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Detection of arthritis-susceptibility loci including Ncf1 and variable effects of the major histocompatibility complex region depending on genetic background in rats.

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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.
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major, histocompatibility, complex, depending, detection, loci, regions, susceptibility, ncf1, effect, variables, arthritis, background, including, genetics, rats
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