFN-, and IL-4 in the spleen at 9 wk of the experiment.It should be noted that IL-429,30 and IFN-31,32 contribute tothe pathogenesis of human lupus nephritis. hDCR3 administrationalso greatly depressed the sera levels of both IL-4 and IFN-at week 9. This could in part explain the favorable effectsof hDCR3 on the condition of the kidney. On the whole, our datasupport the idea that blockade of T cell activity could be thekey mechanism by which hDCR3 prevents renal injury.
The second possible mechanism is that hDCR3 prevents apoptosisin lymphoid organs (e.g., spleen) and kidney. Dysregulationof apoptosis and the clearance of apoptotic products have beenimplicated in the pathogenesis of SLE.4,5,33,39 Xue et al.40showed that acceleration of lymphocytic apoptosis plays a crucialrole in immune pathogenic injury. High apoptotic rates are alsoidentified in severe active glomerular lesions in patients withlupus nephritis.41 The Fas pathway of apoptosis has been shownto be involved in the process of immune tolerance by deletionof unwanted autoreactive T cells and B cells.40,42 DCR3 canbind to FasL and inhibit FasL-induced apoptosis.16 We foundthat hDCR3 overexpression significantly inhibited splenic apoptosisat week 9, which has been shown to contribute to the developmentof lupus nephritis.43 Consistent with these findings, Seeryet al.44 proposed that the use of an apoptosis inhibitor mightbe beneficial in the treatment of patients. Our data showedthat blocking of apoptosis in both the glomerular and tubulointerstitialcompartments of the kidney was associated with lower histopathologicseverity of renal damage. Inhibition of apoptosis in lymphoidtissues (as represented by the spleen) and in the kidney isprobably involved in the beneficial effects of DCR3 on ACGN.
Finally, as regards the third proposed mechanism, deletion ofT cells and/or macrophages attenuates crescentic glomerulonephritis,12,14,36suggesting an essential role for mononuclear leukocytes in thepathogenesis of crescents. The intraglomerular cellular structuresof crescents are formed partly by proliferation of the parietalepithelial cells and partly by mononuclear infiltrates.11,13hDCR3 administration resulted in (1) persistent reduction ofserum MCP-1 levels starting early at week 3 and (2) almost totalabsence of periglomerular and interstitial mononuclear leukocyteinfiltration in the kidney, reducing the formation of crescentsin the glomeruli. Consistent with our data, Roth et al.45 showedthat DCR3 decreases CD4+ T cell and macrophage infiltrationin a rat gliosarcoma model. We believe that this effect mightserve as a crucial and direct mechanism operating locally inthe kidney for the beneficial effect of hDCR3 on ACGN.
Our data show that administration of an hDCR3 plasmid, givenby hydrodynamics-based gene delivery, prevents the developmentof ACGN. We provide evidence that blocking of T cell activation/proliferationand B cell activation could play a role in the mode of actionof hDCR3. Suppression of apoptosis in lymphoid organs and thekidney and inhibition of mononuclear leukocyte infiltrationof the kidney are also highly implicated in its effects.
Induction of ACGN in the Model System
ACGN was induced in 7- to 8-wk-old female (C57BL/6 x DBA/2J)F1 hybrid mice by injection of DBA/2J donor lymphocytes, asdescribed previously.15 Briefly, a cell suspension containinga mixture of donor cells from the thymus, spleen, and lymphnodes (neck, axillary, and inguinal regions) was injected intravenouslythree times at 3- to 4-d intervals. All mice were killed atweek 3 or week 9 after disease induction. Spleen, renal corticaltissue, blood, and urine samples were collected and stored appropriatelyuntil analysis. All animal experiments were performed with theapproval of the Institutional Animal Care and Use Committeeof the National Defense Medical Center, Taiwan, and were consistentwith the National Institutes of Health Guide for the Care andUse of Laboratory Animals.
Plasmid hDCR3
hDCR3 cDNA (sequence data available from GenBank/EMBL/DDBJ underaccession no. AF104419) was produced as described previously.22Briefly, the hDCR3 gene was isolated by the reverse transcriptase–PCRusing the forward primer 5'-CAAGGACCATGAGGGCGCTG-3' and reverseprimer 5'-GTGCACAGGGAGGAAGCGC-3'. The amplified product wascloned 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 intervalat which hDCR3 plasmids were to be administered to the animals,we first injected a single dose of 100 µg/10 g body wthDCR3 plasmid (diluted in 1.6 ml of normal saline) into normal(C57BL/6 x DBA/2J) F1 hybrid mice (n = 10) by hydrodynamics-basedgene delivery through tail vein as described previously.46 Micethat received empty vector (pCMV) served as controls. The animalswere then killed at days 0, 1, 2, 3, 7, 14, and 28 after plasmidadministration, and serum and liver tissues were stored appropriatelyuntil analysis. As shown in Figure 1A, serum hDCR3 levels startedto increase on day 1, peaked on day 2, gradually declined today 14, and returned to baseline levels by day 28, as demonstratedby ELISA. By IHC, the liver showed strong expression of hDCR3(Figure 1B), although there was no signal of staining in thekidney. No staining was seen in the liver (Figure 1B) or kidneyat any time point in mice that were given empty vector. On thebasis of the hDCR3 expression, we decided to deliver the hDCR3plasmid at 14-d intervals in the subsequent therapeutic study.The first dose of hDCR3 plasmid was given on the day when themice received the first shot of lymphocytes for the inductionof the ACGN model and the second after all of the injectionsfor ACGN induction had been given. ACGN mice that were givenempty vector were used as disease controls.
Clinical and Pathologic Evaluation
Collection and assay of blood and urine samples were performedas described previously.47 Urine samples were collected in metaboliccages weekly, and urinary levels of protein were determinedusing a Pierce BCA protein assay kit (Perbio Science, Etten-Leur,Netherlands); serum samples were collected at weeks 3 and week9, respectively, to measure serum levels of BUN and Cr.
For histopathology, the tissues were fixed in 10% buffered formalinand embedded in paraffin. Sections (4 µm) were stainedwith hematoxylin and eosin. One hundred glomeruli were examinedby light microscopy at the magnification of x400 for the slidewith at least two renal tissue sections each. Scoring of theseverity of renal lesions was performed as described previously.47The proportion (percentage) was calculated for the followingthree 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, andincubated with FITC-conjugated goat anti-mouse IgG (Cappel,Durham, NC). Scoring of staining intensity was performed asdescribed previously.47
For IHC, methyl Carnoy solution or formalin-fixed, paraffin-embeddedtissue sections (4 µm) were stained with biotin-labeledmouse anti–-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 theliver and the kidney, frozen sections of the tissues were fixedin acetone for 5 min and incubated with biotin-conjugated anti-hDCR3antibody (Anawrahta, Taipei) as described previously.23 To detectpan-T cells and T helper cells, renal tissues were fixed inperiodate-lysine paraformaldehyde as described previously,48then incubated with biotin-conjugated anti-mouse CD3 (pan-Tcell; Serotec) or CD4 (T helper cell; BioLegend, San Diego,CA) antibodies. Scoring of staining intensity was performedas described previously.15
For the detection of apoptosis, both terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and IHCwith anti-ssDNA were used. For TUNEL, formalin-fixed tissuesections were stained with ApopTag Plus Peroxidase In Situ ApoptosisDetection kit (Chemicon, Temecula, CA) according to the manufacturer’sinstructions. For IHC with anti-ssDNA, formalin-fixed tissuesections were incubated with proteinase K (DAKO, Carpinteria,CA) for 20 min and then were incubated in 50% (vol/vol) formamidein distilled water at 56°C for 20 min, followed by the incubationwith mouse anti-ssDNA mAb (Chemicon) at 4°C overnight, asdescribed previously.34 For scoring, 50 randomly selected glomeruliwere examined, and 20 randomly selected fields of renal tubulesin the cortical area were examined by light microscopy at themagnification of x400, as described previously.49
Flow Cytometry
Splenocytes from the mice were treated with Tris-buffered ammoniumchloride to eliminate erythrocytes; washed; and resuspendedin 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 markersfor T or B cell activation or intracellular cytokines producedby T cells. FITC-conjugated anti-mouse CD3 (17A2), CD4 (G11.5),or CD19 (B cell, 1D3) antibodies and phycoerythrin-conjugatedanti-mouse CD69 (H1.2F3) antibodies (all from BD Biosciences,San Jose, CA) were used for the analysis of surface markersfor 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 x 106 cells in 500 µl/well) in the presence or absenceof 20 ng/ml phorbol nyristate acetate, 1 µM ionomycin,and 4 µM monensin (all from Sigma, St. Louis, MO) for6 h, as described previously.50 The cells were stained withFITC-conjugated anti-mouse CD3 (BD Biosciences) for 1 h at 4°C,fixed in 1% paraformaldehyde (Sigma), and resuspended in 100µl of permeabilization buffer (0.1% saponin, 1% BSA, and0.1% sodium azide; all from Sigma). Cytokine staining was stainedwith phycoerythrin-conjugated anti-mouse IL-2, IFN-, or IL-4antibodies (all from BD Biosciences) for 1 h at 4°C, withFACSCalibur (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 x 105 cells in200 µl/well) in 96-well flat-bottom microtiter platespreviously coated overnight at 4°C with 0.25 µg/mlanti-mouse CD3 (145–2C11) antibodies (BD Biosciences).After 48 h, the cultures were pulsed with 1 µCi of 3H-methylthymidine (Amersham Pharmacia Biotech, Piscataway, NJ) and harvested16 h later, and the incorporated 3H-methyl thymidine was measuredusing a TopCount (Packard, PerkinElmer, Boston, MA) as describedpreviously.23
Real-Time PCR Assay
RNA was extracted from the renal cortex using TriZOL reagents(Invitrogen). For first-strand cDNA synthesis, 1.5 µgof 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, 1x first-strand buffer, 0.4 mMdithiothreitol, 80 U of RNaseout recombinant ribonuclease inhibitor,and 300 U of Superscript II RNase H (Invitrogen) in a totalvolume of 25 µl. Real-time PCR was performed on an ABIPrism 7700 Sequence Detection System (Applied Biosystems, FosterCity, CA). All of the probes and primers were Assays-on-DemandGene expression products (Applied Biosystems). Real-time PCRreactions were performed using 10 µl of cDNA, 12.5 µlof TaqMan Universal PCR Master Mix (Applied Biosystems), and1.25 µl of the specific probe/primer mixture in a totalvolume of 25 µl. The thermal cycler conditions were 1x 2 min at 50°C, 1 x 10 min at 95°C, and 40 cycles ofdenaturation (15 s at 95°C) and combined annealing/extension(1 min at 60°C). The housekeeping gene glyceraldehyde-3-phosphatedehydrogenase 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-, and IL-4 were measured using commercialELISA kits (BD Biosciences) according to the manufacturer’sinstructions. Anti-dsDNA antibody was measured using an anti-mousedsDNA ELISA kit (Alpha Diagnostic, San Antonio, TX). In allELISA, the absorbance at 450 nm was measured using an ELISAplate reader (Bio-Tek, Winooski, VT).
Statistical Analysis
Values are presented as the means ± SEM. Comparison betweentwo groups was performed using t test. P < 0.05 was takenas a statistical difference.
This study was supported by grants from Tri-Service GeneralHospital (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 availableat www.jasn.org.
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Top
Abstract
Introduction
, 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-
-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). 
