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The Journal of Immunology, 2007, 179, 1431-1437
Copyright © 2007 by The American Association of Immunologists, Inc.
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Lack of Reactive Oxygen Species Breaks T Cell Tolerance to Collagen Type II and Allows Development of Arthritis in Mice1

Malin Hultqvist, Johan Bäcklund, Kristin Bauer, Kyra A. Gelderman and Rikard Holmdahl2

Section for Medical Inflammation Research, Lund University, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The view on reactive oxygen species (ROS) in inflammation is currently shifting from being considered damaging toward having a more complex role in regulating inflammatory reactions. We recently demonstrated a role of ROS in regulation of animal models for the autoimmune disease rheumatoid arthritis. Low levels of ROS production, due to a mutation in the Ncf1 gene coding for the Ncf1 (alias p47phox) subunit of the NADPH oxidase complex, was shown to be associated with increased autoimmunity and arthritis severity in both rats and mice. To further investigate the role of ROS in autoimmunity, we studied transgenic mice expressing collagen type II (CII) with a mutation (D266E) in the immunodominant epitope that mimics the rat and human CII (i.e., mutated mouse collagen or MMC). This mutation results in a stronger binding of the epitope to the MHC class II molecule and leads to more pronounced tolerance and resistance to arthritis induced with rat CII. When the Ncf1 mutation was bred into these mice, tolerance was broken, resulting in enhanced T cell autoreactivity, high titers of anti-CII Abs, and development of severe arthritis. These findings highlight the importance of a sufficient ROS production in maintenance of tolerance to self-Ags, a central mechanism in autoimmune diseases such as rheumatoid arthritis. This is important as we, for the first time, can follow the effect of ROS on molecular mechanisms where T cells are responsible for either protection or promotion of arthritis depending on the level of oxygen species produced.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human autoimmune diseases such as rheumatoid arthritis (RA)3 are difficult to dissect due to the complex dependency on both genes (1) and environment (2). Therefore, animal models resembling these diseases have successfully been used to minimize the number of variables, thereby increasing the possibility of identifying genes important for disease development. We have earlier shown the importance of a functional NADPH oxidase complex, regulated via the Ncf1 gene, for the resistance to arthritis in rats and mice (3, 4). The Ncf1 protein (alias p47phox) is one of six heterosubunits of the NADPH oxidase complex responsible for the one electron reduction of oxygen-generating superoxide anions, which serve as precursors for reactive oxygen species (ROS). The formed ROS are mainly used to kill invading pathogens (5) and, therefore, lack of a functional NADPH oxidase complex results in low resistance to bacterial and fungal infections in humans, a disease called chronic granulomatous disease (6). It has also been shown that mice made deficient for Ncf1 through knockout techniques, using 129-derived embryonic stem cells, develop a chronic granulomatous disease phenotype (7). Recently, more focus has been on the immunoregulatory role of ROS, further highlighting the importance of Ncf1 and ROS in autoimmune diseases. Even though in our hands granulocytes and APCs, but not T cells, demonstrate an Ncf1-dependent ROS production (8), we propose that cells capable of making an induced oxidative burst affect the selection and activity of T cells. We have shown that T cells from mice and rats with mutations in the Ncf1 gene display higher levels of cell surface thiols, i.e., have more reduced membrane proteins (8). Furthermore, induction of arthritis by transfer of arthritogenic T cells is crucially dependent on the oxidation status of the cell membrane proteins (8). We have also shown that by increasing the production of ROS from the NADPH oxidase complex, in recipient or donor rats, development of arthritis following adoptive T cell transfer is prevented (9). To investigate the involvement of Ncf1 in mouse models for the highly polygenic disease RA, we used B10.Q mice with a spontaneous mutation in the Ncf1 gene, resulting in a truncated Ncf1 protein and nearly absent oxidative burst (4, 10). The presence of the Ncf1 mutation dramatically increased collagen-induced arthritis (CIA) (11) severity on the B10.Q background (4). The immunodominant region of collagen type II (CII) has earlier been shown to be located at position 260–270 in mouse strains carrying the disease-associated MHC class II molecule Aq (12, 13), as does the B10.Q strain (14), or the human RA-associated DR molecules containing the shared epitope (15, 16). The immunodominant region of CII can be posttranslationally modified since the lysine at position 264 can be hydroxylated, which subsequently can be galactosylated or glucogalactosylated (14), generating several distinct immunodominant epitopes recognized by distinct T cells. Mouse CII differs from rat and human CII in one amino acid at position 266 (aspartic acid (D) vs glutamic acid (E), respectively) resulting in a lower affinity binding to the Aq molecule (17). We have produced a mouse strain expressing transgenic CII in a cartilage-specific manner. The transgenic CII carries the D266E mutation, making the immunodominant peptide identical to rat and human CII i.e., the mutated mouse collagen (MMC) mouse (18). Immunization of the MMC mouse with rat CII allows us to study autoreactive immune responses to CII. On the B10.Q background, these mice are tolerized against CII and protected from CIA (19). Because this model provides an excellent opportunity to study the role of ROS in tolerance induction, we bred the Ncf1 mutation into the MMC mice and studied the effect on autoimmune arthritis. In this study, we show that a mutation in Ncf1, resulting in lower level of ROS, breaks tolerance to tissue-specific Ag and allows the development of chronic autoimmune arthritis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

In these experiments, B10.Q mice were used. Mice with a mutation in Ncf1 have earlier been described (4), as well as MMC-1 mice (here referred to as MMC mice) (18). Briefly, a point mutation in the splice site for exon 8 in Ncf1 (10) was inserted into the B10.Q strain by backcrossing (denoted B10.QNcf1*/* when homozygous for the mutation) (4). The MMC-mutated mouse carries a mutated mouse CII gene, thereby expressing the rat CII260–270 epitope in a cartilage-restricted manner. The MMC mice were backcrossed to B10.Q for 13 generations and kept as heterozygotes (denoted B10.QMMC). The B10.QNcf1*/* and the B10.QMMC were then intercrossed to generate the different littermate variants used; B10.QNcf1*/+.MMC, B10.QNcf1+/+.MMC, B10.QNcf1*/*.MMC, B10.QNcf1*/+, B10.QNcf1+/+, and B10.QNcf1*/*. The mice were sex and age matched (7–12 wk of age) at the start of the experiment. All mice were kept and bred in a climate-controlled environment with a 12-h light/dark cycle, housed in polystyrene cages containing wood shavings, and fed standard rodent chow and water ad libitum in the animal house of Medical Inflammation Research (http://www.inflam.lu.se). All experiments were approved by a local (Malmö/Lund, Sweden) ethical committee (license M70/04).

Evaluation of oxidative burst capacity

The level of intracellular oxidative burst ex vivo was measured in blood from naive mice. Blood was collected in tubes containing heparin and hemolyzed with ammonium chloride (0.84%, pH 7.4). Granulocytes were stained with allophycocyanin-conjugated anti-Gr-1 Ab (RB6-8C5; BD Biosciences) for 30 min (4°C). To determine the level of NADPH activity, we used a modified version of the oxidative burst activity flow cytometry assay previously described (20). Briefly, cells were resuspended in Dulbecco’s complete medium without FCS after staining and incubated for 10 min at 37°C with 3 µM dihydrorhodamine 123 (Molecular Probes and Invitrogen Life Technologies), which, after oxidization by hydrogen peroxide (H2O2), peroxinitrite (ONOO–), and hydroxyl radicals (OH•) to rhodamine 123, emits a bright fluorescent signal upon excitation by blue light. Stimulation with PMA (200 ng/ml; Sigma-Aldrich) for 20 min at 37°C was then performed. Cells were washed with PBS and then acquired on a FACSort (BD Biosciences). Cells were gated on cell-type R-123 fluorescence intensity measured on FL-1 and results are expressed in relative fluorescence units.

Arthritis induction and evaluation

CIA was induced by a s.c. injection into the base of the tail of 100 µg of pepsin-digested rat CII emulsified in CFA (Difco and BD Diagnostic Systems) in a total volume of 50 µl/mouse. The CII was purified from the Swarm rat chondrosarcoma as previously described (21). At day 35, the mice were boosted with a s.c. injection of 50 µg of CII emulsified in IFA (Difco) in a total volume of 50 µl/ mouse. Blood was taken in tubes containing heparin at days 42 and 84. The tubes were then centrifuged at 3500 rpm for 20 min and plasma was transferred to new tubes and stored at –20°C until assayed. Arthritis development was monitored with a macroscopic scoring system of the four limbs ranging from 0 to 15 (1 point for each swollen or red toe, 1 point for midfoot digit or knuckle, 5 points for a swollen ankle). The scores of the four paws were added, yielding a maximum total score of 60 for each mouse (22). Other disease parameters including mean cumulative score (MCS), mean day of onset (MDO), and mean maximum score (MMS) were also evaluated. Ankle joints were taken day 167 after immunization and fixed in 4% paraformaldehyde, decalcified in EDTA, embedded in paraffin, sectioned, and stained with H&E for histology analysis.

Determination of serum levels of COMP

Plasma concentration of cartilage oligomeric matrix protein (COMP) was determined by a competitive ELISA according to an earlier described method (23). Briefly, rat COMP was used for coating the microtiter plates and for preparing the standard curve included in each plate. Plates were blocked with 1% BSA in PBS for 2 h at room temperature. After blocking, plasma coincubated with rabbit polyclonal antiserum against rat COMP (generously provided by Prof. D. Heinegård, Section for Connective Tissue Biology, Lund University, Lund, Sweden) was added and the plates were incubated for 2 h at room temperature. The amount of plasma COMP was estimated after incubation with an alkaline phosphatase-conjugated swine anti-rabbit isotype-specific Ab (DakoCytomation) and phosphatase substrate (Sigma-Aldrich) as substrate followed by detection in a Spectra Max (Molecular Devices) at OD 405 nm.

Ab response to CII

Ab titers against rat CII in plasma were determined with ELISA in 96-well plates (Costar) coated overnight at 4°C with 50 µl/well of 10 µg/ml rat CII or 4 µg/ml the CII-specific epitopes CI, U1, and J1 (prepared as described in Ref. 24) in 50 µl of PBS. All washes were performed with PBS (pH 7.4) containing 0.1% Tween 20. Plasma was diluted in PBS and analyzed in duplicates. The amounts of bound IgG Abs were estimated after incubation with biotin-conjugated isotype-specific Abs (Southern Biotechnology Associates) followed by extravidin-peroxidase (Sigma-Aldrich) and developed with ABTS (Roche Diagnostics) as substrate followed by detection in a Spectra Max at OD 405 nm (Molecular Devices). The relative amount of Abs in plasma was determined in comparison with a positive control of pooled serum. The same standard was used for all experiments and relative amount of IgG could thus be compared between experiments. However, no comparison should be made between levels of Ab isotypes or epitope specific Abs.

Delayed-type hypersensitivity (DTH) in response to CII

Mice were immunized on day 0 with 100 µg of pepsin-digested rat CII in CFA (total volume, 50 µl/mouse). Day 13 after immunization the mice were challenged with an injection of 20 µg of lathyritic CII prepared as earlier described (25) in 0.05 M acetic acid (HAc) in the epidermis of the right external ear. Lathyritic CII was used to circumvent reactivity to pepsin, which is present in minute but not immunogenic quantities in all pepsin-digested CII preparations (26). HAc was injected in the left ear as control. The DTH response was measured after 24, 48, and 72 h and presented as ear thickness after the different challenges.

T cell assays

Mice were immunized s.c. with 150 µg of pepsin-digested rat CII in CFA in a total volume of 150 µl. Ten days later, draining lymph nodes were collected and single-cell suspensions were prepared and restimulated in vitro for determination of Ag-specific IL-2 production and IFN-{gamma} production as previously described (27). Briefly, CII-primed T cells were restimulated with lathyritic CII, synthetic CII peptides, purified protein derivative (PPD) (Leo Pharmaceutical Products), for 96 h. Supernatant (50 µl) was collected from the microtiter plate after 24 and 96 h of restimulation for determination of IL-2 and IFN-{gamma} production. The rat CII peptides used were the CII259–273 with a nonmodified lysine at position 264 (K), CII259–273 with a 5-hydroxy-L-lysine at position 264 (HyK), or CII259–273 with a beta-D-galctopyranosyl-5-hydroxy-L-lysine at position 264 (Gal) that were synthesized as previously described (28). PPD, the immunogenic component of CFA, was used as a positive control. All CII Ags were dissolved in 0.1 M HAc. HAc was also used as a negative control. IL-2 levels were determined by coating microtiter plates with anti-IL-2 Ab (clone Jes6-A12; produced in-house) in PBS overnight. After blocking with 1% BSA in PBS, supernatant was added to the plates. A biotinylated anti-IL-2 Ab (Jes6-5H4; produced in-house) was used as secondary Ab. For detection of IFN-{gamma}, a similar protocol was used with anti- IFN-{gamma} Abs from clones AN18 and R46-A2 (produced in-house and Mabtech). The IL-2 and IFN-{gamma} ELISA was quantified using the dissociation-enhanced lanthanide fluoroimmunoassay technique with europium-labeled streptavidin (Wallac and PerkinElmer Life and Analytical Sciences) as per the manufacturers’ recommendations. The plates were read using a fluorometer (VICTOR; PerkinElmer Life and Analytical Sciences).

Statistics

Quantitative data are expressed as mean ± SEM. Statistical analyses were performed using the Mann-Whitney U test. Significance values are given for the difference between Ncf1 wild-type and Ncf1-mutated mice of each MMC genotype, if nothing else is stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Decreased ROS production reverses resistance to CIA in MMC mice

To investigate the impact of a lower ROS production on the induction and perpetuation of T cell tolerance, we bred the Ncf1 mutation, which dramatically lowers induced ROS production, to B10.Q mice carrying the MMC transgene. First, we confirmed the loss of function of the NADPH oxidase complex as a result of the mutation in the Ncf1 gene. Blood granulocytes from wild-type vs Ncf1-mutated MMC mice were investigated for oxidative burst ex vivo. As expected, Ncf1-mutated mice showed a significantly lowered level of ROS production independent of the presence of the MMC transgene after ex vivo stimulation with PMA (Fig. 1). To clarify the effect of ROS production on tolerance induction and perpetuation, we immunized B10.Q mice, with and without MMC along with the different Ncf1 genotypes, and followed the development of the autoantibody response to CII and eventual development of arthritis. Because the normal, nonmutated Ncf1 variant is dominant, the B10.QNcf1*/+ and B10.QNcf1+/+ mice have similar ROS-producing capacities (Fig. 1) and CIA susceptibility (4). We therefore here denoted them B10.QNcf1+. We have earlier shown that MMC mice on the B10.Q background are completely resistant to CIA (19). The effect of introducing the Ncf1 mutation on both alleles (Ncf1*/*) in MMC mice was therefore striking, resulting in a fully penetrant severe and chronic arthritis with an onset even earlier than that of wild-type B10.Q mice (Fig. 2, A and B, and Table I). However, B10.QNcf1*/*.MMC mice still developed a milder form of arthritis compared with B10.QNcf1*/* mice, indicating that tolerance to self-CII could not be completely overridden by the Ncf1 mutation. MMC-transgenic mice heterozygous for the Ncf1 mutation showed similar arthritis susceptibility as B10.QNcf1+/+ mice (Table I). Arthritis in the Ncf-mutated MMC mice was accompanied by increased levels of COMP, reflecting ongoing cartilage destruction (23) (Fig. 2C). Severe active inflammation and joint destruction in the Ncf1-mutated MMC mice could also be seen on histology sections taken at the end of the experiment (Fig. 2D) in contrast to B10.QNcf1+/+.MMC mice.


Figure 1
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  		FIGURE 1. Mice with a mutation in Ncf1 have lower ROS production. Blood granulocytes from naive mice were analyzed for ROS production in response to PMA ex vivo. Stars indicate difference compared with Ncf1 wild-type mice of respective MMC genotype (n = 5–7). *, p < 0.05; **, p < 0.01.

 

Figure 2
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  		FIGURE 2. Ncf1 mutation breaks resistance to CIA in CII-expressing mice. Arthritis was induced on day 0 by an injection of rat CII emulsified in CFA s.c. in MMC and non-MMC-transgenic mice with (Ncf1+) or without (Ncf1*/*) a functional ROS production. The mice were boosted on day 35 with an injection of rat CII in IFA (represented by vertical line). Arthritis severity (A) and incidence (B) was followed for 102 days. C, Plasma was taken at days 42 and 84 after immunization and analyzed for COMP levels as a measurement of cartilage destruction. Significance values are given in respect to Ncf1+ of corresponding MMC genotype. D, Representative histology taken from MMC-transgenic mice at the end of experiment, stained with H&E (B10.QNcf1*/*, n = 12; B10.QNcf1*/*.MMC, n = 11; B10.QNcf1+, n = 19; B10.QNcf1+.MMC, n = 14). *, p < 0.05; ***, p < 0.001.

 

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  		Table I. Arthritis development in MMC mice

 
Increased levels of anti-CII Abs in Ncf1-mutated MMC-transgenic mice

To investigate the effect on T cell-dependent autoimmunity, we analyzed levels of CII-reactive Abs in blood. Mice with the Ncf1 mutation showed higher Ab levels to CII, measured in plasma day 42 after immunization, in both MMC and non-MMC mice. The MMC mice were analyzed in more detail and we found that the Ncf1 mutation increased the T cell-dependent IgG anti-CII levels at day 42 (Fig. 3A). When titers of different anti-CII IgG isotypes in MMC-transgenic mice were analyzed, we could see that all IgG isotypes tended to be higher in the Ncf1-mutated mice although only IgG2b and IgG2c reached significance (Fig. 3B), suggesting no distinct polarization of the T cell response. We also analyzed the specificity of the anti-CII Abs in the MMC-transgenic mice. The major B cell epitopes have been identified using a series of mAbs from mice (29). The immunodominance of these epitopes seems to be shared between CIA in mice and rats and RA in humans (30, 31, 32, 33). By analyzing the B cell response toward these epitopes, we could see that an increase in levels of Abs to epitopes C1 and U1 could be seen, although the response seems to the J1 epitope shows the opposite pattern (Fig. 3C). Interestingly, Abs to C1 and U1 are arthritogenic and correlate with the development of chronic arthritis (24). No correlation between Ab levels and arthritis severity was observed (Fig. 3D), suggesting that increased Ab production is a primary effect of the Ncf1 mutation and not a secondary effect of arthritis development.


Figure 3
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  		FIGURE 3. Increased autoreactivity in Ncf1-mutated MMC mice. A, Plasma taken at days 42 and 84 from MMC-transgenic mice with (Ncf1+) or without (Ncf1*/*) a functional ROS production was analyzed for IgG Abs directed to CII. Plasma taken from MMC-transgenic mice on day 42 was also analyzed for levels of IgG isotypes to CII (B) and response pattern against major B cell CII epitopes C1, U1, and J1 (C). Results were compared with a standard of positive plasma and expressed as relative titer. Significance values are given for the difference between Ncf1+ and Ncf1*/* of respective MMC genotypes. D, Correlation between arthritis severity (expressed as mean arthritis score) and anti-CII IgG on day 42 after immunization. (B10.QNcf1*/*, n = 12; B10.QNcf1*/*.MMC, n = 11; B10.QNcf1+, n = 19; B10.QNcf1+.MMC, n = 14). *, p < 0.05; **, p < 0.01.

 
The mutation in Ncf1 increases DTH and T cell recall responses

We further investigated T cell-dependent immune functions measured by the DTH response to CII in the MMC mice. In both MMC and non-MMC mice, the Ncf1 mutation increased the DTH reaction (Fig. 4) The DTH reaction was lower in MMC-transgenic mice compared with nontransgenic Ncf1-mutated mice. Because a DTH reaction is a T cell-dependent, but B cell-independent, inflammatory response, this finding again suggests T cells as the main responsible cells of the Ncf1-associated effect on the immune response.


Figure 4
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  		FIGURE 4. Impaired ROS production increases DTH responses. DTH response in Ncf1-mutated MMC-transgenic mice. Mice were immunized with CII in HAc emulsified in CFA. Day 13 after immunization, the mice were challenged by an injection of lathyritic rat CII in the outer ear and the swelling that followed was measured after 24, 48, and 72 h and compared with the control ear injected with HAc. Significance values are given for differences between Ncf1+/+ and Ncf1*/* of each MMC genotype. (B10.QNcf1*/*, n = 12; B10.QNcf1*/*.MMC, n = 14; B10.QNcf1+/+, n = 16; B10.QNcf1+/+.MMC, n = 9). *, p < 0.05.

 
To investigate the T cell responsiveness, lymph node cells from mice immunized 10 days earlier with CII were isolated and stimulated with Ag in vitro. The Ncf1 mutation significantly increased the level of IL-2 production as a measurement of T cell proliferation and IFN-{gamma} secretion in non-MMC as well as MMC-transgenic mice (Fig. 5). As expected, non-MMC Ncf1-mutated mice showed a strong recall response against the CII protein and peptides, with a clear predominance against the galactosylated CII peptide (Gal). In contrast, MMC-transgenic mice with a functional Ncf1 genotype showed a profoundly reduced response against all CII Ags, and nearly all of the remaining response was biased against the galactosylated peptide, as shown before (19). However, introduction of the Ncf1 mutation in MMC-transgenic mice resulted in an overall increase in T cell response, comparable to that seen in wild-type mice. Ncf1-mutated mice tended to mount a stronger IFN-{gamma} response to PPD (the antigenic component of CFA), indicating that a defective oxidative burst also affects T cell responses to heterologous Ags. However, IL-2 production was not affected by the Ncf1 mutation. The similar tendency for an increased IFN-{gamma} response to heterologous Ag was also noted upon in vitro restimulation of OVA-primed mice (data not shown). The response to heterologous Ags, however, is variable, whereas the response to CII is consistent in our experiments.


Figure 5
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  		FIGURE 5. Ncf1 mutation increases T cell responsiveness in MMC-transgenic mice. Recall in vitro response of lymph node cells from mice immunized 10 days earlier with rat CII (B10.QNcf1*/*, n = 9; B10.QNcf1*/*.MMC, n = 9; B10.QNcf1+/+, n = 11; B10.QNcf1+/+.MMC, n = 9) in CFA. Cells were restimulated with 50 µg/ml lathyritic rat CII or 25 µg/ml of the following CII peptides: CII259–273 with a nonmodified lysine at position 264 (K), CII259–273 with a 5-hydroxy-L-lysine at position 264 (HyK), or CII259–273 with a beta-D-galactopyranosyl-5-hydoxy-L-lysine at position 264 (Gal). After 24 and 96 h, supernatants were collected and analyzed for IL-2 (A) and IFN-{gamma} (B) content, respectively. PPD (20 µg/ml) was used as positive control and 0.1 M HAc was used as a negative control. Significance values are given for differences between Ncf1+/+ and Ncf1*/* of each MMC genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Along with the arthritis data, this suggests that a certain level of ROS production is crucial for developing and maintaining T cell tolerance to CII and that we now have an excellent tool for further studies of the mechanism behind the ROS regulation of autoimmunity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The susceptibility to CIA is dependent on the level of T cell tolerance to self-CII. We show here that a lower oxidative burst capacity, due to a mutation in the Ncf1 gene, lowers the tolerance threshold and leads to an increased arthritis susceptibility. This is shown by the fact that MMC mice, expressing an immunodominant CII epitope that binds with higher affinity to the MHC class II molecule, develop CIA if a mutation in Ncf1, lowering oxidative burst capacity, is present.

Clearly, the involvement of oxidative burst to modulate T cell activation and tolerance is a fundamental phenomenon but is likely to be of high complexity in vivo. Thus, we need to unravel the role of oxygen radicals in the context of time, location, and molecular interactions. We were convinced of the importance of a functional ROS production after positional cloning a structural polymorphism in the Ncf1 gene in the rat, showing that a lower oxidative burst increased both T cell responsiveness and arthritis susceptibility (3). The fundamental role of oxidative burst on T cell responsiveness and arthritis susceptibility could be confirmed with a spontaneous mutation in Ncf1 in the mouse. An Ncf1-associated decrease in oxidative burst led to severe and chronic CIA and also spontaneous development of arthritis (4). The exact mechanism behind this effect is likely to be quite complex. We have seen that altered oxidation status of T cell membrane proteins affect arthritogenic capacity (8) but which cellular mechanisms are altered are so far unknown. ROS are involved in numerous cellular functions, primarily bacterial defense (34) but also more subtle regulations of cellular mechanisms such as proliferation (35) and T cell apoptosis and signaling (reviewed in Refs. 36 and 37), but also effects on Ag processing (38) and oxidation of target structures (39) have been reported. Structures important for immunological mechanisms have been suggested to be affected by membrane oxidation status, e.g., linker for activation of T cells (39). The mechanism of action of ROS levels could also be more indirect. Ag presentation could be affected by altered ROS production from the NADPH oxidase complex. Phagosomes are relying on ROS production to regulate protein degradation into peptides that can be presented. Recent data suggest that mice with a deficiency in ROS production from the NADPH oxidase complex have an accelerated Ag degradation due to a more acidic environment in dendritic cell phagosomes, which leads to defective cross-presentation to CD8+ T cells (38). Another intriguing newly reported finding is that redox status regulates editing of the MHC class I repertoire. Presumably, oxidation of the peptide-reactive site is required for optimal peptide loading (40). This might also be the case for MHC class II. It has also been shown that mice lacking Ncf1 have a strong immune suppression due to lack of inhibition of NO that promotes T cell expansion (35).

All of these mechanisms are likely to be affected by a mutation in Ncf1 and this diverse pattern of action of ROS complicates the task of pinpointing its exact role in autoimmunity. Suggestions on how ROS modulate immune responsiveness are apparently quite divergent and sometimes in fact assume the opposite effect, e.g., that a decreased production of ROS will dampen both the inflammatory and immune responsiveness. This could be dependent on differences in vitro vs in vivo or between knockout mice and more genetically controlled mutated mice.

Molecular investigations with RNA or protein expression profiling, even in genetically well-controlled animals with isolated Ncf1 polymorphisms, reveal a quite complex skewing of the expression pattern showing that numerous functions will be changed but does not suggest a single molecular mechanism (47). To reduce the complexity, we focused on extensive and time-consuming crosses of different mouse strains to further investigate the mechanism in its natural environment and in a genetically controlled background. By breeding the Ncf1 mutation into B10.Q mouse strains carrying other mutations or transgenes important for the function of the immune system, we searched to develop relevant tools to study this mutation. One such model used was the MMC mouse strain expressing the immunodominant epitope of CII. When immunized with rat CII, now autologous CII, these mice are partially tolerant and protected from CIA (19). On the B10.Q background, the protection is more complete than in mice on the C3H.Q background and as a consequence B10.QMMC mice are fully resistant to arthritis. In fact, this model better mimics human disease in which a T cell response to the same glycosylated epitope predominates (41), and in which Abs occur specific for the same CII epitopes as in the mouse (30). The finding that the Ncf1 mutation reversed the tolerance and allowed the development of severe arthritis in these otherwise resistant mice shows that oxidation is of fundamental importance for regulating T cell tolerance and susceptibility to autoimmune arthritis. We could not only observe that arthritis developed in Ncf1-mutated mice but we also noticed a stronger T cell responsiveness to CII and to the CII260–270 epitope in its various posttranslational forms. Concomitantly, we also found higher levels of IgG Abs to CII in serum. No particular skewing of the IgG subclasses was observed, indicating that the Ncf1 mutation did not change the phenotype of the underlying T cell response. Interestingly, the Ab response was directed toward CII epitopes known to correlate with arthritis in both mice and humans, and that are targeted by arthritogenic Abs (24, 30, 42).

The enhanced T cells responsiveness, and the increased arthritis susceptibility, was not unique to the MMC mice because the non-MMC mice on the B10.Q background also develop chronic arthritis, (4). However, the Ncf1 effect in the MMC transgene is qualitatively different because it changed the mice from being resistant and completely protected from arthritis to be highly susceptible. In the MMC mouse, the CII peptide is binding with stronger affinity and is therefore better presented to the immune system resulting in a higher degree of tolerance but also by promoting the development of CD4+CD152+ regulatory T cells (43). In the non-MMC mouse, the T cells have the potential to recognize the same peptide if exposed on APCs (44). Thus, the non-MMC mice are likely to develop tolerance as well but less strongly due to ignorance of the Ag in the tissue. Consequently, the same Ncf1-associated mechanisms may operate to enhance arthritis in MMC and non-MMC B10.Q mice. Our hypothesis is that ROS operate as transmitters in the immune synapse, i.e., in the space formed between APCs and T cells where MHC class II, TCR, and costimulatory molecules interact. Since the Ncf1-containing NADPH oxidase complex is expressed in the phagosome and in the cell membrane lipid rafts (45), it is likely that production of ROS will change the redox level in the synapse during T cell activation. Accordingly, we have shown that the T cell membrane proteins are less oxidized in Ncf1-mutated mice without having a lowered redox level in the cytosol (8). Possibly, this will change the T cell activation threshold wherever the T cell interacts with an MHC-Ag complex. Because we see strong effects on the T cell response to CII, which is a self-Ag expressed in thymus (46), it is possible that the Ncf1 mutation operates already during T cell selection in the thymus. Increased oxidative burst from thymic epithelial cells or other APCs could affect the thymic selection, possibly by alteration of oxidation status of proteins on the cell surface. Importantly, however, it is likely that T cells exposed to ROS from various sources like ROS produced from APCs in the periphery or maybe also from bystander cells like granulocytes will also affect the T cell redox status. This would therefore not exclude a role for ROS in regulating general T cell responses at large, i.e., also T cells specific to non-self Ag. Thus, this is an unexpected and possibly important mechanism for the complex function of ROS in vivo.

Taken together, our data suggest that ROS is important for maintaining T cell tolerance to autologous Ags, which has implications also for human disease because the human disease is caused by a similar homologous response. These new findings increase the possibilities to find a pathway in humans that could be targeted to understand the pathogenesis of human disease as well as to develop novel therapeutic treatments.


   Acknowledgments
 
We thank Rebecka Ljungkvist and Carlos Palestro for taking excellent care of the mice. We also thank Emma Mondoc for the help during preparation of histology slides.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by grants from the Anna Greta Crafoord, King Gustaf V’s 80th Year; the Nilsson-Ehle; the Kock and Österlund Foundations; the Swedish Association against Rheumatism; Biovitrum AB; the Swedish Medical Research Council; the Swedish Society for Medical Research; the Strategic Research Foundation and the European Union Projects LSHB-CT-2006-018661 (AUTOCURE), LSHM-CT-2005-005223 (EURAPS), and NEUROPROMISE (LSHM-CT-2005-018637). Back

2 Address correspondence and reprint requests to Dr. Rikard Holmdahl, Medical Inflammation Research, I11 Biomedical Center, Lund University, Lund, Sweden. E-mail address: rikard.holmdahl{at}med.lu.se Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; CII, collagen type II; CIA, collagen-induced arthritis; COMP, cartilage oligomeric protein; DTH, delayed-type hypersensitivity; MMC, mutated mouse collagen; ROS, reactive oxygen species; MCS, mean cumulative score; MDO, mean day of onset; MMS, mean maximum score; HAc, acetic acid; PPD, purified protein derivative. Back

Received for publication March 1, 2007. Accepted for publication May 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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