Implication of oxidative stress as a cause of autoimmune hemolytic anemia in NZB mice

Free Radical Biology & Medicine 48 (2010) 935–944

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Implication of oxidative stress as a cause of autoimmune hemolytic anemia in NZB mice Yoshihito Iuchi a,b,c, Noriko Kibe a,b,c, Satoshi Tsunoda a,b,c, Saori Suzuki a,b,c, Takeshi Mikami a, Futoshi Okada a,b,c, Koji Uchida d, Junichi Fujii a,b,c,⁎ a

Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Yamagata 990-9585, Japan Respiratory and Cardiovascular Diseases Research Center, Research Institute for Advanced Molecular Epidemiology, Yamagata University, Yamagata 990-9585, Japan Global COE Program for Medical Sciences, Japan Society for the Promotion of Science, Yamagata 990-9585, Japan d Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan b c

a r t i c l e

i n f o

Article history: Received 2 October 2009 Revised 14 December 2009 Accepted 6 January 2010 Available online 14 January 2010 Keywords: New Zealand Black mouse Autoantibody Autoimmune hemolytic anemia Reactive oxygen species Red blood cells Free radicals

a b s t r a c t We have recently shown that deficiency of the superoxide dismutase 1 gene (SOD1) causes anemia and autoimmune responses against red blood cells (RBCs) and that transgenic expression of human SOD1 in erythroid cells rescues them. Because these phenotypes observed in SOD1-deficient mice are similar to autoimmune hemolytic anemia (AIHA), a causal connection between reactive oxygen species (ROS) and AIHA was examined using an AIHA-prone New Zealand Black (NZB) mouse. ROS levels in RBCs were high in young NZB mice, compared to control New Zealand White (NZW) mice, and increased during aging. Methemoglobin and lipid peroxidation products were elevated during aging, consistent with the elevated oxidative stress in RBCs of NZB mice. Severity of anemia and levels of intracellular ROS were positively correlated. Levels of antibodies against 4-hydroxynonenal and acrolein were also elevated in NZB mice. Transgenic expression of human SOD1 protein in RBCs of NZB mice suppressed ROS in RBCs and decreased the death rate. When RBCs from C57BL/6 mice were injected weekly into the same strain of mice, production of anti-RBC antibody was observed only in mice that had been injected with oxidized RBCs. Thus, oxidationmediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases. © 2010 Elsevier Inc. All rights reserved.

Autoimmune hemolytic anemia (AIHA) is an antibody-mediated autoimmune disease [1]. Antibodies that specifically recognize red blood cells (RBCs) are produced, accelerate the destruction of RBCs, and cause hemolytic anemia. It is well documented how autoantibody-coated RBCs are destroyed by splenic macrophages. However, the mechanism that initiates abnormal production of the autoantibodies is largely unclear [2]. The New Zealand Black (NZB) strain of mice spontaneously develops AIHA [3] and is a popular animal model used to investigate the mechanism of AIHA and to develop therapeutic treatments. NZB mice Abbreviations: ACR, acrolein; AIHA, autoimmune hemolytic anemia; BSA, bovine serum albumin; CAII, carbonic anhydrase II; CAT, catalase; DHR123, dihydrorhodamine 123; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; GPX, glutathione peroxidase; NAC, N-acetylcysteine; HNE, 4-hydroxy-2-nonenal; HRP, horseradish peroxidase; MetHb, methemoglobin; PBS, phosphate-buffered saline; RBC, red blood cell; ROS, reactive oxygen species; SLE, systemic lupus erythematodes; SOD, superoxide dismutase; TBARS, thiobarbituric acid-reactive substances; TBS, Trisbuffered saline; TBST, TBS containing 0.1% Tween 20; WST-1, 2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium. ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Yamagata 990-9585, Japan. Fax: +81 23 628 5230. E-mail address: [email protected] (J. Fujii). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.01.012

become anemic during aging and show splenomegaly and hepatomegaly due to erythrocyte destruction and extramedullary hematopoiesis [1]. Extensive studies have been performed to clarify the pathway of the antibody generation and to characterize the autoantibodies produced. RBC-bound immunoglobulin G (IgG) autoantibodies are detected from 3 months of age, and the mice develop signs of anemia from about 6 months of age onward [4]. Possible contributions to AIHA by a defect in CD4+CD25+ regulatory T cells [5] and Th1 and Th2 cytokine imbalance have been reported [6]. Meanwhile, peritoneal B-1 cells may be a source of autoantibody-producing cells in NZB mice [7]. The band 3 protein is a dominant T cell epitope that constitutes the major glycosylated membrane protein of RBCs and is highly antigenic [8–10]. Autoantibodies prepared from the surface of RBCs, and many pathogenic monoclonal autoantibodies against RBCs established from NZB mice, specifically recognize the band 3 protein [11–13]. Proteolytic removal of the surface domain, or other modification, exposes the embedded portion of membrane proteins, which the immune system recognizes as new epitopes and activates autoantibody production against them [14]. When the NZB mouse is backcrossed with a mutant mouse lacking the AE1 allele, the gene encoding the band 3 protein, the resultant AE1-deficient NZB mice show an autoimmune response to RBCs and produce an autoantibody

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against glycophorin, another predominant membrane protein of RBCs [15]. This suggests that, in addition to the band 3 protein, RBCs themselves are a preferential antigen in NZB mice. In addition to proteolysis, oxidative modification proceeds during the aging process and generates epitopes that are not present during the removal of the T cells reactive to the self-antigens. Certain lipid peroxidation products, such as malondialdehyde, have been found to be epitopes for innate immunity [16,17]. Thus, oxidative stress may stimulate the immune reaction to susceptible molecules such as polyunsaturated fatty acids, produce autoantibodies against their peroxidized products [18], and be an underlying mechanism for some autoimmune diseases, such as AIHA and systemic lupus erythematosus (SLE) [19,20]. We recently found elevated reactive oxygen species (ROS) levels in RBCs and augmented production of autoantibodies to RBCs in superoxide dismutase 1 gene (SOD1)-knockout mice [21]. Anemia induced by the absence of SOD1 seems to be caused by accelerated hemolysis in the blood plasma and phagocytic removal of RBCs by liver macrophages, namely Kupffer cells [22]. The identified antibodies include those against lipid peroxidation products, 4-hydroxy2-nonenal (HNE) and acrolein (ACR), as well as an erythrocyte protein, carbonic anhydrase II (CAII) [23]. Because both anemia and autoimmune responses in young SOD1−/− mice are totally rescued by local expression of human SOD1 in erythroid cells, these phenotypes are attributable to SOD1 deficiency of the erythroid cells. Based upon these data, we suspect that oxidative stress is a likely mechanism underlying autoimmunity in AIHA via oxidative modification of RBCs. In this communication, we show evidence of elevated oxidative stress in RBCs in NZB mice and prolonged survival of NZB mice that have transgenic overexpression of human SOD1 exclusively in erythroid cells. These findings support the notion that elevation of ROS is a causal factor in the production of antibodies against RBCs and lipid peroxidation products in NZB mice. Materials and methods Animals NZB and New Zealand White (NZW) mice (n = 10 each) purchased from Nippon SLC (Hamamatsu, Japan) were subjected to observation for up to 1 year. Transgenic mice, which were generated previously from the C57BL/6 background strain [23], carry the human SOD1 transgene under the regulation of the GATA1 promoter and, therefore, express human SOD1 protein in erythroid cells only. These hSOD1 transgenic mice were intercrossed with NZB mice and backcrossed to NZB strain mice four times. Female hSOD1-Tg/NZB and the wild-type litters from breeding pairs, which were backcrossed to male NZB mice four times, were also examined for 1 year. The animal room climate was kept under specific-pathogen-free conditions at a constant temperature of 20–22°C with a 12-h alternating light–dark cycle. Animal experiments were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee of Yamagata University. Blood samples were collected in the presence of excess EDTA and contents of cells were counted by an automated hematology analyzer (Celltaca Nihon Kohden, Tokyo, Japan). Detection of ROS and IgG bound to RBCs by flow cytometry RBCs were incubated with 25 μM dihydrorhodamine 123 (DHR123) (Molecular Probes, Eugene, OR, USA) and washed with PBS three times. The IgG bound to RBCs was assessed with RBCs that had been washed with PBS three times, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (100 times dilution; Dako, Kyoto, Japan) [21]. The fluorescence intensity of DHR123 and FITC bound to RBCs was measured using a

fluorescence-activated cell sorter (FACS; FACSCalibur; BD Biosciences, Tokyo, Japan). To standardize the fluorescence intensity among assays on different days, we simultaneously measured the fluorescence in RBCs from normal C57BL/6 mice as the control sample in each experiment and present the relative values in all data. Immunoblot analyses RBCs collected from mice were washed three times with PBS and lysed in 20 mM Tris–HCl, pH 7.4. The lysate was centrifuged at 17,000 g for 10 min in a microcentrifuge. Protein concentrations of the supernatants were determined using a BCA kit (Pierce, Rockford, IL, USA). Total proteins, 30 μg, were separated on 15% SDS–polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Amersham, Tokyo, Japan). The blots were blocked with 0.5% nonfat dry milk in Tris-buffered saline (TBS) and then incubated with the polyclonal antibodies against human Cu,ZnSOD [24], rat glutathione peroxidase 1 [25], or human catalase (Calbiochem) diluted in TBS containing 0.1% Tween 20 (TBST) overnight at 4°C. To detect autoantibodies against RBC proteins in mouse plasma, RBC proteins from NZB and NZW mice were incubated with mouse plasma diluted 1000 times. After being washed twice in TBST for 30 min, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After a wash, the presence of bound HRP was detected by chemiluminescence with an ECL Plus detection reagent (Amersham) and exposure to X-ray films. The intensity of each band was quantified by densitometry (ATTO Densito Graph 4.0; ATTO, Tokyo, Japan). Quantification of antibodies in plasma by ELISA Anti-CAII antibody was quantified by ELISA as described previously [21]. Briefly, each well of a multititer plate (Nunc, Tokyo, Japan) was coated with 10 μg/ml bovine erythrocyte CAII (Sigma) in PBS and allowed to adsorb at 4°C overnight. CAII-coated plates were then blocked with twofold-diluted Immunoblock (Dainippon Sumitomo Pharma, Tokyo, Japan) for 1 h at 37°C. Blood plasma collected from mice was diluted to 1:50 in blocking buffer. After incubation with diluted plasma for 1 h at 37°C, the plates were washed three times with PBS containing 0.05% Tween 20 and then reacted with HRPconjugated anti-mouse IgG (Santa Cruz) for 1 h. The anti-DNA titer was measured by ELISA, using calf thymus double-stranded DNA as the coating antigen [26]. The anti-HNE and anti-ACR titers in the plasma samples were measured by ELISA using HNE-modified BSA and ACR-modified BSA, respectively, as the coating antigens [20,23]. Assays were performed in duplicate and the absorbance was determined at 495 nm. Assay for antibody-dependent complement-mediated hemolysis Blood was collected from wild-type and SOD1-deficient C57BL/6 mice (40 weeks of age). After being washed three times in PBS, RBCs (1 × 107 in 20 µl/well) were incubated with anti-HNE antibody (100 ng/10 µl added; Nikken Seil, Shizuoka, Japan) or PBS for 30 min at 37°C followed by incubation with guinea pig complement (20 µl added; Denka Seiken, Tokyo, Japan) for 2 h at 37°C. After centrifugation for 10 min at 700 g, the absorbance at 405 nm was measured for both supernatant and lysed RBCs in the precipitate. Assay for lipid peroxidation products Thiobarbituric acid-reactive substances (TBARS) were determined as described previously [21]. For each measurement, 1 × 107 RBCs were collected and washed twice with PBS. After the cell pellet was suspended in 0.3 ml of PBS, a 10-μl aliquot of the suspension was retained for use in a protein determination. The cell suspension was

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combined with 0.6 ml of a reagent containing 15% trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, 0.25 M HCl, and 1.8 mM butylhydroxytoluene and mixed thoroughly. The solution was heated for 15 min in boiling water, cooled in ice-cold water, and centrifuged at 10,000 g for 10 min. n-Butanol (250 μl) was mixed with the reaction solution, vortexed, and centrifuged at 10,000 g for 5 min. The absorbance of the n-butanol fraction was measured at 553 nm. TBARS levels were calculated using an extinction coefficient of 1.56 × 105 M−1 cm−1. Assay for methemoglobin (MetHb) content Fresh RBCs collected from mice were lysed in 50 mM Tris–HCl, pH 6.6. Total hemoglobin and MetHb concentrations in the samples were determined spectrophotometrically. MetHb concentration was determined by comparing the absorbance spectra at 630 nm before and after the addition of potassium cyanide to the hemolysates. MetHb content was expressed as a percentage of total hemoglobin concentration. Enzyme assay EDTA-treated blood from NZB mice was washed in cold PBS. The plasma and buffy coat were drawn off directly after each centrifugation. Packed RBCs were washed twice and suspended in 20 mM Tris– HCl, pH 7.4. After centrifugation at 17,000 g for 15 min, the supernatant was collected and used for the enzyme assays. SOD

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activity was determined using WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (Wako) for detection of superoxide anion, as described previously [24]. The reaction mixture contained an appropriate amount of diluted xanthine oxidase (Roche), 0.1 mM xanthine (Wako), 0.025 mM WST-1, 0.1 mM EDTA, 50 mM NaHCO3, pH 10.2, in a total volume of 3 ml. The increase in absorbance at 438 nm was monitored at 25°C for 1 min. One unit was defined as the amount of enzyme required to inhibit 50% of an absorbance change of 0.06/min and was equivalent to 0.8 units, determined by the standard procedure using cytochrome c assay according to the manufacturer's protocol. Glutathione peroxidase (GPX) activity was determined by an indirect assay that linked GPX-mediated oxidation of glutathione with the recycled reduction of oxidized glutathione to reduced glutathione by glutathione reductase, using NADPH as a reductant. Quantification of catalase activity was assayed by measuring the decomposition of H2O2 by monitoring absorbance at 240 nm. The reaction was started by the addition of 30 μg total protein to a reaction buffer containing 50 mM Tris–HCl, pH 7.4, 0.25 mM EDTA, and 10 mM H2O2. Catalase (CAT) activity was defined as the rate of disappearance of H2O2 during the initial 30 s. Intraperitoneal injection of mouse RBCs into mice RBCs were collected from female C57BL/6 mice and washed three times with PBS. Female C57BL/6 mice (6 weeks of age) were divided into four groups (10 mice per group) and were intraperitoneally

Fig. 1. ROS levels are higher in NZB mice even at a young age. (A) Blood collected from NZB and NZW mice at 4 weeks of age was incubated with DHR123 and analyzed by FACS. (B) Relative fluorescence intensity of RBCs from these mice (n = 3) is shown. (C) Blood collected from NZB mice at 5 and 50 weeks of age was incubated with DHR123 and analyzed by FACS. Relative fluorescence intensity of the RBCs from these mice is shown (n = 8 or 9). (D) Detection of IgG bound to RBCs from NZB mice at 5 and 50 weeks of age. Collected erythrocytes were reacted with FITC-labeled anti-mouse IgG, followed by FACS analysis. Relative values of bound IgG are shown (n = 5 or 6).

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injected every week for 50 weeks with PBS (group I), mouse RBCs (3 × 108) (group II), or mouse RBCs (3 × 108) oxidized by treatment with 2 mM hypoxanthine, 50 mU/ml xanthine oxidase for 30 min (groups III and IV). The mice in group IV were administered 1% NAC (N-acetylcysteine) in drinking water.

Statistical analysis Statistical analyses of the data were carried out using analysis of variance followed by a post hoc test when appropriate (⁎P b 0.05; ⁎⁎P b 0.01).

Results Elevation of ROS in RBCs of NZB mice during aging To validate the possible involvement of ROS in the pathogenesis in NZB mice, we first examined intracellular levels of ROS in RBCs of young NZB and NZW mice at 4 weeks of age by FACS analysis using DHR123 as a fluorescent probe for ROS. RBCs from NZB mice showed higher levels of ROS than those from NZW mice (Figs. 1A and 1B). ROS levels were elevated in RBCs at 50 weeks of age, compared with young mice (Fig. 1C), whereas only a slight change was observed in NZW mice during aging (data not shown). Levels of IgG bound to RBCs, corresponding to autoantibodies on the RBC surface, were markedly elevated in NZB mice (Fig. 1D).

Increased oxidation of RBCs and correlation with AIHA To gain insight into the role of oxidative stress in AIHA, we measured oxidation products of lipid and proteins in RBCs of NZB mice. Lipid peroxidation products, as judged by TBARS, were present at significantly higher levels in both RBCs and plasma of NZB mice, compared with those of NZW mice (Fig. 2A). The presence of high levels of MetHb, an oxidized form of hemoglobin, in RBCs of NZB mice (Fig. 2B) also implied that RBCs were under oxidative modification. Because ROS that cause cellular damage may lead to destruction of RBCs and autoantibody production, we examined the correlation between ROS and the IgG bound to RBCs, as well as ROS and anemia in aged NZB mice (Fig. 2C). There was a positive correlation between ROS and IgG-bound RBCs and a negative correlation between ROS and RBC content in the blood of NZB mice at 50 weeks of age. These results suggest the involvement of ROS in anti-RBC autoantibody production and anemia in NZB mice. Antioxidative/redox enzyme status We measured the activity of the major antioxidative enzymes, superoxide dismutase, catalase, and glutathione peroxidase, and assessed the protein levels of their predominant isoforms in RBCs, SOD1 and GPX1, as well as CAT, by immunoblotting (Fig. 3). No significant difference was found in the SOD activity or SOD1 proteins in RBCs of NZB mice compared to NZW mice in either young (10 weeks) or old (50 weeks) mice. Whereas GPX activity

Fig. 2. Elevated lipid peroxidation and methemoglobin content in aged NZB mice. (A) Lipid peroxidation products in RBCs and plasma of NZW and NZB mice were quantified as TBARS. (B) The MetHb content was also measured in their RBCs (50 weeks, n = 3). (C) Levels of bound IgG and RBC content in the blood of NZB mice were plotted against ROS levels evaluated by rhodamine 123 fluorescence of RBCs of each mouse (50 weeks, n = 3).

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Fig. 3. Activities of antioxidative/redox enzymes and their protein levels in RBCs of young and aged NZB mice. Immunoblot analyses using antibodies against SOD1, GPX1, and CAT and activity assay of the proteins from RBCs of NZW and NZB mice were performed at 10 and 50 weeks of age (n = 5).

and GPX1 protein decreased significantly, CAT activity and proteins increased in RBCs of old NZB mice, in comparison with other groups of mice.

autoantibodies bound to RBCs were involved in the complementmediated hemolysis. Suppression of the autoimmune reaction by overproduction of human SOD1 in erythroid cells of NZB mice

Elevation of antibodies against carbonic anhydrase II, 4-hydroxynonenal, and acrolein in plasma of NZB mice Because an autoantibody to CAII, a cytosolic protein of RBCs, and the antibody to RBCs are elevated in SOD1-knockout mice [21,23], we performed immunoblot analyses using blood plasma from NZW and NZB mice as the first antibody source. There was a strongly reactive band around 30 kDa in size, corresponding to CAII, on the RBC protein-blotted membrane incubated with NZB plasma, but only a faint band on that incubated with NZW plasma (Fig. 4A). Quantification of anti-CAII IgG in NZB mouse plasma by ELISA using bovine CAII-coated immunoplates indicated that the antibody was indeed higher in NZB mice than that in NZW (Fig. 4B). Although elevation of anti-double-strand DNA antibody is characteristically observed in blood plasma in SLE, no difference in anti-DNA antibody levels was observed in NZB mice, compared with a marked elevation in (NZB/NZW)F1 mice at 30 weeks of age (data not shown). We also assessed antibodies to two lipid peroxidation products, HNE and ACR, in these mice and found that both anti-HNE and anti-ACR antibodies were significantly higher in NZB mice (Fig. 4B), as seen in SLE patients [20], suggesting the involvement of oxidative stress in the pathogenesis in these mice. Then we examined whether the antiHNE antibody actually induced complement-mediated hemolysis using SOD1-deficient C57BL/6 mice. When RBCs from aged mice were incubated with the anti-HNE antibody followed by incubation with complement, hemolysis was induced more severely in the SOD1-deficient RBCs than in the wild-type RBCs (Fig. 4C). It is also noteworthy that treatment with the complement alone induced hemolysis significantly in the SOD1-deficient RBCs. This suggests that

We tried to confirm the hypothetical role of oxidative stress in the autoimmune reaction by examining whether overproduction of SOD1 in RBCs ameliorates the autoimmune reaction. We have recently generated transgenic mice that carry the hSOD1 transgene under a GATA1 promoter, which enabled production of hSOD1 only in erythroid cells in the C57BL/6 background strain [23]. These hSOD1 transgenic mice were intercrossed with NZB mice and backcrossed to the NZB strain mice four times. The resultant hSOD1-Tg/NZB mice and their wild-type littermates were bred and subjected to observation for 1 year. Because the antibody against human SOD1 was used, band intensity corresponding to hSOD1 was strong (Fig. 5A). However, RBCs of hSOD1-Tg/NZB mice had human SOD1 activity at a level equivalent to endogenous mouse SOD1 (Fig. 5B). Both GPX and CAT activity showed no significant difference between NZB and hSOD1-Tg/NZB mice. There was a trend showing that the elevation of ROS in NZB mice was suppressed in the hSOD1Tg/NZB mice (Fig. 5C). Whereas three of eight NZB mice died because of AIHA, no hSOD1-Tg/NZB mice died in 1 year. Kaplan– Meier analysis showed significant improvement in the survival rate of the hSOD1-Tg/NZB mice (Fig. 5D). This confirmed the involvement of elevated ROS, which were suppressed by hSOD1 protein, in the pathogenesis of AIHA in NZB mice. Oxidation enhances immunogenicity of RBCs in C57BL/6 mice To further confirm the role of oxidative stress in pathogenesis, we examined whether oxidation of RBCs triggered an autoimmune response in normal C57BL/6 mice injected with oxidized RBCs. We

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Fig. 4. Levels of autoantibodies in blood plasma from NZB and NZW mice and anti-HNE antibody-mediated hemolysis. (A) RBC proteins from C57BL/6, NZW, and NZB mice were blotted onto membranes, followed by incubation with blood plasma from NZW and NZB mice. After reaction with HRP-conjugated anti-mouse IgG, positive signals were detected using an ECL Plus kit. (B) Titers of the antibodies against CAII, double strand DNA, HNE, and ACR in blood plasma from NZW (50 weeks) and NZB (50 weeks) were quantified by ELISA (n = 4). (C) RBCs from wild-type and SOD1-deficient C57BL/6 mice (40 weeks) were incubated with or without anti-HNE antibody followed by incubation with guinea pig complement. Percentage hemolysis of the triplicate assay is shown.

collected RBCs from normal C57BL/6 mice, oxidized the RBCs using a hypoxanthine/xanthine oxidase system in vitro, and performed ip injection into female C57BL/6 mice weekly for 50 weeks. FACS analysis indicated that the IgG fraction bound to RBCs was elevated in the plasma of the mice that were injected with the preoxidized RBCs (Fig. 6A). RBC content was low in mice that had been injected with oxidized RBCs (Fig. 6B). When antibodies against HNE and ACR were measured, they were both elevated in the mice that had been injected with oxidized RBCs (Figs. 6C and 6D). However, antibodies were not elevated in the plasma of the mice that were injected either with intact RBCs or with oxidized RBCs together with administration of 1% NAC in the drinking water. These data suggest

that oxidative stress is capable of triggering autoimmune responses in normal mice. Discussion We hypothesized that oxidative stress of RBCs is one of the causes of AIHA and related autoimmune diseases, because SOD1-deficient mice show enhanced oxidation of RBCs, concomitant production of anti-RBC autoantibody, and, ultimately, lupus nephritis-like symptoms in aged mice [21]. This idea was at least partly supported by a recent study that showed rescue of autoimmune-related phenotypes in SOD1-deficient mice by transgenic expression of human SOD1 only

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Fig. 5. Comparison of oxidation state and survival of RBCs between wild-type NZB and hSOD1-Tg/NZB mice. Levels of (A) proteins and (B) activities of major antioxidative enzymes in RBCs of wild-type NZB (NZB) and hSOD1-Tg/NZB (Tg) mice of the same litters were measured at 56–60 weeks of age. (C) ROS levels were quantified by FACS using DHR123 and values are expressed relative to those in RBCs from C57BL/6 mice. (D) Kaplan–Meier analysis of the mice was performed.

in erythropoietic cells under regulation of a GATA1 promoter [23]. To date there has been no strong evidence linking elevated ROS in blood to autoimmune responses in NZB mice. This study provides further supporting data using AIHA-prone NZB mice and provides the first evidence suggesting the involvement of ROS in AIHA of NZB mice. The NZB mouse is the established model animal for AIHA and has been extensively examined from the viewpoint of the pathway of autoantibody production [1]. However, it is still not clear what triggers the immune system to produce autoantibodies. The fact that cleaved membrane proteins, such as band 3, are highly antigenic [8– 10] supports the view that abnormal proteolytic cleavage of the membrane proteins of RBCs is a likely cause of AIHA. However, there is no firm evidence indicating elevated proteolytic activity in RBCs of AIHA patients or AIHA-prone mice, so the initial trigger for the autoimmune response is still obscure. We found that ROS levels were originally higher in NZB mice, even at a young age (4 weeks) (Fig. 1) when levels of autoantibody were still quite low and RBC content was normal. The ROS levels increased as NZB mice aged, whereas they increased only slightly in NZW or C57BL/6 mice (data not shown). A significant correlation was observed between ROS levels in RBCs and anti-RBC antibody levels (Fig. 2C), suggesting the involvement of ROS in autoantibody production. Our hypothesis was further supported by suppression of the AIHA phenotype of NZB mice by transgenic expression of human SOD1 in RBCs (Fig. 5) and by elevation of the IgG bound to RBCs in normal C57BL/6 mice that had received intraperitoneal injection of

oxidized RBCs (Fig. 6). Because supplementation of the antioxidant NAC suppressed autoantibody production in oxidized RBC-injected mice, the redox imbalance induced by oxidized RBCs seems to be involved in autoantibody production. The source of the oxidative stress is still not clear. Elevation of ROS in RBCs could occur either by suppression of the antioxidative/redox system or by activation of ROS generation. Deficiency of glucose 6phosphate dehydrogenase, a rate-determining enzyme of the pentose phosphate pathway, constitutes the majority of anemia. Oxidation of sulfhydryls in RBCs due to a defective supply of NADPH is the suspected cause of the anemia [27]. Other than the genetic defect, a decrease in glucose 6-phosphate dehydrogenase activity has been observed in RBCs under conditions of elevated oxidative stress, such as in preeclampsia [28], iron overload [29], and asbestosis [30] in humans. On the other hand, thus far, no report shows abnormalities in glucose 6-phosphate dehydrogenase in the RBCs of NZB mice. Regarding antioxidation, RBCs carry SOD1, GPX1, and CAT as the major antioxidative enzymes. Analyses of genes for the antioxidative enzymes CAT, SOD, GPX, and glutathione reductase have been performed in 10 inbred mouse strains, including NZB, and nonsynonymous nucleotide polymorphisms were identified in most genes [31]. In this study, the activities and protein levels of CAT, SOD, and GPX in RBCs were similar in NZB and NZW mice at 10 weeks (Fig. 3). However, decreases in GPX1 and concomitant increases in CAT were observed in aged NZB mice at 50 weeks (Fig. 4B). Oxidative stress accelerates senescence and limits the life span of hematopoietic stem

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Fig. 6. Sensitization of C57BL/6 mice with oxidized RBCs. RBCs were oxidized by preincubation with hypoxanthine/xanthine oxidase for 1 h. C57BL/6 mice 5 weeks of age were divided into four groups and were administered vehicle, RBCs, oxidized RBCs, or oxidized RBCs with 1% NAC in their drinking water. (A) IgG-bound RBCs were measured after incubation with FITC-labeled anti-mouse IgG by FACS. (B) RBC contents of the mice were measured (50 weeks, n = 8). Titers of the antibodies against (C) HNE and (D) ACR in blood plasma from the mice were quantified by ELISA (n = 8).

cells [32]. ROS-mediated activation of p38 mitogen-activated protein kinase followed by expression of cyclin-dependent kinase p16Ink4a inhibitors seems to be involved in the aging of hematopoietic stem cells [33]. At the same time, forkhead transcription factors induce CAT and SOD2 in hematopoietic stem cells in response to oxidative stressmediated activation of c-Jun N-terminal kinase [34,35]. Thus, the elevated CAT in RBCs may be a consequence of oxidative stress in hematopoietic stem cells, whereas SOD2, together with other mitochondrial components, is eliminated during RBC maturation from reticulocytes. On the other hand, the GPX1 level decreased in the aged NZB mice. A similar decrease in GPX1 has been observed in SOD1-deficient mouse RBCs [21]. The selenocysteine residue that forms the catalytic center for GPX1 is prone to modification by reactive nitrogen oxide species and inactivation [36,37]. The oxidative modification may accelerate degradation of GPX1 and cause the decrease in RBCs in aged NZB mice and/or SOD1-deficient mice. RBCs, however, have almost normal defenses against oxidative stress such as peroxides in GPX1-deficient mice [38]. Thus, the decrease in the GPX1 level in RBCs may be a consequence, rather than the cause, of elevated oxidative stress in RBCs. However, the decrease in GPX1 in the RBCs of the aged NZB mice was not rescued by overexpressing hSOD1 (Fig. 5). One possibility is that the decrease in GPX1 might be improved by hSOD1 overexpression only at a younger age, but the rescuing ability of hSOD1 fades away during aging of the mice, as we have seen in SOD1-deficient mice [23]. The time point showing comparable levels of GPX1 between NZB and hSOD1-Tg/NZB mice

may reflect the results of the faded rescuing ability of the transgene. The other possibility is that the decrease in GPX1 may be caused by another mechanism, such as a defect in the selenium supply. A pivotal role for selenium has been proposed in the anemia of cattle as well as in lupus nephritis [39,40]. Thioredoxin reductase is another essential selenoprotein in RBCs and thus may contribute to maintenance of the redox state of RBCs in NZB mice. Studies from the viewpoint of selenium availability may provide novel insights into understanding AIHA in NZB mice. The (NZB/NZW)F1 mouse is a well-known model for SLE, and the presence of murine lupus-susceptible loci has been proposed [41]. Two major loci have been identified and are linked to autoimmune hemolytic anemia in NZB mice, but the responsible gene has not yet been defined [42]. An increase in levels of HNE-modified proteins in plasma has been reported in children with autoimmune disease [43]. Analysis of oxidative stress parameters in human plasma and RBCs indicates that plasma concentrations of HNE as well as malondialdehyde and oxidized glutathione increase during aging [44]. Oxidation of phosphatidylserine and interaction with CD36 seem to be essential for phagocytosis by macrophages [45,46]. This implies that oxidized cells can be efficiently phagocytosed by macrophages and that oxidized molecules are exposed as antigens to the immune system. In fact, oxidatively modified lipids are identified as epitopes for innate immunity and are responsible for atherogenesis and other diseases [16,17,47]. Thus, lipid peroxidation products, such as HNE and ACR, on RBC membranes can be bona fide epitopes for autoantibodies and

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responsible for RBC-bound IgG in NZB mice (Figs. 1, 2, 5, and 6). These antibodies, together with the autoantibody to CAII, would be useful as markers for AIHA and/or SLE [21,48]. Superoxide anion diffuses across the RBC membrane via the anion channel, referred to as band 3 protein in RBCs [49], and the band 3 protein is a potent antigen in RBCs [8–10]. It is, thus, hypothesized that the produced anti-band-3 autoantibody binds the band 3 protein and blocks the anion channel activity. Because hemoglobin oxidation produces a large amount of superoxide anion every day in vivo [50], it is possible that dysfunction of the band 3 protein caused by the autoantibody binding would elevate intracellular superoxide anion levels by blocking its release from RBCs, enhancing oxidative stress and eventually causing hemolysis. In conclusion, we have provided evidence of the involvement of oxidative stress in the autoimmune response in NZB mice. Transgenic expression of hSOD1 in the RBCs of NZB mice significantly improved the AIHA fatality rate. The results obtained in this study were consistent with the hypothetical role of ROS in triggering the autoimmune reaction in AIHA in NZB mice. Acknowledgment This work was supported, in part, by the Global COE Program (F03) of the Japan Society for the Promotion of Sciences. References [1] Izui, S. Autoimmune hemolytic anemia. Curr. Opin. Immunol. 6:926–930; 1994. [2] Sokol, R. J.; Hewitt, S. Autoimmune hemolysis: a critical review. Crit. Rev. Oncol. Hematol. 4:125–154; 1985. [3] Helyer, B. J.; Howie, J. B. Spontaneous auto-immune disease in NZB/BL mice. Br. J. Haematol. 9:119–131; 1963. [4] Fagiolo, E.; Toriani-Terenzi, C. Mechanisms of immunological tolerance loss versus erythrocyte self-antigens and autoimmune hemolytic anemia. Autoimmunity 36: 199–204; 2003. [5] Mqadmi, A.; Zheng, X.; Yazdanbakhsh, K. CD4+CD25+ regulatory T cells control induction of autoimmune hemolytic anemia. Blood 105:3746–3748; 2005. [6] Fagiolo, E.; Toriani-Terenzi, C. Th1 and Th2 cytokine modulation by IL-10/IL-12 imbalance in autoimmune haemolytic anaemia (AIHA). Autoimmunity 35:39–44; 2002. [7] Murakami, M.; Yoshioka, H.; Shirai, T.; Tsubata, T.; Honjo, T. Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells. Int. Immunol. 7:877–882; 1995. [8] Beppu, M.; Mizukami, A.; Nagoya, M.; Kikugawa, K. Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes: formation of senescent antigen on erythrocyte surface by an oxidative mechanism. J. Biol. Chem. 265: 3226–3233; 1990. [9] Poole, J. Red cell antigens on band 3 and glycophorin A. Blood Rev 14:31–43; 2000. [10] Shen, C. R.; Youssef, A. R.; Devine, A.; Bowie, L.; Hall, A. M.; Wraith, D. C.; Elson, C. J.; Barker, R. N. Peptides containing a dominant T-cell epitope from red cell band 3 have in vivo immunomodulatory properties in NZB mice with autoimmune hemolytic anemia. Blood 102:3800–3806; 2003. [11] Barker, R. N.; de Sa Oliveira, G. G.; Elson, C. J.; Lydyard, P. M. Pathogenic autoantibodies in the NZB mouse are specific for erythrocyte band 3 protein. Eur. J. Immunol 23:1723–1726; 1993. [12] Chang, N. H.; MacLeod, R.; Wither, J. E.; Autoreactive, B. cells in lupus-prone New Zealand black mice exhibit aberrant survival and proliferation in the presence of self-antigen in vivo. J. Immunol. 172:1553–1560; 2004. [13] de Sá Oliveira, G. G.; Izui, S.; Ravirajan, C. T.; Mageed, R. A.; Lydyard, P. M.; Elson, C. J.; Barker, R. N. Diverse antigen specificity of erythrocyte-reactive monoclonal autoantibodies from NZB mice. Clin. Exp. Immunol. 105:313–320; 1996. [14] Fossati-Jimack, L.; Azeredo da Silveira, S.; Moll, T.; Kina, T.; Kuypers, F. A.; Oldenborg, P. A.; Reininger, L.; Izui, S. Selective increase of autoimmune epitope expression on aged erythrocytes in mice: implications in anti-erythrocyte autoimmune responses. J. Autoimmun. 18:17–25; 2002. [15] Hall, A. M.; Ward, F. J.; Shen, C. R.; Rowe, C.; Bowie, L.; Devine, A.; Urbaniak, S. J.; Elson, C. J.; Barker, R. N. Deletion of the dominant autoantigen in NZB mice with autoimmune hemolytic anemia: effects on autoantibody and T-helper responses. Blood 110:4511–4517; 2007. [16] Chou, M. Y.; Hartvigsen, K.; Hansen, L. F.; Fogelstrand, L.; Shaw, P. X.; Boullier, A.; Binder, C. J.; Witztum, J. L. Oxidation-specific epitopes are important targets of innate immunity. J. Intern. Med 26:479–488; 2008. [17] Hazen, S. L. Oxidized phospholipids as endogenous pattern recognition ligands in innate immunity. J. Biol. Chem 283:15527–15531; 2008. [18] Akagawa, M.; Ito, S.; Toyoda, K.; Ishii, Y.; Tatsuda, E.; Shibata, T.; Yamaguchi, S.; Kawai, Y.; Ishino, K.; Kishi, Y.; Adachi, T.; Tsubata, T.; Takasaki, Y.; Hattori, N.; Matsuda, T.; Uchida, K. Bispecific Abs against modified protein and DNA with oxidized lipids. Proc. Natl. Acad. Sci. USA 103:6160–6165; 2006.

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