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International Immunology Advance Access originally published online on December 22, 2006
International Immunology 2007 19(2):175-183; doi:10.1093/intimm/dxl134
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IL-4R{alpha} polymorphism in regulation of IL-4 synthesis by T cells: implication in susceptibility to a subset of murine lupus

Wakana Shiroiwa1,2, Kazuyuki Tsukamoto1, Mareki Ohtsuji1, Qingshun Lin1,2, Akinori Ida1, Sanki Kodera1, Naomi Ohtsuji1, Kazuhiro Nakamura1, Hiromichi Tsurui1, Katsuyuki Kinoshita2, Hiroyuki Nishimura3, Toshikazu Shirai1 and Sachiko Hirose1

1 Department of Pathology
2 Department of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
3 Toin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, Yokohama, Japan

Correspondence to: S. Hirose; E-mail: sacchi{at}med.juntendo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
To thoroughly understand the role of IL-4 in the pathogenesis of systemic lupus erythematosus (SLE), a prototypic antibody-mediated systemic autoimmune disease, we examined the potential of in vitro IL-4 production by anti-CD3 mAb-stimulated splenic T cells in SLE model of NZB, BXSB and related mouse strains. Unexpectedly, both SLE-prone NZB and BXSB mice had a limited potential to produce IL-4, while disease-free NZW mice had a high potential. Levels in (NZB x NZW) F1 and (NZW x BXSB) F1 were in between. Genome-wide search for quantitative trait loci (QTL) controlling this variation identified a single significant QTL in the vicinity of IL-4R{alpha} gene on chromosome 7. Sequence analysis of IL-4R{alpha} cDNA revealed that there are 17 nucleotide substitutions resulting in eight amino acid changes between NZB and NZW strains. BXSB showed the identical sequence, as did NZB. Thus, it was suggested that the NZW-type polymorphism controls a high potential and the NZB/BXSB-type polymorphism controls a low potential for IL-4 production by T cells. Linkage studies using NZW x (NZW x BXSB) F1 male and (NZB x NZW) F1 x NZW female back-cross mice revealed that the BXSB/NZB-type IL-4R{alpha} polymorphism significantly linked to BXSB, but not to (NZB x NZW) F1 lupus. Thus, the low IL-4-producing phenotype appears to predispose to SLE in BXSB, but not NZB-related strains, suggesting that the role of IL-4 in the pathogenesis may differ between certain subsets of SLE, even if they show similar disease phenotypes.

Keywords: CD4+ T cells, quantitative trait loci, systemic lupus erythematosus, Yaa gene


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
IL-4 is a pleiotropic type I cytokine produced by T cells, mast cells, basophils and NKT cells (1, 2). IL-4 plays a central role in regulating the differentiation of antigen-stimulated naive T cells to develop into IL-4-produing Th2 through IL-4R-mediated signaling (3). Th2 also produce other cytokines such as IL-5, IL-6 and IL-10, and play an important role in humoral immune responses (4). IL-4 antagonizes the macrophage-activating effect of IFN{gamma} secreted by Th1, and thus inhibits cell-mediated immune responses (5). This may be one of the mechanisms by which Th2 function as inhibitors of Th1-mediated immune inflammation.

A wide variety of cell types express IL-4R (3), which is composed of IL-4R{alpha} chain, a member of the type I cytokine receptor family, and a common gamma chain shared with IL-2R (6). IL-4R{alpha} binds IL-4 with high affinity and initiates signaling cascades through activation of the Janus family tyrosine kinases (3). Polymorphisms of IL-4R{alpha} have been reported in both humans (7–12) and mice (13). In humans, the genetic polymorphism with heightened responsiveness to IL-4 was associated with susceptibilities to atopic diseases (7, 8), type 1 diabetes (9, 10), multiple sclerosis (11) and systemic lupus erythematosus (SLE) (12). In mice, sequence analysis of IL-4R{alpha} cDNA revealed two allotypes of IL-4R{alpha}, i.e. BALB/c type and C57BL/6 type, with eight amino acid substitutions, leading to differences in the dissociation rate of IL-4 (13). Baker et al. (14) showed that susceptibility to experimental allergic encephalomyelitis (EAE) was most tightly linked to the interval linked to IL-4R{alpha} gene. In our earlier studies, one of the alleles responsible for anti-phospholipid syndrome, a characteristic disease feature of (NZW x BXSB) F1 mice (15), was linked to the IL-4R{alpha} gene (16). Collectively, it is highly plausible that the IL-4R{alpha} polymorphism may predispose to some autoimmune diseases.

Other molecules possibly involved in production of and/or responsiveness to IL-4 are T cell membrane proteins encoded by T cell Ig mucine (Tim) family genes (17, 18). Th1 and Th2 express Tim-3 and Tim-1, respectively, and thus, interaction of Tim proteins and their ligands is suggested to be involved in regulation of Th1–Th2 balance. Intriguingly, there are notable polymorphisms in genes encoding Tim-1 and Tim-3 (17, 18), and such polymorphisms in humans were reported to be linked to asthma and other allergic diseases (17). In studies of the mouse strains congenic for the Tim-containing interval, evidence showed that the allelic variation in the Tim family genes controls the production of IL-4 by T cells and that the genotype for high IL-4 production is linked to the airway hyperreactivity (19).

The Th1–Th2 balance may be an important immunologic factor in the pathogenesis of autoimmune diseases. There were reports showing that cell-mediated autoimmune diseases, such as EAE and insulin-dependent diabetes mellitus, are exacerbated by Th1 polarization, while IL-4-mediated Th2 polarization ameliorates these diseases (20–22). However, in case of SLE, a prototypic antibody-mediated autoimmune disease, roles of Th1-type and Th2-type cytokines have been controversial, and opposite results were reported in studies using two separate murine SLE models such as (NZB x NZW) F1 and BXSB mice (23, 24).

(NZB x NZW) F1 mice spontaneously develop a systemic autoimmune disease closely resembling human SLE in female predominance (25). They produce high-affinity IgG auto-antibodies to a variety of nuclear components and deposition of formed immune complexes (ICs) causes tissue inflammation, including severe lupus nephritis. There is evidence that CD4+ T cells play a critical role for production of high-affinity IgG anti-DNA antibodies in (NZB x NZW) F1 mice (26, 27). When we treated these mice with mAbs to IL-4 and to IL-12, a Th1 inducer (28), the former, but not the latter, ameliorated the disease (23). Others also reported that administration of mAb to IL-6 and IL-10 markedly delayed the onset of disease (29, 30), suggesting that the predominance of Th2 response underlies the (NZB x NZW) F1 disease. However, as there is a report indicating that neutralization of IFN{gamma} ameliorated the (NZB x NZW) F1 disease (31), the regulatory role of Th1-type and Th2-type cytokines for SLE in these mice appears more complex.

BXSB mice, another SLE model, develop IC-type lupus nephritis in association with the production of various auto-antibodies, as found in (NZB x NZW) F1 mice. In contrast to (NZB x NZW) F1 mice, however, the disease occurs much earlier and is more severe in males due to the involvement of a mutant Y chromosome-linked autoimmune acceleration gene (Yaa) (32). The exact role of Yaa is yet unknown; however, Yaa was shown to enhance overall humoral autoimmune responses and promote Th1 responses over Th2 against self-antigens (33, 34). There is evidence that IL-4-deficient BXSB mice did develop as severe lupus nephritis as that seen in wild-type BXSB mice (24), and that the constitutive expression of the IL-4 transgene in B cells protected against the lupus nephritis in the Yaa-induced lupus model (35). Taken collectively, it appears that the role of IL-4 in the pathogenesis of SLE may differ between Yaa-induced lupus and (NZB x NZW) F1 lupus.

In the present studies, we found that the potential of naive T cells to differentiate into IL-4-producing Th2 is tightly linked to IL-4R{alpha} polymorphism in mice. Based on this finding, we then examined the role of IL-4 in the pathogenesis of SLE in BXSB-related and NZB-related mouse strains, using linkage studies. Our data strongly support the idea that there are subsets of SLE, in which IL-4 dependency in the pathogenesis differs.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
NZB, NZW, (NZB x NZW) F1, BXSB, (NZW x BXSB) F1, BALB/c and C57BL/6 mice, originally obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan), were maintained in our animal facility. Back-cross mice were obtained by crossing female (NZB x NZW) F1 mice with male NZW mice or by crossing female NZW mice with male (NZW x BXSB) F1 mice. All mice used were housed under identical conditions and all experiments were performed in accordance with our institutional guidelines. Male mice were used in studies with BXSB and their crosses with Yaa allele. For analysis of (NZB x NZW) F1 disease, female mice were used because of female predominance of the disease.

In vitro cytokine production and proliferation assay
Splenic T cells were sorted using flow cytometry, as followed. Spleen cells were double stained with {alpha}-galactosyl ceramide ({alpha}-GalCer)-loaded mouse dimeric CD1d:IgG (DimerX I, BD PharMingen, San Jose, CA, USA), followed by FITC-labeled anti-mouse IgG and allophycocyanin-labeled anti-CD3 mAb, and cells positive for CD3 and negative for CD1d dimmer were sorted. An aliquot of 5 x 106 spleen cells or sorted splenic T cells per well was cultured in vitro in 96-well flat-bottomed plates pre-coated with anti-CD3 mAb (10 µg ml–1) in RPMI1640 containing 10% FCS, L-glutamine, penicillin–streptomycin, 2-mercaptoethanol and HEPES. Culture supernatants were harvested 2 days after stimulation, and IL-4 and IFN{gamma} levels were assayed using the ELISA system (BD PharMingen), according to manufacturer's instructions. Proliferative responses of splenic T cells in response to immobilized anti-CD3 mAbs were determined based on the levels of [3H]thymidine (1 µCi per well) incorporated in the last 12 h of a 3-day culture.

Genotyping for microsatellite markers and PCR–SSCP for IL-4R{alpha} polymorphism
DNA was extracted from the mouse tail tissue. Genotyping for microsatellite markers was done using PCR. Microsatellite primers were purchased from Research Genetics (Huntsville, AL, USA). PCRs were run in a 20-µl volume containing 40 ng of genomic DNA. A three-temperature PCR protocol (94, 65 and 72°C) was used for 35 cycles in a GeneAmp 9800 Thermal Cycler (PerkinElmer Applied Biosystems, Warrington, UK). PCR products were diluted 2-fold with loading buffer consisting of xylene cyanol and bromophenol blue dyes in 50% glycerin and were electrophoresed on 18% polyacrylamide gels. After electrophoresis, products in the gels were visualized with ethidium bromide staining.

For genotyping of IL-4R{alpha} polymorphism, PCR–SSCP was done using primers designed to amplify fragments from nucleotide positions 1087 to 1261. The 5' and 3' primers used were 5'-CGCTGTATGGAGCTGTTTGA-3' (positions 1087–1106) and 5'-CCTCCAACAAGTCGGAAAAC-3' (positions 1242–1261), respectively. After the initial denaturation at 95°C for 5 min, PCR was done using a three-temperature protocol (95, 55 and 72°C) for 40 cycles, with GeneAmp reagents and AmpliTaq Gold DNA polymerase (PerkinElmer Applied Biosystems). PCR products were denaturated at 98°C for 10 min, immediately cooled on ice and electrophoresed on a 10% polyacrylamide gel in 0.5x TBE buffer at a constant current at 10°C for 2 h. Single-stranded DNA fragments in the gel were visualized with silver staining.

Sequencing of IL-4R{alpha} gene
Total RNA was isolated from spleen cells using ISOGEN (Nippon Gene Co. Ltd, Tokyo, Japan). First-strand cDNA was synthesized using a random hexamer, and nucleotide sequence analysis of IL-4R cDNA was done using a BigDye terminator cycle sequencing ready reaction kit (PerkinElmer Applied Biosystems) as recommended by the manufacturer. The primer pairs used were as follows: forward 5'-ATGGGGCGGCTTTGCACC-3' (positions 19–43) and reverse 5'-CCTGAGCATGTCACCTGAGA-3' (positions 1176–1195); forward 5'-CATCATTCAGGATGCACAGG-3' (positions 853–871) and reverse 5'-GAGTTTGTGCAGGCAGTGAA-3' (positions 1747–1766); forward 5'-AAGAAGAGGAGCCTCCAAGC-3' (positions 1598–1617) and reverse 5'-GTCACTCTCCTGTTCCCAGC-3' (positions 2384–2403).

Measurement of proteinuria
The onset of renal disease was monitored by biweekly testing for proteinuria, as described (36). Due to the contamination of semen, background levels of total urinary proteins in male mice were higher than those in female mice. Thus, female mice with urinary proteins of 111 mg per 100 ml or more and male mice with those of 333 mg per 100 ml or more in repeated tests were considered as having a positive proteinuria.

Histopathology and tissue immunofluorescence
For histopathological examination, kidney tissues were fixed in 4% PFA, embedded in paraffin, sectioned 4-µm thick and stained with periodic acid–Schiff and hematoxylin. For immunofluorescence examination, kidney tissues were embedded in Tissue-Tek OCT compound and frozen in liquid nitrogen. Frozen kidney sections were stained with Alexa 488-labeled macrophage-specific mAb F4/80 (37) (Dainippon Sumitomo Pharma, Osaka, Japan) for 60 min at room temperature. Color images were obtained using laser scanning microscopy (LSM 510 META Ver. 3.2, Carl Zeiss Co. Ltd, Germany). Images for F4/80 were obtained using FITC channel and images for auto-fluorescence were obtained using rhodamine channel, as a counter stain to define the tissue structure. Final images were obtained by superimposing those images, assigning green to FITC and red to rhodamine.

Statistics
The quantitative trait loci (QTL) analysis for the amount of IL-4 produced in vitro by anti-CD3 mAb-stimulated spleen cells was done using the computer programs of MAPMAKER/EXP and MAPMAKER/QTL. The likelihood ratio statistics (base-10 LOD score) of ≥1.9 and ≥3.3 were used as the threshold for statistically suggestive and significant linkage, respectively. Student's t-test and the {chi}2 test were used for comparisons of cytokine levels and of the incidence of proteinuria, respectively. P values of <5% were considered to have statistical significance.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In vitro cytokine production by anti-CD3 mAb-stimulated splenic T cells from SLE-prone and related mouse strains
To compare the potential to produce IL-4 of FACS-sorted splenic T cells among 2-month-old NZB, NZW, BXSB and their F1 hybrid mice, splenic T cells were first stimulated with immobilized anti-CD3 mAbs in vitro, and amounts of IL-4 in culture supernatants were measured using ELISA (Fig. 1). Unexpectedly, T cells from lupus-prone NZB and BXSB mice showed a limited potential to produce IL-4. In contrast, NZW T cells produced a large amount of IL-4. Amounts produced by (NZB x NZW) F1 or (NZW x BXSB) F1 T cells were intermediate between the parental T cells. The results did not differ significantly between studies using sorted T cells and whole spleen cells (data not shown). There were no significant differences in amounts of IFN{gamma} produced in the same culture condition among NZB, NZW and BXSB T cells. Proliferative responses of anti-CD3 mAb-stimulated T cells also did not differ significantly among these strains of mice.


Figure 1
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  		Fig. 1. Amounts of IL-4 and IFN{gamma} produced in vitro by splenic T cells and their proliferative responses, when stimulated with immobilized anti-CD3 mAb. FACS-sorted splenic T cells from 2- to 3-month old mice were cultured in vitro in anti-CD3 mAb-coated culture plates for 2 days and the amounts of IL-4 and IFN{gamma} in culture supernatants were analyzed. Proliferative responses to anti-CD3 mAb stimulation were determined based on the levels of [3H]thymidine incorporation. Mean and SE in triplicate culture are shown. Results are representative of three independent experiments. Asterisks indicate a statistical significance (*P < 0.05, **P < 0.01).

 
QTL controlling the potential for IL-4 production
To determine the genetic basis of the variation in IL-4-producing potential, we examined the amount of IL-4 secreted in cultures of immobilized anti-CD3 mAb-stimulated spleen cells obtained from 142 (NZB x NZW) F1 x NZW back-cross mice at 2 months of age. Genome-wide QTL analysis, using 117 polymorphic microsatellite markers, revealed a single significant QTL on chromosome 7 (Fig. 2A), with a peak LOD score of 4.3, locating between polymorphic microsatellite markers, D7Mit255 (61 cM from the centromere) and D7Mit103 (63.5 cM), in the close vicinity of IL-4R{alpha} gene (62 cM) (Fig. 2B).


Figure 2
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  		Fig. 2. QTL analysis for the amount of IL-4 produced in vitro by anti-CD3 mAb-stimulated spleen cells in 142 (NZB xNZW) F1 x NZW back-cross mice. (A) Genome-wide QTL scans on each chromosome except sex chromosome. Dotted lines show LOD score with suggestive linkage (LOD 1.9) and with significant linkage (LOD 3.3). (B) QTL scans of chromosome 7. The LOD score curve is shown on the right with the scale at the bottom. Map positions of microsatellite markers are arranged from the centromere to the telomere on the left of the chromosome line.

 
IL-4R{alpha} gene polymorphism
Given that the IL-4R{alpha} gene per se acts to control the IL-4-producing potential of spleen cells, IL-4R{alpha} gene must be polymorphic among NZB, NZW and BXSB strains. To clarify this issue, nucleotides of the IL-4R{alpha} cDNAs in these mouse strains were sequenced. As shown in Fig. 3(A), there are 12 exons in the IL-4R{alpha} gene (38), and due to 17 nucleotide substitutions in the coding regions, there were two allotypes in IL-4R{alpha} with three amino acid substitutions in extracellular and five in cytoplasmic domains, in keeping with the report of Schulte et al. (13). The sequence of the IL-4R{alpha} cDNA of NZW was identical to that of BALB/c (BALB/c type), and the sequences of NZB and BXSB were identical to that of C57BL/6 (C57BL/6 type), except for a single-nucleotide change in position 954 (G in the former and A in the latter) with no amino acid substitution. Thus, using primers covering nucleotide positions from 1086 to 1261, NZW/BALB/c-type and NZB/BXSB/C57BL/6-type IL-4R{alpha} polymorphisms were identified by PCR–SSCP, as shown in Fig. 3(B).


Figure 3
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  		Fig. 3. IL-4R{alpha} gene polymorphism. (A) Structure of IL-4R{alpha} gene and comparison of IL-4R{alpha} cDNA sequences among various mouse strains. (B) PCR–SSCP for polymorphic IL-4R{alpha} gene. PCR was performed with primer pairs covering nucleotide positions from 1087 to 1261. PCR–SSCP was carried out in various mouse strains, as described in Methods.

 
Correlation between IL-4R{alpha} polymorphism and IL-4 production
To our knowledge, there have been no reports documenting the correlation between IL-4-producing potential of T cells in response to signals via TCR and the IL-4R{alpha} polymorphism. We therefore compared the amounts of IL-4 produced in vitro by FACS-sorted, immobilized anti-CD3 mAb-stimulated splenic T cells from BALB/c, NZW, C57BL/6, NZB and BXSB strains of mice. As shown in Fig. 4(A), BALB/c and NZW belonged to a high IL-4 producer and C57BL/6, NZB and BXSB belonged to a low IL-4 producer. To further confirm the correlation between the IL-4 production and the IL-4R{alpha} polymorphism, we examined IL-4R{alpha} genotypes and the IL-4-producing potential of spleen cells from 142 (NZB x NZW) F1 x NZW and 30 NZW x (NZW x BXSB) F1 back-cross mice. As shown in Fig. 4(B and C), the progeny with the heterozygous NZB/NZW and the NZW/BXSB genotypes for IL-4R{alpha} produced a significantly lower amount of IL-4 than did the progeny with the homozygous NZW/NZW genotype.


Figure 4
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  		Fig. 4. Association of IL-4R{alpha} gene polymorphism with in vitro IL-4 production. (A) Comparisons of IL-4-producing potential of FACS-sorted splenic T cells stimulated with anti-CD3 mAb between mouse strains aged 2–3 months with either BXSB-type or C57BL/6-type IL-4R{alpha} polymorphism. Mean and SE in triplicate culture are shown. Representative results of three independent experiments are shown. (B) Comparisons of IL-4-producing potential of spleen cells stimulated with anti-CD3 mAb between the two groups of (NZB x NZW) F1 x NZW back-cross progeny with NZW/NZW homozygous and NZB/NZW heterozygous genotypes of IL-4R{alpha} polymorphism. Horizontal bars represent mean and SD. (C) Comparisons of IL-4-producing potential of spleen cells stimulated with anti-CD3 mAb between the two groups of NZW x (NZW x BXSB) F1 back-cross progeny with homozygous NZW/NZW-type and heterozygous NZW/BXSB-type IL-4R{alpha} polymorphism. Horizontal bars represent mean and SD.

 
Association between lupus nephritis and IL-4R{alpha} polymorphism
To examine the possible involvement of the IL-4R{alpha} polymorphism in susceptibility to lupus nephritis, we examined the association between positive proteinuria and several microsatellite markers including the IL-4R{alpha} genotype on chromosome 7. In studies using 219 (NZB x NZW) F1 x NZW back-cross and 117 NZW x (NZW x BXSB) F1 back-cross mice, although there was no significant association in the former back-cross progeny (data not shown), significant association was observed in the latter, with a peak {chi}2 value of 15.125 at the IL-4R{alpha} gene (Table 1). Figure 5 compares the cumulative incidence of proteinuria sequentially examined in NZW x (NZW x BXSB) F1 back-cross progeny with separate IL-4R{alpha} polymorphisms. The progeny carrying the heterozygous NZW/BXSB genotype for IL-4R{alpha} with the low IL-4-producing potential showed a significantly higher incidence of proteinuria than did the progeny with the homozygous NZW/NZW genotype after 4 months of age onward.


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  		Table 1. Association of positive proteinuria and IL-4R{alpha} locus on chromosome 7 in NZW x (NZW x BXSB) F1 back-cross progeny

 

Figure 5
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  		Fig. 5. Association of IL-4R{alpha} gene polymorphism with the cumulative incidence of proteinuria in 117 NZW x (NZW x BXSB) F1 back-cross mice. Mice were separated into two groups, i.e. NZW/BXSB heterozygotes and NZW/NZW homozygotes for the genotype of IL-4R{alpha} gene polymorphism, and the incidence of proteinuria was sequentially compared between the two groups. The number of mice examined is shown in parentheses. Asterisks indicate statistical significance (*P < 0.005, **P < 0.0001).

 
Similar studies were carried out on the association between the serum level of IgG anti-DNA antibodies and several microsatellite markers on chromosome 7. However, there was no significant association in both NZW x (NZW x BXSB) F1 back-cross and (NZB x NZW) F1 x NZW back-cross mice (data not shown).

Difference in glomerular immunopathology of lupus nephritis between (NZB x NZW) F1 and BXSB mice
In histopathology, as shown in Fig. 6, glomerular changes in lupus nephritis are basically similar between (NZB x NZW) F1 and BXSB mice, showing diffuse glomerulonephritis with mesangiocapillary proliferation and PAS-positive mesangial matrices in enlarged glomeruli. However, one noticeable difference was observed in tissue immunofluorescent studies. In BXSB mice, there were considerable numbers of F4/80+ macrophages mainly infiltrating into the mesangial area of an affected glomerulus, while in (NZB x NZW) F1 mice, they were scarcely found within the glomerulus, but instead abundant in pericapsular inflammatory sites.


Figure 6
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  		Fig. 6. Histopathological periodic acid–Schiff and hematoxylin staining (PASH) and tissue immunofluorescent findings (F4/80 staining) of renal glomeruli in 6-month-old mice. Histopathologically, glomeruli of both (NZB x NZW) F1 and BXSB mice show diffuse mesangiocapillary lupus glomerulonephritis. Note that, in immunofluorescence, F4/80+ macrophages are infiltrated into the inflamed glomerulus of BXSB, while they are virtually absent inside the glomerulus, and localized in pericapsular inflammatory regions in (NZB x NZW) F1 mice. The bar in the histopathology figures represents 50 µm. Representative figures obtained from three mice in each strain are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In studies of the role of IL-4 in the pathogenesis of SLE, we found that the potential of T cells to produce IL-4, upon stimulation with immobilized anti-CD3 mAbs, was markedly down-regulated in SLE-prone BXSB and NZB mouse strains, while their potential to produce IFN{gamma} was intact. In contrast, T cells from the normal strain NZW had high potential to produce both IL-4 and IFN{gamma}. In genome-wide screening, a single QTL tightly linked to IL-4R{alpha} gene on chromosome 7 was found to control such T cell potential.

The IL-4R{alpha} gene was reported to be polymorphic between Th1-polarized C57BL/6 and Th2-polarlized BALB/c mice (13). Our sequence analysis of IL-4R{alpha} cDNA showed that NZW mice had the sequence identical to that of BALB/c, while SLE-prone NZB and BXSB mice had the sequence identical to that of C57BL/6, except for a single-nucleotide change with no amino acid substitution. Because the dissociation rate of IL-4 differs between the polymorphic IL-4R structures (13), the polymorphic IL-4R{alpha} gene may differentially control the response of CD4+ T cells to IL-4 and regulate their IL-4-producing potential.

Mclntire et al. (19) reported that polymorphic Tim family genes are involved in regulation of IL-4 synthesis by T cells, and that the polymorphism controlling high IL-4 synthesis is associated with airway hyperreactivity in mice. In our studies, however, there was no significant linkage between the IL-4-producing potential of T cells in response to immobilized anti-CD3 mAbs and the chromosomal interval containing Tim family genes on chromosome 11 (Fig. 2A). The exact reason for this discrepancy is unknown; however, Mclntire et al. (19) measured IL-4-producing potential of lymph node T cells prepared from keyhole limpet hemocyanin (KLH)-immunized mice in vitro in the presence of KLH, whereas we examined the potential of anti-CD3 mAb-stimulated splenic T cells from untreated mice. It may be possible that signals through polymorphic Tim molecules regulate the potential of IL-4 synthesis by antigen-primed CD4+ T cells, whereas IL-4R-mediated signals regulate the differentiation of naive T cells into IL-4-producing Th2. This is in agreement with the finding of Noben-Trauth et al. (39) that CD4+ T cells from IL-4R{alpha}-deficient mice display a strikingly diminished capacity to produce IL-4 upon stimulation with immobilized anti-CD3 mAbs. Thus, in response to signals through TCRs, naive CD4+ T cells may differentiate into T cells with potential to produce IL-4, where IL-4 functions as an autocrine growth factor to generate mature Th2 through IL-4R-mediated signaling. Our present studies suggested that this process is affected by the polymorphic IL-4R.

Upon stimulation with immobilized anti-CD3 mAbs, not only Th2 but also NKT cells may be activated to produce IL-4. Taniguchi et al. (40) provided evidence that NKT cells produce large amounts of IL-4 and IFN{gamma}, soon after activation with {alpha}-GalCer or anti-CD3 mAb in vitro, and that repeated in vivo administrations of {alpha}-GalCer in mice result in Th2 polarization (40). However, in contrast to Th2, NKT cells can secrete considerable amounts of IL-4 in IL-4R{alpha}-deficient mice (39). Thus, it seems likely that the IL-4R{alpha} polymorphism regulates CD4+ T cells, but not NKT cells, to differentiate into IL-4-producing Th2. This notion is in keeping with the result of our genome-wide analysis showing that variations in amounts of IL-4 produced by {alpha}-GalCer-stimulated NKT cells in mice was not linked to the polymorphic IL-4R{alpha} locus on chromosome 7 (K. Tsukamoto, submitted for publication).

The present study is the first to show the genetic linkage between the SLE susceptibility and the IL-4R{alpha} polymorphism with a low IL-4-producing phenotype. Significance of this linkage was observed in SLE particularly of Yaa-bearing BXSB and related mouse strains, suggesting that Th1 mainly contribute to the disease in BXSB-related mice. This notion is consistent with the finding that IL-4-deficient mutant BXSB and wild-type BXSB mice both develop indistinguishable disease (24). Evidence showed that the transgene-mediated over-expression of IL-4 ameliorates the disease in (NZW x C57BL/6.Yaa) F1 mice, another Yaa-dependent SLE model (35). Thus, it is highly plausible that the Th1-type response is preferentially responsible for the Yaa-related lupus, while IL-4-mediated Th2 polarization is protective. This protective mechanism was suggested to be due to the IgG subclass deviation of auto-antibodies in Yaa-induced disease. Th2 are very efficient helper cells for production of antibodies especially of IgG1 and IgE, while Th1 can encourage B cells to produce IgG2a and IgG3 (41). Indeed, Yaa-related lupus models have high serum levels of IgG2a and IgG3 (33, 34), with nephritogenic potential (42).

In the present studies, however, there was no significant association between serum anti-DNA antibody levels and the BXSB genotype for IL-4R{alpha} polymorphism (data not shown). The reason for this lack of association remains unknown. The low IL-4-producing phenotype may be unlinked to the auto-antibody production, or its influence may be under the threshold for identification, because levels of the pathogenic high-affinity auto-antibody production in SLE are controlled by additional susceptibility genes including the MHC class II haplotype (34, 43). In any instance, however, our present studies suggested that the low IL-4-producing phenotype is related to the increase in macrophage function in BXSB-related mice. One unique immunological abnormality in Yaa-induced disease is an age-associated increase in the monocyte/macrophage population, in parallel with disease progression (44–46). The present tissue immunofluorescence studies revealed the infiltration of significant numbers of F4/80+ macrophages within the inflamed glomeruli of BXSB mice, in keeping with the observation in SLE-prone MRL/lpr mice (47). However, these intraglomerular infiltrates were virtually absent in (NZB x NZW) F1 mice, although they were abundant in pericapsular parenchymal tissues. Thus, the effector phase mechanism of glomerular injury probably differs between these two mouse strains, and macrophage-derived inflammatory cytokines may be more critical in the pathogenesis of glomerulonephritis in Yaa-bearing BXSB than in (NZB x NZW) F1 mice. It appears that the down-regulation of IL-4 synthesis in BXSB-related mouse strains promotes such macrophage-mediated glomerular injuries, while its up-regulation protects against the disease, as evidenced by studies of IL-4-transgenic (NZW x C57BL/6.Yaa) F1 mice (35).

In contrast to the findings in BXSB-related mice, there was no significant linkage between the IL-4R{alpha} polymorphism with a low IL-4-producing phenotype and the lupus nephritis in (NZB x NZW) F1 mice. Erb et al. (48) reported that the transgene expression of IL-4 leads to the expansion of autoreactive B cells, resulting in the increased production of auto-antibodies and autoimmune-type kidney damages in mice with C3H background. Thus, IL-4 may play a role in the pathogenesis of a certain subset of SLE. The finding that treatment with anti-IL-4 mAb prevented the onset of SLE in (NZB x NZW) F1 mice (23) may suggest the involvement of IL-4 in the pathogenesis of lupus in these mice. Although the IL-4R{alpha} polymorphism for low IL-4 phenotype did not associate with SLE in (NZB x NZW) F1 mice in the present studies, a more precise analysis may be needed to clarify the disease correlation with high IL-4 phenotype. There is indeed a report indicating that the IL-4R{alpha} polymorphism with increased receptor function was associated with SLE in human subjects (12). However, the pathogenesis of (NZB x NZW) F1 lupus appears more complex in terms of the Th1–Th2 balance, because neutralization of IFN{gamma} was reported to ameliorate the (NZB x NZW) F1 disease (31). Importantly, (NZB x NZW) F1 mice produce high-affinity anti-DNA antibodies mainly of IgG2a and IgG2b, but not IgG1 and IgG3 (49, 50); thus, neither Th1 nor Th2 alone may be responsible for Ig subclass switching and affinity maturation of self-reactive B cells.

SLE is a complex, multigenic autoimmune disease with a wide spectrum of clinical manifestations and immunological abnormalities, in which a different set of susceptibility and modifying genes separately controls each specific aspect of diverse SLE phenotypes (51, 52). Our present studies provided evidence that the genetic mechanism for cytokine dependency of SLE is variable in different subsets of SLE. This observation added a notion that the same SLE phenotype such as high-affinity pathogenic auto-antibodies and lupus nephritis in different individuals may develop under the control of different sets of susceptibility genes. One must note this genetic heterogeneity in studies of SLE susceptibility in different races and ethnic groups.


   Acknowledgements
 
We thank Y. Jiang for helpful contribution in genetic studies on lupus nephritis in (NZB x NZW) F1 x NZW back-cross mice. This work was supported in part by Grant-in-Aid for Scientific Research (B), for Scientific Research on Priority Areas and for Center of Excellence Research from the Ministry of Education, Science, Technology, Sports and Culture, Japan, and grant from The Organization for Pharmaceutical Safety and Research, Japan.


   Abbreviations
 
EAE, experimental allergic encephalomyelitis
{alpha}-GalCer, {alpha}-galactosyl ceramide
IC, immune complex
KLH, keyhole limpet hemocyanin
QTL, quantitative trait loci
SLE, systemic lupus erythematosus
Tim, T cell Ig mucine
Yaa, Y chromosome-linked autoimmune acceleration gene

   Notes
 
Transmitting editor: K. Yamamoto

Received 31 March 2006, accepted 23 November 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Seder RA and Paul WE. (1994) Acquisition of lymphokine-producing phenotype by CD4+ T-cells. Annu. Rev. Immunol. 12:635.[CrossRef][Web of Science][Medline]
  2. Yoshimoto T and Paul WE. (1994) CD4pos, NK1.1pos T-cells promptly produce interleukin-4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179:1285.[Abstract/Free Full Text]
  3. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. (1999) The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17:701.[CrossRef][Web of Science][Medline]
  4. Mosmann TR and Coffman RL. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145.[CrossRef][Web of Science][Medline]
  5. Powrie F and Coffman RL. (1993) Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol. Today 14:270.[CrossRef][Web of Science][Medline]
  6. Kondo M, Takeshita T, Ishii N, et al. (1993) Sharing of the interleukin 2 (IL-2) receptor {gamma} chain between receptors for IL-2 and IL-4. Science 262:1874.[Abstract/Free Full Text]
  7. Hershey GK, Friedrich MF, Esswein LA, Thomas ML, Chatila TA. (1997) The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor. N. Engl. J. Med. 337:1720.[Abstract/Free Full Text]
  8. Mitsuyasu H, Izuhara K, Mao X-Q, et al. (1998) Ile50Val variant of IL-4Ralpha upregulates IgE synthesis and associates with atopic asthma. Nat. Med. 9:119.
  9. Mitel DB, Valdes AM, Lazzeroni LC, Reynolds RL, Erlich HA, Noble JA. (2000) Association of IL-4R haplotypes with type I diabetes. Diabetes 51:3336.
  10. Steck AK, Bugawan TL, Valdes AM, et al. (2005) Association of non-HLA genes with type 1 diabetes autoimmunity. Diabetes 54:2482.[Abstract/Free Full Text]
  11. Mirel DB, Barcellos LF, Wang J, Hauser SL, Oksenberg JR, Erlich HA. (2004) Analysis of IL4R haplotypes in predisposition to multiple sclerosis. Genes Immun. 5:138.[CrossRef][Web of Science][Medline]
  12. Kanemitsu S, Takabayashi A, Sasaki Y, et al. (1999) Association of interleukin-4 receptor and interleukin-4 promoter gene polymorphisms with systemic lupus erythematosus. Arthritis Rheum. 42:1298.[CrossRef][Web of Science][Medline]
  13. Schulte T, Kurrle R, Röllinghoff M, Gessner A. (1997) Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes. J. Exp. Med. 186:1419.[Abstract/Free Full Text]
  14. Baker D, Rasenwasser OA, O'Neill JK, Turk JL. (1995) Genetic analysis of experimental allergic encephalomyelitis in mice. J. Immunol. 155:4046.[Abstract]
  15. Hang LM, Izui S, Dixon FJ. (1981) (NZW x BXSB) F1 hybrid. A model of acute lupus and coronary vascular disease with myocardial infarction. J. Exp. Med. 154:216.[Abstract/Free Full Text]
  16. Ida A, Hirose S, Hamano Y, et al. (1998) Multigenic control of lupus-associated antiphospholipid syndrome in a model of (NZW x BXSB) F1 mice. Eur. J. Immunol. 28:2694.[CrossRef][Web of Science][Medline]
  17. Kuchroo VK, Umetsu DT, DeKruyff RH, Freeman GJ. (2003) The TIM gene family: emerging roles in immunity and disease. Nat. Rev. Immunol. 3:454.[CrossRef][Web of Science][Medline]
  18. Wills-Karp M, Belkaid Y, Karp CL. (2003) I-Tim-izing the pathways of counter-regulation. Nat. Immunol. 4:1050.[CrossRef][Web of Science][Medline]
  19. Mclntire JJ, Umetsu SE, Akbari O, et al. (2001) Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat. Immunol. 2:1109.[CrossRef][Web of Science][Medline]
  20. Liblau RS, Singer SM, McDevitt HO. (1995) Th1 and Th2 CD4+T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol. Today 16:34.[CrossRef][Web of Science][Medline]
  21. O'Garra A, Steinman L, Gijbels K. (1997) CD4+ T-cell subsets in autoimmnity. Curr. Opin. Immunol. 9:872.[CrossRef][Web of Science][Medline]
  22. Hill N and Sarvetnick N. (2002) Cytokines: promotes and dampeners of autoimmunity. Curr. Opin. Immunol. 14:791.[CrossRef][Web of Science][Medline]
  23. Nakajima A, Hirose S, Yagita H, Okumura K. (1997) Roles of IL-4 and IL-12 in the development of lupus in NZB/W F1 mice. J. Immunol. 158:1466.[Abstract]
  24. Kono DH, Balomenos D, Park MS, Theofilopoulos AN. (2000) Development of lupus in BXSB mice is independent of IL-4. J. Immunol. 164:38.[Abstract/Free Full Text]
  25. Howie JB and Helyer BJ. (1968) The immunology and pathology of NZB mice. Adv. Immunol. 9:215.[Medline]
  26. Wofsy D and Seaman WE. (1985) Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4. J. Exp. Med. 161:378.[Abstract/Free Full Text]
  27. Sekigawa I, Ishida Y, Hirose S, Sato H, Shirai T. (1986) Cellular basis of in vitro anti-DNA antibody production: evidence for dependence of IgG-class anti-DNA antibody synthesis in the (NZB x NZW) F1 hybrid. J. Immunol. 136:1247.[Abstract]
  28. Trinchieri G. (1993) Interleukin-12 and its role in the generation of TH1 cells. Immunol. Today 14:335.[CrossRef][Web of Science][Medline]
  29. Finck BK, Chan B, Wofsy D. (1994) Interleukin 6 promotes murine lupus in NZB/NZW F1 mice. J. Clin. Invest. 94:585.[Web of Science][Medline]
  30. Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M. (1994) Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J. Exp. Med. 179:305.[Abstract/Free Full Text]
  31. Jacob CO, van der Meide PH, McDevitt HO. (1987) In vivo treatment of (NZB x NZW) F1 lupus-like nephritis with monoclonal antibody to {gamma}-interferon. J. Exp. Med. 166:798.[Abstract/Free Full Text]
  32. Andrew BS, Eisenberg RA, Theofilopoulos AN, et al. (1978) Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148:1198.[Abstract/Free Full Text]
  33. Takahashi S, Fossati L, Iwamoto M, et al. (1996) Imbalance towards Th1 predominance is associated with acceleration of lupus-like autoimmune syndrome in MRL mice. J. Clin. Invest. 97:1597.[Web of Science][Medline]
  34. Izui S, Ibnou-Zekri N, Fossati-Jimack L, Iwamoto M. (2000) Lessons from BXSB and related mouse models. Int. Rev. Immunol. 19:447.[Medline]
  35. Santiago M-L, Fossati L, Jacquet C, Müller W, Izui S, Reininger L. (1997) Interleukin-4 protects against a genetically linked lupus-like autoimmune syndrome. J. Exp. Med. 185:65.[Abstract/Free Full Text]
  36. Miura-Shimura Y, Nakamura K, Ohtsuji M, et al. (2002) C1q regulatory region polymorphism down-regulating murine C1q protein levels with linkage to lupus nephritis. J. Immunol. 169:1334.[Abstract/Free Full Text]
  37. Austyn JM and Gordon S. (1981) F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11:805.[Web of Science][Medline]
  38. Mosley B, Beckmann MP, March CJ, et al. (1989) The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 59:335.[CrossRef][Web of Science][Medline]
  39. Noben-Trauth N, Shultz LD, Brombacher F, Urban JF Jr, Gu H, Paul WE. (1997) An interleukin 4 (IL-4)-independent pathway for CD4+T cell. IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. Natl Acad. Sci. USA 94:10838.[Abstract/Free Full Text]
  40. Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. (2003) The regulatory roles of V{alpha}14 NKT cells in innate and acquired immune response. Annu. Rev. Immunol. 21:483.[CrossRef][Web of Science][Medline]
  41. Snapperm CM and Mond JJ. (1993) Towards a comprehensive view of immunoglobulin class switching. Immunol. Today 14:15.[CrossRef][Web of Science][Medline]
  42. Lemoine R, Bemey T, Shibata T, et al. (1992) Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins. Kidney Int. 41:65.[Web of Science][Medline]
  43. Hirose S, Jiang Y, Hamano Y, Shirai T. (2000) Genetic aspects of inherent B-cell abnormalities associated with SLE and B-cell malignancy: lessons from New Zealand mouse models. Int. Rev. Immunol. 19:389.[Medline]
  44. Wofsy D, Kerger CE, Seaman WE. (1984) Monocytosis in the BXSB model for systemic lupus erythematosus. J. Exp. Med. 159:629.[Abstract/Free Full Text]
  45. Merino R, Fossati L, Lacour M, Lemoine R, Higaki M, Izui S. (1992) H-2-linked control of the Yaa gene-induced acceleration of lupus-like autoimmune disease in BXSB mice. Eur. J. Immunol. 22:295.[Web of Science][Medline]
  46. Amano H, Amano E, Santiago-Raber M-L, et al. (2005) Selective expansion of a monocyte subset expressing the CD11c dendritic cell marker in the Yaa model of systemic lupus erythematosus. Arthritis Rheum. 52:2790.[CrossRef][Web of Science][Medline]
  47. Itoh J, Nose M, Takahashi S, et al. (1993) Induction of diffuse types of glomerulonephritis by monoclonal antibodies derived from MRL/lpr lupus mouse. Am. J. Pathol. 143:1436.[Abstract]
  48. Erb KJ, Rüger B, von Brevern M, Ryffel B, Schimpl A, Rivett K. (1997) Constitutive expression of interleukin (IL)-4 in vivo causes autoimmune-type disorders in mice. J. Exp. Med. 185:329.[Abstract/Free Full Text]
  49. Hirose S, Wakiya M, Kawano-Nishi Y, et al. (1993) Somatic diversification and affinity maturation of IgM and IgG anti-DNA antibodies in murine lupus. Eur. J. Immunol. 23:2813.[Web of Science][Medline]
  50. Nakajima A, Azuma M, Kodera S, et al. (1995) Preferential dependence of autoantibody production in murine lupus on CD86 co-stimulatory molecule. Eur. J. Immunol. 25:3060.[Web of Science][Medline]
  51. Shirai T and Hirose S. (2000) Genetics of SLE; a sine qua non for identification. Int. Rev. Immunol. 19:289.[Medline]
  52. Shirai T, Nishimura H, Jiang Y, Hirose S. (2002) Genome screening for susceptibility loci in systemic lupus erythematosus. Am. J. Pharmacogenomics 2:1.[CrossRef][Medline]

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