Detection of arthritis-susceptibility loci including Ncf1 and variable effects of the major histocompatibility complex region depending on genetic background in rats.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 2, February 2009, pp 419–427 DOI 10.1002/art.24292 © 2009, American College of Rheumatology Detection of Arthritis-Susceptibility Loci, Including Ncf1, and Variable Effects of the Major Histocompatibility Complex Region Depending on Genetic Background in Rats Carola Rintisch, Michael Förster, and Rikard Holmdahl Objective. To characterize the arthritismodulating effects of 3 non–major histocompatibility complex (MHC) quantitative trait loci (QTLs) in rat experimental arthritis in the disease-resistant E3 strain, and to investigate the disease-modulating effects of the MHC region (RT1) in various genetic backgrounds. Methods. A congenic fragment containing Ncf1 along with congenic fragments containing the strongest remaining loci, Pia5/Cia3 and Pia7/Cia13 on chromosome 4, were transferred from the arthritis-susceptible DA strain into the background of the completely resistant E3 strain. The arthritis-regulatory potential of the transferred alleles was evaluated by comparing the susceptibility to experimental arthritis in congenic rats with that in E3 rats. The RT1u haplotype from the E3 strain was transferred into the susceptible DA strain (RT1av1), and various F1 and F2 hybrids were generated to assess the effects of RT1 on arthritis susceptibility. Results. The DA allele of Ncf1 did not break the arthritis resistance of the E3 rats, although it led to enhanced autoimmune B cell responses, as indicated by significantly elevated levels of anticollagen antibodies in congenic rats. Introgressing Pia5 and Pia7 loci on chromosome 4 broke the resistance to arthritis, and the MHC locus on chromosome 20 in DA rats enhanced arthritis when RT1 interacted with E3 genes. Conclusion. The findings in these congenic lines confirm the existence of 3 major QTLs that regulate the severity of arthritis and are sufficient to induce the transformation of a completely arthritis-resistant rat strain into an arthritis-susceptible strain. This study also reveals a dramatic difference in the arthritisregulatory potential of the rat MHC depending on genetic background, suggesting that strong epistatic interactions occur between MHC and non-MHC genes. Rheumatoid arthritis (RA) is a polygenic autoimmune disease characterized by chronic inflammation in the joints that eventually leads to destruction of the bone and cartilage (1). Although efforts have largely been directed to the understanding of the pathophysiologic mechanisms underlying RA, there is still little known about the genetic factors contributing to RA. Recent studies based on various gene-finding strategies have identified the first genes implicated in the susceptibility to RA (2–4). However, most susceptibility genes are yet to be discovered. Among the difficulties involved in gene identification are factors inherent in this complex trait, such as variable penetrance, variable relative risk associated with the disease allele, epistasis, genetic heterogeneity of human populations, and complex interactions between environmental and genetic factors (5). Therefore, animal models are attractive tools for the study of RA, because use of such models not only overcomes genetic complexities but also permits studies under stable environmental conditions. We have previously reported the location of 12 non–major histocompatibility complex (MHC) loci (Pias 2–8, Pia10, and Pias 12–15) that regulate the severity of pristane-induced arthritis (PIA) in F2 offspring of the 100%-susceptible DA and 100%-resistant E3 inbred rat strains (6–8). To Supported by the Swedish Research Council, the Swedish Association Against Rheumatism, the Swedish Foundation for Strategic Research, and the European Union Sixth Framework Programme (MUGEN Network grant LSHG-CT-2005-005203 and EURATools project contract LSHG-CT-2005-019015). Carola Rintisch, MS, Michael Förster, MS, Rikard Holmdahl, MD, PhD: Lund University, Lund, and Karolinska Institute, Stockholm, Sweden. Address correspondence and reprint requests to Rikard Holmdahl, MD, PhD, Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden. E-mail: Rikard.Holmdahl@ki.se. Submitted for publication May 19, 2008; accepted in revised form November 3, 2008. 419 420 RINTISCH ET AL positionally clone the underlying gene and study the effect of an arthritis-regulating quantitative trait locus (QTL), we have produced congenic strains in which the regulatory loci were transferred from the arthritisresistant E3 strain into the arthritis-susceptible DA strain. With this strategy, we recently identified the gene controlling the major QTL for PIA (Pia4) as the Ncf1 gene (9). However, little is known about the impact that this gene and other QTLs play in the genetic context of the arthritis-resistant E3 rat. In the present study, we constructed congenic rat strains in which 3 major arthritis-promoting QTLs from the DA strain (Pia4/Cia12, Pia5/Cia3, and Pia7/Cia13) (6,10–12) were transferred into the completely arthritisresistant E3 rat strain (7). These congenic rats were evaluated for the susceptibility to and severity of arthritis, which was induced by 2 different arthritogenic agents: native type II rat collagen emulsified in Freund’s incomplete adjuvant (IFA), and the mineral oil pristane (13,14). In addition, we investigated the influence of a fourth QTL, Pia1, which includes the RT1 region (the MHC in rats). Although Pia1 was not identified in previous linkage studies of (DA ⫻ E3)F2 hybrids in models of collagen-induced arthritis (CIA) and PIA (6,7), we obtained some evidence that this absence of identifiable Pia1 was partly due to poor genotyping, which hampered its detection. In this study, we reevaluated the influence of Pia1 in rat PIA and found a significant impact of this QTL on arthritis severity, but also found that the effects were dependent on the genetic background of the animals. MATERIALS AND METHODS Animals. The DA/ZtmRhd (short DA) and E3/ ZtmRhd (short E3) rats used in this study originated from Zentralinstitut für Versuchstierkunde (Hannover, Germany) and were bred for more than 20 generations by brother/sister mating in the animal facility in Lund, Sweden, in a climatecontrolled environment with 12-hour light/dark cycles. In the same facility, crossbreeding to produce all congenic rats, as well as various F1 and F2 hybrids, was performed. The congenic rats, E3.DA-Pia4 (D12Wox5 ⫻ D12Rat26; N10F5), E3.DAPia4/Pia5/Pia7, referred to as short E3.DA-Pia457 (D4Wox22 ⫻ D4Got132 ⫻ D12Wox5 ⫻ D12Rat26; N10F5), and DA.E3-RT1u/u (D20Rat45 ⫻ D20Rat47; ⬎N15), were obtained through conventional backcross breeding with negative selection of all known PIA QTLs and positive selection for the target QTLs, using microsatellite markers. The purity of the genetic background of E3.DA-Pia457–congenic rats was confirmed using 256 microsatellite markers covering all 20 autosomes and the X-chromosome. Six female E3.DA-Pia457 rats and 6 male DA rats were intercrossed to produce F1 hybrids, which were further intercrossed to produce 638 (E3.DA-Pia457 ⫻ DA)F2 hybrids. An additional 6 female E3.DA-Pia457 rats were intercrossed with DA.E3-RT1u/u rats to obtain F1 hybrids. Rats were housed in polystyrene cages containing wood shavings and were fed standard rodent chow and water ad libitum. The rats were free from common pathogens, including Sendai virus, Hantaan virus, corona virus, reovirus, cytomegalovirus, and Mycoplasma pulmonis. All experiments were approved by the local ethics committees (Malmö/Lund, Sweden). Genotyping. DNA was prepared from toe biopsy samples by alkaline lysis, with amplification using fluorescencelabeled microsatellite markers in a Multiplex polymerase chain reaction carried out in accordance with a standard protocol. Results were analyzed on a MegaBACE 1000 (Amersham Pharmacia Biotech, Buckinghamshire, UK). (The sequences for the microsatellite markers used for genotyping of the congenic fragments from E3 and DA rats were retrieved from the Ensembl database at http://www.ensembl.org/Rattus_ norvegicus/index.html.) Induction and evaluation of arthritis. Lathyritic type II collagen (CII) was purified from Swarm rat chondrosarcoma, which had been grown in male rats receiving ␤ aminopropionitrile monofumaratic salt in drinking water during a tumor-growing period, as previously described (15,16). CIA was induced by a single intradermal injection of lathyritic rat CII dissolved in 0.1M acetic acid and emulsified in IFA. In the case of PIA, disease was induced by a single intradermal injection of pristane (2,6,10,14-tetramethylpentadecane; ACROS Organics, Kenilworth, NJ) at the base of the tail. (Injection volumes varied between experiments, as indicated in the Figure legends and Table 1.) Arthritis was induced in rats at ages 7–9 weeks, and all 4 limbs were monitored for arthritis development using a macroscopic scoring system. Briefly, 1 point was given for each swollen and red toe, 1 point for each affected midfoot, digit, or knuckle, and 5 points for a swollen ankle (maximum score per limb 15). Arthritis development was examined in the rats every second or third day for 1 month after induction of the disease (17). Blood sampling and detection of antibodies. Peripheral blood was collected on the termination day by cutting the tip of the tail. Blood samples were assayed for IgG antibodies against rat CII using a Europium3⫹-linked sandwich enzymelinked immunosorbent assay (ELISA). For detection of total IgG and all subclasses, ELISA plates were coated with polyclonal mouse anti-rat IgG. After blocking with 2% bovine serum albumin, the plates were washed, and serum samples were added. The mixture was then incubated with biotinlabeled mouse anti-rat monoclonal antibodies, followed by Eu3⫹-labeled streptavidin (in Assaybuffer; Wallac, Turku, Finland). For final detection of antibodies, enhancement solution (Wallac) was added, and fluorescence emissions were read using a Victor/Wallac protocol (Wallac). Statistical analysis. The StatView software program (Stata, College Station, TX) was used for all statistical analyses. The incidence of arthritis was analyzed by Fisher’s exact test, and the nonparametric Mann-Whitney U test (comparison of 2 groups) or Kruskal-Wallis test (comparison of 3 groups) was used in all other statistical analyses. P values less than 0.05 were considered significant. QUANTITATIVE TRAIT LOCI AND MHC EFFECTS ON RAT ARTHRITIS 421 Table 1. Susceptibility to pristane-induced arthritis (PIA) and collagen-induced arthritis (CIA) in E3.DA-Pia457–congenic rats compared with DA rats* Disease, strain, immunogen PIA E3.Pia457 200 l pristane 150 l pristane DA, 150 l pristane CIA E3.Pia457, 150 g CII DA, 100 g CII Days to onset† Maximal arthritis score‡ Incidence Mean ⫾ SD Minimum–maximum Mean ⫾ SD Minimum–maximum 17/44 6/8 17/18 21 ⫾ 4 20 ⫾ 2 14 ⫾ 2 13–27§ 18–24§ 12–16 18 ⫾ 15 13 ⫾ 7 33 ⫾ 10 1–48 5–24 15–47 12/24 23/24 23 ⫾ 6 16 ⫾ 2 16–35§ 14–25§ 24 ⫾ 17 38 ⫾ 15 2–59 4–60 * Values for DA rats are representative results from previous, unpublished experiments. † First day of visible signs of arthritis (arthritis score ⬎0) among only rats that developed disease throughout the experiment. ‡ Maximal obtained clinical score of the severity of arthritis during the experiment. § The maximum day is also the termination day. RESULTS Arthritis susceptibility in E3.DA-Pia457 triplecongenic rats. Since the E3 rat strain is 100% resistant to arthritis, we sought to investigate whether the resistance could be broken by introducing alleles from the arthritissusceptible DA strain into the E3 background. To achieve this, we generated E3.DA-Pia4–congenic rats that harbored a DA fragment at chromosome 12 (Pia4), and E3.DA-Pia457–congenic rats that harbored a DA fragment at both chromosome 4 (Pia5 and Pia7) and chromosome 12. These congenic rats, as well as the E3 parental rats, were immunized with a single dose of the mineral oil pristane and then evaluated for the development of arthritis. Three independent experiments were performed, and all yielded similar results; the data were therefore pooled for further analyses. These experiments showed that all of the immunized E3 rats (n ⫽ 17) and all of the immunized E3.DA-Pia4–congenic rats (n ⫽ 16) remained completely protected from the development of PIA, but the resistance to PIA was broken in E3.DA-Pia457 rats, since 17 of 44 developed mild arthritis after a single injection of 200 l pristane (P ⬍ 0.0001 versus parental E3 rats) (Figure 1A). The first visual signs of arthritis (disease onset) in individual E3.DA-Pia457 rats were observed between day 13 and day 27 (the latter being the termination day) (Figure 2), and the maximal arthritis score ranged from 1 to 48. A summary of these results and additional data from the arthritis-susceptible DA strain are shown in Table 1. In general, E3-congenic rats developed arthritis later, and the arthritis was somewhat milder than that in DA rats, although the maximal arthritis score in some individual E3-congenic rats was the same as that in DA rats. As a second method of studying the difference between E3 rats and E3.DA-Pia457–congenic rats, we chose a CIA model in which rats were immunized with lathyritic CII emulsified in IFA. As in PIA, all of the CII-immunized E3 rats (n ⫽ 12) and E3.DA-Pia4 singlecongenic rats (n ⫽ 21) were resistant to CIA, but 50% of the E3.DA-Pia457 triple-congenic rats (12 of 24) developed arthritis (P ⬍ 0.0001 versus parental E3 rats) (Figure 1B). The first macroscopic signs of arthritis in individual E3.DA-Pia457 rats were observed between day 16 and day 35 (the latter being the termination day), and the maximal arthritis score ranged from 2 to 59 (Table 1). When we analyzed the serum levels of anti-CII IgG on day 35 after the onset of CIA, we detected a significantly enhanced level of antibodies in E3.DAPia457 triple-congenic rats compared with that in E3.DA-Pia4 single-congenic rats and E3 parental rats (each P ⬍ 0.0001 versus the triple-congenic strain) (Figure 1C). Interestingly, we also registered a significantly elevated level of antibodies in E3.DA-Pia4 singlecongenic rats compared with that in E3 parental rats (P ⬍ 0.0013). Taken together, these results clearly demonstrate that the single QTL Pia4, which includes the arthritis-promoting Ncf1 allele, had no effect on arthritis susceptibility or severity in the genetic background of the resistant E3 strain. However, by transferring 3 major QTLs for PIA from the susceptible DA strain, and thus generating E3.DA-Pia457 triplecongenic rats, we could break the tolerance to PIA, as well as CIA, in the E3 rat. Varied importance of the MHC in different genetic backgrounds. Because the arthritis incidence was still surprisingly low in E3.DA-Pia457 triple-congenic 422 RINTISCH ET AL . Figure 2. Arthritic hind paw from an E3.DA-Pia457–congenic rat 20 days after pristane administration (bottom), compared with a normal paw from a nonimmunized rat (top). Figure 1. Development of arthritis in E3.DA-Pia457–congenic rats. A, Arthritis incidence after a single injection of 200 l pristane. None of the E3 rats (0 of 17) and none of the E3.DA-Pia4 rats (0 of 16) developed pristane-induced arthritis (PIA), whereas 17 of 44 E3.DAPia457 rats developed macroscopic signs of PIA. B, Mean arthritis score after a single injection of type II collagen (CII). None of the E3 rats (0 of 12) and none of the E3.DA-Pia4 rats (0 of 21) developed collagen-induced arthritis (CIA), whereas 50% of the E3.DA-Pia457 rats (12 of 24) developed CIA. Bars show the mean ⫾ SEM pooled results from 3 independent experiments. C, Levels of anti-CII IgG antibodies after development of CIA. E3.DA-Pia4 rats, although resistant to CIA, produced significantly more anti-CII IgG antibodies compared with E3 rats. E3.DA-Pia457 rats developed CIA and produced significantly higher levels of anti-CII IgG antibodies compared with both E3 and E3.DA-Pia4 rats. Results are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. Circles indicate outliers. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.0001, versus E3 and/or E3.Pia4. rats, we investigated the role of a fourth QTL, Pia1, in PIA. Pia1, which includes the RT1 region in rats, is a QTL that has been difficult to reproduce in rat models of PIA and was not found in linkage studies of F2 hybrids generated with the E3 strain (RT1u/u) and the DA strain (RT1av1/av1) (6,7). However, when the data from the previous F2 intercross experiments in a considerably low number of the rats (n ⫽ 153) were reanalyzed, we found that only 91 rats were in fact genotyped in the RT1 region. The remaining 62 animals had unknown genotypes and had a significantly lower arthritis score than the other rats (P ⬍ 0.0001). We therefore sought to reevaluate the importance of RT1 in PIA. However, we did not have EJ.DA-RT1av1/av1–congenic rats. By using the reciprocal DA.E3-RT1u/u–congenic strain as a breeding partner, we could determine the importance of RT1 in different genetic backgrounds. In addition, since sex-specific differences in the MHC have been reported in rats and in humans, we analyzed the female and male rats separately. We first investigated the influence of RT1u and av1 RT1 in rats with a DA background. We immunized homozygous DA.E3-RT1u/u– and heterozygous DA.E3- QUANTITATIVE TRAIT LOCI AND MHC EFFECTS ON RAT ARTHRITIS 423 Figure 3. Influence of RT1 in the development of pristane-induced arthritis (PIA) in different genetic backgrounds. A and B, Mean arthritis score after injection of 100 l pristane in male congenic rats (A) and female congenic rats (B) in the DA.E3-RT1u/u (short u/u) and DA.E3-RT1u/av1 (short u/av1) groups. There was no difference between RT1u/av1-heterozygous and RT1u/u-homozygous rats, among males or among females. Data in A and B are the mean ⫾ SEM pooled results from 2 experiments. C and D, Mean arthritis score in male hybrids (C) and female hybrids (D) in the (E3.DA-Pia457 ⫻ DA)F1 (short u/av1) and (E3.DA-Pia457 ⫻ DA.E3-RT1u/u)F1 (short u/u) groups. In male F1 hybrids, a significant difference between RT1u/av1-heterozygous and RT1u/u-homozygous rats was observed, since heterozygous male rats had more severe PIA in the early phase of disease. Female F1 hybrids with the RT1u/av1 haplotype had significantly more severe arthritis after pristane injection compared with female F1 hybrids with the RT1u/u haplotype. This difference was more pronounced than in male rats, and a significantly higher mean arthritis score could be observed in these female rats from day 12 to day 27. Data in C and D are the mean ⫾ SEM pooled results from 4 independent experiments. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.0001, versus RT1u/u. RT1u/av1–congenic rats with pristane, and followed up the disease course for 24 days. There were no differences in arthritis incidence, number of days to onset of arthritis, or disease severity between heterozygous and homozygous congenic rats among the males (Figure 3A) or among the females (Figure 3B). We then investigated the influence of RT1 in rats with a mixed background from E3 and DA. To achieve this, we generated F1 hybrids from (E3.DA-Pia457 ⫻ DA.E3-RT1u/u) rats and compared them with F1 hybrids from (E3.DA-Pia457 ⫻ DA) rats. In this genetic setup, all rats were DA/E3 heterozygous for the entire genome except for the Pia4, Pia5, and Pia7 loci, at which positions all rats were DA homozygous. Both groups differed only at the RT1 region, in which F1 hybrids from (E3.DA-Pia457 ⫻ DA.E3-RT1u/u) were RT1u/u homozy- gous, and F1 hybrids from (E3.DA-Pia457 ⫻ DA) were RT1u/av1 heterozygous. In 3 independent experiments, we immunized the F1 hybrid rats with pristane, and after 27 days of followup, we detected a significant difference in the mean arthritis score between rats with the RT1u/u haplotype and those with the RT1u/av1 haplotype, both among males (Figure 3C) and among females (Figure 3D). Since we obtained evidence of a varying impact of RT1 on PIA depending on the genetic background, we subsequently tried to identify and investigate genes in the background genome that epistatically interact with the MHC. Therefore, we produced 638 F2 hybrids from an (E3.DA-Pia457 ⫻ DA) background, and then immunized these F2 hybrids with pristane and followed up this group over 35 days. Preliminary data, after genotyping of the RT1 region, showed a significant impact of the 424 Figure 4. Influence of RT1 in (E3.DA-Pia457 ⫻ DA)F2 hybrids, as assessed by the mean arthritis score in males (A) and females (B) after injection of 150 l pristane. In both male and female F2 hybrids, a significant difference between the RT1 genotypes was observed. Rats with the homozygous RT1av1/av1 haplotype had more severe pristaneinduced arthritis (PIA) compared with RT1u/av1-heterozygous rats. At the same time, RT1u/av1-heterozygous rats had more severe PIA compared with RT1u/u-homozygous rats. Data are the mean ⫾ SEM pooled results from 7 independent experiments. ⴱⴱⴱ ⫽ P ⬍ 0.0001 between homozygous and heterozygous groups. RINTISCH ET AL factors in PIA, we generated F1 hybrids by reciprocally crossing the E3.DA-Pia457 males with the DA females, and reciprocally crossing the DA females with the E3.DA-Pia457 males. E3.DA-Pia457–congenic rats had been generated by an initial outcross between an E3 male and a DA female, followed by 2 subsequent backcrosses to E3 females, thus ensuring that both the sex chromosomes and the mitochondria were derived from the E3 strain. We performed 4 independent experiments, and all yielded similar results; the data were therefore pooled for further analyses. We observed only a slight tendency toward a higher arthritis severity score in the early phase of PIA in F1 hybrid males from a DA mother (Figure 5A). In F1 hybrid female rats, we detected a small, but significant, difference in the severity of PIA in the early phase (days 12–16). Moreover, similar to the male hybrids, female offspring from a DA mother had a higher mean arthritis score compared with offspring from an E3.DA-Pia457 mother (Figure 5B). At the time of this experiment, there were no mitochondrial sequence data available from the DA and E3 rat strains, and also the knowledge on imprinted genes was limited; therefore, we could not conclusively address the cause of the observed maternal effect. Nevertheless, this effect was more pronounced in female F1 hybrids, and male rats showed almost no difference in arthritis severity. Therefore, we could exclude the Y chromosome as a carrier of PIA-regulating genes. Because the observed effects were only very small and brief, further experiments are needed to verify a possible epigenetic effect in F1 hybrids. DISCUSSION RT1 region on PIA severity in F2 hybrids. Male F2 hybrids with the RT1u/u haplotype had a significantly lower mean arthritis score compared with rats with the RT1u/av1 haplotype, while the latter group had lower arthritis severity than that in rats with the RT1av1/av1 haplotype (Figure 4A). The same pattern was also seen in female F2 hybrids (Figure 4B). Since the rest of the genome had not been typed at the time of these experiments, we could not investigate epistatic genes. Nevertheless, our results clearly demonstrate the importance of the MHC (Pia1) in the development of PIA, and in addition, we could show that this effect was dependent on the genetic background. Epigenetic effects in F1 hybrids. To investigate the effect of sex chromosomes as well as epigenetic The DA allele of Ncf1, which exerts a major influence in enhancing arthritis severity, was found to be silent in the E3 background. To isolate the cause of this disease resistance, 2 additional DA loci were introgressed to the arthritis-resistant E3 strain, and this triple-congenic strain of rat could then be shown to develop arthritis. Two different arthritis models, PIA and CIA, were used to assess the arthritis-regulating effect of this triple-congenic strain. In the CIA model, immunization with rat CII emulsified in IFA induced an antigen-specific autoimmune response in diseasesusceptible strains, including generation of pathogenic antibodies that were able to transfer arthritis (18). In PIA, the nonimmunogenic mineral oil pristane was injected, causing an arthritis that was dependent on polyclonal activation of T cells, as shown by depletion of QUANTITATIVE TRAIT LOCI AND MHC EFFECTS ON RAT ARTHRITIS 425 Figure 5. Influence of sex chromosomes and epigenetic factors in F1 hybrids with pristane-induced arthritis (PIA). A, Mean arthritis score in male (E3.DA-Pia457 ⫻ DA)F1 and (DA ⫻ E3.DA-Pia457)F1 offspring after injection of 150 l pristane. Male rats from a DA mother showed a tendency toward an increased arthritis score in the early phase of PIA compared with rats from an E3.DA-Pia457 mother. B, Mean arthritis score in female (E3.DA-Pia457 ⫻ DA)F1 and (DA ⫻ E3.DA-Pia457)F1 offspring. Female rats from a DA mother had a significantly higher arthritis score in the early phase of PIA (days 12–16). Data are the mean ⫾ SEM pooled results from 4 independent experiments. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, versus (E3.DA-Pia457 ⫻ DA)F1. ␣/␤ T cells and disease transfer though CD4⫹ T cells (19,20). Although each experimental model appears to involve different physiologic pathways in the introduction of arthritis, they share a high similarity in their genetic control, as has been observed in linkage analyses in which a strikingly high number of QTLs were found to be overlapping and the effects of isolated loci were observed to be similar between CIA and PIA in congenic rats (7,21). Congenic strains have a great potential for use in the identification of arthritis-regulatory genes. Moreover, once the whole rat genome has been sequenced (22), one can expect an acceleration in the number of identified genes within the next decade. Currently, one arthritis-regulating gene in the rat has been positionally identified, the Ncf1 gene (9), and another mapping study has identified a gene complex on chromosome 4, including 7 genes of the APLEC complex (23). At present, almost all mapping studies involve congenic strains that have been generated by introduction of an arthritis-suppressing locus from a resistant strain into the genetic background of a susceptible strain, and as a result, nearly complete protection from experimentally induced arthritis has been observed in those congenic strains. However, that does not necessarily mean only one locus is involved in the regulation of arthritis; rather, it provides evidence for the existence of a complex interaction of multiple arthritis-enhancing loci in the susceptible DA rat. To provide evidence of such interactions, we established reciprocal congenic rats in which arthritis- promoting loci derived from the DA rat were transferred to the completely resistant E3 rat. Using these crossbred strains, we were able to show that the Pia4 locus alone, including the gene Ncf1, is not sufficient to break the disease resistance in the E3.DA-Pia4 rat. Only after 2 additional loci were transferred was the tolerance of the E3 rat partially broken. However, only 40–50% of those triple-congenic rats developed arthritis, indicating that DA alleles from additional, minor QTLs for PIA and CIA must be required to completely break the resistance in E3 rats. RT1, which is the MHC region in rats, appears to be a likely candidate for this role. Although previous linkage studies of (E3 ⫻ DA)F2 hybrids did not identify the MHC as a major QTL in either PIA or CIA in DA and E3 rats, we had obtained some evidence of its importance in PIA. We therefore performed a series of experiments with rats of various genetic backgrounds, with groups segregated according to haplotypes of the rat MHC. In rats with a pure DA background, we could not detect any significant influence of RT1. This finding is in concordance with that of a previous study using various MHC-congenic rats with a Lewis background (14). Only one MHC haplotype, RT1f, was found to have a significant influence on the development of PIA, whereas other haplotypes, including av1 and u, did not show any effect on the incidence or severity of PIA. Interestingly, between (E3.DA-Pia457 ⫻ DA)F1 and (E3.DA-Pia457 ⫻ DA.E3-RT1u/u)F1 hybrids, there was a highly significant difference in the effect of RT1, thus proving the existence of important interacting 426 genes. We sought to identify the interacting loci, and performed a large F2 intercross experiment with (E3.DA-Pia457 ⫻ DA)F2 hybrids. Surprisingly, as in the F1 hybrids, we could detect a significant influence of the 2 different alleles of RT1 (u and av1) in the F2 hybrids, which was not seen in previous studies with (E3 ⫻ DA)F2 hybrids (6,7). This could be explained by several possibilities. First, there may be an epistatic effect of Pia4, Pia5, or Pia7, at which positions 2 DA alleles might be needed to enhance the arthritis-regulating effect of RT1. Second, it is also possible that neutralization of the strongest QTL (Pia4) led to the detection of minor QTLs that previously had been masked by the strong influence of Pia4 on arthritis development. At the same time, we could point to substantial gaps in the genotyping in the first linkage studies, and these gaps could have partially hampered the identification of Pia1 in those studies. However, all of these effects do not explain the absence of a significant difference in DA.E3-RT1– congenic rats. Further genotyping of the whole genome of F2 hybrid rats will provide important knowledge on additional MHC interacting loci, which will be relevant for the study of human RA. RA is not a single disease entity, but rather a heterogeneous syndrome caused by different pathways involving B cells and autoantibodies, T cells, and the cytokine network, as well as the fibroblast and many other cell types (1). This is also reflected in the genetic heterogeneity of RA. The first, and perhaps most important, association in RA was found to be the so-called shared epitope, a group of related epitopes found in DR4 and DR1 alleles that have identical antigenbinding pockets (24). Since then, genetic studies of RA in humans have revealed associations of several genes in addition to HLA. Many of those associations were found only in subpopulations of RA patients, after stratification for the presence of rheumatoid factor (RF) or anti–cyclic citrullinated peptide (anti-CCP) antibodies. Indirectly, this corresponded to the stratification of the HLA haplotypes, because the occurrence of anti-CCP and RF was found to be associated with the shared epitope (25,26). Similarly, an association with the C5TRAF haplotype has been found only in RF-positive patients (27), and an association with IRF5 has been found mainly in anti-CCP–negative patients (28). With more and more genes implicated in the pathogenesis of RA, the need for more thorough analyses of stratification and genetic interaction is growing. Our present study addresses the importance of interacting loci that could either prevent or facilitate the detection of the allelic effects of candidate genes in a large RINTISCH ET AL outcross population. We show that the arthritispromoting effect of the Ncf1 DA allele was completely masked by arthritis-protecting E3 alleles of other genes. These genetic interactions could partially explain why it has been challenging to study the genetic association of NCF1 in human RA, and the E3.DA-congenic rat might be an excellent tool to identify those loci. However, another reason why conducting association studies of NCF1 has been a difficult task is the high complexity of the human NCF1 region, with 2 nonfunctional pseudogenes and evidence of an additional functional copy, all of which are in close proximity to NCF1 (29). Our results underscore the importance of the genetic context when studying the effect of one particular locus. The effect of Ncf1, the major gene regulating arthritis in DA rats, was observed to be completely neutralized in E3 rats. In contrast, we detected no arthritis-regulatory potential of the rat MHC in DA rats; however, in various mixed genetic backgrounds, we could observe an effect of the MHC, suggesting that strong epistatic interactions are taking place between MHC and non-MHC genes. Furthermore, our findings confirm the existence of 3 major QTLs regulating experimentally induced arthritis. By introducing these 3 QTLs derived from the susceptible DA strain into the genetic background of the resistant E3 strain, the arthritis resistance of the completely protected E3 strain was partially broken. At the same time, our results show the importance of other minor QTLs and the relevance of generating reciprocal congenic lines to evaluate the full arthritis-regulating effect of a QTL. In a broader context, our study highlights the advantage of using congenic strains and identifies the potential difficulties in finding a gene, such as Ncf1, that directly plays a strong regulatory role in arthritis, particularly in terms of the multiple interactions of resistant genes in outbred or wild rats or in human populations. ACKNOWLEDGMENTS We thank the technicians at Medical Inflammation Research (Lund University, Lund, Sweden), particularly Carlos Palestro and Isabelle Bohlin, for taking excellent care of the animals. We thank Dr. Peter Olofsson and Jonatan Tuncel for providing the E3.DA-Pia457–congenic rats and DA.E3RT1u/u–congenic rats, respectively. AUTHOR CONTRIBUTIONS Dr. Holmdahl had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. QUANTITATIVE TRAIT LOCI AND MHC EFFECTS ON RAT ARTHRITIS Study design. Rintisch, Holmdahl. Acquisition of data. Rintisch, Förster. Analysis and interpretation of data. Rintisch, Holmdahl. Manuscript preparation. Rintisch, Förster, Holmdahl. Statistical analysis. Rintisch. 15. 16. REFERENCES 1. Firestein GS. Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J Clin Rheumatol 2005;11(3 Suppl):S39–44. 2. Worthington J. Investigating the genetic basis of susceptibility to rheumatoid arthritis. J Autoimmun 2005;25 Suppl:16–20. 3. Plenge RM, Seielstad M, Padyukov L, Lee AT, Remmers EF, Ding B, et al. TRAF1-C5 as a risk locus for rheumatoid arthritis: a genomewide study. N Engl J Med 2007;357:1199–209. 4. The Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007;447:661–78. 5. Lander ES, Schork NJ. Genetic dissection of complex traits. Science 1994;265:2037–48. 6. Vingsbo-Lundberg C, Nordquist N, Olofsson P, Sundvall M, Saxne T, Pettersson U, et al. Genetic control of arthritis onset, severity and chronicity in a model for rheumatoid arthritis in rats. Nat Genet 1998;20:401–4. 7. Olofsson P, Lu S, Holmberg J, Song T, Wernhoff P, Pettersson U, et al. A comparative genetic analysis between collagen-induced arthritis and pristane-induced arthritis. Arthritis Rheum 2003;48: 2332–42. 8. Olofsson P, Holmberg J, Pettersson U, Holmdahl R. Identification and isolation of dominant susceptibility loci for pristane-induced arthritis. J Immunol 2003;171:407–16. 9. Olofsson P, Holmberg J, Tordsson J, Lu S, Akerstrom B, Holmdahl R. Positional identification of Ncf1 as a gene that regulates arthritis severity in rats. Nat Genet 2003;33:25–32. 10. Griffiths MM, Wang J, Joe B, Dracheva S, Kawahito Y, Shepard JS, et al. Identification of four new quantitative trait loci regulating arthritis severity and one new quantitative trait locus regulating autoantibody production in rats with collagen-induced arthritis. Arthritis Rheum 2000;43:1278–89. 11. Nordquist N, Olofsson P, Vingsbo-Lundberg C, Petterson U, Holmdahl R. Complex genetic control in a rat model for rheumatoid arthritis. J Autoimmun 2000;15:425–32. 12. Remmers EF, Longman RE, Du Y, O’Hare A, Cannon GW, Griffiths MM, et al. A genome scan localizes five non-MHC loci controlling collagen-induced arthritis in rats. Nat Genet 1996;14: 82–5. 13. Larsson P, Kleinau S, Holmdahl R, Klareskog L. Homologous type II collagen–induced arthritis in rats: characterization of the disease and demonstration of clinically distinct forms of arthritis in two strains of rats after immunization with the same collagen preparation. Arthritis Rheum 1990;33:693–701. 14. Vingsbo C, Sahlstrand P, Brun JG, Jonsson R, Saxne T, Holmdahl R. Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 427 histocompatibility complex and non-major histocompatibility complex genes. Am J Pathol 1996;149:1675–83. Smith BD, Martin GR, Miller EJ, Dorfman A, Swarm R. Nature of the collagen synthesized by a transplanted chondrosarcoma. Arch Biochem Biophys 1975;166:181–6. Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol 1982;82 Pt A:33–64. Holmdahl R. Genetic analysis of mouse models for rheumatoid arthritis. Adolph KW, editor. In: Textbook of human genome methods. Boca Raton (FL): CRC Press; 1997. p. 215–38. Griffiths MM, Cannon GW, Leonard PA, Reese VR. Induction of autoimmune arthritis in rats by immunization with homologous rat type II collagen is restricted to the RT1av1 haplotype. Arthritis Rheum 1993;36:254–8. Kleinau S, Klareskog L. Oil-induced arthritis in DA rats: passive transfer by T cells but not with serum. J Autoimmun 1993;6: 449–58. Holmberg J, Tuncel J, Yamada H, Lu S, Olofsson P, Holmdahl R. Pristane, a non-antigenic adjuvant, induces MHC class II-restricted, arthritogenic T cells in the rat. J Immunol 2006;176: 1172–9. Joe B. Quest for arthritis-causative genetic factors in the rat. Physiol Genomics 2006;27:1–11. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, et al. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 2004;428: 493–521. Lorentzen JC, Flornes L, Eklow C, Backdahl L, Ribbhammar U, Guo JP, et al. Association of arthritis with a gene complex encoding C-type lectin–like receptors. Arthritis Rheum 2007;56: 2620–32. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis: an approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30: 1205–13. Van Gaalen FA, van Aken J, Huizinga TW, Schreuder GM, Breedveld FC, Zanelli E, et al. Association between HLA class II genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum 2004;50:2113–21. Gran JT, Husby G, Thorsby E. The association between rheumatoid arthritis and the HLA antigen DR4. Ann Rheum Dis 1983; 42:292–6. Potter C, Eyre S, Cope A, Worthington J, Barton A. Investigation of association between the TRAF family genes and RA susceptibility. Ann Rheum Dis 2007;66:1322–6. Sigurdsson S, Padyukov L, Kurreeman FA, Liljedahl U, Wiman AC, Alfredsson L, et al. Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis. Arthritis Rheum 2007;56:2202–10. Heyworth PG, Noack D, Cross AR. Identification of a novel NCF-1 (p47-phox) pseudogene not containing the signature GT deletion: significance for A47 degrees chronic granulomatous disease carrier detection. Blood 2002;100:1845–51.