X-linked immunodeficient mice spontaneously produce lupus-related anti-RNA helicase A autoantibodies, but are resistant to pristane-induced lupus

Minoru Satoh1,2, Akiei Mizutani1, Krista M. Behney1, Yoshiki Kuroda1, Jun Akaogi1, Hideo Yoshida1, Dina C. Nacionales1, Michito Hirakata3, Nobutaka Ono4 and Westley H. Reeves1,2

1 Division of Rheumatology and Clinical Immunology, Department of Medicine, and 2 Department of Pathology, Immunology and Laboratory Medicine, University of Florida,Gainesville, FL 32610-0221, USA 3 Department of Medicine, Keio University School of Medicine, Tokyo 160-0016, Japan 4 First Department of Pathology, Fukushima Medical College, Fukushima 960-1247, Japan

Correspondence to: M. Satoh, Division of Rheumatology and Clinical Immunology, University of Florida, PO Box 100221, Gainesville, FL 32610-0221, USA. E-mail: satohm{at}medicine.ufl.edu
Transmitting editor: K. Yamamoto


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine lupus can occur spontaneously or be induced by hydrocarbons, such as pristane. Spontaneous disease in MRL and NZB/W F1 mice is suppressed by the xid (X-linked immunodeficiency) mutation, which greatly diminishes T cell-independent type 2 responses as well as the number of peritoneal B1 cells. The present study asked whether lupus induced by i.p. injection of pristane likewise is inhibited by the xid defect. Male CBA/N (xid) mice were refractory to the induction of autoantibodies by pristane, whereas 23% of pristane-treated male CBA/CaJ controls produced anti-nRNP/Sm, -Su and/or -OJ (isoleucyl tRNA synthetase) antibodies. Unexpectedly, 43% (12 of 28) of the xid mice spontaneously produced anti-nuclear antibodies that proved highly specific for the lupus antigen RNA helicase A (RHA). Strikingly, this specificity was absent in CBA/CaJ mice (none of 51). Moreover, pristane treatment suppressed the production of anti-RHA antibodies when administered prior to the onset of autoantibody production, but enhanced anti-RHA levels when given after the onset of autoantibody production, suggesting that pristane interferes with anti-RHA production at an early stage. Large amounts of IgG1 anti-RHA autoantibodies were detected in the sera of xid mice, whereas pristane-induced anti-nRNP/Sm and -Su autoantibodies were almost exclusively IgG2a. Cytokine production within the peritoneal cavity reflected the predominant isotypes: IL-12 and IFN-{gamma} predominated in pristane-treated mice, whereas IL-4 and IL-6 were more predominant in untreated xid mice. The spontaneous production of anti-RHA by xid mice and its suppression by pristane treatment at the level of autoantibody induction supports the idea that lupus autoantibodies may be generated through a variety of mechanisms.

Keywords: autoantibodies, autoimmunity, cytokines, inflammation


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mutations of Bruton’s tyrosine kinase (Btk) cause severe B cell immunodeficiency (X-linked agammaglobulinemia, XLA) and profound hypogammaglobulinemia in humans (1). However, the murine Btk mutation xid (X-linked immunodeficiency) results in a less severe immunodeficient state characterized by mild hypogammaglobulinemia, the inability to mount immune responses to a class of T cell-independent antigens (e.g. pneumococcal polysaccharide) and a nearly complete absence of peritoneal B1 cells (1). Lupus is characterized by B cell hyperactivity and autoantibody production (2). Curiously, although there is strong evidence that most of the autoantibodies characteristic of lupus are both T cell dependent and produced by conventional B cells (24), the xid mutation greatly suppresses both autoantibody production and disease in mice prone to spontaneous lupus (5). This is probably explained by the importance of Btk in the maturation of conventional B cells (6).

Paradoxically, B cell immunodeficiency in humans is associated with an increased incidence of antibody-mediated autoimmune diseases (7). Despite extremely low Ig levels, XLA is associated with a dermatomyositis-like syndrome precipitated by chronic echoviral infections (8). The suppression of autoimmunity by Btk mutations in mice and the association of such mutations with autoimmunity in humans could be explained by interspecies differences in the role of Btk in B cell maturation. Alternatively, it could reflect the possibility that different pathways to autoantibody production are variably dependent on Btk. Using an inducible model of lupus initiated by i.p. injection of certain hydrocarbons, such as pristane, we have obtained evidence that there may be more than one pathway to lupus autoantibody production (9). In the present study, we investigated the effect of xid on the pathogenesis of autoantibodies in pristane-induced lupus. The ability of pristane to induce lupus autoantibodies was nearly abrogated by xid. However, immunodeficient CBA/N (xid) mice spontaneously produced high levels of autoantibodies to the double-stranded RNA binding protein RNA helicase A (RHA), a specificity associated with spontaneous lupus in humans (10) as well as NZB/W F1 mice (11). These data strengthen the association of B cell immunodeficiency with humoral autoimmunity and support for the notion that different subsets of lupus-associated autoantibodies are produced by dissimilar mechanisms.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Treatment of mice
Four-week-old male CBA/CaHN-Btkxid/J (CBA/N, xid) and control CBA/CaJ male mice were purchased from Jackson Laboratory (Bar Harbor, ME), and housed in a virus-free conventional animal facility with barrier cages. The CBA/CaJ strain of mice is considered to have the same genetic background as the CBA/N mice and has been used as a control for CBA/N strain (www.jax.org) (12). At 3 months of age, 20–24 mice per group received 0.5 ml i.p. of pristane (Sigma, St Louis, MO) or sterile PBS (13). Sera were collected from the tail vein before injection, at 2 and 4 weeks, and monthly thereafter. Additional mice received the same treatment followed by immunoprecipitation analysis of autoantibodies at a single time point. In other experiments, 10-month-old mice with anti-RHA detectable by immunoprecipitation received either pristane (n = 5) or PBS (n = 3). Sera were obtained before and at monthly intervals after treatment. This study was approved by the IACUC of University of Florida.

Ig levels
Total levels of IgG1, IgG2a, IgG2b, IgG3, IgA and IgM were measured by sandwich ELISA as described (14).

Radiolabeling and immunoprecipitation
Autoantibodies to cellular proteins in murine sera were analyzed by immunoprecipitation of [35S]methionine/cysteine-radiolabeled K562 cell extract using 4 µl murine serum as described (13). Specificity of autoantibodies was confirmed using reference sera for anti-nRNP/Sm, -Su, -OJ and -RHA (15).

Autoantibody ELISAs
A standard protocol for the anti-nRNP/Sm ELISA was used (14). Sera were tested at a 1:500 dilution and OD405 was converted to units using a standard curve based on the anti-U1-70K mAb 2.73 (14). Similarly, anti-RHA antigen capture ELISA was performed using monospecific IgG from a patient with systemic lupus erythematosus (SLE). A standard curve was generated with a prototype anti-RHA positive serum (titer 1:250,000) from an xid mouse. The OD produced by 1:250, 1:2,500, 1:25,000, 1:250,000 and 1:2,500,000 diluted serum was assigned a value of 1000, 100, 10, 1, 0.1 and 0.01 U respectively. Alkaline phosphatase-conjugated rat mAb to mouse IgG1, IgG2a, IgG2b or IgG3 (1:1000; BD PharMingen, San Diego, CA) were used to detect IgG subclass-specific antibodies. To detect total IgG autoantibodies, an equal mixture of each of the anti-subclass antibodies (1:1000 dilution) was used.

Anti-single stranded DNA antibody and -chromatin ELISAs were performed as described using heat-denatured calf thymus DNA (Sigma) and chicken chromatin respectively as antigens (14). OD405 was converted to units based on a standard curve produced by serial dilutions of pooled sera from MRL/lpr mice: 1:500 dilution = 1000 U, 1:5000 = 100 U, 1:50,000 = 10 U, 1:500,000 = 1 U and 1:5,000,000 = 0.1 U. Usually, the standard is clearly positive at a 1:500,000 dilution. Since anti-chromatin antibodies are produced sporadically in some non-autoimmune strains of mice, the mean + 3 SD of 10 blank wells was used as cut-off for positive and negative.

Fluorescent anti-nuclear antibodies (ANA)
Fluorescent ANA of mouse sera were tested using L929 (mouse fibroblast) cells at a dilution of 1:40 as previously described. The fluorescent ANA titer was obtained using the ImageTiter emulation system (RhiGene, Des Plaines, IL) (16).

Cytokine ELISA
ELISAs for IL-4, IL-6, IL-10, IL-12, IL-18, IFN-{gamma} and tumor necrosis factor-{alpha} were performed using antibody pairs for various mouse cytokines (BD PharMingen) (14). After incubation with biotinylated cytokine-specific antibodies, 100 µl/well of 1:1000 streptavidin–alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL) was added for 30 min at 22°C and the reaction developed.

In vitro cytokine production
At 6 months after PBS or pristane treatment (9 months of age), mice were euthanized with CO2 and the peritoneal cavity was lavaged with 5 ml of DMEM 4.5 g/l glucose + 10% FCS and 10 U/ml heparin using a 5-ml plastic syringe and 18-G needles; spleen was also removed. Peritoneal cells were collected by centrifuging at 1200 r.p.m. for 10 min. Spleen was crushed into a Cell Strainer (70 µm Nylon; Falcon, Franklin Lakes, NJ), and then single-cell suspensions of peritoneal and spleen cells were prepared lysing erythrocytes in 0.15 M NH4Cl 1.0 mM, KHCO3 0.1 mM and Na2EDTA, pH 7.4. Samples were cultured in 24-well cell culture plates (106 cells/well/1 ml DMEM, 4.5 g/l glucose, 10% FCS, 10 mM HEPES, 1% glutamine and 0.1% penicillin–streptomycin) without stimulation or in the presence of 10 ng/ml or 10 µg/ml of lipopolysaccharide (LPS; Salmonella Minnesota; Sigma) or 1 µg/ml of anti-CD3 (BD PharMingen) respectively. Samples were harvested after 48 h culture at 37°C with a 5% CO2 atmosphere. Samples were frozen at –80°C until analysis.

Statistical analysis
Frequencies and levels of autoantibodies were compared by Fisher’s exact test (two-sided unless otherwise stated) and the Mann–Whitney test respectively. Changes in levels of anti-RHA following treatment were compared by the Wilcoxon matched-pairs test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Since the xid mutation suppresses autoantibody formation and the development of spontaneous lupus in B/W and MRL/lpr mice, it was of interest to see if this defect had a similar effect in an inducible model of lupus.

CBA mice are susceptible to pristane-induced lupus
Spontaneous production of IgG anti-chromatin and anti-single-stranded DNA antibodies was uncommon in both CBA/N (xid) mice and CBA/CaJ controls (Fig. 1, PBS treatment). Pristane frequently induced IgG anti-chromatin antibodies in CBA/CaJ mice (18 of 19 pristane-treated mice, Fig. 1, top). IgG anti-single-stranded DNA antibodies were also induced (13 of 19 pristane-treated mice, Fig. 1, bottom). This result indicated that CBA/CaJ mice are susceptible to pristane-induced lupus. Pristane also induced IgG anti-chromatin autoantibodies in four of 19 CBA/N (xid) mice (Fig. 1, top). These data suggest that although not completely refractory, xid mice are relatively resistant to the induction of autoantibodies by pristane in comparison with the CBA/CaJ control.



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Fig. 1. Levels of IgG anti-chromatin and -single-stranded DNA antibodies by ELISA. Levels of IgG anti-chromatin and -single-stranded DNA antibodies 6 months after treatment were measured in sera diluted 1:500 (ELISA). Chicken chromatin (1 µg/ml) and heat-denatured calf thymus DNA (3 µg/ml) were used as antigen respectively.

 
Spontaneous ANA production by xid mice
In the fluorescent ANA test, it was found unexpectedly that sera from many PBS-treated CBA/N mice contained ANA that stained the nuclei of L929 cells in a speckled pattern (Fig. 2A–C). In contrast, CBA/CaJ sera were uniformly negative (Fig. 2D). Fifty percent (12 of 24) of PBS-treated CBA/N mice had a positive ANA in a speckled pattern (titer >= 1:80 by titration emulation, Fig. 3 ). In contrast, none of the PBS-treated CBA/CaJ controls had a positive ANA. The frequency and titer of the ANA were not increased by pristane in CBA/N mice, whereas high titers of ANA were induced by pristane in 14 of 19 (74%) of CBA/CaJ mice (Fig. 3). Some pristane-treated xid mice showed a homogeneous peripheral pattern of fluorescence, consistent with the induction of anti-chromatin antibodies (Fig. 1). These observations indicated that xid mice spontaneously produce autoantibodies and suggested that the production of these autoantibodies is not substantially enhanced by pristane treatment.



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Fig. 2. ANA by immunofluorescence. IgG ANA were tested using sera obtained 6 months after the treatment by immunofluorescence using L929 (mouse fibroblast) cells. Many CBA/N mice spontaneously produced ANA in a speckled pattern (A–C), in contrast to negative staining by CBA/CaJ mouse sera (D). Serum dilution, 1:40

 


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Fig. 3. ANA titers in CBA/N (xid) and CBA/CaJ mice. IgG ANA titers (immunofluorescence) were measured using the ImageTiter system. Fifty percent (12 of 24) of CBA/N mice spontaneously produced speckled ANA at a titer of >=1:80. Pristane treatment did not affect the frequency or titer. In contrast, none of the PBS-treated CBA/CaJ control mice produced ANA. Pristane induced ANA in 14 of 19 (74%) of CBA/CaJ mice.

 
Xid mice spontaneously produce autoantibodies to RHA
To identify the ANA produced spontaneously by CBA/N mice, sera were tested by immunoprecipitation (Fig. 4). Ten of 12 ANA+ sera from PBS-treated CBA/N mice immunoprecipitated a 140-kDa protein (Fig. 4A, lanes 1–7), which was identified to be RHA based on its size and reactivity on immunoblots with rabbit anti-RHA antisera (not shown). In contrast, none of the ANA xid sera immunoprecipitated RHA, suggesting that the speckled ANA pattern exhibited by PBS-treated CBA/N mice is largely (or entirely) due to anti-RHA autoantibodies. Levels of anti-RHA antibodies in CBA/N mice were comparable to or even higher than those in NZB/W F1 mice (11). In CBA/CaJ mice, pristane induced the production of anti-Su, -nRNP/Sm and -OJ autoantibodies (Fig. 4B), as seen in other non-autoimmune strains following pristane treatment. In contrast, none of the pristane-treated CBA/N mice produced these autoantibodies (not shown). These data indicate that xid mice spontaneously produce autoantibodies to the lupus autoantigen RHA, but are refractory to the induction of other lupus autoantibodies by pristane.



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Fig. 4. Immunoprecipitation analysis of autoantibodies. Sera from many untreated CBA/N mice immunoprecipitated RHA (A, lanes 1–7), whereas others were negative (lanes 8–9). In CBA/CaJ mice, pristane induced anti-Su (B, lanes 2 and 4) and -nRNP/Sm (lane 5) as seen in other normal strains, as well as -OJ antibodies (lane 6).

 
Pristane treatment inhibits the spontaneous production of anti-RHA
The frequencies of autoantibodies in CBA/N versus CBA/CaJ mice are compared in Table 1. Forty-three percent of PBS-treated CBA/N mice produced anti-RHA compared with 17% in the pristane-treated group (P = 0.048, one-sided Fisher’s exact test). The levels of anti-RHA antibodies also were lower in pristane-treated CBA/N mice versus PBS-treated controls (P = 0.03, Mann–Whitney test, Fig. 5). None of the CBA/CaJ mice produced anti-RHA, regardless of treatment. However, in CBA/CaJ mice, pristane induced anti-nRNP/Sm (three of 23 mice) and -Su (three of 23). One mouse was positive for both anti-nRNP/Sm and -Su. An additional mouse produced anti-OJ autoantibodies, a specificity associated with polymyositis. Thus, a total of six of 23 pristane-treated CBA/CaJ mice produced these disease-associated autoantibodies versus none of 28 PBS-treated CBA/CaJ mice (P = 0.006, one-sided Fisher’s exact test). In contrast, none of the pristane-treated CBA/N mice produced anti-nRNP/Sm, -Su or -OJ (P = 0.01, CBA/N versus CBA/CaJ, one-sided Fisher’s exact test), suggesting that xid mice are resistant to the induction of these autoantibodies.


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Table 1. Autoantibodies in CBA/N and CBA/CaJ mice by immunoprecipitation
 


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Fig. 5. Levels of autoantibodies to RHA in Xid mice. Levels of IgG autoantibodies to RHA in CBA/N sera (6 months after treatment) were compared (ELISA). Levels of anti-RHA were lower in pristane treated mice than in PBS controls (P = 0.03, Mann–Whitney test). Serum dilution 1:500.

 
Pristane inhibits anti-RHA autoantibody production at the induction stage
The data shown in Fig. 5 and Table 1 suggest that pristane treatment of young (pre-autoimmune) CBA/N mice inhibits the production of anti-RHA antibodies. This could be due to the prevention of anti-RHA autoantibody induction or could reflect an action at the stage of B cell maturation and antibody secretion. To distinguish between these possibilities, CBA/N mice with pre-existing anti-RHA antibodies were treated with either pristane or PBS and anti-RHA antibody levels were measured 2 months later (Fig. 6). As shown in Fig. 6(A), pristane treatment increased the levels of anti-RHA antibodies by 140–1473% in all five mice (P = 0.04, Wilcoxon matched-pairs test), whereas the changes were minimal in the PBS-treated group. The percentage change in anti-RHA antibody levels was also greater in the pristane-treated group than in the PBS-treated controls (P = 0.04, Mann–Whitney test) (Fig. 6B). This finding suggests that the inhibition of anti-RHA antibody production observed in young mice is due to an effect of pristane at the stage of autoantibody induction rather than at the stage of B cell maturation.



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Fig. 6. Pristane treatment increases the levels of established anti-RHA autoantibodies. Xid mice that spontaneously produced anti-RHA antibodies were treated with pristane (n = 5) or PBS (n = 3). IgG anti-RHA levels were measured before and 2 months after treatment (ELISA). ODs were converted into units as described in Methods. (A) Levels of anti-RHA by ELISA. Levels of anti-RHA antibodies before and 2 months after pristane (left) or PBS (right) treatment are shown. Levels of anti-RHA increased in the pristane group (P = 0.04 by Wilcoxon matched-pair test), but not in the PBS group. (B) Changes in levels of anti-RHA antibodies. Percent change in anti-RHA antibodies 2 months after treatment was calculated as: (U anti-RHA antibodies 2 months after treatment – U before treatment/U before treatment). Anti-RHA levels were significantly increased in all pristane-treated mice compared with controls (P = 0.04, Mann–Whitney).

 
Pristane has a modest effect on total IgG1 and IgG2a levels in xid mice
Consistent with previous observations, CBA/N mice had severely reduced levels of IgG1 and IgG2a compared with CBA/CaJ (P < 0.001, Mann–Whitney test, Fig. 7A). Although the IgG1 and IgG2a levels increased following pristane treatment in both CBA/N (P < 0.005, Mann–Whitney) and CBA/CaJ (P < 0.001) mice, levels in the xid mice were still very low compared with those induced by pristane in CBA/CaJ controls (P < 0.001, Mann–Whitney).



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Fig. 7. (A) Total serum Ig levels by ELISA. Serum total IgG1 and IgG2a levels 3 months after the treatment were measured by ELISA. Mean values with SD of 24 (PBS group) or 19 (pristane group) mice are shown for each group. (B) Ratio of serum IgG2a:IgG1 in CBA mice. Total serum IgG1 and IgG2a levels were measured (ELISA) 3 months after the treatment and IgG2a:IgG1 ratios were calculated. Pristane treatment increased the ratio in both strains.

 
Pristane treatment increased the ratio of IgG2a (IFN-{gamma}-dependent isotype) to IgG1 (IL-4-dependent isotype) 3 months after treatment in CBA/CaJ mice in comparison with PBS-treated controls (Fig. 7B, P < 0.0001, Mann–Whitney). Although there was a trend toward a higher ratio in pristane-treated CBA/N mice, as well, it did not reach statistical significance due to the heterogeneity of the response in this strain. Interestingly, all but one of the mice positive for anti-RHA autoantibodies, whether PBS or pristane-treated, had low ratios of total IgG2a/IgG1 in the serum (Fig. 7B, closed circles).

Isotype distribution of anti-RHA versus anti-nRNP/Sm and Su autoantibodies
To investigate whether the isotype skewing apparent in the total Ig levels was reflected in autoantibody production, the isotype distribution of anti-RHA antibodies was compared with that of anti-nRNP/Sm and -Su (Fig. 8). IgG1 anti-RHA antibodies were predominant in four of 14 CBA/N mice (PBS or pristane-treated, Fig. 8 A, RHA #1 and #2 are representative examples). IgG2a was predominant in the remaining 10 mice, but was accompanied by significant amounts of IgG1 as well (Fig. 8A, RHA #3 is representative). It is noteworthy that significant amounts of IgG1 anti-RHA antibodies were seen despite the fact that total IgG1 levels were considerably lower than total IgG2a in CBA/N mice (mean IgG1 0.07 mg/ml versus mean IgG2a 0.21 mg/ml, P < 0.0001 by Mann–Whitney test, see Fig. 7 A). In contrast, nearly all anti-nRNP/Sm and -Su autoantibodies induced by pristane in CBA/CaJ mice were IgG2a (Fig. 8B, nRNP/Sm#1, Su#1 and Su#2). These data suggested that the production of anti-RHA and anti-nRNP/Sm/Su may depend differentially on cytokines: IFN-{gamma} may be important for the inducible and predominantly IgG2a anti-nRNP/Sm/Su response, whereas IL-4 could play a role in the spontaneous anti-RHA response.



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Fig. 8. Subclasses of autoantibodies in CBA mice. Subclasses of anti-RHA versus anti-nRNP/Sm and -Su antibodies at 6 months were examined by ELISA. The CBA/N mice produced significant levels of IgG1 anti-RHA antibodies. In contrast, nearly all antibodies to nRNP/Sm and Su induced by pristane in CBA/CaJ mice were IgG2a. Serum dilution 1:500.

 
Effect of pristane on cytokines
In view of the isotype differences, levels of cytokines produced spontaneously in culture by peritoneal cells in pristane- versus PBS-treated mice, were evaluated. CBA/N and CBA/CaJ mice showed widely divergent patterns of cytokine production (Fig. 9). In the case of 9-month-old CBA/N mice, peritoneal cells spontaneously produced IL-6, but not IL-12, whereas after pristane treatment, less IL-6 was produced and high levels of IL-12 were apparent (Fig. 9A, left). In contrast, peritoneal cells from CBA/CaJ mice showed the opposite response to pristane: IL-6 production was low initially and increased after pristane treatment, whereas IL-12 (also low initially) decreased even further after pristane treatment (Fig. 9A, right).



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Fig. 9. Cytokine production by peritoneal cells. Peritoneal cells from PBS versus pristane-treated CBA/N and CBA/CaJ mice 6 months after the treatment were cultured at 106/ml for 48 h without stimulation (A) or with LPS (10 ng/ml, B) or anti-CD3 mAb (1 µg/ml, C) stimulation. Cytokine levels in culture supernatants were measured by sandwich ELISA. Representative data from two separate experiments, median of four mice per group, are shown. (A) Spontaneous cytokine production by peritoneal cells. Cells from PBS-treated CBA/N mice produced IL-6, whereas cells from pristane-treated mice produced less IL-6 and high levels of IL-12. Cells from CBA/CaJ mice showed the opposite pattern. (B) LPS stimulated cytokine production. Cells from PBS-treated CBA/N mice produced mainly IL-6, whereas cells from pristane-treated CBA/N mice tended to produce IL-12. (C) Anti-CD3 stimulated cytokine production. Cells from PBS-treated CBA/N mice produced IL-4, whereas cells from pristane-treated CBA/N or CBA/CaJ mice did not. The cells from pristane-treated CBA/N mice produced substantially more IFN-{gamma}.

 
Cytokine production following stimulation of CBA/N and CBA/CaJ peritoneal cells in vitro with LPS (Fig. 9B) or anti-CD3 antibodies (Fig. 9C) was also examined. Peritoneal cells from PBS-treated CBA/N mice produced large amounts of IL-6 with LPS stimulation (Fig. 9B) and IL-4 with anti-CD3 stimulation (Fig. 9C), whereas peritoneal cells from pristane-treated CBA/N mice produced larger amounts of IL-12 (Fig. 9B) and IFN-{gamma} (Fig. 9C). Production of IL-6, IL-12 and IFN-{gamma} by peritoneal cells from CBA/CaJ mice was considerably less than that of CBA/N mice. These data indicate that the xid mutation leads to markedly altered patterns of cytokine production in CBA mice, and that pristane treatment shifts the cytokine balance toward the production of IFN-{gamma} and IL-12, rather than IL-6 and IL-4.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SLE is a prototype systemic autoimmune disease caused by the interplay of multiple genes and environmental factors. Alteration in a particular gene may induce lupus either directly or indirectly by modifying susceptibility to environmental factors (17). The present study demonstrated that mutation of the Btk gene can promote the induction of lupus autoantibodies. The xid mutation induces spontaneous production of lupus-related anti-RHA autoantibodies in association with disordered cytokine production, while at the same time antagonizing the onset of spontaneous (5) or pristane-induced lupus. Defective function of B1 and/or conventional B cells caused by the xid mutation could play a major role in suppressing autoimmunity in murine lupus. However, other effects associated with xid mutation, such as modified macrophage function, cytokine production, dysregulation of apoptosis or aberrant immune responses to microorganisms may also contribute (18,19).

B cell hyperactivity characterized by polyclonal hypergammaglobulinemia and specific autoantibody production to various cellular antigens are characteristic of human and murine SLE (2). Xid mice have a unique autoimmune syndrome that combines severely reduced levels of serum Ig and a highly restricted autoimmune response to RHA with little evidence of recognition of other autoantigens. RHA is a nuclear protein that binds to double-stranded DNA, double-stranded RNA and highly structured viral RNAs such as adenoviral RNAs (20), and functions as a shuttle protein between the nucleus and cytoplasm (21). Autoantibodies to RHA are found in ~8% of patients with SLE, but rarely encountered in other diseases (M. Satoh et al., unpublished) (10,15). It also is found in ~25% of B/W lupus mice (11).

Autoimmunity develops frequently among patients with B cell immunodeficiency. Cases of immunodeficiency with a strong family history of autoimmunity, have been reported (22). Juvenile rheumatoid arthritis, rheumatoid arthritis and dermatomyositis have been reported in XLA (23,24), the human equivalent of the mouse xid defect. It has been suggested that at least some of these autoimmune-like features are associated with susceptibility to microbial infection. Arthritis in XLA is associated with mycoplasma infection and dermatomyositis with persistent echovirus infection (23,24). It is possible that aberrant immune responses to microorganisms also play a role in autoimmunity in xid mice. Although the mice used in the present study were not exposed to exogenous pathogens and were free of mouse hepatitis virus, Sendai virus or murine herpes virus, involvement of endogenous retroviruses present in virtually all strains of mice cannot be excluded. It is of particular interest that the target of autoimmunity in xid mice is limited to the viral RNA-binding protein, RHA. In addition to effects on B cell development, Btk also has effects on apoptosis (18). Btk interacts with Fas and controls Fas-mediated apoptosis by competing for binding to FADD (19), suggesting that dysfunctional Btk might accelerate Fas-mediated apoptosis. It has been suggested that various autoantigenic proteins including RHA (10) are cleaved during apoptosis and accumulate in apoptotic blebs, possibly inducing autoimmunity (25). Thus, abnormal Fas-mediated apoptosis caused by the xid mutation may contribute to autoimmunity in xid mice.

Cytokines play an essential role in the pathogenesis of autoantibody production and lupus-like disease in human and mouse. Recent studies suggest a critical role of Th1 cytokines, in particular IFN-{gamma}, in the pathogenesis of lupus in MRL/lpr mice and B/W mice (26,27). Cytokine transgenic mice overexpressing IL-4, IL-6 or IFN-{gamma} all spontaneously produce ANA (2830). Induction of ANA also has been reported in patients treated with IFN-{alpha} or anti-tumor necrosis factor-{alpha} antibodies (31). These observations suggest that unbalanced cytokine production alone can promote autoantibody formation. Data from our laboratory suggest a critical role of IL-6 in the production of IgG anti-double-stranded DNA and -chromatin antibodies (9), whereas IFN-{gamma} appears to play an important role in the induction of anti-nRNP/Sm autoantibodies in pristane-induced lupus (32). Anti-RHA appears to be regulated differently. Spontaneous production of anti-RHA antibodies is associated with IL-4/IL-6 production in B/W (11) and in xid mice. Pristane treatment shifted the cytokine balance toward IL-12/IFN-{gamma} and suppressed production of anti-RHA in both strains. The xid mutation shifts spontaneous cytokine production toward IL-4/IL-6 (Fig. 8A), potentially contributing to the development of autoimmunity. Cytokines may affect presentation of specific autoantigen via induction of proteases (33), altered post-translational modification (34), regulation of immune responses to microorganism (35) or effects on apoptosis (36).

Pristane induces hypergammaglobulinemia and weak anti-chromatin antibody responses, but suppresses anti-RHA antibody production if given early (Table 1 and Fig. 5). However, unexpectedly, pristane treatment increased levels of anti-RHA antibodies when administered after their establishment (Fig. 6 ). Thus, pristane suppresses or enhances anti-RHA antibody production depending on timing of its administration. The present study and data from B/W mice (11) suggest that IL-4/IL-6 may play a critical role in the ‘induction’ of anti-RHA, whereas the levels are controlled by different mechanisms. These data strongly suggest that the different sets of cytokines may be responsible for either triggering autoantibody production or maintaining it. Similarly, opposite effects of IFN-{gamma} in collagen-induced arthritis at different stages of the disease have been reported (37). Despite the apparent involvement of cytokines, it is also possible that pristane has additional direct effects on B cells or other immune cells. For example, pristane may eliminate B1 cells from the peritoneal cavity (38), with a possible impact on the maintenance of tolerance (39).

Although it has been proposed that autoantibody formation is a random ‘stochastic’ process (40), our model suggests that the autoantibody phenotype may differ in predictable ways in a cytokine-dependent manner. Viewed this way, autoantibodies may be important markers for the ongoing pathological process. This hypothesis is consistent with the heterogeneous phenotype of lupus even among individuals within a single family (41).


    Acknowledgements
 
The present work was supported by NIH grants R01-AR44731 and AI44074.


    Abbreviations
 
ANA—anti-nuclear antibody

Btk—Bruton’s tyrosine kinase

LPS—lipopolysaccharide

SLE—systemic lupus erythematosus

RHA—RNA helicase A

xid—X-linked immunodeficiency

XLA—X-linked agammaglobulinemia


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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