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Blockade of IL-18 Receptor Signaling Delays the Onset of Autoimmune Disease in MRL-Fas^lpr Mice¹

Department of Hematology, Nephrology and Rheumatology, Kinki University School of Medicine, Osaka, Japan

	Abstract

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Autoimmune disease in Fas-deficient MRL-Fas^lpr mice is dependent on infiltrating autoreactive leukocytes and autoantibodies, and IFN- plays an important role in the pathogenesis. As IL-18 is capable of inducing IFN- production in T cells, we hypothesized that signaling through IL-18R is involved in the pathogenesis. To investigate the impact of IL-18 in this autoimmune disease, we generated an MRL-Fas^lpr strain deficient in IL-18R. Compared with the wild-type strain, IL-18R-deficient MRL-Fas^lpr mice survived longer and showed a significant reduction in renal pathology, including glomerular IgG deposits, proteinuria, and serum anti-DNA Abs. Intrarenal transcripts encoding IFN-, TNF-, IL-12, and IL-10, which have been linked to nephritis, were all markedly reduced. Skin lesions, lymphadenopathy, and lung pathology characteristic of the MRL-Fas^lpr mouse disease were diminished in IL-18R-deficient MRL-Fas^lpr mice. Thus, we conclude that IL-18R signaling is critical to the pathogenesis of autoimmune disease in MRL-Fas^lpr mice.

	Introduction

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The MRL-Fas^lpr mice spontaneously develop a systemic autoimmune disease characterized by large quantities of autoantibodies (anti-DNA, anti-Sm, etc.), immune complex-type glomerulonephritis (lupus nephritis), skin lesions, marked lymphadenopathy and splenomegaly, arthritis, and vasculitis, and closely resembling human systemic lupus erythematosus (SLE)³ (1). Tissue injury in autoimmune disease is mediated by autoantibodies and autoreactive lymphocytes (2, 3).

Fas mutation and MRL background genes in MRL-Fas^lpr mice induce rapid and severe renal dysfunction. Kidney disease, which is evident at 3 mo of age and results in 50% mortality at 5–8 mo, is complex and consists of glomerular, interstitial, and perivascular components (4, 5). Each component is infiltrated by leukocytes, including macrophages and CD4, CD8, and CD4^–, CD8^–, B220⁺ double-negative (DN) T cells. Furthermore, MRL-Fas^lpr mice, which are deficient in these T cell populations, do not develop autoantibodies and immune complex kidney disease (5, 6, 7). Thus, it is clear that T cells are required for autoimmune disease. The T cell-mediated mechanism responsible for inciting MRL-Fas^lpr kidney disease requires IFN-. IFN- has been reported to be a key cytokine in murine lupus by leading to glomerular injury and by inducing apoptosis of renal parenchymal cells (8). In addition, IFN- or IFN- receptor-deficient MRL-Fas^lpr mice have been shown to be protected from autoimmune disease (8, 9).

IL-18 is a cytokine that is secreted mainly from macrophages, induces IFN- production from Th1 cells and splenocytes, and enhances NK cell activity (10, 11). IL-18 also has a synergistic effect with IL-12, another IFN--inducible cytokine, on those functions (12, 13). IL-1-converting enzyme (caspase-1) cleaves the inactive precursor of IL-18 (pro-IL-18) to a bioactive mature form of IL-18 that recognizes a heterodimeric receptor, and that consists of unique (IL-1R-related protein (IL-1Rrp)) and nonbinding (IL-1R-accessory protein-like) signaling chains (14, 15, 16). Like IL-18-deficient mice, IL-18R-deficient (IL-1Rrp^–/–) mice also suppress IFN- and NK cell activity (17). It has been reported that elevation of serum IL-18 is correlated with disease activity in both human and murine lupus (18, 19, 20). Based on these findings, we hypothesized that blockade of IL-18R signaling would reduce autoimmune disease in MRL-Fas^lpr mice. To address this hypothesis, we generated and analyzed MRL-Fas^lpr mice genetically deficient in IL-18R (IL-1Rrp^–/–). Compared with IL-18R-intact MRL-Fas^lpr mice, IL-18R-deficient mice did not develop autoimmune kidney disease, elevated anti-DNA Ab, or accumulation of leukocytes in the kidney and lungs. Thus, IL-18 R signaling is critical to the pathogenesis of autoimmune lupus.

	Materials and Methods

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Mice

MRL/MpJ-Fas^lpr/Fas^lpr (MRL-Fas^lpr) mice were purchased from Japan SLC (Shizuoka, Japan). IL-18R-deficient (IL-1Rrp^–/–) mice (129/SvJ) were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan). The IL-18R gene was inactivated in these mice by transfecting embryonic stem cells with a replacement vector containing a disrupted murine IL-18R gene (17). All mice were maintained in our specific pathogen-free animal facility.

Generating IL-18R-deficient MRL-Fas^lpr mice

We constructed an IL-18R-deficient MRL-Fas^lpr strain using a backcross-intercross breeding scheme (21). MRL-Fas^lpr mice were mated with IL-18R-deficient mice to yield heterozygous F₁ offspring. We intercrossed F₁ mice and screened the progeny by PCR amplification of tail genomic DNA for the Fas^lpr mutation and IL-18R using specific primers (13, 22). Double-homozygous (Fas^lpr/Fas^lprIL-18R^–/–) N₁F₁ progeny were backcrossed to MRL-Fas^lpr mice. B₁ progeny, homozygous for Fas^lpr mutation and heterozygous for IL-18R (IL-18R^+/–), were intercrossed, and mice homozygous for the IL-18R-deficient mutation were selected by PCR typing for continued backcrossing. After three generations of backcross-intercross matings, this breeding scheme generated a colony of MRL-Fas^lpr mice (94% MRL-Fas^lpr background) homozygous and heterozygous for the mutated IL-18R. We analyzed mice in this backcross generation because we have established that the expression levels of autoimmune disorders and survival rates (50% mortality at 6 mo of age in the wild-type vs 8 mo of age in the third generation of IL-18R-intact MRL-Fas^lpr mice) in these mice are not so different from those in the wild-type MRL-Fas^lpr strain (8.21). The IL-18R-deficient and intact MRL-Fas^lpr of the third backcross generation are referred to hereafter as IL-18R^–/– MRL-Fas^lpr and IL-18R^+/+ MRL-Fas^lpr, respectively.

Survival, proteinuria, and blood urea nitrogen (BUN)

Survival were assessed in IL-18R^–/– MRL-Fas^lpr and IL-18R^+/+ MRL-Fas^lpr mice that died of disease spontaneously and those sacrificed due to general debility. Urines were examined before, and tissues such as kidney, spleen, and lymph nodes were obtained after the moribund mice were sacrificed. Urinary protein levels were assessed semiquantitatively using albumin reagent strips (Hema-Combistix; Bayer/Sankyo, Tokyo, Japan) on a monthly basis. Grades of proteinuria were expressed as 0 (none), 1 (30–99 mg/dl), 2 (100–299 mg/dl), 3 (300–999 mg/dl), and 4 (1000 or more mg/dl). Mice exhibiting grade 2 or more proteinuria were regarded as positive. When mice became positive for proteinuria, tests were repeated. To evaluate the renal function, we also analyzed BUN by using an autoanalyzer (model 917; Hitachi, Tokyo, Japan).

Gross pathology

Skin pathology and lymphadenopathy were scored monthly beginning at 3 mo of age. Skin lesions, which consisted of alopecia and scab formation, were scored from 0 to 3 based on the number of lesions and area (0, none; 1, <0.5 cm; 2, two or more, <0.5 cm; 3, multiple, >0.5 cm). Lymphadenopathy (cervical, brachial, and inguinal) was scored on a scale of 0–3 by evaluating the number and size of palpable nodes (0, no nodes; 1, one small node (<0.5 cm); 2, two small-to-moderate nodes (0.5–1.0 cm); 3, three or more moderate-to-large nodes (>1.0 cm)). Splenomegaly was determined by spleen weight.

Renal histopathology and immunopathology

Kidney tissues were either snap-frozen in OCT compound (Miles Scientific, Naperville, IL) for cryostat sectioning or fixed in 10% neutral-buffered formalin. Formalin-fixed tissues were embedded in paraffin, 4-µm-thick sections were stained with periodic acid Schiff, and the glomerular, tubular, and perivascular pathology was evaluated morphometrically by light microscopy (21). The glomerular pathology of 50 glomerular cross-sections (gcs) per kidney was scored on a semiquantitative scale: 0, normal (35–40 cells/gcs); 1, a few lesions with slight proliferative change and mild hypercellularity (41–50 cells/gcs); 2, moderate hypercellularity (51–60 cells/gcs), segmental and/or diffuse proliferative change, hyalinosis, and moderate exudates; and 3, severe hypercellularity (>60 cells/gcs) with segmental or global sclerosis and/or severe necrosis, crescent formation, and heavy exudation. Tubular pathology was evaluated by counting the percentage of tubules exhibiting damage (dilation, atrophy, or necrosis) among 200 randomly selected tubules. Perivascular inflammatory cell infiltration was evaluated by scoring the number of cell layers surrounding 10 randomly chosen inter- and intralobular arteries (0, none; 1, <5 layers surrounding less than half of the vessel; 2, 5–10 layers surrounding more than half of the vessel; 3, >10 layers surrounding more than half of the vessel). To examine IgG deposits within renal glomeruli, kidney cryostat sections (4 µm thick) were stained with FITC-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 Ab (Serotec, Oxford, U.K.). Fluorescence intensity within glomerular capillary walls was scored on a scale of 0–3 (0, none; 1, weak; 2, moderate; 3, strong). At least 10 glomeruli per section were analyzed. Scoring was performed by two investigators using coded slides.

Cryostat sections of kidney tissues were also stained using an immunoperoxidase method for T cells with anti-CD4, anti-CD8, and anti-B220 rat anti-mouse mAbs (BD Pharmingen, San Diego, CA), and for macrophages with F4/80 hybridoma culture supernatant (HB198; American Type Culture Collection, Manassas, VA). As a specificity control, normal rat IgG was used as the primary rat Ab. Numbers of T cells and macrophages within the kidney were expressed as number of cells per glomerulus or number of cells per field, as described previously (21). Scoring was performed by two investigators using coded slides.

Lung histopathology

Lungs were fixed in formalin, sectioned (4 µm), stained with H&E, and evaluated by light microscopy (21). The perivascular leukocyte infiltration was evaluated by counting the leukocyte infiltrates surrounding 10 random vessels (0, none; 1, less than three layers surrounding <50% of vessel; 2, three to six cell layers surrounding >50% of vessel; 3, more than six layers). Peribronchiolar leukocyte infiltration was evaluated by counting the cells surrounding 10 random bronchi (0, none; 1, less than three layers surrounding <50% of bronchi; 2, three to six cell layers surrounding >50% of bronchi; 3, more than six layers).

Flow cytometric analysis

Single-cell suspensions were obtained from spleens and cervical lymph nodes by gently dispersing the tissues with a glass tissue grinder. After lysis of RBC by distilled water, the suspended cells were washed in RPMI 1640 medium and subjected to the flow cytometric analysis (FACSCalibur; BD Biosciences, San Jose, CA) using the following Abs: rat anti-mouse CD4 to detect CD4 T cells, rat anti-mouse CD8 to detect CD8 T cells, rat anti-mouse B220 to detect DN CD4^–/CD8^– T cells, and rat anti-mouse CD21/35 to detect B cells (all purchased from BD Pharmingen).

Serum IgG

Serum IgG levels were measured by ELISA. Plates coated with goat anti-mouse Ig Ab (Southern Biotechnology Associates, Birmingham, AL) were developed with alkaline phosphatase (ALP)-conjugated isotype-specific anti-Ig Abs (Southern Biotechnology Associates). Sera at dilutions of 1/100 to 1/72,900 were analyzed for IgG concentrations using standard curves generated with Mouse Standard Panel (Southern Biotechnology Associates).

Histochemistry for peanut agglutinin (PNA)

To detect germinal center (GC) cells, PNA histochemistry was performed as described previously (23). Briefly, spleens were taken from both IL-18R^–/– MRL-Fas^lpr mice and IL-18R^+/+ MRL-Fas^lpr mice at 6 mo of age and snap-frozen in OCT compound. Cryostat sections of spleen were stained with biotinylated PNA (Vector Laboratories, Burlingame, CA).

Anti-DNA Abs

Ig class-specific anti-DNA Abs were measured by ELISA as described previously (24). Each well of flat-bottom 96-well plates (Costar, Cambridge, MA) was coated with calf thymus DNA (Sigma-Aldrich, St. Louis, MO), dried at 37°C, and washed in isotonic PBS (pH 7.4). Nonspecific protein binding was blocked by coating the plate with 10% goat serum in PBS. After 100 µl of each serum sample appropriately diluted with PBS was added and the preparation was incubated for 2 h at 37°C, each well was washed and allowed to react with 100 µl of appropriately diluted ALP-conjugated goat anti-mouse Ig, IgG1, IgG2a, IgG2b, or IgG3 Abs (Valeant Pharmaceuticals, Costa Mesa, CA) for 2 h at 37°C. ALP activity was measured using an ALP substrate kit (Wako Pure Chemical, Osaka, Japan) containing phenyl phosphate and 4-aminoantipyrine. Pooled sera from 6-mo-old MRL-Fas^lpr mice were used as standards. Titers of the standard sera were defined as follows: for total IgG, 1000 U/ml; IgG1, 170 U/ml; IgG2a, 340 U/ml; IgG2b, 220 U/ml; and IgG3, 270 U/ml. Absorbance at 490 nm for each well was measured by a microplate reader (Benchmark; Bio-Rad, Hercules, CA). Ab titers were determined from absorbance using a standard curve constructed for each IgG subclass.

Measurements of mRNA cytokine transcripts in the kidney

To examine levels of expression of TNF-, IFN-, IL-10, and IL-12 in the kidney, mRNA transcripts in the kidney isolated from IL-18R^–/– MRL-Fas^lpr and IL-18R^+/+ MRL-Fas^lpr were quantitatively measured using the real-time quantitative PCR method. Total RNA was extracted from the renal cortex using Isogen (Nippon Gene, Tokyo, Japan). The RT reaction was performed with RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Grand Island, NY). The resulting RT product was used as a cDNA template for PCR analysis. Real-time quantitative PCR analysis was performed by using an ABI 7700 sequence detector system (Applied Biosystems, Foster City, CA). A fluorescent dye, FAM-labeled primer, was used as the target hybridization probe for TNF-, IFN-, IL-10, and IL-12 (Applied Biosystems), and another fluorescent dye, VIC-labeled primer (Applied Biosystems), was used as the control hybridization probe for 18S ribosomal RNA (Applied Biosystems). The thermal cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s, and 60°C for 1 min for denaturing and annealing, respectively. All PCR were run in triplicate.

Statistics

Statistical significance was evaluated by two-tailed unpaired Student’s t test. Survival curves were determined using log-rank two-tailed test.

	Results

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Survival

IL-18R^–/– MRL-Fas^lpr mice survived significantly longerthan IL-18R^+/+ MRL-Fas^lpr mice (Fig. 1). The 50% mortality in the IL-18R^+/+ MRL-Fas^lpr mice was 8 mo of age, while only 9% of IL-18R^–/– MRL-Fas^lpr mice were dead at this age (n = 43 and 26, respectively; p < 0.05). Similarly, the majority (83%) of the IL-18R^+/+ MRL-Fas^lpr mice were dead at 12 mo of age, as compared with a minority (43%) of the IL-18R^–/– MRL-Fas^lpr mice. All the moribund IL-18R^+/+ MRL-Fas^lpr and IL-18R^–/– MRL-Fas^lpr mice exhibited severe proteinuria, and kidney tissues obtained after the mice were sacrificed showed severe renal pathology (n = 28 and 12, respectively). We noted that in the third generation of IL-18R-intact MRL-Fas^lpr mice, the 50% mortality was only minimally prolonged compared with that in the wild-type strain (8 vs 6 mo of age, respectively). Thus, survival in the IL-18R^+/+ MRL-Fas^lpr strain (third generation) was similar to that in the wild-type MRL-Fas^lpr strain.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Comparison of percentages of survival between IL-18R-deficient and IL-18R-intact MRL-Faslpr mice. IL-18R-deficient MRL-Faslpr mice survived longer than IL-18R-intact MRL-Faslpr mice (n = 26 and 43, respectively). The difference was significant (p < 0.05).

IL-18R^–/– MRL-Fas^lpr mice were protected from proteinuria (Fig. 2A and B). As shown in Fig. 2A, urinary protein levels in the IL-18R^–/– MRL-Fas^lpr mice (n = 20–26) were significantly reduced, compared with those in the IL-18R^+/+ MRL-Fas^lpr mice (n = 15–43) (grades of proteinuria at 6 mo of age, 2.0 ± 0.5 vs 1.6 ± 0.4, respectively; p < 0.05). Fig. 2B shows the cumulative incidence of proteinuria. IL-18R^+/+ MRL-Fas^lpr mice (n = 15–43) began to develop proteinuria at 4 mo of age and the cumulative incidence reached 95% at 10 mo of age, while the incidence (62%) in the IL-18R^–/– MRL-Fas^lpr mice (n = 20–26) was significantly lower than that in IL-18R^+/+ MRL-Fas^lpr mice at this age. However, both the urinary protein level and incidence of proteinuria in the IL-18R^–/– MRL-Fas^lpr mice were progressively increased with advancing age. Therefore, blockade of IL-18R signaling in MRL-Fas^lpr mice reduces, but does not prevent, proteinuria. To evaluate the renal function more accurately, we also measured BUN. As shown in Table I, BUN levels in the IL-18-deficient MRL-Fas^lpr mice were significantly reduced, compared with findings in IL-18-intact MRL-Fas^lpr mice at 7 mo of age (n = 6/group).

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Comparisons of cumulative incidences among proteinuria, urinary protein levels, skin lesions, and lymphadenopathy. A, Urinary protein levels (*, p < 0.05). B, Cumulative incidences of proteinuria (p < 0.05). C, Skin lesion (*, p < 0.005). D, Lymphadenopathy (*, p < 0.05; **, p < 0.005; ***, p < 0.01). IL-18R-deficient MRL-Faslpr mice (n = 20–26), IL 18R-intact MRL-Faslpr mice (n = 15–43). Values are the mean ± SD.

View this table: [in this window] [in a new window]   Table I. Comparison of BUN between IL-18-deficient MRL-Faslpr mice and IL-18-intact MRL-Faslprmicea

Skin lesions, lymphadenopathy, and splenomegaly are gross pathologic features characteristic of systemic autoimmune disease in MRL-Fas^lpr mice. The severity of skin lesions were 2-fold less in IL-18R^–/– MRL-Fas^lpr mice (n = 20–26) as compared with the findings in IL-18R^+/+ MRL-Fas^lpr mice (n = 15–43) evaluated at 6, 8, and 10 mo of age (p < 0.05) (Fig. 2C). Similarly, lymphadenopathy was 2-fold less in IL-18R^–/– MRL-Fas^lpr mice (n = 20–26) as compared with that in IL-18R^+/+ MRL-Fas^lpr mice (n = 15–43) at 4, 6, 8, and 10 mo of age (p < 0.005, p < 0.01, p < 0.01, and p < 0.05, respectively) (Fig. 2D). Splenomegaly was diminished in IL-18R^–/– MRL-Fas^lpr mice, as compared with IL-18R^+/+ MRL-Fas^lpr mice; the mean spleen weight in IL-18R^–/– MRL-Fas^lpr mice was one-half that in IL-18R^+/+ MRL-Fas^lpr mice (0.54 ± 0.21 g vs 1.12 ± 0.35 g, respectively; n = 8; p < 0.05).

Renal pathology

Renal disease including glomerular, tubular, and perivascular pathology was diminished in the IL-18R^–/– MRL-Fas^lpr strains at 6 mo of age. Therefore, we compared renal disease in groups of IL-18R^–/– and IL-18R^+/+ MRL-Fas^lpr strains at 6 mo of age (n = 8/group; Figs. 3 and 4). Fig. 3 compares representative renal pathological findings between the two groups of mice. IL-18R^+/+ MRL-Fas^lpr mice at 6 mo of age showed glomerular hypercellularity, glomerulosclerosis, and crescent formation, damage in 27% of tubules, and abundant leukocytes surrounding vessels. In contrast, the renal glomeruli, tubules, and vasculature in IL-18R^–/– MRL-Fas^lpr mice at 6 mo of age remained almost intact (n = 8; p < 0.05 to 0.001; Fig. 4A). Because kidney disease in MRL-Fas^lpr mice consists of an infiltration of leukocytes, including macrophages (F4/80⁺) and CD4, CD8, and DN (B220⁺, CD21/35^–) T cells, we compared leukocytes in IL-18R^–/– MRL-Fas^lpr mice at 6 mo of age with age-matched IL-18R^+/+ MRL-Fas^lpr mice. In IL-18R^+/+ MRL-Fas^lpr mice at 6 mo of age, there were numerous kidney-infiltrating macrophages and CD4, CD8, and DN T cells in the periglomerular, interstitial, and perivascular areas. In contrast, the amount of these infiltrates was significantly less marked in IL-18R^–/– MRL-Fas^lpr mice (n = 8; p < 0.05 to 0.001; Fig. 4B).

View larger version (185K): [in this window] [in a new window]   FIGURE 3. Histopathological findings of the kidney. A, Kidney tissue from an IL-18R-intact MRL-Faslpr mouse at 6 mo of age. Note the sclerotic glomeruli (short, wide arrow, grade 3), damaged tubules (star), and perivascular leukocytic infiltrates (long arrow, grade 2). B, Kidney tissue from an IL-18R-deficient MRL-Faslpr mouse at a comparable age. Note the glomerulus (short arrow, grade 1) and perivascular leukocytic infiltrates (long arrow, grade 1). Periodic acid Schiff hematoxylin stain, x400.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Differences in histopathological scores of renal disease and extent of inflammatory cell infiltrates between IL-18R-deficient and IL 18R-intact MRL-Faslpr mice. A, Scores of glomerular and tubular damages and perivascular inflammatory cell infiltrates (n = 8; *, p < 0.001; **, p < 0.05). B, Numbers of infiltrating cells and macrophages in the kidneys (n = 8; *, p < 0.001; **, p < 0.05; ***, p < 0.005; #, p < 0.01). Values are the mean ± SD.

The deposition of IgG in glomeruli is a characteristic feature of renal disease in MRL-Fas^lpr mice. Table II shows the extents of glomerular deposition of IgG in the two groups of mice at 6 mo of age. Compared with the findings in IL-18R^+/+ MRL-Fas^lpr mice, glomerular deposits of IgG and IgG2a in IL-18R^–/– MRL-Fas^lpr mice were significantly less marked (n = 8/group; p < 0.05).

View this table: [in this window] [in a new window]   Table II. Comparisons of fluorescence intensities of glomerular deposits of IgG between IL-18R-deficient MRL-Faslpr mice and IL-18R-intact MRL-Faslpr micea

Nephritogenic cytokines, including TNF-, IFN-, IL-10, and IL-12, are up-regulated in the kidney in advance of injury and increase with progressive renal damage in MRL-Fas^lpr mice (8, 21, 25). To determine whether blockade of IL-18R signaling alters these cytokine expressions in the kidneys, we compared the amounts of these cytokine transcripts (TNF-, IFN-, IL-10, and IL-12) in the kidneys between IL-18R^–/– MRL-Fas^lpr mice and IL-18R^+/+ MRL-Fas^lpr mice at 6 mo of age (Fig. 5). The levels of expression of TNF-, IFN-, IL-10, and IL-12 mRNA in IL-18R^–/– MRL-Fas^lpr mice were significantly lower than those in IL-18R^+/+ MRL-Fas^lpr mice (n = 8/group; p < 0.001, p < 0.01, p < 0.01, and p < 0.005, respectively). Thus, we conclude that IL-18R^–/– MRL-Fas^lpr mice are protected from kidney disease via the mediation of nephritogenic cytokines.

View larger version (50K): [in this window] [in a new window]   FIGURE 5. Reduction of IFN-, TNF-, IL-12, and IL-10 transcription in the kidney from IL-18R-deficient MRL-Faslpr mice. Transcript levels for IFN-, TNF-, IL-10, and IL-12 were assessed by RT-PCR in the kidney. A, IFN- (n = 8; *, p < 0.001); B, TNF- (n = 8; **, p < 0.01); C, IL-10 (n = 8; **, p < 0.01); D, IL-12 (n = 8; ***, p < 0.005). Values are the mean ± SD.

Massive lymphadenopathy with expansion of DN T cells is characteristic of MRL-Fas^lpr mice. To determine whether blockade of IL-18R signaling reduces the lymphadenopathy in MRL-Fas^lpr mice, we compared the number of splenic and lymph node cells between IL-18R^–/– MRL-Fas^lpr mice and IL-18R^+/+ MRL-Fas^lpr mice at 6 mo of age (n = 6/group). Total number of cells, B cells, and each subset of T cells from both spleen and lymph node in IL-18R^–/– MRL-Fas^lpr mice were significantly reduced, as compared with those in IL-18R^+/+ MRL-Fas^lpr mice (p < 0.05 to 0.0001; Fig. 6, A and B). In addition, PNA-positive cells in the GC were diminished in IL-18R^–/– MRL-Fas^lpr mice, compared with those in IL-18R^+/+ MRL-Fas^lpr mice (Fig. 6C). Furthermore, total serum IgG levels in IL-18R^–/– MRL-Fas^lpr mice were significantly reduced, compared with those in IL-18R^+/+ MRL-Fas^lpr mice (p < 0.0001; Fig. 6D).

View larger version (72K): [in this window] [in a new window]   FIGURE 6. Comparisons of lymphadenopathy and serum IgG between IL-18R-deficient and IL-18R-intact MRL-Faslpr mice at 6 mo of age. A, Cell numbers of different cellular subsets in the spleen (n = 6; *, p < 0.0005; **, p < 0.05; ***, p < 0.0001). B, Cell numbers of different cellular subsets in the lymph nodes (n = 6; *, p < 0.0001; **, p < 0.05). C, Photographs of spleen sections stained for PNA, as a marker of GC. Decreased PNA-positive cell populations (arrow) in the IL-18R-deficient MRL-Faslpr spleen compared with those in the IL-18R-intact MRL-Faslpr spleen; x400. D, The elevated serum IgG levels in IL-18R-deficient MRL-Faslpr mice were reduced compared with those in IL-18R-intact MRL-Faslpr mice (n = 6; *, p < 0.0001). Values are the mean ± SD.

Elevated anti-DNA Ab levels are characteristic of MRL-Fas^lpr mice. To determine whether IL-18R signaling is required for the increase in serum anti-DNA Ab, we evaluated serum anti-DNA Ab isotype (total IgG, IgG1, IgG2a, IgG2b, and IgG3) concentrations in the two groups of MRL-Fas^lpr mice at 6 mo of age (Fig. 7). Total IgG and IgG2a anti-DNA Ab concentrations were significantly lower in IL-18R^–/– MRL-Fas^lpr mice than in IL-18R^+/+ MRL-Fas^lpr mice (n = 8; p < 0.03, p < 0.02, respectively). In contrast, no significant difference was observed in the concentrations of IgG1, IgG2b, or IgG3 anti-DNA Abs between the two groups.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Comparisons of serum levels of anti-DNA Abs of different IgG subclasses between IL-18R-deficient and IL-18R-intact MRL-Faslpr mice (n = 8; *, p < 0.03; **, p < 0.02). Values are the mean ± SD.

Pulmonary disease was reduced in IL-18R^–/– MRL-Fas^lpr mice compared with IL-18R^+/+ MRL-Fas^lpr mice. The lung pathology in MRL-Fas^lpr mice showed a progressive influx of leukocytes surrounding the vasculature. Similar to the findings in the kidney, IL-18R^–/– MRL-Fas^lpr mice showed no increase in accumulation of leukocytes in the perivascular or peribronchiolar areas at 6 mo of age compared with IL-18R^+/+ MRL-Fas^lpr mice (n = 8; p < 0.05) (Fig. 8).

View larger version (90K): [in this window] [in a new window]   FIGURE 8. Comparisons of pulmonary disease between IL-18R-deficient and IL-18R-intact MRL-Faslpr mice. A, Scores of perivascular and peribronchiolar leukocyte infiltrates (n = 8; *, p < 0.05). B, Histopathologic findings of IL-18R-deficient (left) and IL-18R-intact MRL-Faslpr mice (right). Note the perivascular leukocyte infiltrates (arrowhead), and peribronchiolar leukocytic infiltrates (arrow). H&E stain, x400.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-18R-deficient MRL-Fas^lpr mice showed reduced systemic pathology, including skin, lung, and kidney disease. Recently, administration of a cDNA coding for the murine IL-18 precursor has been reported to reduce lymphoproliferation, IFN- production, and renal damage, and prolongs survival in MRL-Fas^lpr mice (26). It has also been reported that in caspase-1 transgenic mice, overproduction of IL-18 by keratinocytes in the skin induces atopic dermatitis-like chronic dermatitis by inducing the accumulation of mast cells, which release chemical mediators such as histamine (27). Furthermore, administration of IL-18 in synergy with IL-2 induces lethal lung injury in mice, and this lethal lung injury was shown to be suppressed completely in IL-18R-deficient mice or partially in IFN--deficient mice (28). Thus, it seems likely that IL-18-induced cytokines, chemokines, and inflammatory cell infiltrates play an important pathogenic role in the autoimmune tissue destruction.

We found that IL-18R-deficient MRL-Fas^lpr mice showed a significant reduction in IFN- transcription in the kidney and serum levels of IgG2a anti-DNA Abs, the isotype switching of which is dependent on IFN-. Glomerular deposits of IgG2a were also reduced in these mice. There are reports demonstrating that MRL-Fas^lpr mice genetically depleted of IFN- or IFN-R show a significant reduction in humoral and histopathologic characteristics of autoimmune disease (8, 29, 30). Thus, blockade of IL-18 signaling appears to predominantly inhibit IFN- expression, leading to reduction in IgM to IgG2a class switching of anti-DNA Abs and resultant kidney disease in MRL-Fas^lpr mice. Therefore, although many cytokines have been linked to the progression of autoimmune kidney disease in MRL-Fas^lpr mice, IFN- appears to be a key cytokine that plays an important role in the pathogenesis in MRL-Fas^lpr mice (8, 31). Involvement of IFN- in the pathogenesis of autoimmune disease in NZB/W F₁ mice, another murine lupus model, has also been suggested, in which mice treated with IFN- show accelerated disease manifestations (32), while, conversely, treatment with anti-IFN- Ab or soluble IFN-R delays the disease progression (32, 33). Because complement-fixing IgG2a anti-DNA Abs are predominant among other isotypes of anti-DNA Abs in these mice, these are thought to be major nephritogenic Abs in these two lupus models (29). Although our results support a central role of IFN- in the pathogenesis of murine lupus model, it is also worth noting that serum IFN- levels in human lupus are reduced or essentially absent, and that human T cells stimulated via the TCR or by mitogen produce markedly reduced amounts of IFN- (34, 35). Thus, there is a dichotomy between the murine lupus model and human lupus.

Overexpression of IL-18R in lymphocytes in MRL-Fas^lpr mice has been reported to be linked to leukocyte hyperresponsiveness and the elevated production of IFN- (36). In the present study, however, it was evident that IL-18R-deficiency can significantly reduce IFN- expression in the kidney. Thus, although IL-18R is overexpressed in MRL-Fas^lpr mice, it seems feasible that IFN- production is down-regulated in these mice via the blocking of IL-18 binding to IL-18R.

We found that intrarenal TNF- was significantly reduced in IL-18R-deficient MRL-Fas^lpr mice. In MRL-Fas^lpr mice, TNF- can be detected in the kidney and in the circulation before the onset of renal injury, and continues to increase as the renal damage progresses with aging (8, 37). Such circulating and intrarenal TNF- levels are also reduced markedly in IFN-R-deficient MRL-Fas^lpr mice (8). Together with the finding that IL-18 can induce macrophages to produce proinflammatory cytokines, including TNF- (38, 39), these results strongly suggest that blockade of IL-18 signaling is causally related to down-regulation of TNF-. Because IL-18 can also induce some chemokines, such as IL-8, MIP-1, and MCP-1 (39), which are thought to be linked to the pathogenesis of lupus (40, 41, 42), IL-18 is thought to play a central role in the observed cytokine and chemokine abnormalities in MRL-Fas^lpr mice.

In this study, there was a substantial variability in the renal pathology. IL-18R-deficient congenic MRL-Fas^lpr mice used in the present studies were identical with the inbred MRL-Fas^lpr mice across 94% of the genome. Although they showed almost the same disease features as wild-type MRL-Fas^lpr mice did, it is possible that the variability may be due to the involvement of genetic factors derived from IL-18R-deficient 129 mice. To clarify this issue, we are now continuing further backcrossing.

Although disruption of the IL-18R gene markedly reduced the autoimmune disease, the preventive effect was not complete. There appear to be some IL-18R-independent mechanisms leading to autoimmune tissue destruction. Clearly, additional studies are required to determine the relationship between IL-18 and other cytokines involved in the pathogenesis of autoimmune tissue destruction. Studies on the effects of IL-12 on MRL-Fas^lpr mice have shown that administration of IL-12 elicits autoimmune kidney injury by fostering the accumulation of IFN--secreting T cells in the kidney (43). In IL-12p40-deficient MRL-Fas^lpr mice, there was a delay in renal and systemic pathology (44). However, similar to the findings in IL-18R-deficient MRL-Fas^lpr mice in the present studies, the protective effect of IL-12 gene disruption on the MRL-Fas^lpr disease was not complete. Because IL-18 and IL-12 are known to synergistically induce T cells and macrophages to produce IFN-, blockade of both IL-12 and IL-18 may be a more powerful approach to treat SLE.

	Acknowledgments

 
We are grateful to Dr. T. Shirai for critically reviewing this manuscript. We also thank K. Niki and K. Furukawa for technical assistance.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant-in-aid for scientific research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Koji Kinoshita, Department of Hematology, Nephrology and Rheumatology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka, 589-8511, Japan. E-mail address: kinoshita{at}int3.med.kindai.ac.jp

³ Abbreviations used in this paper: SLE, systemic lupus erythematosus; ALP, alkaline phosphatase; BUN, blood urea nitrogen; DN, double negative; GC, germinal center; gcs, glomerular cross-sections; IL-1Rrp, IL-1R-related protein; PNA, peanut agglutinin.

Received for publication November 11, 2003. Accepted for publication August 5, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Theofilopoulos, A. N., F. J. Dixon. 1981. Etiopathogenesis of murine SLE. Immunol. Rev. 55:179.[Medline]
2. Pisetsky, D. S., G. A. McCarty, D. V. Peters. 1980. Mechanisms of autoantibody production in autoimmune MRL mice. J. Exp. Med. 152:1302.[Abstract/Free Full Text]
3. Santoro, T. J., J. P. Portanova, B. L. Kotzin. 1988. The contribution of L3T4⁺ T cells to lymphoproliferation and autoantibody production in MRL-lpr/lpr mice. J. Exp. Med. 167:1713.[Abstract/Free Full Text]
4. Foster, H. M., V. R. Kelley. 1999. Lupus nephritis: update on pathogenesis and disease mechanisms. Semin. Nephrol. 19:57.[Medline]
5. Pen, S. L., M. P. Madaio, D. P. Hughes, I. N. Crispe, M. J. Owen, L. Wen, A. C. Hayday, J. Craft. 1996. Murine lupus in the absence of T cells. J. Immunol. 156:4041.[Abstract]
6. Chesnutt, M. S., B. K. Fink, N. Kileen, M. K. Connolly, H. Goodman, D. Wofsy. 1998. Enhanced lymphoproliferation and diminished autoimmunity in CD4-deficient MRL/lpr mice. Clin. Immunol. Immunopathol. 87:23.[Medline]
7. Peng, S. L., J. Craft. 1996. T cells in murine lupus: propagation and regulation of disease. Mol. Biol. Rep. 23:247.[Medline]
8. Schwarting, A., T. Wada, K. Kinoshita, G. Tesch, V. R. Kelley. 1998. IFN- receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas^lpr mice. J. Immunol. 161:494.[Abstract/Free Full Text]
9. Balomenos, D., R. Rumold, A. N. Theofilopoulos. 1998. Interferon- is required for lupus-like autoimmune syndrome in MRL mice. J. Clin. Invest. 101:364.[Medline]
10. Okamura, H., H. Tsutsui, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori, et al 1995. Cloning of a new cytokine that induces IFN- production by T cells. Nature 378:88.[Medline]
11. Kohno, K., J. Kataoka, T. Ohtsuki, Y. Suemoto, I. Okamoto, M. Usui, M. Ikeda, M. Kurimoto. 1997. IFN--inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158:1541.[Abstract]
12. Micallef, M. J., T. Ohtsuki, K. Kohno, F. Tanabe, S. Ushio, M. Namba, T. Tanimoto, K. Torigoe, M. Fujii, M. Ikeda, et al 1996. Interferon--inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon- production. Eur. J. Immunol. 26:1647.[Medline]
13. Yoshimoto, T., K. Takeda, T. Tanaka, K. Ohkusu, S. Kashiwamura, H. Okamura, S. Akira, K. Nakanishi. 1998. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN- production. J. Immunol. 161:3400.[Abstract/Free Full Text]
14. Gu, Y., K. Kuida, H. Tsutsui, G. Ku, K. Hsiao, M. A. Fleming, N. Hayashi, K. Higashino, H. Okamura, K. Nakanishi, et al 1997. Activation of interferon- inducing factor mediated by interleukin-1 converting enzyme. Science 275:206.[Abstract/Free Full Text]
15. Torigoe, K., S. Ushio, T. Okura, S. Kobayashi, M. Taniai, T. Kunikata, T. Murakami, O. Sanou, H. Kojima, M. Fujii, et al 1997. Purification and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272:25737.[Abstract/Free Full Text]
16. Born, T. L., E. Thomassen, T. A. Bird, J. E. Sims. 1998. Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J. Biol. Chem. 273:29445.[Abstract/Free Full Text]
17. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, S. Akira. 1999. Generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162:5041.[Abstract/Free Full Text]
18. Wong, C. K., C. Y. Ho, E. K. Li, L. S. Tam, C. W. Lam. 2002. Elevated production of interleukin-18 is associated with renal disease in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 130:345.[Medline]
19. Esfandiari, E., I. B. McInnes, G. Lindop, F. P. Huang, M. Field, M. Komai-Koma, X. Wei, F. Y. Liew. 2001. A proinflammatory role of IL-18 in the development of spontaneous autoimmune disease. J. Immunol. 167:5338.[Abstract/Free Full Text]
20. Wong, C. K., C. Y. Ho, E. K. Li, C. W. Lam. 2000. Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 9:589.[Abstract/Free Full Text]
21. Kinoshita, K., G. Tesch, A. Schwarting, R. Maron, A. H. Sharpe, V. R. Kelley. 2000. Costimulation by B7-1 and B7-2 is required for autoimmune disease in MRL-Fas^lpr mice. J. Immunol. 164:6046.[Abstract/Free Full Text]
22. Wu, J., T. Zhou, J. He, J. D. Mountz. 1993. Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene. J. Exp. Med. 178:461.[Abstract/Free Full Text]
23. Masuda, A., T. Kasajima. 1999. Follicular dendritic cell dysfunction and disorganization of lymphoid structures in MRL/lpr mice. Lab. Invest. 79:849.[Medline]
24. Kinoshita, K., B. S. Yoo, Y. Nozaki, M. Sugiyama, S. Ikoma, M. Ohno, M. Funauchi, A. Kanamaru. 2003. Retinoic acid reduces autoimmune renal injury and increases survival in NZB/W F₁ mice. J. Immunol. 170:5793.[Abstract/Free Full Text]
25. Yin, Z., G. Bahtiyar, N. Zhang, L. Liu, P. Zhu, M. E. Robert, J. McNiff, M. P. Madaio, J. Craft. 2002. IL-10 regulates murine lupus. J. Immunol. 169:2148.[Abstract/Free Full Text]
26. Bossu, P., D. Neumann, E. Del Giudice, A. Ciaramella, I. Gloaguen, G. Fantuzzi, C. A. Dinarello, E. Di Carlo, P. Musiani, P. L. Meroni, et al 2003. IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease. Proc. Natl. Acad. Sci. USA 100:14181.[Abstract/Free Full Text]
27. Yamanaka, K., M. Tanaka, H. Tsutsui, T. S. Kupper, K. Asahi, H. Okamura, K. Nakanishi, M. Suzuki, N. Kayagaki, R. A. Black, et al 2000. Skin-specific caspase-1 transgenic mice show cutaneous apoptosis and preendotoxin shock condition with a high serum level of IL-18. J. Immunol. 165:997.[Abstract/Free Full Text]
28. Okamoto, M., S. Kato, K. Oizumi, M. Kinoshita, Y. Inoue, K. Hoshino, S. Akira, A. N. McKenzie, H. A. Young, T. Hoshino. 2002. IL-18 in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines and NK cells in the pathogenesis of interstitial pneumonia. Blood 99:1289.[Abstract/Free Full Text]
29. Peng, S. L., J. Moslehi, J. Craft. 1997. Roles of interferon– and interleukin-4 in murine lupus. J. Clin. Invest. 99:1936.[Medline]
30. Haas, C., B. Ryffel, M. Le Hir. 1997. IFN- is essential for the development of autoimmune glomerulonephritis in MRL/lpr mice. J. Immunol. 158:1585.
31. Faust, J., J. Menke, J. Kriegsmann, V. R. Kelley, W. J. Mayet, P. R. Galle, A. Schwarting. 2002. Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Fas^lpr mice with autoimmune lupus nephritis. Arthritis Rheum. 46:3083.[Medline]
32. Jacob, C. O., P. H. van der Meide, H. O. McDevitt. 1987. In vivo treatment of (NZB x NZW)F₁ lupus-like nephritis with monoclonal antibody to interferon. J. Exp. Med. 166:798.[Abstract/Free Full Text]
33. Ozmen, L., D. Roman, M. Fountoulakis, G. Schmid, B. Ryffel, G. Garotta. 1995. Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon- receptor inhibits the onset of glomerulonephritis. Eur. J. Immunol. 25:6.[Medline]
34. Handwerger, B. S., V. Rus, L. da Silva, C. S. Via. 1994. The role of cytokines in the immunopathogenesis of lupus. Springer Semin. Immunopathol. 16:153.[Medline]
35. Horwitz, D. A., J. D. Gray, S. C. Behrendsen, M. Kubin, M. Rengaraju, K. Ohtsuka, G. Trinchieri. 1998. Decreased production of interleukin-12 and other Th1-type cytokines in patients with recent-onset systemic lupus erythematosus. Arthritis Rheum. 41:838.[Medline]
36. Neumann, D., E. Del Giudice, A. Ciaramella, D. Boraschi, P. Bossu. 2001. Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain. J. Immunol. 166:3757.[Abstract/Free Full Text]
37. Yokoyama, H., B. Kreft, V. R. Kelley. 1995. Biphasic increase in circulating and renal TNF- in MRL-lpr mice with differing regulatory mechanisms. Kidney Int. 47:122.[Medline]
38. Gracie, J. A., R. J. Forsey, W. L. Chan, A. Gilmour, B. P. Leung, M. R. Greer, K. Kennedy, R. Carter, X. Q. Wei, D. Xu, et al 1999. A proinflammatory role for IL-18 in rheumatoid arthritis. J. Clin. Invest. 104:1393.[Medline]
39. Puren, A. J., G. Fantuzzi, Y. Gu, M. S. Su, C. A. Dinarello. 1998. Interleukin-18 (IFN-inducing factor) induces IL-8 and IL-1 via TNF production from non-CD14⁺ human blood mononuclear cells. J. Clin. Invest. 101:711.[Medline]
40. Rovin, B. H., L. Lu, X. Zhang. 2002. A novel interleukin-8 polymorphism is associated with severe systemic lupus erythematosus nephritis. Kidney Int. 62:261.[Medline]
41. Kaneko, H., H. Ogasawara, T. Naito, H. Akimoto, S. Lee, T. Hishikawa, I. Sekigawa, Y. Tokano, Y. Takasaki, S. I. Hirose, H. Hashimoto. 1999. Circulating levels of -chemokines in systemic lupus erythematosus. J. Rheumatol. 26:568.[Medline]
42. Tesch, G. H., S. Maifert, A. Schwarting, B. J. Rollins, V. R. Kelley. 1999. Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas^lpr mice. J. Exp. Med. 190:1813.[Abstract/Free Full Text]
43. Schwarting, A., G. Tesch, K. Kinoshita, R. Maron, H. L. Weiner, V. R. Kelley. 1999. IL-12 drives IFN--dependent autoimmune kidney disease in MRL-Fas^lpr mice. J. Immunol. 163:6884.[Abstract/Free Full Text]
44. Kikawada, E., D. M. Lenda, V. R. Kelley. 2003. IL-12 deficiency in MRL-Fas^lpr mice delays nephritis and intrarenal IFN- expression, and diminishes systemic pathology. J. Immunol. 170:3915.[Abstract/Free Full Text]

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