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Published ahead of print on October 12, 2005
J Am Soc Nephrol 16: 3264-3272, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2004111014
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Basic Immunology and Pathology

Interferon-{beta}: A Therapeutic for Autoimmune Lupus in MRL-Faslpr Mice

Andreas Schwarting*, Kathrin Paul*, Stefan Tschirner*, Julia Menke*, Torsten Hansen{dagger}, Walburgis Brenner{ddagger}, Vicki Rubin Kelley§, Manfred Relle* and Peter R. Galle*

* First Department of Medicine, {dagger} Institute of Pathology, and {ddagger} Department of Urology, Johannes Gutenberg-University Mainz, Mainz, Germany; and § Renal Division, Brigham & Women’s Hospital, Boston, Massachusetts

Address correspondence to: Dr. Andreas Schwarting, First Department of Medicine, Johannes-Gutenberg University of Mainz, Langenbeckstrasse 1, Mainz 55131, Germany. Phone: +49-6131-17-2666; Fax: +49-6131-17-6621; aschwart{at}mail.uni-mainz.de

Received for publication November 30, 2004. Accepted for publication August 18, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Type I interferons are associated with lupus. Genes that are regulated by IFN-{alpha} are upregulated in pediatric lupus patients. Gene deletion of the IFN-{alpha}/{beta} receptor in experimental lupus-like NZB mice results in reduced disease activity. Conversely, IFN-{beta} is a well-established treatment in multiple sclerosis, another autoimmune disease. For determining whether IFN-{beta} treatment is harmful or beneficial in lupus, MRL-Faslpr mice were injected with this type I IFN. Treatment was initiated in MRL-Faslpr mice with mild and advanced disease. IFN-{beta} was highly effective in prolonging survival and ameliorating the clinical (renal function, proteinuria, splenomegaly, and skin lesions), serologic (autoantibodies and cytokines), and histologic parameters of the lupus-like disease in mice that had mild and advanced disease. Several underlying mechanisms of IFN-{beta} therapy involving cellular (decreased T cell proliferation and infiltration of leukocytes into the kidney) and humoral (decrease in IgG3 isotypes) immune responses and a reduction in nephrogenic cytokines were identified. In conclusion, IFN-{beta} treatment of lupus nephritis in MRL-Faslpr mice is remarkably beneficial and suggests that IFN-{beta} may be an appealing therapeutic candidate for subtypes of human lupus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hallmark of systemic lupus erythematosus (SLE) includes inflammation in multiple organs and pathogenic autoantibodies (1). Nephritis is common in lupus, often resulting in renal failure (1). Lupus nephritis is mediated by the infiltration of T cells and of macrophages and the deposition of autoantibodies in the capillary walls (2). Advancing tubulointerstitial pathology and an increase in infiltrating leukocytes are unfavorable prognostic indicators (3). Current immunosuppressive therapies for lupus are limited and plagued by harmful side effects, highlighting the need for new therapeutic strategies (4).

In the attempt to identify therapeutic agents for lupus, numerous reports highlight the interferons. Interferons are tempting therapeutic targets, notably topical, and perhaps controversial. Type I interferons are distinct signatures in peripheral blood leukocytes and glomeruli in patients with lupus (5,6). However, enhanced gene expression of type I IFN does not necessarily indicate that these molecules promote disease. For example, type I interferons, more specific IFN-{beta}, is a well-established therapeutic agent for patients with multiple sclerosis during active flares and experimental autoimmune diseases such as exogenous allergic encephalomyelitis and collagen-induced arthritis in rodents (7–9).

The MRL-Faslpr mice are well suited to test novel therapeutic approaches for lupus nephritis. This strain develops a glomerular, tubulointerstitial, and perivascular kidney disease; arthritis; lymphadenopathy; splenomegaly; and circulating autoantibodies, including antineutrophil antibodies (ANA) and anti-dsDNA antibodies (10). The disease tempo in MRL-Faslpr is sufficiently slow to dissect the pathogenesis and sufficiently rapid to be efficient in testing new therapies (11–13).

Using the MRL-Faslpr strain, we and others reported that IFN-{gamma} is required for lupus nephritis, as IFN-{gamma} receptor–deficient MRL-Faslpr mice were protected from fatal kidney disease (14,15). Subsequently, we determined that cytokines that are upstream of IFN-{gamma} (IL-12 and IL-18) mediate lupus nephritis: Overexpressing IL-12 in MRL-Faslpr kidneys leads to a massive infiltration of CD4+ that are dependent on IFN-{gamma}, and upregulation of IL-18 in the MRL-Faslpr kidneys correlates with disease severity (16,17).

Other members of the interferons are potent immunomodulators. In contrast to IFN-{gamma}, a type II IFN, IFN-{beta}, belongs to the ancient family of so-called type I interferons identified originally as potent antiviral cytokines. Subsequently, IFN-{beta} was identified as a modifier of immune reactions (18). For instance, IFN-{beta} downregulates IFN-{gamma} and IL-12 in vitro and in vivo and modulates numerous other cytokine pathways (19–21). Nevertheless, it is not clear whether IFN-{beta} drives or thwarts lupus.

In this study, we examined the therapeutic impact of IFN-{beta} in lupus nephritis in MRL-Faslpr mice. We now report that IFN-{beta} is highly effective for treating minimal and well-established lupus nephritis by modulating both cellular and Ig-related pathogenetic factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
MRL-Faslpr mice were purchased from the Jackson Laboratory and kept in a germ-free condition in the animal facility of the University of Mainz.

Treatment
We categorized MRL-Faslpr mice as having mild or advanced disease as detailed in Table 1.


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  		Table 1. Categories of mild and advanced lupus disease in MRL-Fasmice

 
Mild Disease.
MRL-Faslpr mice (3 mo of age) without clinical signs of nephritis were treated with 103 IU of IFN-{beta} or PBS subcutaneously for 5 d/wk. This procedure, which was repeated twice after an interval of 3 wk, was based on the dose calculated according to the efficacious amount per kilogram in patients with multiple sclerosis.

Advanced Disease.
MRL-Faslpr mice with nephritis (5 mo of age) received subcutaneous injections of 103 IU of IFN-{beta} or PBS every second day for 4 wk. MRL-Faslpr mice that were treated with IFN-{beta} or PBS were killed at 6 mo of age. Of note, 50% mortality occurs in mice at 5 to 6 mo of age.

Lymphadenopathy
Lymphadenopathy (cervical, brachial, and inguinal) was assessed weekly. We palpated the lymph node and scored them using a grade of 0 to 3 (0 = none; 1 = small, at one site; 2 = moderate, at two sites; 3 = large, at three or more different sites). In addition, we assessed the lymphadenopathy at time of killing by measuring and counting the number of enlarged lymph nodes to calculate the mean size of lymph nodes per mouse.

Skin Lesions
Skin lesions were assessed weekly. We scored the skin lesions by gross pathology using a grade of 0 to 3 (0 = none; 1 = mild [snout and ears]; 2 = moderate, <2 cm [snout, ears, and intrascapular]; and 3 = severe, <2 cm [snout, ears, and intrascapular]).

Proteinuria and Serum Urea
The mice were tested semiquantitatively for proteinuria with albumin test strips (Albustix; Miles, Naperville, IL) in weekly intervals (0 = none; 1 = 30 to 100 mg/dl; 2 = 100 to 300 mg/dl; 3 = 300 to 1000 mg/dl; 4 = >1000 mg/dl). Serum urea was measured using commercially available UREA/BUN kinetic UV-Test (cat. no. 1982486001V8; Roche, Mannheim, Germany).

Histopathology
The kidneys were either frozen in OCT (Tissue Tek, Sakura, Zoeterwoude, The Netherlands) for frozen sections or fixed in 10% neutral buffered formalin. Paraffin sections (4 µm) were stained with hematoxylin and periodic acid-Schiff reagent. We evaluated glomerular pathology by assessing 50 glomerular cross-sections (gcs) per kidney and scored each glomerulus on a semiquantitative scale: 0 = normal (35 to 40 cells/gcs); 1 = mild (glomeruli with few lesions showing slight proliferative changes, mild hypercellularity [41 to 50 cells/gcs], and/or minor exudation); 2 = moderate (glomeruli with moderate hypercellularity [50 to 60 cells/gcs], including segmental and/or diffuse proliferative changes, hyalinosis, and moderate exudate); and 3 = severe (glomeruli with segmental or global sclerosis and/or exhibiting severe hypercellularity [>60 cells/gcs], necrosis, crescent formation, and heavy exudation).

Damaged tubules (%; consisting of dilation and/or atrophy and/or necrosis) were determined in 400 randomly selected renal cortical tubules per kidney (x400). Perivascular cell accumulation was determined semiquantitatively by scoring the number of cell layers surrounding the majority of vessel walls (score: 0 = none, 1 = <5, 2 = 5 to 10, and 3>10). Scoring was evaluated using coded slides.

Immunostaining
The phenotype of the infiltrating cells was analyzed by immunohistochemistry of frozen sections, as recently described (22): Macrophages by staining with F4/80 (HB198; American Type Culture Collection, Rockville, MD) and T cells by anti-CD4, anti-CD8, and anti-B220 rat anti-mouse antibodies (PharMingen, San Diego, CA). To distinguish B220-positive, double-negative (DN; CD3+CD4–CD8–) T cells from B cells, additional sequential slides were stained with an antibody against a shared B cell isotope of CD21 and CD35 (7G6; PharMingen). The specific DN T cells were therefore defined as CD4–CD8–B220+ CD21/CD35–. The controls contained normal rat IgG, idiotypic controls, or rabbit serum as a substitute for the first antibody (23).

Ig Deposits in the Kidney
To examine the intrarenal IgG precipitations, we stained 4-µm frozen sections with FITC-conjugated anti-mouse IgG, IgG1, IgG2a, IgG2b, and IgG3 (Dako, Hamburg, Germany) at 37°C for 30 min. The extent of the IgG precipitation was assessed by titrating the antibodies on serial tissue sections using dilution steps. At least 50 glomeruli on coded slides were analyzed using a fluorescence microscope (Axiophot, Zeiss, Germany).

Determination of IgG Subgroups in Cell Supernatant
Splenocytes that were derived from 2-mo-old MRL-Faslpr mice (106 cells/ml) were cultured in DMEM/FCS in the presence or absence of 2500 IU/ml IFN-{beta} for 18 h. IgG subgroups in the supernatant of stimulated splenocytes were detected by ELISA technique (Southern Biotechnology Associates, Birmingham, AL). Briefly, ELISA flat-bottomed plates were coated overnight at 4°C with peroxidase-conjugated goat anti-mouse capture antibodies to IgG1, IgG2a, IgG2b, and IgG3. After washing with PBST (PBS that contains 0.05% Tween buffer), unspecific binding sites were blocked with 1% BSA in PBS. The supernatants were diluted 1:100 in 1% BSA and incubated at room temperature for 1 h. After further washing steps with PBST, the substrate solution was added and developed.

Determination of Circulating ANA and Anti–Double-Stranded DNA Antibodies
ANA were detected using the Hep2 cell ANA kit (The Binding Site, Birmingham, UK). Briefly, serum samples (1:40 dilution) were incubated with the Hep2 substrate, washed, and incubated with fluorescein-conjugated anti-mouse IgG (Cappel, Qbiogene, Heidelberg, Germany; 1:100 dilution). ANA titers were determined by serial dilution steps using a fluorescence microscope (Axiophot). Anti–double-stranded DNA antibodies were analyzed using a commercially available ELISA system (Pharmacia Diagnostic Art. No. 14148/14196). Briefly, serum samples (1:200 dilution) were incubated on a microplate that was precoated with recombinant plasmid dsDNA for 30 min, washed, and incubated with enzyme-labeled secondary antibody. After further washing steps, the substrate solution was added and developed.

Cytokine ELISA.
The serum concentration of IFN-{gamma} and IL-4 in mice that were treated with PBS or IFN-{beta} was detected in duplicate by ELISA technique according to the manufacturer’s instructions (R&D, Wiesbaden, Germany). Mouse sera were diluted 1:3 with the calibrator diluent solution of the kit and analyzed using peroxidase-coupled antibodies against mouse IFN-{gamma} or mouse IL-4. The absorption of the samples was detected in a microplate-ELISA reader at {lambda} = 450 nm.

RNAse Protection Assay.
For detecting intrarenal cytokine mRNA, a multiprobe RNAse protection assay system was applied (Riboquant; PharMingen). Briefly, total RNA was extracted from tissue samples (Qiagen RNeasy, cat. no. 74104) and hybridized to labeled multiprobe template set (mCK-2b, cat. no. 45051, or mCK-3, cat. no. 45003P; PharMingen). After RNAse treatment, remaining "RNase-protected" probes were purified, resolved on denaturing polyacrylamide gels, and quantified by autoradiography. Five samples per treatment group were analyzed and quantified using densitometry relative to the housekeeping genes.

Flow Cytometry (FACS).
To determine the cell phenotype of splenocytes under IFN-{beta} therapy in vitro, we performed FACS analysis. For determining the ratio of "double negative" T cells over B220+ B cells, dual staining combined FITC-conjugated anti-mouse CD3 antibodies with PE-marked anti-mouse B220 antibodies. Similarly, the ratio of CD4+ to CD8+ T cells (FITC-marked anti-mouse CD4 and PE-marked anti-mouse CD8 antibodies) were analyzed. The following antibodies were used: PE anti-mouse CD8 (0.2 mg/ml, cat. no. 01045A; Pharmingen), PE anti-mouse CD45R/B220 (RAB-6B2, cat. no. 01125A, 0.2 mg/ml; Pharmingen), FITC anti-mouse CD3 Molecular Complex (17A2, 0.5 mg/ml, cat. no. 555274; Pharmingen), and FITC anti-mouse CD4 (L3T4/RM4–5, 0.5 mg/ml, cat. no. 01064; Pharmingen). Cells (105) were stained with the FITC-marked antibody at 4°C for 40 min, followed by washing steps, and staining with the PE-marked antibody at similar conditions and fixed in 1% formalin.

MTT Proliferation Assay.
The dimethylthiazol-tetrazolium bromide (MTT) proliferation assay is a colorimetric test for the nonradioactive detection of cell proliferation and cell activity (Cell Proliferation Kit; Roche) (24). We stimulated freshly isolated MRL-Faslpr splenocytes (100,000 cells/well) with medium, phytohemagglutanine (PHA), phorbolmyristate acetate (PMA), and TNF-{alpha} in the presence or absence of IFN-{beta} overnight. Tubular epithelial cells (TEC) were cultured for 24 h before stimulation with IFN-{gamma} or nephritic serum in the presence or absence of IFN-{beta}. After addition of 10 µl/well of the MTT-salt solution and incubation at 37°C for 4 h, 100 µl/well of the solubilization solution was added and kept in the incubator overnight. We measured the absorption with an ELISA reader at {lambda} = 550 nm. In addition to the MTT proliferation assay, the thymidine proliferation assay was performed, as recently described (25).

Statistical Analyses
Data were analyzed using the Kruskal Wallis test for comparing survival curves and the Mann-Whitney test to compare group means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{beta} Is a Therapeutic Agent for Mild and Advanced Lupus Nephritis
Initially, MRL-Faslpr mice (3 mo of age) with mild lupus (Table 1) were treated with IFN-{beta} (n = 14) or PBS (n = 11) for 3 mo. IFN-{beta} dramatically reduced proteinuria (50%) as compared with the PBS-injected group (Figure 1A). In addition, IFN-{beta} treatment led to a remarkable improvement of renal function reflected by an almost 50% reduction of serum urea levels (P < 0.001; Figure 1B). Although greatly reduced, renal function in MRL-Faslpr mice that received IFN-{beta} therapy remained slightly above the values in mice with normal kidneys (Fas-intact MRL and BALB/c mice; P < 0.01; Figure 1B).



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  		Figure 1. Treatment of mild lupus nephritis with IFN-{beta} reduces proteinuria and improves renal function in MRL-Faslpr mice. (A) IFN-{beta} dramatically reduced proteinuria (50%) as compared with the PBS-injected group. Proteinuria measured semiquantitatively in weekly intervals; **P < 0.001, Mann-Whitney, at 6 mo of age. (B) Serum urea levels were reduced by almost 50% in MRL-Faslpr mice that were treated with IFN-{beta} as compared with MRL-Faslpr mice that were treated with PBS at 6 mo of age. Values are mean ± SD (n = 10 per group; *P < 0.001, Mann-Whitney). However, the treated mice did not return to baseline values in Fas-intact MRL-++ and normal BALB/c mice of the same age (n = 5 per group; **P < 0.001; Mann-Whitney).

 
The difference in survival was even more striking. When MRL-Faslpr mice were given IFN-{beta}, only 10% as compared with 45% in the PBS-treated group succumbed (Figure 2A). Of note, the mortality (50%) in the control group was similar to unmanipulated MRL-Faslpr mice between 5 and 6 mo of age. Thus, IFN-{beta} is a therapeutic agent for MRL-Faslpr mice with mild lupus nephritis.



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  		Figure 2. Treatment of lupus nephritis with IFN-{beta} improves survival of MRL-Faslpr mice. (A) Mild nephritis starting with 14 mice (IFN-{beta}) or 11 mice (PBS): 10% of MRL-Faslpr mice only died at 6 mo of age under IFN-{beta} therapy compared with 45% mortality in the control group similar to untreated mice (15) (*P < 0.001). (B) Advanced lupus nephritis: Whereas only 25% of 5-mo-old proteinuric MRL-Faslpr mice in the PBS group (n = 8) survived until 6 mo, 67% survived under IFN-{beta} therapy (n = 12; *P < 0.001, Kruskal Wallis test).

 
The therapeutic impact of IFN-{beta} on mild nephritis prompted us to test the hypothesis that IFN-{beta} is a therapeutic agent for advanced lupus nephritis in MRL-Faslpr mice. When MRL-Faslpr mice with advanced nephritis (Table 1) received an injection of IFN-{beta}, survival was extended by 42%. Survival in the IFN-{beta} group (67%, n = 12) was increased as compared with the PBS group (25%, n = 8; P < 0.001; Figure 2B). Taken together, IFN-{beta} is highly effective in reducing mortality even in the treatment of MRL-Faslpr mice with advanced lupus nephritis (Figure 2B).

Reduced Kidney Pathology and Decreased Infiltrating Cells in Kidneys (Macrophages and T Cells) of IFN-{beta}–Treated MRL-Faslpr Mice
To determine the mechanisms underlying the improvement of renal function after IFN-{beta} treatment, we examined the histopathology in mice with mild disease at start of treatment. Renal (tubular and glomerular) pathology was reduced in IFN-{beta}–treated compared with PBS-treated MRL-Faslpr mice (Figure 3A). The reduction in tubular, glomerular, and perivascular pathology assessed histologically was >50% in MRL-Faslpr kidneys after IFN-{beta} therapy (P < 0.001; Figure 3B). In particular, IFN-{beta}–treated MRL-Faslpr mice had markedly diminished perivascular and peritubular infiltrate, tubular atrophy, glomerular hypercellularity, glomerulosclerosis, and crescent formation. Furthermore, we detected a reduction in leukocytic kidney infiltrates, including macrophages and T cells (CD4+, CD8+, and CD4–CD8–B220+ [DN]; data not shown). Thus, IFN-{beta} halts the progression of lupus nephritis by preventing renal leukocytic infiltration in MRL-Faslpr mice.



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  		Figure 3. IFN-{beta} treatment reduces kidney pathology and cell infiltration. (A) Representative examples of reduced cell infiltration into the kidney of MRL-Faslpr mice during IFN-{beta} treatment (hematoxylin and eosin staining, light microscopy). (B) Histologic analysis of tubular, glomerular, and perivascular pathology in IFN-{beta}–treated compared with PBS-treated MRL-Faslpr mice. IFN-{beta}–treated MRL-Faslpr mice had markedly diminished perivascular infiltrate, tubular, and glomerular pathology. Data are mean ± SD, n = 6 per group; *P < 0.001, Mann-Whitney. (C) Representative photograph of perivascular proliferating cells (dark red–labeled cells) in the kidney of IFN-{beta}–and PBS-treated MRL-Faslpr mice (Ki-67 staining, x200).

 
Decreased Local Proliferation of Perivascular Macrophages and T Cells in IFN-{beta}–Treated Kidneys
We determined whether the decreased accumulation of macrophages and T cells in IFN-{beta}–treated MRL-Faslpr kidneys in mice with mild disease was a result of a decline in local proliferation. These mice were evaluated at 5 mo of age using in situ detection of Ki-67 expression. The majority of proliferating cells were localized surrounding vessels (>15%) as compared with proliferating cells surrounding glomeruli and tubules in the PBStreated MRL-Faslpr kidneys (P < 0.001). The number of proliferating cells in the perivascular areas decreased markedly in mice that were treated with IFN-{beta} as compared with PBS (28 ± 15/section versus 9 ± 5/section; P < 0.01; n = 6 per group; Figure 3). Because only a few of the cells surrounding tubules (<1%) and glomeruli (<1%) were proliferating in MRL-Faslpr mice, the number of proliferating cells was not substantially reduced in the IFN-{beta}–treated MRL-Faslpr group (P > 0.2; n = 5). Of course, it must be appreciated that the proliferation measurements in tissue sections are a reflection of the level of cell division at the time these tissues were removed. Taken together, our data suggest that decreased local proliferation of kidney-infiltrating cells surrounding vessels in MRL-Faslpr that were treated with IFN-{beta} is responsible at least in part for the reduction in leukocytic accumulation.

IFN-{beta} Reduces Serum- and Kidney-Deposited Ig
Because there was a dramatic reduction in tubular and glomerular pathology in IFN-{beta}–treated MRL-Faslpr kidneys, we determined whether IFN-{beta} altered glomerular Ig deposits. We detected a reduction in glomerular deposition of total IgG and IgG3 in IFN-{beta}–treated MRL-Faslpr mice (*P < 0.02, **P < 0.05; Figure 4, A and B). To determine whether IFN-{beta} directly reduced the IgG isotype profile, freshly isolated MRL-Faslpr splenocytes were treated in vitro with IFN-{beta}, and alterations in IgG isotypes in the supernatant were measured quantitatively by ELISA technique. It is interesting that IFN-{beta} stimulation reduced IgG3 production by 80%, whereas other IgG isotypes remained unaffected (Figure 4C). This may be of particular relevance, as IgG3 is considered to be a "nephritogenic" Ig (26). However, serum levels of IgG subtypes and circulating ANA and anti–double-stranded DNA antibodies were similar in both treatment groups (Table 2).



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  		Figure 4. IFN-{beta} treatment reduces glomerular IgG precipitations. Fluorescence microscopy showed a notable reduction of total glomerular IgG precipitations and reduced deposition of IgG3 subtype in IFN-{beta}–treated in comparison with PBS-treated MRL-Faslpr mice. Immunostaining with FITC-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3. (A) Representative examples of IgG immunofluorescence in the kidney. (B) The extent of IgG precipitations was assessed by titrating the antibodies on serial tissue sections using dilution steps. Data are mean ± SD, n = 9 per group; *P < 0.02, **P < 0.05, Mann-Whitney. The titer given is the lowest titer at which staining was observed. (C) IFN-{beta} selectively reduces IgG 3 production in vitro. Splenocytes that were derived from 2-mo-old MRL-Faslpr mice (106 cells/ml) were cultured in DMEM/FCS in the presence or absence of 2500 IU/ml IFN-{beta} for 18 h. IgG isotypes in the supernatant were quantified by ELISA technique in triplicate. The experiment was repeated twice. IFN-{beta} stimulation reduced IgG3 production by 80%, whereas other IgG isotypes remained unaffected (values given are mean ± SD; ***P < 0.002; Mann-Whitney). Magnification, x200 in A.

 

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  		Table 2. IFN-{beta} treatment does not affect IgG subtypes or autoantibody levels in the circulation of MRL-Fasmicea

 
IFN-{beta} Suppresses Extrarenal Lupus Manifestation in MRL-Faslpr
Striking, the massive splenomegaly characteristic of MRL-Faslpr mice (6 mo of age) was not detectable in MRL-Faslpr mice that were treated with IFN-{beta} (Figure 5). In fact, IFN-{beta} reduced the spleen size of nephritic MRL-Faslpr mice to normal levels (1.0 ± 0.25 versus 0.5 ± 0.29, untreated versus treated mice; Figure 5).



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  		Figure 5. IFN-{beta} normalizes splenomegaly in MRL-Faslpr mice. (A) Splenomegaly characteristic of lymphoproliferation in MRL-Faslpr mice occurred in PBS-treated mice. In contrast, IFN-{beta} reduced the spleen size in nephritic MRL-Faslpr mice to normal (photo, representative examples). Accordingly, spleen weight was reduced by 50% under IFN-{beta} therapy (1.0 ± 0.25 versus 0.5 ± 0.29, untreated versus treated mice, mean ± SD; *P < 0.001, Mann-Whitney). (B) IFN-{beta} has an antiproliferative effect on MRL-Faslpr splenocytes. MRL-Faslpr splenocytes were stimulated with medium, phorbolmyristate acetate (PMA), phytohemagglutinine (PHA), and TNF-{alpha} in the presence or absence of IFN-{beta} overnight. Proliferation was measured as 3H-thymidine incorporation in a scintillation counter (counts per minute) in triplicate. Values are mean ± SD for three separate experiments (*P < 0.001, Mann-Whitney). (C) Antiproliferative effect of IFN-{beta} on splenocytes is strain dependent. Splenocytes from MRL-Faslpr and NZB/W mice that were 2 to 3 mo of age were stimulated with medium, PMA, or PHA in the presence or absence of IFN-{beta}. The effects of IFN-{beta} are given as percentage difference of the mean cell proliferation (with IFN-{beta}/without IFN-{beta}), MTT-proliferation assay, data are mean of triplicate, two independent experiments.

 
As a possible explanation for this macroscopic finding, we determined that there was a marked antiproliferative effect of IFN-{beta} on stimulated MRL-Faslpr splenocytes in vitro (Figure 5B). In addition, this antiproliferative effect was dose dependent (IFN-{beta} concentrations used between 50 and 1500 U; data not shown). To examine whether the Fas mutation was responsible for the antiproliferative effect of IFN-{beta} on MRL-Faslpr splenocytes, we performed similar proliferation experiments with splenocytes from Fas intact, age- and sex-matched MRL-+/+ mice (2 mo of age). IFN-{beta} similarly downregulated the proliferation of both Fas-intact and Fas-deficient splenocytes by 29 and 25%, respectively (data not shown). Thus, the antiproliferative effect of IFN-{beta} is not mediated by the Fas receptor mutation. To investigate further whether the antiproliferative effect of IFN-{beta} was restricted to the MRL-Faslpr mouse strain, we performed proliferation assays with freshly isolated splenocytes from both MRL-Faslpr and another spontaneous lupus mouse model, the NZB/W mice, 2 to 3 mo of age (Figure 5C). It is interesting that IFN-{beta} downregulates proliferation of splenocytes from MRL-Faslpr mice and upregulates proliferation of splenocytes from NZB/W mice (Figure 5C).

Finally, the skin lesions and lymphadenopathy, characteristic of the MRL-Faslpr strain, were ameliorated in mice that were treated with IFN-{beta} as compared with PBS (Figure 6). Thus, the therapeutic benefit of IFN-{beta} was obvious macroscopically (Figure 6).



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  		Figure 6. IFN-{beta} treatment ameliorates skin disease and lymphadenopathy in MRL-Faslpr mice. (A and B). Representative photographs. (C) Note the bulging lymph nodes in the PBS as compared with barely detectable lymph nodes in the IFN-{beta}–treated mice; mean size of lymph nodes per mouse was calculated by measuring the size and counting the number of enlarged lymph nodes (mean ± SD; P < 0.05). (D) PBS-treated MRL-Faslpr mice had characteristic skin lesions, whereas IFN-{beta} treatment halted the progression of skin lesions (mean ± SD; P < 0.001, Mann-Whitney).

 
IFN-{beta} Suppresses Intrarenal Cytokine Expression in MRL-Faslpr Mice
Because the beneficial effects of IFN-{beta} in the therapy of multiple sclerosis have been attributed to a shift in cytokine expression (downregulation of IL-12 and IFN-{gamma} and upregulation of IL-4) (19), we examined the cytokine pattern in the serum of IFN-{beta}–versus PBS-treated MRL-Faslpr mice. However, we did not detect any alteration in circulating IL-12, IFN-{gamma}, and IL-4 in mice that were treated with IFN-{beta} as compared with PBS (P > 0.05; n = 6 per group). In addition, although IL-10 has been reported as an IFN-{beta}–induced regulator molecule (7), we did not detect differences in IL-10 serum levels between the treatment groups (IFN-{beta}–versus PBS-treated mice, 404 ± 250 and 520 ± 320 pg/ml, respectively; n = 7 per group; P > 0.05). Thus, these findings suggest that IFN-{beta} treatment does not ultimately lead to a shift in the Th1/Th2 cytokine response.

To determine whether IFN-{beta} has an impact on local cytokines within the kidney, we evaluated intrarenal cytokine mRNA levels by RNAse protection. We detected a downregulation of three cytokines that are hallmarks of renal inflammation and fibrosis—IFN-{gamma}, TNF-{alpha}, and TGF-{beta}—in IFN-{beta}–treated MRL-Faslpr mice (30, 22, and 39%, respectively). In contrast, IL-18, another proinflammatory cytokine involved in the nephritis of MRL-Faslpr mice, was not different between the treatment groups (Figure 7).



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  		Figure 7. IFN-{beta} suppresses intrarenal cytokine expression in MRL-Faslpr mice. Using the multiprobe RNAse protection assay system, we analyzed the quantity of renal cytokine mRNA expression (n = 5 per group). Intrarenal expression of IFN-{gamma}, TNF-{alpha}, and TGF-{beta} mRNA was downregulated in IFN-{beta}–treated MRL-Faslpr mice as compared with the PBS group. In contrast, IL-18 mRNA in the kidneys remained unaffected (*P < 0.001, Mann-Whitney).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we tested the hypothesis that IFN-{beta} halts autoimmune disease in MRL-Faslpr mice. We now report that treatment of MRL-Faslpr mice with IFN-{beta} was highly effective in prolonging survival and ameliorating the serologic and histologic parameters of the lupus-like disease, even when initiated at the advanced nephritic stage. We identified several mechanisms involved in the protection against autoimmune tissue injury: (1) IFN-{beta} downregulated proliferation of MRL-Faslpr splenocytes resulting in normalized spleen size; (2) IFN-{beta} decreased trafficking of leukocytes (macrophages and T cells) into the kidney and reduced perivascular proliferation of kidney infiltrating cells; and (3) IFN-{beta} influenced the humoral immune response by reducing intrarenal IgG precipitations. Finally, IFN-{beta} modulated intrarenal cytokine expression at the mRNA level. Clearly, IFN-{beta} modulates the immune system at different sites. Because the autoimmune kidney destruction in MRL-Faslpr mice results from both humoral and cellular mechanisms (10–16), the combined impact on cellular and humoral immune events make IFN-{beta} an ideal potential therapeutic at least for certain subtypes of human SLE.

In contrast with our findings, Santiago-Raber et al. (27) recently reported that type-I IFN receptor deficiency reduced lupus-like disease in another autoimmune disease mouse model, the NZB mice. They demonstrated a decreased frequency of IFN-{gamma}–producing T cells and IgG2a autoantibody levels in the type I IFN receptor–deficient NZB mice, suggesting a downregulation of IFN-{gamma} signaling. Thus, it is conceivable that genetic targeting of the type I IFN receptor in NZB mice partially reduced type II IFN–mediated effects. In fact, a cross-talk between IFN-{gamma} and IFN-{alpha}/IFN-{beta} signaling has been reported (28,29). Thus, blocking type I IFN receptor may affect both type I and type II IFN. The type II IFN, IFN-{gamma}, however, is well established as a critical factor in the development of lupus (14,15). Further support for the interplay of the type I and type II IFN in murine lupus is supported by a recent study in type I and/or type II IFN receptor–deficient MRL-Faslpr mice. Whereas a deficiency in type II IFN receptor protected MRL-Faslpr mice from the development of lymphadenopathy and autoimmune renal disease, the lack of the type I IFN receptor resulted in increased lymphoproliferation, autoantibody production, and renal disease (30). However, caveats in knockout approaches focus on the possibility that other genes substitute in the development for the deficient cytokine signaling. In this study, we tested the application of IFN-{beta} in a mild and severe autoimmune lupus-like setting and clearly detail the beneficial effects of IFN-{beta} in the effector phase of the disease. It will be of interest to dissect the signaling pathways of type I IFN in the various lupus-prone models.

IFN-{beta} is a pleiotropic cytokine with a broad spectrum of actions that are capable of enhancing and suppressing immune reactions (27,30). Several reports confirm the IFN-{beta}–induced antiproliferative effects in various cell lines (31,32). In particular, the anti-tumor impact of IFN-{beta} seems to be mediated by induction of apoptosis (31–33). Similarly, in MRL-Faslpr mice, a mutation in the Fas receptor leads to reduced elimination of activated and autoreactive T cells by apoptosis, leading to an extensive accumulation of lymphocytes (34). In our study, IFN-{beta} downregulates this "uncontrolled" lymphoproliferation in MRL-Faslpr splenocytes. In the NZB/W lupus mouse model, however, we found an increased proliferation of splenocytes by IFN-{beta}, reflecting the diverse findings of type I receptor blockade in MRL-Faslpr and NZB/W mice (27,30). Whereas Rep et al. (35) report that IFN-{beta} inhibits the proliferation of T cells and reduces the secretion of IFN-{gamma} and TNF-{alpha} depending on the dose in patients with multiple sclerosis, Zipp et al. (36) detected no evidence of apoptosis induction by IFN-{beta} on myelin specific autoreactive T cells. Moreover, even a protective effect of IFN-{beta} on T cells was described (37–39). Similarly, in the MRL-Faslpr mouse model, IFN-{beta} seems to counteract IFN-{gamma}–induced TEC death (unpublished observation). Thus, at present, it is unclear whether the signaling pathways for antiproliferative and antiapoptotic actions of IFN-{beta} are interlinked, and further studies are required to understand better the regulation of the multitude of pro- and anti-inflammatory functions of IFN-{beta} in various lupus-prone models and among various cell types. Therefore, we are currently investigating the different signal transduction pathways that are activated by IFN-{beta} in splenocytes and TEC from MRL-Faslpr and NZB/W mice.

The beneficial effects of IFN-{beta} in the therapy of multiple sclerosis have been attributed to a shift in the Th1/Th2 cytokine response (18–21). IFN-{beta} treatment of MRL-Faslpr mice did not affect serum levels of IFN-{gamma}, IL-12, IL-4, and IL-10, respectively. However, intrarenal mRNA levels of IFN-{gamma}, TNF-{alpha}, and TGF-{beta} were greatly reduced by IFN-{beta}. How can we explain this discrepancy? We suggest that IFN-{beta} affects the local, intrarenal, rather than the systemic inflammatory response, because IFN-{beta} treatment of MRL-Faslpr mice resulted in reduced infiltration of inflammatory cells into the kidney. Further support for this concept is supported by a recent study by Teige et al. (40) reporting that endogenous IFN-{beta} acts as a limiting factor for macrophage activation and TNF-{alpha} production using an IFN-{beta} knockout approach in a murine model of encephalomyelitis. Thus, blocking macrophage activation may limit local T cell activation and cytokine production and, in turn, lead to reduced renal pathology.

IFN-{beta} treatment led to a reduction in glomerular deposition of Ig despite unchanged circulating levels of Ig. This gives further support to the concept that the extent of circulating autoantibodies does not correlate exclusively with kidney damage, as we previously reported in monocyte chemoattractant protein-1–deficient MRL-Faslpr mice (22). The binding of immune complexes to Fc{gamma}RIII expressed by mesangial cells triggers a proinflammatory response in the kidney (production of IL-12, IL-6, and TNF-{alpha}) leading to kidney damage (41,42), suggesting that IFN-{beta} downregulates Fc{gamma}RIII expression. Taken together, our results indicate that IFN-{beta} suppresses lupus pathologies by modulating the local inflammatory response. However, it was reported recently that prolonged application of IFN-{alpha} in the NZB/W model resulted in dramatic acceleration of lupus nephritis (43). Therefore, it is important to unravel the signaling events of IFN-{beta} in the different lupus-prone mouse models and identify subtypes of human lupus that may profit from application or blockade of type I IFN.


   Acknowledgments
 
This work was supported by Deutsche Forschungsgemeinschaft Grant Schw 785/2-1, the Stiftung Innovation Rheinland-Pfalz and SFB 548 (to A.S.), and National Institutes of Health Grants RO1-DK 36149 and 52369 (to V.R.K.).

We gratefully thank Michaela Blanfeld and Ursel Dang for excellent technical support.


   Footnotes
 
See related editorial, "More Targeted Treatments for Lupus Nephritis: Is the Future (Nearly) Here?," on pages 3146–3148.

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 30, 2004. Accepted for publication August 18, 2005.




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