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International Immunology Advance Access originally published online on August 16, 2007
International Immunology 2007 19(8):935-942; doi:10.1093/intimm/dxm054
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Ifi202, an IFN-inducible candidate gene for lupus susceptibility in NZB/W F1 mice, is a positive regulator for NF-{kappa}B activation in dendritic cells

Moriyasu Yamauchi1,2, Masayuki Hashimoto1, Kenji Ichiyama1, Ryoko Yoshida1, Toshikatsu Hanada1, Tatsushi Muta3, Shizuo Komune2, Takashi Kobayashi1 and Akihiko Yoshimura1

1 Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation
2 Department of Otorhinolaryngology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
3 Laboratory of Cell Recognition and Response, Graduate School of Life Science, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan

Correspondence to: A. Yoshimura; E-mail: yakihiko{at}bioreg.kyushu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies and lupus nephritis. The [New Zealand black (NZB) x New Zealand white (NZW)]F1 (BWF1) mouse has been recognized as an important animal model of human SLE. The Th1-prone phenotype of BWF1 mice has been shown to contribute to the development of the lupus. However, the molecular basis for Th1 skewing in BWF1 mice has not been clarified. We noticed that IL-6, IL-12 and other proinflammatory cytokines as well as I{kappa}B-{zeta} induction were higher in mature bone marrow-derived dendritic cells (BMDCs) from NZB and BWF1 mice than those from NZW mice. The expression of an IFN-inducible gene Ifi202, a candidate gene for lupus, was almost undetectable in NZW BMDCs. Thus, we hypothesized that Ifi202 is involved in elevated IL-12 production from BWF1 BMDCs. Overexpression of Ifi202 enhanced the LPS-induced I{kappa}B-{zeta}, IL-12p40 and NF-{kappa}B promoter activities, while anti-sense (AS) RNA against Ifi202 strongly suppressed them in a monocytic cell line, RAW 264.7. Furthermore, overexpression of Ifi202 enhanced LPS-induced IL-12p40 and I{kappa}B-{zeta} mRNA induction while Ifi202 AS RNA suppressed these in RAW 264.7 cells. In addition, forced expression of Ifi202 enhanced IL-12p40 mRNA induction in NZW BMDCs. Thus, Ifi202 is an important NF-{kappa}B activator in DCs and involved in IL-12 production, which may account for a Th1-prone phenotype of BWF1 mice.

Keywords: dendritic cell, IL-12, interferon, SLE


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is considered to be the prototypic systemic autoimmune disease. The common denominator among SLE patients is IgG autoantibody production, and the hallmark of this disease is elevated serum levels of anti-nuclear antibodies (1). SLE is considered to be a complex of genetic trait with contributions from MHC genes and multiple non-MHC genes. Hybrids of New Zealand black (NZB) and New Zealand white (NZW) mice (BWF1) develop a severe immune complex-mediated glomerulonephritis associated with high serum levels of IgG anti-nuclear autoantibodies, and these mice are considered to be an excellent model for human SLE (2–5). Since neither NZB nor NZW mice develop severe lupus-like renal disease, genes from both parents must be involved in the full expression of F1 disease.

Contributions of several cytokines, such as IFN-{gamma}, IFN-{alpha}/ß, IL-6, IL-10 and IL-12, to development of lupus in the BWF1 mice have been reported (6–12). Administration of rIL-6 accelerated the disease and blockade of IL-6R inhibited the onset of kidney disease in BWF1 mice (10, 13). IL-6 affects a variety of biological functions, such as Ig production, the acute phase inflammatory response and plasmacytoma generation by regulating cell growth, differentiation and survival (14). In addition, high levels of IFN{gamma} have been implicated in the disease progression in BWF1 mice because IFN{gamma}-treated animals have accelerated development of a fatal immune complex glomerulonephritis, while administration of mAbs specific for IFN{gamma} resulted in significant remission (7). These evidences suggest that a Th1/Th2 balance, especially, the IFN{gamma} levels, is critical for the development of autoimmune diseases, but the molecular basis for the Th1-prone phenotype of BWF1 mice has not been clarified.

An IFN-inducible gene, Ifi202 (encoding the protein Ifi202), has been identified as a susceptible gene for lupus (15). Ifi202 is a member of IFN-inducible p200 family of proteins. Generation of B6.Nba2 mice congenic for the Nba2 interval, which contains NZB allele of the Ifi202 gene on the C57BL/6 background, has identified Ifi202 as a major candidate gene for lupus susceptibility (15). But the molecular mechanisms by which increased levels of Ifi202 enhance the development of autoimmunity are currently unknown.

Dendritic cells (DCs) include a heterogeneous family of professional antigen-presenting cells involved in the initiation of immunity and in immunological tolerance. Ishikawa et al. (16). found that CD11b+CD11c+ cells were markedly increased in the thymus, spleen and peripheral blood in aged BWF1 mice. DCs in the thymus produce high levels of B lymphocyte chemoattractant (BLC/CXCL13) and recruit B cells into the thymus, which has been shown to be linked to breaking immune tolerance in the thymus (17). We have also reported that SOCS1-deficient mice developed SLE-like autoimmune diseases and that DCs and B cells were accumulated in the thymus. SOCS1-deficient DCs induce stronger Th1 responses, including higher IFN{gamma} production from T cells (18, 19).

We suspected that DCs in BWF1 mice might play a critical role in the increased production of proinflammatory cytokines and the induction of enhanced Th1-type responses as SOCS1-deficient DCs. IL-6, IL-12 and other proinflammatory cytokine levels were higher in mature bone marrow-derived dendritic cells (BMDCs) from NZB and BWF1 mice than from NZW mice. Consistent with previous findings, Ifi202 was rapidly induced in NZB- and BWF1-BMDCs but not in NZW-BMDCs in response to LPS. Therefore, we hypothesized that Ifi202 protein positively regulates proinflammatory gene expression. Overexpression of Ifi202 enhanced the LPS-induced IL-12p40 and I{kappa}B-{zeta} promoter activities, while knockdown of Ifi202 reduced them in a monocytic cell line RAW 264.7 cells. Ifi202 also enhanced NF-{kappa}B promoter activity, suggesting that Ifi202 protein directly enhances transcriptional activity of the NF-{kappa}B in monocytic cells. Taken together, we propose that Ifi202 positively regulates production of IL-12 from DCs, which contributes to the progression of lupus in BWF1 mice.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
NZB, BWF1, NZW and C57BL/6 mice, originally obtained from the Nippon SLC Corporation (Shizuoka, Japan), were maintained in our animal facility at Kyushu University. Female mice aged 2 months were used.

Cell culture
RAW 264.7 cells were maintained in DMEM (Sigma) containing 10% FBS, antibiotics and nonessential amino acids (GIBCO). BMDCs were generated from bone marrow according to the method described by Inaba et al. (20) with modifications. Briefly, bone marrow samples from the femurs and tibiae of mice were cultured in RPMI 1640 with 10% FBS, antibiotics and 10 mM HEPES (Sigma) containing 20 ng ml–1 granulocyte macrophage colony-stimulating factor (GM-CSF) (Pepro Tech) or the culture supernatant from a J558L cell line transfected with the murine GM-CSF cDNA for 7 days with replenishment of the medium every other day.

Plasmids
cDNA fragment encoding Ifi202 was obtained from RAW 264.7 cell RNA by reverse transcription (RT)-PCR and cloned into pCMV14 (Sigma) and pRcCMV (Invitrogen) in the appropriate (sense) and inverted [anti-sense (AS)] orientation, respectively (21).The I{kappa}B-{zeta} promoter reporter plasmid was described previously (22). IL-12p40 promoter region (about 700 bp) was isolated by genomic PCR using primers 5'-GAGCTCAGTATGGGTACCCAAAAGAGTG-3' and 5'-CTCGAGTGCTTTGCTGCGAGCTGCCT-3' as described (23) and sub-cloned into the pGVB-luciferase reporter vector.

Luciferase reporter assay
Reporter plasmids and control lacZ plasmid were transiently co-transfected into RAW 264.7 cells with ether control, Ifi202, or AS-Ifi202 expression vectors using FuGENE HD transfection reagent (Roche). After stimulation with 10 ng ml–1 LPS for 6 h, luciferase activities and ß-galactosidase were measured. Relative luciferase activities normalized with lacZ activity are shown.

Retroviral constructs and transduction to RAW 264.7 cells
The sense Ifi202 was sub-cloned into pGCD{Delta}NSamI/E retroviral vector, and the transfection was performed as described previously (24). RAW 264.7 cells were retrovirally transduced with the virus supernatants. GFP-positive cells were sorted by a cell sorter, FACSAria (BD Biosciences), and used for the assays.

Transfection into RAW 264.7 cells and BMDCs by electroporation
The expression vectors for control empty or ASIfi202 were co-transfected with pEGFP vector into RAW 264.7 cells by MicroPorator (Digital Bio Technology) according to the manufacturer's instructions. GFP-positive cells were sorted by a cell sorter and immediately used for gene expression analysis with RT-PCR. For NZW-BMDCs, 1.8 x 107 cells were mixed with 18 µg of empty or Ifi202/pCMV14 plasmid together with 18 µg of GFP plamid, then pulsed twice at 2000 V for 10 ms. After 6 h incubation in the presence of GM-CSF, GFP-positive cells were sorted by FACSAria, and then collected cells were stimulated with 100 ng ml–1 LPS for 3 h. Transfection efficiency was >60% judged by GFP fluorescence.

Western blot
Total cells were lysed in lysis buffer A [20 mM HEPES (pH 7.3), 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM dithiothreitol, 1 mM sodium vanadate, 1 mM phenylmethylsulphonylfluoride and 1% aprotinin]. The total cell extracts were resolved by SDS-PAGE, and the proteins were detected by immunoblotting as described (25, 26).

RNA isolation and gene expression analysis
Total cellular RNA was isolated from RAW 264.7 cells and BMDCs using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR was carried out using the one-step RT-PCR kit (Applied Biosystems) according to the manufacturer's instructions. The following primers were used: Ifi202 (exon)(F: cca acc gta act taa ggt cat c, R: cta gga tgc cac tgc tgt tg), Ifi202 (3'UTR)(F: ggt atg tat ggg tct ctg gc, R: gga aat ttc cac cat tga att ggg gt), tumor necrosis (TNF){alpha} (F: tga act tcg ggg tga tcg gtc, R: agc ctt gtc cct tga aga gga c), IL-12p35 (F: gac cac aga tga cat ggt gaa gac, R: gga gct cag ata gcc cat ca), IL-12p40 (F: gta gag gtg gac tgg act cc, R: gca gac aga gac gcc att cc), IFNß (F: tcc aag aaa gga cga aca ttc g, R: tga gga cat ctc cca cgt caa), IL-6 (F: cct ctc tgc aag aga ctt cc, R: act cct tct gtg act cca gc), I{kappa}B-{zeta} (F: gct caa cct ggc tta ctt cta cgg, R: cgg aag cct tct gct tgt tgc ttc), G3PDH (F: acc aca gtc cat gcc atc ac, R: tcc acc acc ctg ttg ctg ta). The PCR products were visualized in ethidium bromide-stained agarose gels.

Antibodies and reagents
The anti-ERK2 (C-14), anti-I{kappa}B{alpha} (C-21), anti-Stat1 (E-23) and anti-Stat3 (C-20) antibodies were purchased from Santa Cruz Biotechnology. The anti-phospho-ERK1/2 (9106), anti-phospho-JNK (9255), anti-phospho-p38 (9216), anti-JNK (9252), anti-p38 (9212), anti-phospho I{kappa}B{alpha} (9246), anti-phospho Akt (4058) and anti-Akt (9272) antibodies were obtained from Cell signaling. The LPS (Escherichia coli serotype 055:B5) was from Sigma Chemical.

High-density oligonucleotide microarray analysis
Total RNA was extracted by standard methods using a TRIzol reagent. cRNA preparation and microarray hybridization were carried out according to the instructions from the supplier (Affymetrix) using Genechip HG-U95Av2. Scanned output files were analyzed using the probe level analysis package, Microarray Suite MAS 5.0 (Affymetrix).

Statistical analysis
For statistical analysis, we performed the Student's t test, and a 95% confidence limit was taken to be significant, defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Higher IL-12 production from NZB- and BWF1-BMDCs than from NZW-BMDCs
Previous observations suggest a Th1-prone condition of BWF1 mice (6, 7, 27). To define the molecular basis for this, we first examined the phenotypes of matured BMDCs from New Zealand mice. Bone marrow cells were prepared from mice and cultured in the presence of GM-CSF for 6 days, and maturation was induced by incubation with LPS for 24 h. The number, morphology and CD11c expression of BMDCs from BWF1 mice did not differ from those from parental mice (data not shown). In response to LPS, the expressions of CD40, CD80 and CD86 on BMDCs were equally enhanced among these three strains (data not shown).

To identify the genes that would be involved in preferential Th1 induction by BWF1-BMDCs, we compared LPS-inducible genes in BWF1-BMDCs with those in NZW-BMDCs using microarray analysis. Among LPS-inducible genes, which were >3 fold higher after 2 h LPS stimulation in BWF1-BMDCs than without LPS stimulation, several cytokines, such as IL-12p40, TNF{alpha}, IFNß and some chemokines were higher in BWF1-BMDCs than in NZW-BMDCs (Table 1). Attention was given to IL-12 since it is the most important cytokine secreted from DCs that induces Th1 differentiation. We confirmed IL-12 levels by ELISA. Consistent with the result of microarray analysis, BWF1-BMDCs secrete higher levels of IL-12p70 than NZW-BMDCs in response to LPS (Fig. 1A). Interestingly, NZB-BMDCs secreted more IL-12p70 than BWF1-BMDCs, suggesting that the IL-12 levels are regulated by genes inherited in the NZB strain. The TNF{alpha} and IL-6 levels were also higher in the order of NZB-, BWF1- and NZW-BMDCs, but the differences were not as strong as with IL-12. The IL-10 levels were also higher in NZB- and BWF1-BMDCs than in NZW-BMDCs.


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  		Table 1. List of LPS-inducible genes whose expression levels were more than two times higher in BWF1-BMDC than NZW-BMDC

 

Figure 1
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  		Fig. 1. Cytokine production profiles in BMDCs from NZB, BWF1 and NZW mice. (A) Cytokine production in BMDCs from New Zealand mice. BMDCs from indicated mice were cultured with 0, 1 or 10 ng ml–1 LPS for 24 h. IL-6, IL-12p70, TNF{alpha} and IL-10 levels in supernatants were determined by ELISA. (B) BMDCs were stimulated with LPS (10 ng ml–1) for the indicated periods. The levels of mRNA transcripts for Ifi202, IL-6, IL-12p35, IL-12p40, I{kappa}B-{zeta}, TNF{alpha} and the internal control gene G3PDH were analyzed by RT-PCR.

 
Ifi202 gene expression of NZB-, BWF1- and NZW-BMDCs
It has been shown that Ifi202 is the lupus susceptibility gene in the NZB strain (15), but the molecular function of Ifi202 has not been clarified. The Ifi202 gene encodes a 52 kDa protein p202, which is an inducible transcriptional modulator (28–30) belonging to the p200 protein family. The ability of p202 to modulate the transcription of genes depends on its ability to bind and inhibit the transcriptional activity of factors, such as E2Fs, NF-{kappa}B, c-Myc, AP-1 and p53 (21, 30–33).

As reported, our microarray and RT-PCR analysis also confirmed the absence of expression of Ifi202 in NZW-BMDCs (Table 1 and Fig. 1B) (15). In contrast, the expression levels of Ifi202 were highly induced by LPS in NZB-BMDCs and those in BWF1-BMDCs were intermediate between those in NZB- and NZW-BMDCs. The expression levels of Ifi202 were well correlated to the levels of the proinflammatory cytokines, such as IL-6 and IL-12p40 subunits, especially at late stage of induction (Fig. 1B). Thus, we suspected that Ifi202 is the regulator of IL-6 and IL-12 expression. In addition, we examined the expression of I{kappa}B-{zeta}, which has been implicated in the expression of IL-6 and IL-12p40 (34) because it was also low in NZW-BMDCs in microarray data. As shown in Fig. 1(C), I{kappa}B-{zeta} was induced very rapidly in NZB-BMDCs within 1 h of LPS stimulation and lasted for up to 12 h. In contrast, I{kappa}B-{zeta} was expressed weakly in NZW-BMDCs and decreased thereafter. BMDCs from BWF1 mice were intermediate between them. Thus, I{kappa}B-{zeta} expression seems to be regulated by a factor present in the NZB strain.

TLR-signal transduction of BMDCs from NZB, BWF1 and NZW mice
To examine the mechanisms by which NZB- and BWF1-BMDCs produce higher levels of IL-12 than from NZW-BMDCs, we then compared the LPS-induced intracellular signaling pathways. LPS signaling regulates gene transcription via NF-{kappa}B, AP-1 and other transcriptional factors through the activation of p42/44 ERK, JNK and the p38 Mitogen activated protein kinase (MAPK) family as well as the I-{kappa}B kinase (IKK) complexes (35). STAT1 and STAT3 were also activated by LPS, but this activation is a late event, probably due to autocrine activation by cytokines including IFNß, IL-6 and IL-10. As shown in Fig. 2(A), no significant differences in STAT1 and STAT3 activation were observed among three strains. The phosphorylation of I{kappa}B{alpha} and degradation of I{kappa}B{alpha} were not different, suggesting that IKK activation was similar among the three strains. The phosphorylation levels of Jun kinase (JNK) and p38 were not altered either. We noticed the presence of a consistently higher activation of ERK and reduced Akt phosphorylation in NZW-BMDCs in repeated experiments. PI3K and ERK pathways have been shown to be involved in the regulation of IL-12 induction (36, 37). Therefore, we examined the effect of PI3K and ERK inhibitors (Fig. 2B). As reported, the PI3K inhibitor, Wortmannin, reduced Akt activation and enhanced IL-12p70 production. And the MEK inhibitor, PD98059, suppressed ERK activation and increased the IL-12p70 levels. However, the ratios of IL-12p70 levels in NZB-, BWF1- and NZW-BMDCs were not changed by these inhibitors (Fig. 2C), suggesting that these signaling pathways of Toll-like receptor (TLR4) were not a direct cause of the differences in IL-12 production levels.


Figure 2
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  		Fig. 2. TLR signaling and the effect of kinase inhibitors on IL-12 production. (A) LPS signaling in BMDCs from New Zealand mice. BMDCs from NZB, BWF1 and NZW mice were stimulated with LPS (10 ng ml–1) for indicated periods. Whole-cell lysates were prepared and subjected to western blot analysis using antibodies specific for the indicated molecules. (B) IL-12p70 production by BMDCs in response to LPS stimulation in the presence of kinase inhibitors. BMDCs were cultured with 10 ng ml–1 LPS for 24 h in the presence of the indicated inhibitors. The inset shows the phosphorylation of ERK and Akt to confirm the effect of inhibitors. The IL-12p70 levels in the supernatants were determined by ELISA. (C) Normalized data of IL-12p70 levels in BWF1- and NZW-BMDCs to that in NZB-BMDCs.

 
Ifi202 enhanced the LPS induced I{kappa}B-{zeta} and IL-12p40 expression
To test the possibility that Ifi202 is such a regulator of I{kappa}B-{zeta}, we examined the effects of forced expression of Ifi202 on I{kappa}B-{zeta} levels. We used a monocytic cell line RAW 264.7 cells because gene transfer efficiency in BMDCs was too low to detect reporter activity and mRNA levels. To analyze the transcriptional regulation of the I{kappa}B-{zeta} gene, we used reporter plasmids containing mouse I{kappa}B-{zeta} promoter fused to a luciferase reporter gene (22). Overexpression of Ifi202 enhanced the I{kappa}B-{zeta} promoter-reporter activity in a dose-dependent manner (Fig. 3A). Furthermore, the similar effect of Ifi202 was observed in the experiments of IL-12p40 promoter-reporter activity (Fig. 3A). It has been reported that I{kappa}B-{zeta} and IL-12p40 promoter activations are dependent on NF-{kappa}B (22, 23). Thus, we examined the effect of Ifi202 on NF-kB transcriptional activity. As shown in Fig. 3(A), Ifi202 enhanced LPS-mediated NF-{kappa}B transcriptional activity, but not IFN{gamma}-mediated STAT1 measured by GAS-promoter, in RAW 264.7 cells. We next examined the effect of Ifi202 on NF-{kappa}B activation by the TLR signaling components. Ifi202 enhanced the TRAF6 and p65-mediated activation of NF-{kappa}B transcriptional activity (Fig. 3B). These results suggest that Ifi202 modulates NF-{kappa}B transcriptional activity by acting downstream of p65.


Figure 3
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  		Fig. 3. Effect of forced expression of Ifi202 in a monocytic cell line RAW 264.7 cells. (A and B) RAW 264.7 cells were transfected with the indicated reporter plasmid and increasing amounts of an expressing vector for Ifi202 together with control lacZ plasmid. After 36 h, cells were stimulated with 10 ng ml–1 LPS for 6 h (A) or without stimulation (B), and then luciferase activity was measured and normalized based on ß-galactosidase activity. (C) GFP-positive RAW 264.7 cells infected with either control or Ifi202 retrovirus vectors were FACS sorted and stimulated with LPS (100 ng ml–1) for the indicated periods. The levels of mRNA transcripts for indicated genes were analyzed by RT-PCR. (D) NZW-BMDCs cells (1.8 x 107) were transfected with an expressing vector for Ifi202 together with control GFP plasmid. After 6 h, GFP-positive cells were sorted and then stimulated with 100 ng ml–1 LPS for 3 h. The levels of mRNA transcripts for indicated genes were analyzed by RT-PCR.

 
Then to validate the effect of Ifi202, we stably expressed Ifi202 in RAW 264.7 cells and examined the LPS-induced gene expression by RT-PCR (Fig. 3C). TNF{alpha} levels were constantly high probably because we used retrovirus to introduce Ifi202 gene. Overexpression of Ifi202 resulted in hyperinduction of I{kappa}B-{zeta} and slightly higher induction of IL-12p40 mRNAs in response to LPS stimulation (Fig. 3C). Then we transiently expressed Ifi202 in NZW-BMDCs and examined the LPS-induced IL-12p40 and TNF{alpha} expression (Fig. 3D). Overexpression of Ifi202 caused the hyperinduction of IL-12p40 mRNAs in NZW-BMDCs in response to LPS stimulation, while TNF{alpha} levels were not strongly affected. These data suggest that the expression of I{kappa}B-{zeta} and IL-12p40 is positively regulated by Ifi202.

Suppression of Ifi202 reduced I{kappa}B-{zeta} and IL-12 expression
To verify the effect of Ifi202, we knocked down the expression of Ifi202 using ASIfi202 (32) (Fig. 4). ASIfi202 expectedly suppressed the I{kappa}B-{zeta} and IL-12p40 as well as NF-{kappa}B reporter activities (Fig. 4A). Overexpression of ASIfi202 reduced the TRAF6 and p65-mediated activation of NF-{kappa}B transcriptional activities (Fig. 4B). Furthermore, knockdown of Ifi202 expression down-regulated the I{kappa}B-{zeta} mRNA expression in RAW 264.7 cells (Fig. 4C). IL-6 and IL-12p40 mRNA levels were also suppressed by ASIfi202. Taken together, these observations indicate that Ifi202 positively regulates LPS-induced expression of I{kappa}B-{zeta}, and IL-12p40 expression, through the enhanced NF-{kappa}B transcriptional activities.


Figure 4
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  		Fig. 4. Effect of knockdown of Ifi202 using ASIfi202. (A and B) RAW 264.7 cells were transfected with the indicated reporter plasmid and increasing amounts of an expressing vector for ASIfi202 together with control lacZ plasmid. After 36 h, the cells were stimulated with 10 ng ml–1 LPS for 6 h (A) or without stimulation (B), and then luciferase activity was measured and normalized based on ß-galactosidase activity. (C) Expression vectors for control empty or ASIfi202 were introduced into RAW 264.7 cells by electroporation and GFP-positive cells were sorted. The cells were stimulated with LPS (100 ng ml–1) for the indicated periods, and then the levels of mRNA transcripts for indicated genes were analyzed by RT-PCR.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Impaired Th1/Th2 balance, especially increased IFN{gamma} levels, in BWF1 mice has been shown to be critical for the development of autoimmune diseases (6). However, the molecular basis for the Th1-prone phenotype of BWF1 mice has not been clarified. In this study, we demonstrated that DCs from NZB and BWF1 mice has a higher potential to produce IL-12, which can partly account for a Th1-prone phenotype of BWF1 mice. This enhanced IL-12 production is accompanied with up-regulated expression of I{kappa}B-{zeta} mRNA in response to LPS stimulation. Another I{kappa}B-{zeta}-dependent cytokine IL-6 was also higher in NZB- and BWF1-BMDCs. We propose that Ifi202 is deeply involved in these phenotypes.

An IFN-inducible gene, Ifi202, has been identified as a susceptible gene for lupus (15). However, molecular mechanisms by which increased levels of Ifi202 enhance the development of autoimmunity are currently unknown. In this study, we showed the role of Ifi202 in the augmentation of LPS-induced cytokine production. We demonstrated that Ifi202 enhances the induction of I{kappa}B-{zeta} as well as IL-12p40 by modulating the transcriptional activities of NF-{kappa}B.

The up-regulated expression of I{kappa}B-{zeta} was followed by increased induction of IL-6 and IL-12p40, thus higher production of these cytokines may be due to a higher induction of I{kappa}B-{zeta}. In fact, the up-regulation of I{kappa}B-{zeta} was more prominent than other cytokines whose induction is dependent on NF-{kappa}B. Alternatively, since both IL-12p40 and IL-6 promoters contain NF-{kappa}B-binding sites, Ifi202 may directly augment these cytokine productions. Further study is necessary to dissect these two possibilities.

Ifi202 was recently shown to prevent apoptosis induced by p53 (21, 32). Higher levels of cytokines, including IL-6 and IL-12, could be due to resistance against apoptosis induced by LPS. However, we did not observe strong differences in the viability among BMDCs from three strains.

Molecular mechanism how Ifi202 up-regulates NF-{kappa}B remains to be investigated. It has been reported that Ifi202 binds to NF-{kappa}B but rather suppresses NF-{kappa}B transcriptional activity (30). We believe that the effect of Ifi202 on NF-{kappa}B is cell type dependent. We did not observe any up-regulation of NF-{kappa}B reporter activity in HEK293-TLR4 cells. Thus, monocyte-specific genes may cooperatively regulate Ifi202 activity on NF-{kappa}B.

It is possible that a gene that is specifically expressed in the NZW strain is involved in the reduction of IL-12 expression. We noticed that the IL-4R{alpha} chain is more highly expressed in NZW-BMDCs than in BWF1-BMDCs (Table 1). Since IL-4 has been shown to be involved in the suppression of macrophage activation and Th2-induction, a high level of IL-4R expression in NZW-DCs may account for Th2-type responses in NZW mice in vivo. However, IL-4 is a maturation factor for BMDCs, and IL-4 was not present in our culture conditions of BMDCs. Therefore, it is unlikely that IL-4 is involved in the suppression of IL-12 expression in NZW-BMDCs.

In conclusion, we found a novel function of Ifi202, which can explain a Th1-prone phenotype of BWF1 mice. This study also supports that Ifi202 could be a therapeutic target in human SLE.


   Acknowledgements
 
We thank T. Yoshioka for technical assistance and Y. Nishi for manuscript preparation. This work was supported by special grants-in-aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan for A.Y. and T.K; the Yamanouchi Foundation for Research on Metabolic Disorders; the Takeda Science Foundation; the Kato Memorial Foundation; the Kanae Foundation for the Promotion of Medical Science and the Uehara Memorial Foundation.


   Abbreviations
 
AS, anti-sense
BMDC, bone marrow-derived dendritic cell
DC, dendritic cell
GM-CSF, granulocyte macrophage colony-stimulating factor
NZB, New Zealand black
NZW, New Zealand white
RT, reverse transcription
SLE, systemic lupus erythematosus
TNF, tumor necrosis

   Notes
 
Transmitting editor: T. Watanabe

Received 24 March 2007, accepted 24 April 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kotzin BL. Systemic lupus erythematosus. Cell (1996) 85:303.[CrossRef][ISI][Medline]
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