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• W2040928908 abstract "Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Faslpr mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Faslpr mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Faslpr and IL-12p40/IL-23 knockout MRL-Faslpr mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Faslpr mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism. Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Faslpr mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Faslpr mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Faslpr and IL-12p40/IL-23 knockout MRL-Faslpr mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Faslpr mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism. Systemic lupus erythematosus (SLE) is an autoimmune disease that involves increased production of autoantibodies, immune complex deposition in various organs, complement activation, and leukocyte infiltration. Until now the major cause of disease initiation remains unknown. The role of genetics in disease development is controversial. In addition to some widely known hereditary functional disorders regarding the early components of the classical complement pathway (complement 1q, complement 1r/complement 1s, complement 4, or complement 2),1.Barilla-LaBarca M.L. Atkinson J.P. Rheumatic syndromes associated with complement deficiency.Curr Opin Rheumatol. 2003; 15: 55-60Crossref PubMed Scopus (63) Google Scholar numerous susceptibility loci for SLE have been identified in the last years. Recently, polymorphisms in the Stat4 gene have been described as risk factors for SLE.2.Remmers E.F. Plenge R.M. Lee A.T. et al.STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus.N Engl J Med. 2007; 357: 977-986Crossref PubMed Scopus (763) Google Scholar Several of the polymorphisms showed a strong association with double-stranded DNA autoantibodies, nephritis, early age of diagnosis,3.Taylor K.E. Remmers E.F. Lee A.T. et al.Specificity of the STAT4 genetic association for severe disease manifestations of systemic lupus erythematosus.PLoS Genet. 2008; 4: e1000084Crossref PubMed Scopus (148) Google Scholar and lower interferon (INF)-α activity in peripheral blood mononuclear cells in SLE patients.4.Kariuki S.N. Kirou K.A. MacDermott E.J. et al.Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-alpha in lupus patients in vivo.J Immunol. 2009; 182: 34-38Crossref PubMed Scopus (178) Google Scholar However, the impact of these polymorphisms on the functions of the immune system and the pathophysiology of SLE remain unknown. Stat4 was first isolated in 19945.Yamamoto K. Quelle F.W. Thierfelder W.E. et al.Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.Mol Cell Biol. 1994; 14: 4342-4349Crossref PubMed Scopus (200) Google Scholar, 6.Zhong Z. Wen Z. Darnell Jr, J.E. Stat3 and Stat4: members of the family of signal transducers and activators of transcription.Proc Natl Acad Sci USA. 1994; 91: 4806-4810Crossref PubMed Scopus (330) Google Scholar and its expression is restricted in myeloid cells, thymus, and testis.6.Zhong Z. Wen Z. Darnell Jr, J.E. Stat3 and Stat4: members of the family of signal transducers and activators of transcription.Proc Natl Acad Sci USA. 1994; 91: 4806-4810Crossref PubMed Scopus (330) Google Scholar In the resting human T cells, Stat4 expression is low, however, expression can be upregulated by activation with phytohemagglutinin. Stat4 is predominantly activated by interleukin (IL)-127.Bacon C.M. Petricoin III, E.F. Ortaldo J.R. et al.Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.Proc Natl Acad Sci USA. 1995; 92: 7307-7311Crossref PubMed Scopus (355) Google Scholar and Stat4-deficient mice showed an impaired Th1 differentiation with reduced INF-γ production and an impaired cell-mediated immune response (8, Ivashkiv, 2004 #18). Stat4-deficient lymphocytes showed an impaired response to IL-12, with a reduced proliferative response.8.Kaplan M.H. Sun Y.L. Hoey T. et al.Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice.Nature. 1996; 382: 174-177Crossref PubMed Scopus (1026) Google Scholar In numerous studies, the role of cytokines, especially that of INF-γ, IL-12, and IL-18, has been intensively investigated in mouse models for SLE disease development. The MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) mouse is a model of SLE characterized by many clinical (for example, glomerulonephritis, arthritis) and immunological features (for example, autoantibody formation) similar to human SLE.9.Andrews B.S. Eisenberg R.A. Theofilopoulos A.N. et al.Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains.J Exp Med. 1978; 148: 1198-1215Crossref PubMed Scopus (1275) Google Scholar Renal injury in MRL-Faslpr mice is complex and consists of glomerular, interstitial, and perivascular disease mediated by immune complex deposition and infiltration of monocytes and lymphocytes. INF-γ is crucial for disease development and drives the autoimmune kidney destruction in MRL-Faslpr(ref. 10.Schwarting A. Wada T. Kinoshita K. et al.IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1998; 161: 494-503PubMed Google Scholar). IL-12 is important for the regulation of INF-γ production, and IL-12 secreted by renal tubular epithelial cells (TECs) has been shown to promote the autoimmune kidney destruction by expanding IFN-γ-secreting T cells.11.Schwarting A. Tesch G. Kinoshita K. et al.IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1999; 163: 6884-6891PubMed Google Scholar, 12.Fan X. Oertli B. Wuthrich R.P. Up-regulation of tubular epithelial interleukin-12 in autoimmune MRL-Fas(lpr) mice with renal injury.Kidney Int. 1997; 51: 79-86Abstract Full Text PDF PubMed Scopus (55) Google Scholar IL-18, another potent IFN-γ-inducing cytokine expressed by TECs and mononuclear cells, is related to the IL-1 family in terms of its structure, receptor family, and function (see below).13.Dinarello C.A. Interleukin-18.Methods. 1999; 19: 121-132Crossref PubMed Scopus (384) Google Scholar IL-18 is involved in Th1 clone development and IFN-γ production in T cells and therefore involved in inflammatory and autoimmune disorders.13.Dinarello C.A. Interleukin-18.Methods. 1999; 19: 121-132Crossref PubMed Scopus (384) Google Scholar, 14.Puren A.J. Fantuzzi G. Dinarello C.A. Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells.Proc Natl Acad Sci USA. 1999; 96: 2256-2261Crossref PubMed Scopus (304) Google Scholar The importance of IL-18 in the lupus-like syndrome of MRL-Faslpr mice was demonstrated in a study showing that the overexpression of an IL-18 receptor accessory chain (IL-18Rβ) on lymphocytes from MRL-Faslpr mice could account for the hyperresponsiveness of these cells to IL-18, resulting in enhanced IFN-γ secretion.15.Neumann D. Del Giudice E. Ciaramella A. et al.Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain.J Immunol. 2001; 166: 3757-3762Crossref PubMed Scopus (34) Google Scholar Furthermore, a correlation of tubular IL-18 expression and disease activity could be shown.16.Faust J. Menke J. Kriegsmann J. et al.Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis.Arthritis Rheum. 2002; 46: 3083-3095Crossref PubMed Scopus (96) Google Scholar Thus, IL-18 has many functional properties that are similar to that of IL-12. In mice deficient for IL-18, IFN-γ production was suppressed despite the presence of IL-12.17.Takeda K. Tsutsui H. Yoshimoto T. et al.Defective NK cell activity and Th1 response in IL-18-deficient mice.Immunity. 1998; 8: 383-390Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar These data suggest that there is an important interplay between IL-12 and IL-18 for optimal IFN-γ production. Although functionally similar, differences in the downstream signaling pathway of IL-12 and IL-18 receptors have been reported.18.Dinarello C.A. Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process.Am J Clin Nutr. 2006; 83: 447S-455SPubMed Google Scholar In contrast to IL-18, IL-12 is an important activator of Stat4 and signals via the receptor-associated Janus kinases, Janus kinases 2 and TYK2.19.Sinigaglia F. D'Ambrosio D. Panina-Bordignon P. et al.Regulation of the IL-12/IL-12R axis: a critical step in T-helper cell differentiation and effector function.Immunol Rev. 1999; 170: 65-72Crossref PubMed Scopus (125) Google Scholar Thus, although IL-12 and IL-18 seem to act synergistically, they use independent pathways of intracellular signaling. In the present study, we report that targeting Stat4 in severe lupus nephritis reveals compensatory proinflammatory mechanisms proven by two approaches: the knockout approach of Stat4 gene and the knockdown approach of the Stat4 mRNA. Knocking out the Stat4 gene does not affect clinical features compared with wild-type (WT) mice, whereas treatment with antisense (AS) oligonucleotides for a period of 3 weeks ameliorates the advanced lupus nephritis in MRL-Faslpr mice. Interestingly, we show that IL-18 functions independently of IL-12 to incite kidney injury in MRL-Faslpr mice. We conclude that the compensatory upregulation of IL-18 in the Stat4 knockout approach mediates kidney disease in MRL-Faslpr mice. However, the AS oligonucleotide treatment to knock down Stat4 temporarily during advanced renal injury resulted in an amelioration of kidney diseases. To evaluate the relevance of Stat4 in initiation and acceleration of kidney and systemic disease in MRL-Faslpr mice, we generated Stat4-/-MRL-Faslpr mice. We did not detect a difference in the survival of Stat4-/- MRL-Faslpr mice compared with WT mice (Figure 1a). Furthermore, we did not determine a difference in lymphadenopathy and splenomegaly in Stat4-/- compared with Stat4+/+MRL-Faslpr mice (data not shown) and renal damage (renal function; Figure 1b). The severity of glomerular, interstitial, and perivascular pathologies was similar in Stat4-/- and Stat4+/+MRL-Faslpr mice (data not shown). Similarly, there was no difference in the number of infiltrating leukocytes (CD4+, CD8+, CD45/B220+, and F4/80+ cells; Figure 1c). However, we detected significantly higher serum levels of IL-18 and IL-12, but no difference in INF-γ serum levels in Stat4-/- compared with Stat4+/+MRL-Faslpr mice (Figure 1d). The analysis of immunoglobulin (Ig) G subclasses revealed significantly higher serum titres of IgG1 in Stat4-/- mice, whereas serum levels of IgG2a, IgG2b, and IgG3 showed comparable values in Stat4-/- and Stat4+/+MRL-Faslpr mice (Figure 1e). Taken together, deletion of Stat4 did not result in an improvement of renal disease in Stat4-/-MRL-Faslpr mice. However, we detected a compensatory upregulation of IL-18 and IL-12 in Stat4-/-MRL-Faslpr mice, suggesting that IL-18 independent of IL-12-Stat4 signaling mediates kidney injury. To confirm the efficacy of oligonucleotide treatment, splenocytes from Balb/c Stat4+/+ mice were stimulated with IL-12, resulting in a significant increase in INF-γ production (Figure 2). After co-incubation with IL-12 and AS oligonucleotides, the level of INF-γ was markedly reduced than stimulation with IL-12 alone. Co-stimulation with IL-12 and anti-IL-18 showed decreased INF-γ levels than stimulation with IL-12. Thus, AS oligonucleotides are capable of sufficient inhibition of IL-12-induced INF-γ production. To determine whether Stat4 modulates disease activity and progression, we treated MRL-Faslpr mice at 5 months of age with severe kidney and systemic disease using Stat4 AS oligonucleotides compared with mice treated with missense (MS) oligonucleotides and phosphate-buffered saline for control. After 3 weeks of treatment, renal function was improved in AS oligonucleotide-treated compared with MS oligonucleotide-treated MRL-Faslpr mice, as determined by proteinuria, blood urea nitrogen (BUN), and creatinine (Figure 3a). In correlation to our clinical findings, we detected less glomerular damage (Figure 3b) and a decreased number of infiltrating leukocytes in kidneys of AS oligonucleotide-treated vs MS oligonucleotide-treated MRL-Faslpr mice (Figure 3c). Immunostaining revealed a tendency to a lower number of CD4+ infiltrating cells glomerularly and interstitially. Furthermore, a significantly lower number of glomerular and interstitial F4/80+, CD8+, and CD45/B220+ cells could be found in mice treated with AS oligonucleotides compared with MS oligonucleotide-treated mice or controls (Figure 3c). As expected from our in vitro experiments, we detected decreased INF-γ levels following AS oligonucleotide treatment compared with MS oligonucleotide-treated and control MRL-Faslpr mice (AS: 20.3±16.3; MS: 36.3±10.5; controls: 26.3±26.3 pg/ml). Surprisingly, we found significantly higher levels of IL-18 in mice treated with AS oligonucleotides compared with mice MS oligonucleotide- or phosphate-buffered saline-treated mice (Figure 3d). Treatment with AS or MS oligonucleotides had no significant effect on serum levels of IgG and its subclasses. The role of IgGs in mediating kidney and systemic disease is discussed controversially; our findings would support the concept that the extent of circulating IgGs and autoantibodies does not exclusively correlate with kidney damage.20.Tesch G.H. Schwarting A. Kinoshita K. et al.Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis.J Clin Invest. 1999; 103: 73-80Crossref PubMed Scopus (227) Google Scholar, 21.Lin L. Peng S.L. Interleukin-18 receptor signaling is not required for autoantibody production and end-organ disease in murine lupus.Arthritis Rheum. 2005; 52: 984-986Crossref PubMed Scopus (12) Google Scholar In conclusion, Stat4 knockout did not affect the disease initiation and acceleration, whereas temporary treatment with AS oligonucleotides ameliorates the disease activity and progression in MRL-Faslpr mice. To clarify the underlying mechanisms of our in vivo approach, we investigated the INF-γ production in splenocytes isolated from MRL-Faslpr mice. Incubation with IL-12 led to very high levels of INF-γ production (Figure 4a). By comparison, IL-12-induced INF-γ production in splenocytes of MRL-Faslpr mice treated with AS oligonucleotides was greatly reduced, and not affected by MS treatment. Interestingly, splenocytes of MRL-Faslpr mice showed a higher baseline production of INF-γ compared with BALB/c mice. Stimulation with AS oligonucleotides and anti-IL-18 was able to reduce INF-γ production below baseline levels compared with stimulation with AS oligonucleotides alone. In conclusion, Stat4-signaling and IL-18 appear to be the most potent mediators of INF-γ production. Using splenocytes of MRL-Faslpr Stat4-/- and Stat4+/+ mice, we determined that the INF-γ production in response to IL-18 was comparable in MRL-Faslpr Stat4-/- and Stat4+/+ mice. Moreover, following stimulation with IL-12, IL-18 production in MRL-Faslpr was independent of Stat4. In addition, the IFN-γ and IL-18 expression could be blocked by anti-IL-18 treatment. Thus, the in vitro data support the importance of IL-18 in initiation and progression of lupus nephritis in MRL-Faslpr. On the basis of the results in MRL-Faslpr Stat4-/- mice and AS oligonucleotide treatment, we evaluated the relevance of IL-18 during kidney disease in MRL-Faslpr mice by overexpressing IL-18 locally in the kidney of MRL-Faslpr mice using ‘IL-18-carrier cells’. ‘IL-18-carrier cells’ constitutively secrete high levels of IL-18 in culture as compared with ‘carrier cells’ (Figure 5a). Furthermore, we confirmed successful gene transfer of IL-18 into MRL-Faslpr TECs by the presence of EGFP (Figure 5a). Of note, in the IL-18 overexpression study, we analyzed mice at day 28 after ‘carrier cell’ implantation, unless otherwise stated. Intrarenal IL-18 transcripts increased following ‘IL-18-carrier cell’ implantation as compared with ‘carrier cell’ implantation and untreated kidneys at 3 months of age (Figure 5a, right panel). Following intrarenal IL-18 overexpression, we detected massive accumulation of leukocytes within the implant site that extended into the adjacent intrarenal area (Figure 5b). Furthermore, we detected a notable increase in glomerular and tubular damage following ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation (Figure 5b). The increase in kidney pathology is accompanied by accumulation of multiple leukocyte populations in the kidney following ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation (Figure 5c). To determine whether the increase in renal pathology and intrarenal leukocytes following overexpression of IL-18 was accompanied by impaired renal function, we compared proteinuria and serum BUN levels. In fact, proteinuria and BUN levels rose in mice following ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation to levels comparable to that of nephritic MRL-Faslpr mice at 5 months of age. Thus, intrarenal overexpression of IL-18 initiates kidney disease in MRL-Faslpr mice. To determine whether intrarenal IL-18 expression could be responsible for systemic disease in MRL-Faslpr mice, we assessed several cytokines in the circulation, as well as the impact of IL-18 overexpression on contralateral kidney and lung pathology. We detected a rise in circulating IL-18, IFN-γ, and tumor necrosis factor-α following intrarenal ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation (Figure 6a) that are comparable to levels detected in MRL-Faslpr mice with advanced kidney disease (data not shown). Interestingly, other pro-inflammatory cytokines, such as IL-12 and IL-1β, were not affected by the intrarenal IL-18 overexpression. To establish whether these enhanced cytokine levels were a reflection of accelerated systemic disease, we evaluated intrarenal leukocytes in the contralateral kidney. Interestingly, we detected an increase of multiple leukocyte populations in the contralateral kidney following ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation (Figure 6b). Moreover, we could detect an accumulation of perivascular and interstitial leukocytes in the lung following intrarenal ‘IL-18-carrier cell’ compared with ‘carrier cell’ implantation (intrapulmonary 2.25±0.89 vs 1.17±0.41, *P<0.05; perivascular 2.75±0.46 vs 1.83±0.75, *P<0.05; peribronchial 2.63±0.52 vs 2.00±0.63). Thus, the rise in the level of circulating cytokines and increased leukocyte infiltration in the contralateral kidney and lung suggests that local intrarenal IL-18 expression initiates systemic disease in MRL-Faslpr mice. To clarify whether the presence of IL-12 and/or IL-23 is required for the IL-18 induced nephritis, we implanted ‘IL-18-carrier cells’ into IL-12p40/IL-23-/- and intact MRL-Faslpr mice. We detected a comparable influx of leukocytes in IL-12p40/IL-23-/- and MRL-Faslpr mice assessed at the implantation site, periglomerular, perivascular, and interstitial (Figure 7a). Furthermore, circulating IL-18 and IFN-γ levels were increased whereas level of IL-12 was not detectable (data not shown). The increase in intrarenal leukocytes in IL-12p40/IL-23-/- and MRL-Faslpr was accompanied by impaired renal function. Proteinuria and BUN levels rose to levels comparable to nephritic MRL-Faslpr mice at 5 months of age (Figure 7b). Thus, IL-18 initiates and accelerates kidney disease in MRL-Faslpr mice independent of IL-12p40 and/or IL-23. In the present study, we dissected the role of Stat4 on lupus nephritis by two approaches. Stat4 gene deletion does not affect disease initiation and progression in MRL-Faslpr compared with WT mice, whereas temporary treatment with AS oligonucleotides ameliorates advanced lupus nephritis in MRL-Faslpr mice. On the basis of the finding that in both approaches a compensatory IL-18 upregulation was detectable, studies using gene transfer to overexpress IL-18 in MRL-Faslpr and IL-12p40/IL-23-/-MRL-Faslpr mice reveal a critical role for IL-18 in mediating kidney and systemic disease in MRL-Faslpr mice. In MRL-Faslpr mice, INF-γ deficiency led to the absence of lupus disease,10.Schwarting A. Wada T. Kinoshita K. et al.IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1998; 161: 494-503PubMed Google Scholar as well as in NZB/NZW mice, another mouse model for lupus.22.Haas C. Ryffel B. Le Hir M. IFN-gamma is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice.J Immunol. 1997; 158: 5484-5491PubMed Google Scholar In experiments using a mouse model for SLE based on NZB/NZW mice, Stat4-/- New Zealand mixed mice developed accelerated kidney disease with an increase in proteinuria and mortality despite decreased IFN-γ serum levels.23.Jacob C.O. Zang S. Li L. et al.Pivotal role of Stat4 and Stat6 in the pathogenesis of the lupus-like disease in the New Zealand mixed 2328 mice.J Immunol. 2003; 171: 1564-1571Crossref PubMed Scopus (92) Google Scholar, 24.Singh R.R. Saxena V. Zang S. et al.Differential contribution of IL-4 and STAT6 vs STAT4 to the development of lupus nephritis.J Immunol. 2003; 170: 4818-4825Crossref PubMed Scopus (125) Google Scholar In contrast, administration of exogenous IFN-γ accelerates renal disease in (NZB × NZW)F1 mice. Surprisingly, we did not detect any amelioration or acceleration of lupus disease in MRL-FaslprStat4-/- mice, moreover, Stat4 deficiency had no impact on INF-γ production. These differences between MRL-Faslpr and NZB/NZW mice could be explained by the different role of type I IFNs in these models: in NZB/NZW models, type-I IFN receptor deficiency reduced lupus-like disease,25.Santiago-Raber M.L. Baccala R. Haraldsson K.M. et al.Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice.J Exp Med. 2003; 197: 777-788Crossref PubMed Scopus (437) Google Scholar whereas the type I IFN, IFN-β, turned out to be therapeutic in MRL-Faslpr mice.26.Schwarting A. Paul K. Tschirner S. et al.Interferon-beta: a therapeutic for autoimmune lupus in MRL-Faslpr mice.J Am Soc Nephrol. 2005; 16: 3264-3272Crossref PubMed Scopus (58) Google Scholar In human SLE patients, Stat4 polymorphisms were recently described in patients who showed decreased IFN-α activity in peripheral blood mononuclear cells but increased expression of IFN-α-induced genes.4.Kariuki S.N. Kirou K.A. MacDermott E.J. et al.Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-alpha in lupus patients in vivo.J Immunol. 2009; 182: 34-38Crossref PubMed Scopus (178) Google Scholar Stat4 showed a dominant influence on the sensitivity of peripheral blood mononuclear cells to serum IFN-α and seems to be an important mediator in type-I IFN pathway. In fact, Stat4 deficiency does not mediate initiation of lupus nephritis in MRL-Faslpr mice. Thus, we speculated that different cytokines might be responsible for this observation and we focused on IL-18. We choose to focus on IL-18 as lymphocytes from MRL-Faslpr mice have been shown to be hyperresponsive to IL-18, resulting in enhanced IFN-γ secretion. The importance of IL-18 in the lupus-like syndrome of MRL-Faslpr mice was demonstrated in a study showing that lymphocytes of MRL-Faslpr mice are hyperresponsive to IL-18, which results in enhanced IFN-γ secretion.15.Neumann D. Del Giudice E. Ciaramella A. et al.Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain.J Immunol. 2001; 166: 3757-3762Crossref PubMed Scopus (34) Google Scholar Further support for the concept of IL-18 as a sufficient inducer of a TH1 response comes from recent studies in our lab on overexpression and blocking of IL-18. Local intrarenal overexpression of IL-18 incites lupus nephritis and systemic disease, a blockade of IL-18 activation ameliorates kidney and systemic disease (manuscript submitted). IL-18 receptor-α-deficient MRL-Faslpr mice have markedly reduced autoimmune disease,27.Kinoshita K. Yamagata T. Nozaki Y. et al.Blockade of IL-18 receptor signaling delays the onset of autoimmune disease in MRL-Faslpr mice.J Immunol. 2004; 173: 5312-5318Crossref PubMed Scopus (46) Google Scholar and IL-18 complementary DNA vaccination protects MRL-Faslpr mice from spontaneous lupus-like autoimmune disease.28.Kitching A.R. Tipping P.G. Kurimoto M. et al.IL-18 has IL-12-independent effects in delayed-type hypersensitivity: studies in cell-mediated crescentic glomerulonephritis.J Immunol. 2000; 165: 4649-4657Crossref PubMed Scopus (43) Google Scholar Furthermore, IL-18-deficient mice show an ameliorated kidney disease in a model of induced autoimmune nephritis (nephrotoxic serum nephritis).29.Sugiyama M. Kinoshita K. Kishimoto K. et al.Deletion of IL-18 receptor ameliorates renal injury in bovine serum albumin-induced glomerulonephritis.Clin Immunol. 2008; 128: 103-108Crossref PubMed Scopus (13) Google Scholar In addition, it has been shown that tubular IL-18 expression correlates with disease activity in MRL-Faslpr mice.16.Faust J. Menke J. Kriegsmann J. et al.Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis.Arthritis Rheum. 2002; 46: 3083-3095Crossref PubMed Scopus (96) Google Scholar We found elevated levels of IL-18 in MRL-FaslprStat4-/- mice and, therefore, we speculate that IL-18 bypasses the Stat4 gene defect in MRL-FaslprStat4-/- mice and leads to similar levels of INF-γ compared with WT. The results of our in vitro approach support that both Stat4 and IL-18 contribute to spontaneous INF-γ production and that IL-18 works independently of IL-12 to promote a sufficient IFN-γ production to mediate disease. Bearing in mind the limitations of performing studies in gene-targeted mice,30.Lin J.H. Applications and limitations of interspecies scaling and in vitro extrapolation in pharmacokinetics.Drug Metab Dispos. 1998; 26: 1202-1212PubMed Google Scholar we used AS oligonucleotide treatment as a posttranscriptional modification of gene expression to knock down Stat4 in vivo. We found that AS oligonucleotide therapy resulted in an amelioration of advanced kidney disease. This technique offers a realistic option as a therapeutic treatment in human SLE patients. AS oligonucleotides have mainly been tested in the field of oncology in phase II and III studies. A combination with oblimersen sodium, a Bcl-2 AS oligonucleotide, and rituximab was shown to be effective in patients with recurrent B-cell non-Hodgkin's lymphoma.31.Pro B. Leber B. Smith M. et al.Phase II multicenter study of oblimersen sodium, a Bcl-2 antisense oligonucleotide, in combination with rituximab in patients with recurrent B-cell non-Hodgkin lymphoma.Br J Haematol. 2008; 143: 355-360Crossref PubMed Scopus (79) Google Scholar In contrast, aprinocarsen, a protein kinase C-alpha AS oligonucleotide, did not enhance survival and other efficacy measures in patients with advanced non-small cell lung cancer treated with gemcitabine and cisplatin.32.Paz-Ares L. Douillard J.Y. Koralewski P. et al.Phase III study of gemcitabine and cisplatin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleotide, in patients with advanced-stage non-small-cell lung cancer.J Clin Oncol. 2006; 24: 1428-1434Crossref PubMed Scopus (110) Google Scholar Future studies will likely prove the efficacy of AS oligonucleotide treatment and give a rationale for the application in patients with autoimmune disease. In the present study, treating MRL-Faslpr mice for 3 weeks resulted in the amelioration of lupus nephritis. However, we also observed a concomitant increase in serum levels of IL-18. One possibility for the differing effect of the inhibition of Stat4 signaling is the time of blockade. The increased levels of IL-18 in the AS oligonucleotide-treated group suggest that extended treatment periods will have no sufficient effect because of the compensatory disease-mediating IL-18 upregulation. Therefore, future studies should evaluate a combination therapy using Stat4 and IL-18 blockade. In conclusion, Stat4-independent factors can drive autoimmune lupus nephritis in Stat4-/-MRL-Faslpr mice, while blocking Stat4 temporarily in advanced nephritis can ameliorate disease suggesting time-dependent compensatory proinflammatory mechanisms. The AS oligonucleotide treatment to block Stat4 in mice with lupus-like disease suggests a clinically relevant therapy that should be further investigated for potential and immediate benefits to human SLE patients. To investigate the influence of Stat4 in early stages of murine SLE, we generated Stat4-deficient (-/-) MRL-Faslpr mice. MRL-Faslpr mice and Stat4-/- BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, Maine), and Stat4-/- MRL-Faslpr mice were derived by a series of genetic backcrosses using the backcross–intercross scheme. MRL-Faslpr mice were mated with Stat4-/- Balb/c mice to yield heterozygous F1 offspring. We intercrossed F1 mice and screened the progeny by PCR amplification of tail genomic DNA for the Faslpr mutation (primer: WT sense 5′-CCAACTCAGACAGCAACTGG-3′, AS 5′-GCTCTTTGAGCAGGGATG-3′) and Stat4 (using the linked neomycin resistance (neo) gene, primer: neo sense: 5′-ATTGAACAAGATTTGCAC-3′, neo AS: 5′-CGTCCAGATCATCCTGATC-3′). Double homozygous (Faslpr/Faslpr Stat4-/-) N1F1 progeny were backcrossed with MRL-Faslpr/Faslpr mice. B1 progeny, homozygous for the Faslpr mutation and heterozygous for the Stat4, was intercrossed, and mice homozygous for the Stat4-/- mutation were selected by PCR typing for continued backcrossing. After five generations of backcross matings, this breeding scheme generated a colony of MRL-Faslpr mice (>95–97.8% MRL background) homozygous and heterozygous for the mutated Stat4. We analyzed the F6 generation as it was previously established that there are sufficient MRL-Faslpr background genes even by the F3 generation to result in phenotypic changes characteristic of the MRL-Faslpr strain.33.Kikawada E. Lenda D.M. Kelley V.R. IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.J Immunol. 2003; 170: 3915-3925Crossref PubMed Scopus (125) Google Scholar Moreover, we performed a genome scan to ensure >95% MRL background in used mice (Supplementary Figure 1). MRL-Faslpr Stat4+/+ (n=22) and MRL-Faslpr Stat4-/- (n=13) were under observation for 6 months to determine the effect of Stat4 deficiency in disease development. MRL-Faslpr Stat4+/+ mice were littermate controls. IL-12p40-deficient (-/-) MRL-Faslpr mice were derived by a series of 10 genetic intercross/backcross matings as previously described.10.Schwarting A. Wada T. Kinoshita K. et al.IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(lpr) mice.J Immunol. 1998; 161: 494-503PubMed Google Scholar Download .jpg (.11 MB) Help with files Supplementary Figure 1 The use of mice in this study was reviewed and approved by the Standing Committee on Animals at the University of Mainz. All of the animal experiments in this study were performed in Germany. Mice were bred and housed in our pathogen-free facilities at Johannes Gutenberg University in Mainz. To investigate the effect of Stat4 blockade in mice with manifest lupus nephritis, we treated MRL-Faslpr mice with AS oligonucleotides, MS oligonucleotides (each kindly provided by Professor Neurath, Mainz, Germany) or phosphate-buffered saline (control).34.Hildner K.M. Schirmacher P. Atreya I. et al.Targeting of the transcription factor STAT4 by antisense phosphorothioate oligonucleotides suppresses collagen-induced arthritis.J Immunol. 2007; 178: 3427-3436Crossref PubMed Scopus (50) Google Scholar MRL-Faslpr mice were purchased from the Jackson Laboratory. We started oligonucleotide treatment at 5 months of age. Oligonucleotides were administered intraperitoneally three times per week for 3 weeks. Each group consisted of 12 mice. Proteinuria and hematuria were measured with a dipstick (Multistix: Bayer Diagnostics, Bridgend, UK). We assessed urinary protein levels and hematuria only semiquantitatively on a monthly basis beginning at 2 months of age. On the days of analysis, dipstick proteinuria measurements were made in individual mouse in the morning and repeated in the evening. If the measurements were inconsistent, the mouse was reassessed on the following day and in addition, all mice were reassessed twice a week.33.Kikawada E. Lenda D.M. Kelley V.R. IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.J Immunol. 2003; 170: 3915-3925Crossref PubMed Scopus (125) Google Scholar We measured BUN levels using a colorimetric analysis kit (Infinity, Thermo Electron, Melbourne, Victoria, Australia), and serum creatinine using the creatinine reagent kit and Creatinine Analyzer 2 (Beckman Coulter, Galway, Ireland) according to the manufacturer's instructions. Levels of IL-18, INF-γ, IL-12, and IL-1β in serum were quantified using the enzyme-linked immunosorbent assay technique according to the manufacturer's instructions (OpEIA mouse kit, BD Bioscience, San José, CA). IgG subclasses: levels of IgG and its subclasses (IgG1, IgG2a, IgG2b, IgG3) have been determined by enzyme-linked immunosorbent assay technique (Alkaline phosphatase-based enzyme-linked immunosorbent assay, SouthernBiotech, Birmingham, AL) Body weight was measured once a week. After killing, organ weight was measured and the size of lymph nodes was scored (0=none, 0.5=small, 1=moderate, 2=large, 3=very large). We fixed kidneys in 10% neutral-buffered formalin for 24 h, and stained paraffin sections (4 μm) with periodic acid-Schiff's reagent. We evaluated kidney pathology as previously described.33.Kikawada E. Lenda D.M. Kelley V.R. IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.J Immunol. 2003; 170: 3915-3925Crossref PubMed Scopus (125) Google Scholar Briefly, we assessed glomerular pathology by scoring each glomerulus on a semiquantitative scale: 0=normal (35–40 cells/glomerular cross-sections (gcs)); 1=mild (glomeruli with few lesions showing slight proliferative changes, mild hypercellularity (41–50 cells/gcs)); 2=moderate (glomeruli with moderate hypercellularity (51–60 cells/gcs), including segmental and/or diffuse proliferative changes, hyalinosis); 3=severe (glomeruli with segmental or global sclerosis and/or severe hypercellularity (>60 cells/gcs), necrosis, crescent formation). We scored 20 gcs per kidney. Interstitial/tubular pathology was assessed semiquantitatively on a scale of 0–3 in 10 randomly selected high-power fields (hpf). We determined the largest and average number of infiltrates and damaged tubules, and adjusted the grading system accordingly: 0=normal, 1=mild, 2=moderate, 3=severe. Perivascular cell accumulation was determined semiquantitatively by scoring the number of cell layers surrounding the majority of vessel walls (score: 0=none, 1=<5 cell layers, 2=5–10 cell layers, 3=>10 cell layers). Kidney tissue was processed and stained for the presence of F4/80, CD4, CD8, and B220, as previously described for the kidney.35.Schwarting A. Moore K. Wada T. et al.IFN-gamma limits macrophage expansion in MRL-Fas(lpr) autoimmune interstitial nephritis: a negative regulatory pathway.J Immunol. 1998; 160: 4074-4081PubMed Google Scholar We determined the number of positive cells in 10 randomly selected hpf. To check the specificity and efficacy of the oligonucleotides used in the in vivo treatment, splenocytes of BALB/c and MRL-Faslpr mice were isolated as previously descried26.Schwarting A. Paul K. Tschirner S. et al.Interferon-beta: a therapeutic for autoimmune lupus in MRL-Faslpr mice.J Am Soc Nephrol. 2005; 16: 3264-3272Crossref PubMed Scopus (58) Google Scholar and stimulated with IL-12, IL-18 (R&D Systems, Minneapolis, MN), goat anti-IL-18 (Santa Cruz Biotechnologies, Santa Cruz, CA), with or without the addition of either Stat4 AS or Stat4 MS oligonucleotides (kindly provided by Professor Dr Neurath, Mainz, Germany, and modified by Carl Roth GmbH, Karlsruhe, Germany).34.Hildner K.M. Schirmacher P. Atreya I. et al.Targeting of the transcription factor STAT4 by antisense phosphorothioate oligonucleotides suppresses collagen-induced arthritis.J Immunol. 2007; 178: 3427-3436Crossref PubMed Scopus (50) Google Scholar We isolated and cultured TECs derived from MRL-Faslpr mice at 2–3 months of age, as previously reported.16.Faust J. Menke J. Kriegsmann J. et al.Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis.Arthritis Rheum. 2002; 46: 3083-3095Crossref PubMed Scopus (96) Google Scholar For the retroviral gene transfer, we used a helper-free retrovirus packaging cell line (PT67; Clontech Laboratories, Mountain View, CA). The complete IL-18 precursor complementary DNA was kindly provided by Professor Dr Neurath (Johannes Gutenberg University of Mainz, Germany). This recombination-incompetent retroviral vector (pLEGFP-N1, Clontech Laboratories Inc.) containing the murine IL-18 gene was introduced into PT67 cells containing proviral sequences necessary for the ‘packing’ (for example, encapsidation) of the virus.36.Danos O. Mulligan R.C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.Proc Natl Acad Sci USA. 1988; 85: 6460-6464Crossref PubMed Scopus (793) Google Scholar Using the supernatant of PT67-IL-18 cells, we infected TECs with the recombinant retrovirus in the presence of polybrene. To verify gene transfer into TECs, (1) vector-containing cells were detected by fluorescence (green fluorescent protein, part of the vector) (2) neomycin-resistant TECs were selected using G418 (Sigma, St Louis, MO; 800 μg/ml), and (3) IL-18 was measured in supernatants collected 5 days after infection by enzyme-linked immunosorbent assay. For control experiments, TECs were infected with the pLEGFP-N1 retroviral vector without the gene of interest. TECs genetically modified to express a gene are termed ‘carrier cells’, for example, TEC-producing IL-18 as ‘IL-18-carrier cells’; TECs containing the vector without insert as ‘carrier cells’. We placed ‘carrier cells’ under the renal capsule of MRL-Faslpr IL-12/IL-23-/-MRL-Faslpr and MRL+/+ mice at 2 months of age. We tested the viability of the TECs immediately before implantation using trypan blue (Invitrogen, Carlsbad, CA) exclusion staining, viability was >90%. The animals were anesthetized and the left kidney was exposed through a left flank incision. ‘Carrier cells’ (1 × 106 in 50 μl of Hanks balanced salt solution w/o Ca) (Sigma) were micropipetted under the capsule of the dorsal surface of the left kidney. Data were compared using SPSS 14 (Chicago, IL). The significance was determined by Student's unpaired t-test or Mann–Whitney U-test. P-values <0.05 were considered significant. This work was supported by the German Research Foundation (DFG) Grant Schw 752 2/2 and by the National Institutes of Health grant DK36149 (V.R.K.)). Figure 1. The genome scan revealed a >95% MRL background in MRL-Faslpr mice used in this study. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .doc (.03 MB) Help with doc files Supplementary Figure Legend" @default.
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