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Identification of new quantitative trait loci in mice with collagen-induced arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 11, November 2004, pp 3721–3728
DOI 10.1002/art.20624
© 2004, American College of Rheumatology
Identification of New Quantitative Trait Loci in
Mice With Collagen-Induced Arthritis
Kristin Bauer, Xinhua Yu, Patrik Wernhoff, Dirk Koczan, Hans-Juergen Thiesen,
and Saleh M. Ibrahim
Objective. Collagen-induced arthritis (CIA) in the
mouse is one of the most widely used autoimmune
experimental models, with many features similar to
rheumatoid arthritis. This study sought to identify
potential genetic regulatory mechanisms of CIA in
major histocompatibility complex–matched (H2-q) F2
hybrid mice.
Methods. We used 126 polymorphic markers to
perform simple sequence-length polymorphism analysis
on 290 F2 hybrids of arthritis-susceptible (DBA/1J) and
arthritis-resistant (FVB/N) inbred mouse strains. The
major clinical traits (disease severity and onset) were
assessed, and serum antibodies specific to type II collagen (CII) were determined by enzyme-linked immunosorbent assay in 270 F2 mice. Lymph nodes from 94
F2 mice were used to test the ratio of CD4 to CD8 by
fluorescence-activated cell sorter analysis, and cell proliferation was determined by XTT test.
Results. Two quantitative trait loci (QTLs) identified in previous studies were confirmed; these were
severity-controlling Cia2 and onset-controlling Cia4 on
chromosome 2. Moreover, we identified 5 new QTLs, 1
for CII-specific IgG2a antibodies on chromosome 5, 2
controlling the CII-specific IgG1 antibody response on
chromosomes 10 and 13, 1 for the CD4:CD8 ratio on
chromosome 2, and 1 for cell proliferation (measured by
XTT test) on chromosome 16. Complement component
C5 was identified as the probable main candidate gene
for the QTLs Cia2 and Cia4. F2 mice carrying a
2-basepair deletion of C5, the FVB/N allele, had low
incidence and less severe disease as compared with
those carrying the DBA/1J allele.
Conclusion. This genome scan provides additional evidence confirming the role of C5 as a probable
candidate gene for Cia2 and Cia4 loci, and identifies new
QTLs controlling new traits in autoimmune arthritis.
Rheumatoid arthritis (RA) is a complex, multifactorial polygenic disease that is influenced by age and
sex as well as hormonal and environmental factors (1).
Susceptibility to RA is controlled by genetic factors, as
indicated by many studies showing higher rates of disease concordance in monozygotic twins than in dizygotic
twins, and higher incidence in offspring of RA patients
(2). The genetic contribution to RA susceptibility is
estimated to be as much as 60%, of which the HLA–
DRB1 locus is thought to account for 30–50% (3,4).
Identification of genetic loci regulating RA is complicated by genetic heterogeneity and incomplete penetrance, as well as environmental factors (1). Thus,
genetic analysis of well-defined experimental models of
autoimmune arthritis has the potential to markedly
accelerate the genetic investigation of RA.
Experimental models of arthritis, such as
collagen-induced arthritis (CIA) in mice, have been used
extensively for studying the roles of autoimmunity and
inflammation in the pathogenesis of arthritis. As is the
case in humans, most studies indicate that both non–
major histocompatibility complex (MHC) genes and
MHC genes are associated with the susceptibility to
CIA. Indeed, the main quantitative trait locus (QTL)
identified, Cia1, is located on chromosome 17 and
includes the MHC. However, susceptibility/severity of
the disease varies significantly among inbred strains and
even among those sharing the same MHC haplotype.
For example, inbred DBA/1J mice are markedly suscep-
Supported by grants from EUROME (EU FP5) and BMBF
(FKZ 01ZZ0108).
Kristin Bauer, MSc, Xinhua Yu, MSc, Patrik Wernhoff, PhD,
Dirk Koczan, PhD, Hans-Juergen Thiesen, MD, Saleh M. Ibrahim,
MD, PhD: Institute for Immunology, University of Rostock, Rostock,
Germany.
Ms Bauer and Mr. Yu contributed equally to this work.
Address correspondence and reprint requests to Saleh M.
Ibrahim, MD, PhD, Institute of Immunology, Rostock University,
Schillingallee 70, 18055 Rostock, Germany. E-mail: saleh.ibrahim@
med.uni-rostock.de.
Submitted for publication February 12, 2004; accepted in
revised form August 10, 2004.
3721
3722
BAUER ET AL
tible to CIA, whereas FVB/N mice are completely
resistant; both strains carry the H2-q haplotype.
The conservative estimate for the quantification
of the non-MHC susceptibility genes is suggested to be
more than 20. Recently, a few studies localized QTLs
that regulate CIA in mice, mostly in crosses involving the
DBA/1J strain (5,6). These QTLs (Cia2, Cia3, Cia4,
Cia6, Cia7, and Cia8) are located on chromosomes 2, 6,
7, and 10. Other QTLs identified in crosses involving
other strains are located on chromosomes 1, 3, 5, 8, 13,
14, 17, 18, 19, and X (7–9).
We assume, along with others, that there are
additional QTLs contributing to the susceptibility to
CIA and to additional disease traits that have not been
identified. We therefore set out to identify these QTLs
in a genome scan of the F2 generation between the
FVB/N and DBA/1J inbred mouse strains. Herein we
report the identification of QTLs controlling the severity
of disease, onset of disease, autoantibody response,
CD4:CD8 ratio, and cell proliferation (measured by
XTT test) in mice with CIA.
MATERIALS AND METHODS
Mice, immunization, and scoring. All animals used in
this study were obtained from The Jackson Laboratory (Bar
Harbor, ME) and were housed at the animal facility at the
University of Rostock. All procedures and assays were preapproved by the local Animal Care Committee. CIA was induced
in control and experimental animals according to established
protocols. In brief, DBA/1J, FVB/N, (DBA/1J ⫻ FVB/N)F1,
and (DBA/1J ⫻ FVB/N)F2 progeny were immunized at 8–12
weeks of age at the base of the tail with 125 ␮g of bovine type
II collagen (CII; Chondrex, Redmond, WA) dissolved in 50 ␮l
of 0.1M acetic acid and mixed with an equal volume (50 ␮l) of
Freund’s complete adjuvant (Freund’s incomplete adjuvant
with 4 mg/ml Mycobacterium tuberculi; Difco, Detroit, MI).
Mice were followed up for 14 weeks postimmunization.
The clinical scoring of arthritis was commenced from
18 days after immunization. Animals were monitored 3 times
weekly for signs of CIA. An arthritis index was assigned to each
mouse by using the following criteria: 0 ⫽ no signs of arthritis;
1 ⫽ swelling and redness in a single joint; 2 ⫽ inflammation in
multiple joints; and 3 ⫽ severe swelling, joint erosion, and/or
ankylosis. Each paw was scored on a scale of 0–3, with the
index being the sum of the scores for all 4 paws. The severity
trait was defined as the maximum arthritis index score observed in each individual mouse. The onset trait was calculated
as described previously (6). In brief, the earliest day of
observed onset after immunization was given a score of 50 and
the latest day of observed onset was given a score of 0. The
onset score for each of the remaining mice was calculated as
follows: 50 ⫻ (day after latest observed onset day ⫺ the
observed onset day)/(day after latest observed onset day ⫺ day
after the earliest observed onset day). Healthy mice were given
an onset score of 0.
Genomic screening. For the genetic analysis, 126 informative microsatellite markers covering the genome to the
Table 1. Summary of arthritis susceptibility in DBA/1J, FVB/N, F1,
and F2 mice*
Strain
Incidence,
no. (%)
Onset,
days
Maximum
severity score
DBA†
FVB†
F1
F2
15/15 (100)
0/15 (0)
13/60 (21.7)
85/290 (29.3)
39.1 ⫾ 11.7
–
62.1 ⫾ 18.1
44.5 ⫾ 19.9
7.91 ⫾ 3.08
–
3.15 ⫾ 2.99
4.51 ⫾ 3.48
* Except where indicated otherwise, values are the mean ⫾ SD.
† Only male mice were tested.
extent of 92%, with an average intermarker distance of 11.5
cM, were used. The intermarker distance ranged from 5.1 cM
to 27 cM for the different chromosomes according to the
Mouse Genome Informatics marker positions. The accuracy of
our loci order and interval maps was verified by comparing the
genetic map calculated from our data with the Mouse Genome
Informatics map. Microsatellite markers were chosen from
The Jackson Laboratory mouse database (available at their
Web site at http://www.jax.org) and were tested if they were
informative for the DBA/1J and FVB/N strains. (A complete
list of markers is available by request.) Primers for informative
microsatellite markers were ordered from Metabion (PlaneggMartinsried, Germany).
The genomic DNA used for genotyping the mice was
isolated from a 1-cm tail clip by using standard isolation
protocols (10). Genomic DNA from each animal was genotyped for 126 microsatellite loci (Mouse Genome Informatics,
Bar Harbor, ME) by using polymerase chain reaction (PCR)
amplification. Genomic DNA (20 ng) was amplified in a final
volume of 10 ␮l containing HotStar Taq polymerase (0.25
units; Qiagen, Chatsworth, CA), primers (0.1 ␮M each), 50
mM KCl, 10 mM Tris, 2.5 mM MgCl2, 0.2 mM dNTP, and 0.02
␮M M13-IRD700 or M13-IRD800 (Li-Cor, Lincoln, NE).
Amplification conditions were as follows: 95°C for 15 minutes,
followed by 2 cycles of 94°C for 30 seconds, 57°C for 1 minute,
72°C for 1 minute, then another 37 cycles of 94°C for 30
seconds, 55°C for 1 minute, 72°C for 1 minute, and a final
extension at 72°C for 7 minutes. The reactions were performed
using a GeneAmp PCR System 9700 cycler (Applied Biosystems, Foster City, CA). The PCR products were resolved on
denaturing polyacrylamide gels, and were detected by using a
Li-Cor Model 4200L automated DNA sequencer. The genotypes were scored independently by at least 2 people (KB and
XY), or using the Saga software supplied by Li-Cor.
Measurement of antibodies, CD4:CD8 ratio, and cell
proliferation by XTT test. Blood was obtained from mice at the
thirtieth day after immunization; sera were prepared and used
for antibody determination. Antibodies to CII were determined by enzyme-linked immunosorbent assay (ELISA).
Maxisorp 96-well plates (Nunc, Hanover Park, IL) were coated
with bovine CII (0.25 ␮g of CII per well). Anti-IgG1 or
anti-IgG2a secondary antibodies (for Th2- and Th1-supported
isotypes, respectively) were used to determine the collagenspecific IgG1 or IgG2a antibody response. A serial dilution
(1:50 to 1:50,000) of the pooled sera of F2 mice was used for
choosing the dilution factor for the test, the concentration of
which provided the median optical density value used for
positive sample determination. Pooled sera were used as
standard to make the adjustment among the different plates.
At day 90 after immunization, lymph nodes of the mice
QUANTITATIVE TRAIT LOCI IN COLLAGEN-INDUCED ARTHRITIS
Figure 1. Plots of quantitative trait loci (QTLs), on chromosomes 2, 5,
and 16, containing non–major histocompatibility complex–linked loci
for the disease traits of (collagen-induced) arthritis severity, CD4:CD8
ratio, IgG2a response level, and cell proliferation (by XTT test).
Log-likelihood values were determined by using R/qtl software. Logarithm of odds (LOD) scores are presented in relation to positions of
the marker loci along the chromosome. Horizontal bar indicates the
median LOD score as the cutoff for a positive association. a–c show
newly identified QTLs, while d shows a previously reported QTL that
was confirmed in this study. a, Trmq3 (T cell ratio modifier QTL 3) on
chromosome 2, controlling the CD4:CD8 ratio trait. b, Cia27 on chromosome 5, controlling the IgG2a phenotype. c, Lp1 (lymphocyte proliferation 1) on chromosome 16, by XTT, controlling the cell proliferation trait.
d, Cia2 on chromosome 2, controlling the severity phenotype.
were used to make single-cell suspensions. CD4 and CD8
cells were determined by fluorescence-activated cell sorter
(FACS) analysis as described before (11). Briefly, cells (1 ⫻
106) were washed twice with FACS buffer, then incubated
with anti-CD8–fluorescein isothiocyanate and anti-CD4–
phycoerythrin for 20 minutes in the dark, followed by washing
twice. FACScan and FACSCalibur (Becton Dickinson, Mountain View, CA) were used for these analyses. Lymphocytes
were gated using forward and side scatter, and 10,000 lymphocytes were counted. The percentage of CD4 cells was divided
by the percentage of CD8 cells to determine the CD4:CD8 ratio.
Lymph nodes from the mice at the ninetieth day after
3723
immunization were also used to make single-cell suspensions
for the XTT cell proliferation test, which was done according
to the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). Briefly, 4 ⫻ 105 lymph node cells were cultured
in 100 ␮l medium per well in 96-well plates and were incubated
at 37°C and 5% CO2 with concanavalin A (1 ␮g) stimulation.
After 24 hours, a mixture of 0.1 ml PMS (an electron-coupling
reagent) and 5 mg/5 ml XTT was added to each well with a
volume of 50 ␮l and the cells were incubated for an additional
16 hours for XTT formazan production. The spectrophotometric absorbance of the samples was determined with an ELISA
reader at 450 nm.
Linkage analysis. All linkage analyses were made with
QTX Map Manager software (12) and the imputation model in
R/qtl software package (13,14). The order of the loci was
obtained from the Mouse Genome Informatics map (available
at their Web site at http://www.informatics.jax.org). The results
for severity of arthritis, onset of arthritis, collagen-specific
antibody responses, lymph node CD4:CD8 ratio, and XTT cell
proliferation were used as phenotypes. Continuous trait values
were checked for normal distribution, and logarithmic values
were used when necessary. With regard to the significant and
suggestive linkage threshold values, we followed the guidelines
resulting from the permutation test (number of permutations ⫽ 1,000). The associations between marker and phenotype were also tested by F statistic (analysis of variance
[ANOVA]) using KaleidaGraph software). The 2-locus interaction analyses were performed using the scan-two function in
R/qtl software, with Harley-Knott regression. The scan-two
functions perform a 2-dimensional genome scan with a 2-QTL
model. The logarithm of odds (LOD) combined values
(LODjoint) represent a comparison of a full model with covariates (y ⫽ ␮ ⫹ ␤q1 ⫹ ␤q2 ⫹ ␤q1 ⫻ q2 ⫹ A␥ ⫹ Z␦q1 ⫹ Z␦q2 ⫹
Z␦q1 ⫻ q2 ⫹ ⑀) with a null model (y ⫽ ␮ ⫹ A␥ ⫹ ⑀). The
genome-wide significance levels for the scan-two results were
established using permutation tests (number of permutations ⫽ 1,000).
RESULTS
Arthritis penetrance and variance. Table 1 presents the results of the experiment determining the
incidence and severity of CIA in DBA/1J control mice
Table 2. Summary of the quantitative trait loci (QTLs) identified*
QTL
Chromosome
Peak
LOD
score
Significance
level†
Flanking markers
Position,
cM
Confidence
interval, cM
Trait
Cia2‡
Cia4‡
Cia27
Cia28
Cia29
Lp1
Trmq3
2
2
5
13
10
16
2
D2Mit81
D2Mit81
D5Mit277
D13Mit144
D10Mit96
D16Mit138
D2Mit398
12.08§
10.57§
4.28§
3.2¶
3.63#
3.8#
5.03§
3.4
3.5
3.4
3.4
3.4
3.5
3.42
D2Mit81–D2Mit367
D2Mit81–D2Mit367
D5Mit201–D5Mit425
D13Mit13–D13Mit144
D10Mit261–D10Mit14
D16Mit60–D16Mit139
D2Mit61–D2Mit395
2
4
60
52
52
31
65
6
10
38
15
28
38
14
Severity
Onset
IgG2a
IgG1
IgG1
Cell proliferation by XTT
CD4:CD8
* LOD ⫽ logarithm of odds; Lp1 ⫽ lymphocyte proliferation 1; Trmq3 ⫽ T cell ratio modifier QTL 3.
† Significance level (P ⬍ 0.05) determined according to permutation test (n ⫽ 1,000).
‡ Reference.
§ P ⬍ 0.01 (highly significant).
¶ P ⬍ 0.1 (suggestive).
# P ⬍ 0.05 (significant).
3724
BAUER ET AL
Table 3. Analysis of variance for chromosomes with evidence of
linkage*
Phenotype,
loci, marker
Severity
Chromosome 2 Cia2
D2Mit81
D2Mit367
D2Mit61
D2Mit398
D2Mit395
D2Mit145
D2Mit148
Onset
Chromosome 2 Cia4
D2Mit81
D2Mit367
D2Mit61
D2Mit398
D2Mit395
D2Mit145
D2Mit148
IgG2a
Chromosome 5 Cia27
D5Mit348
D5Mit387
D5Mit54
D5Mit201
D5Mit277
D5Mit425
D5Mit31
D5Mit143
IgG1
Chromosome 13 Cia28
D13Mit16
D13Mit275
D13Mit19
D13Mit13
D13Mit144
IgG1
Chromosome 10 Cia29
D10Mit248
D10Mit126
D10Mit20
D10Mit261
D10Mit96
D10Mit14
Cell proliferation by XTT
Chromosome 16 Lp1
D16Mit131
D16Mit101
D16Mit60
D16Mit138
D16Mit139
D16Mit153
D16Mit52
CD4:CD8
Chromosome 2 Trmq3
D2Mit81
D2Mit367
D2Mit61
D2Mit398
D2Mit395
D2Mit145
D2Mit148
Position, cM
F†
P
13.5
26.2
34
57.9
66.9
98.5
105
29.66
25.18
14.11
4.06
4.82
1.48
0.32
⬍0.0001
⬍0.0001
⬍0.0001
0.018
0.0087
0.227
0.722
13.5
26.2
34
57.9
66.9
98.5
105
21.51
21.84
15.97
3.72
3.03
1.93
0.1
⬍0.0001
⬍0.0001
⬍0.0001
0.026
0.049
0.146
0.902
8
15
28
42
58
65
78
86
4.28
4.57
4.27
8.42
9.73
9.58
4.1
1.24
0.014
0.011
0.014
0.00028
⬍0.0001
⬍0.0001
0.017
0.29
10
16
24
35
48
1.12
0.08
1.26
3.2
5.88
0.329
0.923
0.285
0.042
0.003
7
21
35
47
56
65
3.68
1.16
4.13
5.17
5.28
1.21
0.026
0.313
0.017
0.006
0.005
0.3
4.3
17
23.4
31
43.1
56.8
66.8
0.95
3.04
5.68
9.51
6.76
7.48
7.01
0.391
0.052
0.0047
0.00018
0.0018
0.00098
0.00147
13.5
26.2
34
57.9
66.9
98.5
105
1.58
3.21
2.39
14.47
9.83
6.72
4.37
0.212
0.044
0.097
⬍0.0001
0.00014
0.00189
0.015
* See Table 2 for definitions.
† Test of rank, 2 degrees of freedom.
and (DBA/1J ⫻ FVB/N)F1 and F2 experimental animals.
As expected, collagen-immunized DBA/1J mice had an
arthritis incidence of 100% (15 of 15) with a mean ⫾ SD
arthritis index of 7.91 ⫾ 3.08 and a mean ⫾ SD time to
arthritis onset of 39.1 ⫾ 11.7 days. However, FVB/N
mice did not develop arthritis. The (DBA/1J ⫻ FVB/
N)F1 progeny had an arthritis incidence of 21.7% (13 of
60), an arthritis index of 3.15 ⫾ 2.99, and time to arthritis
onset of 62.1 ⫾ 18.1 days. The (DBA/1J ⫻ FVB/N)F2
animals had an arthritis incidence of 29.3% (85 of 290)
with an arthritis index of 4.51 ⫾ 3.48 and time to arthritis
onset of 44.5 ⫾ 19.9 days (Table 1).
Identification of arthritis QTLs. The linkage
analysis was performed by combining the genotype data
from the genome scan with 126 informative microsatellite markers and the phenotype data on the antibody
responses, CD4:CD8 ratio, and cell proliferation (determined by XTT test). Two hundred seventy mice were
used to determine the collagen-specific IgG1 and IgG2a
levels. The IgG1 values ranged from 0.063 to 2.702
(mean ⫾ SD 0.414 ⫾ 0.518). For IgG2a, the optical
density values at 405 nm among the samples ranged
from 0.068 to 0.963 (mean ⫾ SD 0.472 ⫾ 0.198). The
CD4:CD8 ratio values ranged from 0.81 to 3.83 (mean ⫾
SD 1.77 ⫾ 0.536). In addition, the cell proliferation as
measured by XTT was also defined as a phenotype.
These absorbance values ranged from 0.17 to 2.65
(mean ⫾ SD 1.32 ⫾ 0.64).
Seven QTLs were found in the screen, located on
chromosomes 2, 5, 10, 13, and 16 (Figure 1 and Table 2).
Two of the identified QTLs had been discovered in
previous studies and were confirmed herein (5). Cia2,
controlling the severity phenotype on chromosome 2,
had highly significant evidence of linkage to marker
D2Mit81 (LOD score of 12.1). The second locus, Cia4,
controlling the onset phenotype and located on chromosome 2, also had highly significant evidence of linkage to
marker D2Mit81 (LOD score of 10.6). Novel QTLs were
identified on chromosomes 2, 5, 10, 13, and 16. Cia27 on
chromosome 5 was identified as a QTL controlling the
CII-specific IgG2a phenotype; it had significant evidence of linkage to marker D5Mit277 (LOD score of
4.3). We identified 2 loci controlling the CII-specific
IgG1 antibody phenotype: Cia28 on chromosome 13,
having suggestive evidence of linkage to marker
D13Mit144 (LOD score of 3.2), and Cia29, showing
significant evidence of linkage to marker D10Mit96
(LOD score of 3.6). Trmq3 (T cell ratio modifier QTL
3), controlling the CD4:CD8 ratio, was found on chromosome 2 with significant evidence of linkage to marker
D2Mit398 (LOD score of 5). Finally, QTL Lp1 (lymphocyte proliferation 1) on chromosome 16, controlling the
cell proliferation measured by XTT test, had significant
QUANTITATIVE TRAIT LOCI IN COLLAGEN-INDUCED ARTHRITIS
Table 4.
3725
Two-locus interaction analysis*
Phenotype
Position 1,
marker 1 (loci)
Position 2,
marker 2 (loci)
LODjoint
LOD significance
level†
IgG1
IgG2a
Onset
Onset
Onset
Onset
Onset
Severity
Severity
D1Mit111 (Cia9)
D5Mit425 (Cia27)
C5 (Cia4)
C5 (Cia4)
C5 (Cia4)
C5 (Cia4)
C5 (Cia4)
C5 (Cia2)
C5 (Cia2)
D13Mit144 (Cia28)
D10Mit96 (Cia29)
D2Mit395 (Trmq3)
D10Mit261 (Cia8)
D11Mit4 (C1qbp)
D13Mit144 (Cia28)
D16Mit138 (Lp1)
D5Mit425 (Cia27)
D10Mit261 (Cia8)
9.12
10.34
12.14
13.55
13.23
10.95
12.4
14.94
15.98
8.63
8.57
9.37
9.37
9.37
9.37
9.37
10.63
10.63
* Logarithm of odds (LOD) combined values were calculated by 2-locus analysis in R/qtl (13), with
Haley-Knott regression. See Table 2 for definitions.
† Significance levels (P ⬍ 0.05) determined according to permutation tests (number of permutations ⫽
1,000).
evidence of linkage to marker D16Mit138 (LOD score
of 3.8).
To confirm the existence of these loci, a 2-point
analysis (F statistic, ANOVA) was performed. The data
from this analysis (Table 3) yielded similar results, thus
demonstrating an association between the linkage analysis results and the findings by ANOVA. Cia2 was
associated with marker D2Mit81 (F ⫽ 29.66, P ⬍
0.0001), Cia4 with D2Mit81 (F ⫽ 21.51, P ⬍ 0.0001),
Cia27 with D5Mit277 (F ⫽ 9.73, P ⬍ 0.0001), Cia28 with
D13Mit144 (F ⫽ 5.88, P ⫽ 0.003), Cia29 with D10Mit96
(F ⫽ 5.28, P ⫽ 0.005), Trmq3 with D2Mit398 (F ⫽ 14.47,
P ⬍ 0.0001), and Lp1 with D16Mit138 (F ⫽ 9.51, P ⫽
0.00018).
Genome-wide 2-locus interaction analyses were
performed on all phenotypes using R and R/qtl software
(13,14). The disease phenotypes for onset and severity
showed significant interactions with the Cia2 and Cia4
loci, marker C5, on chromosome 2, together with Cia8,
Cia27, Cia28, C1qbp, Lp1, and Trmq3 (Table 4). Two
significant interactions were found for the phenotypes
controlling antibody levels for IgG1 and IgG2a, respectively. The IgG1 levels showed a linkage between the
Cia9 locus on chromosome 1 and Cia28 on chromosome
13 (LODjoint ⫽ 9.12). The Cia27 locus on chromosome 5
and Cia29 on chromosome 10 controlled the IgG2a
levels (LODjoint ⫽ 10.34).
C5 as a candidate gene for CIA. In the linkage
analysis, we confirmed 2 loci on chromosome 2, Cia2
and Cia4. Hemolytic complement (Hc), or complement
component C5, is a gene that has been mapped to the
region of this genomic interval. A 2-basepair deletion in
exon 5 of the C5 gene, which leads to deficiency of the
molecule, has been previously described (15). The association of C5 deficiency with resistance against CIA has
also been reported previously (5,7,16–20). Therefore, we
investigated whether the resistance of the FVB/N strain
to the induction of CIA could be the result of C5
deficiency. Using genotyping on polyacrylamide gels, we
showed that the 2-basepair deletion in the C5 gene is
present in the FVB/N mouse strain.
We then genotyped all F2 mice for this deletion
and analyzed the incidence and severity of arthritis
among different groups genotyped for C5. The analyses
showed that both incidence and severity were clearly
decreased in mice deficient in C5. The incidence in
C5⫺/⫺ mice was highly significantly lower than in
C5⫹/⫹ mice (incidence 8.7% in C5⫺/⫺ versus 53.2% in
C5⫹/⫹; P ⬍ 0.001 by chi-square test) and C5-deficient
Figure 2. Chromosomes with previously published Cia quantitative
trait loci (QTLs) (thick, striped vertical bars) (positions determined
according to original reference and the Mouse Genome Informatics
database) (see refs. 5–9) were compared with all QTLs that were
identified in this study (Cia27–Cia29, Lp1, and Trmq3). Thin, solid
vertical bars represent chromosomal regions to which linkage of
microsatellite markers was found. Cia2 and Cia4 on chromosome 2
were previously identified and were confirmed. Cia27 on chromosome
5, Cia28 on chromosome 13, Cia29 on chromosome 10, Trmq3 on
chromosome 2, and Lp1 on chromosome 16 are newly identified QTLs.
The application used for visualization is available (under Fischer G,
et al) at the Web site http://www.ensembl.pzr.uni-rostock.de/Mus_
musculus/expressionview.
3726
BAUER ET AL
mice also had highly significantly lower disease severity
than did mice with sufficient C5 (mean ⫾ SD arthritis
index 0.11 ⫾ 0.39 in C5⫺/⫺ versus 3.0 ⫾ 3.9 in C5⫹/⫹;
P ⬍ 0.001 by Mann-Whitney test). Although a few
C5-deficient mice get disease, the severity is very low.
This suggests that the resistance to CIA in these mice is
mainly caused by C5 deficiency. However, because C5heterozygous mice also still develop disease (incidence
27.8% in C5⫹/⫺ versus 53.2% in C5⫹/⫹ [P ⬍ 0.001 by
chi-square test]; mean ⫾ SD arthritis index 1.1 ⫾ 2.3 in
C5⫹/⫺ versus 3.0 ⫾ 3.9 in C5⫹/⫹ [P ⬍ 0.001 by
Mann-Whitney test]), the contribution of an additional
gene cannot be completely excluded.
DISCUSSION
In this study, we identified 7 QTLs controlling the
pathogenesis of CIA, of which 2 had been identified
before and the other 5 are new QTLs (Table 2 and
Figure 2). These QTLs were confirmed by ANOVA test
(Table 3).
The susceptibility and resistance of DBA/1J and
FVB/N mouse strains, respectively, to the induction of
CIA is well documented. As observed in previous studies, the DBA/1J mice in these experiments had an
arthritis incidence of 100%, whereas the FVB/N mice
were completely resistant (0% incidence). As expected
and previously seen in similar crosses, the (DBA/1J ⫻
FVB/N)F1 progeny were susceptible to CIA (Table 1).
However, there was incomplete penetrance of the susceptible phenotype (21% incidence). A comparison of
the severity (arthritis) indexes between the F2 progeny
and the parental DBA/1J showed significant differences
(P ⬍ 0.0001), suggesting the existence of protective
FVB/N loci.
Two genomic intervals (QTLs) were associated
with CIA severity and onset (Figure 2), overlapping with
known loci previously linked to CIA, i.e., Cia2 and Cia4
located on chromosome 2. This locus seems to be a
common locus for CIA, as confirmed by our data.
Despite having many candidate genes in the locus on
chromosome 2 (Cia2), such as Ptgs1, Dpp4, and Ssb (5),
it is most likely that the main susceptibility gene is
complement C5 (Hc). This gene is an important mediator of inflammation, and its role in the disease process of
CIA is well documented (16–20). The implication of C5
in the resistance exhibited by SWR/J mice (5) and NOD
mice (7) to CIA induction has been described before,
and it has also been identified as a possible candidate
gene for serum/antibody-transferred arthritis (21).
Nevertheless, in both, the SWR ⫻ DBA/1J cross
and our current FVB/N ⫻ DBA/1J cross, there is a clear
effect on disease susceptibility and severity in the het-
erozygous form of the gene. This suggests that there is a
dose effect of the gene, although there is evidence that
levels of C5 are comparable in heterozygous mice and
homozygous C57BL/10.Q mice (7). Another explanation
is that there are 2 susceptibility genes operating within
this locus, a possibility that will be explored with the use
of congenic mice, which are currently being generated.
The first new QTL identified by us, Cia29, is
located on chromosome 10 and controls the collagenspecific IgG1 antibody trait. When considering the peak
(52 cM) of this QTL and taking into account the
confidence interval, it extends quite close to the peak of
the known QTL Cia8 (40.4 cM) (Figure 2), a locus
identified previously as a disease severity QTL (6). This
locus appears to be a common locus for autoimmune
diseases, including CIA (6), experimental allergic encephalomyelitis (EAE) (22), and systemic lupus erythematosus (23). Cia29 could be the same locus as Cia8,
although in our cross, the linkage with severity was weak.
Assuming that this is the same locus, then Cia8/29 is
indeed a major locus contributing to autoimmune inflammation. There are a number of candidate genes
found in this region that have been implicated in the
modulation of CIA susceptibility, such as the macrophage migration inhibitory factor Mif (24), the autoimmune regulator aire (25), and perforin (Bauer K, et al:
unpublished data). The autoantibody-linked Pgia26 locus, identified in proteoglycan-induced arthritis, was also
mapped to chromosome 10, but this was at a locus more
at the beginning of the chromosome, at 16 cM (26).
Perhaps the most interesting finding from this
new genome scan has been the identification of Cia27 on
chromosome 5, a QTL controlling the collagen-specific
IgG2a level. It is the first time that this genomic interval
on chromosome 5 has been identified in association with
autoimmune antibodies and arthritis. We were able to
show that F2 mice carrying the DBA allele in this
interval produced more IgG2a than did F2 mice with the
FVB allele in this area (the latter had levels corresponding to the IgG2a levels in the parental mice) (data not
shown). We also observed in our experiments that the
IgG2a level was associated with the severity of the
disease (data not shown). Previous studies also showed
that IgG2a is one of the important isotypes contributing
to the pathogenesis of arthritis (27). Candidate genes
found in the region of the peak marker that would be
predicted to have an influence on the inflammation
process of the disease are CXCL9 and CXCL10. A
recent report by Ruschpler and coworkers provides this
assumption. They showed an up-regulated expression of
CXCL9 as well as CXCL10 in inflammatory synovial
tissues of patients with rheumatoid arthritis (28). Furthermore, the receptor of CXCL9 and CXCL10,
QUANTITATIVE TRAIT LOCI IN COLLAGEN-INDUCED ARTHRITIS
CXCR3, was also found to be highly expressed in
synovial mast cells. Earlier studies assigned CXCR3
protein primarily to the Th1 lymphocytes (29–32). The
presence of this T cell subset is consistent with the
changes in the IgG2a level, because we actually choose
this IgG isotype to detect the Th1 pathway, which is
known to be required for the induction of CIA in DBA/1
mice (33).
Previous studies also identified QTLs that control
autoantibody production in arthritis models. With regard to CIA in a cross between Balb/c and DBA/1J
strains, 4 QTLs have been found on chromosomes 3, 17,
18, and X, and these are known as mCia2, mCia1,
mCia18, and mCia13, respectively (34). QTLs on chromosomes 1, 2, 7, 8, 10, 11, 16, and 18, controlling
autoantibody production in proteoglycan-induced arthritis, have also been described before in a cross
between Balb/c and DBA/2 (24). Furthermore, in the
Lyme disease model, IgG-controlling loci have been
identified in a C3H ⫻ C57BL/6 cross on chromosome 9
(Bb9) and chromosome 12 (Bb6) (35). In addition,
different antibody QTLs have been described in rat
arthritis models (36), such as Ciaa3, a locus that regulates the production of IgG anticollagen antibodies,
identified in a linkage study of a (DA ⫻ BN)F2 cross
(37). In the region of chromosome 5, in which we found
Cia27, different loci linked to autoimmune disease traits
have been mapped: for Lyme disease, Bb2 and Bb3 (35),
for arthritis, Pgia16, Cia13, and Cia14 (34,38), and also
recently one for EAE, Eae26. However, no locus with
linkage to an antibody phenotype has been reported on
chromosome 5.
In our experiments, the CD4:CD8 ratio of lymph
node cells always changed in different stages of the
immunization. DBA mice had a higher CD4:CD8 ratio
than did FVB mice (⬃4:2.5) without immunization;
however, after immunization with CII, the difference in
the ratio between the 2 mouse strains started to decrease
with age (data not shown). The existence of the Trmq3
locus, which is linked to the CD4:CD8 phenotype and
located on chromosome 2, suggests there is a gene
controlling the development of T cells in this area.
Trmq3 overlapped with Trmq2, a QTL associated with
the CD4:CD8 ratio in a C57BL/6 ⫻ DBA/2 cross (39). In
our study, we could show that this phenotype is probably
associated with susceptibility to CIA. Interesting genes
that seem to regulate CD4⫹ and CD8⫹ populations
have been mapped to chromosome 2, such as notch1 and
bcl-x (39,40). Increased expression of notch1 seems to
enhance the size of the CD8⫹ population and bcl-x
induction appears to be required for B7–CD28 interaction that again affects the CD4:CD8 ratio (39). Unexpectedly, the FVB, not the DBA allele, increased the
3727
ratio in our experiments, since the lymph node cells were
obtained from mice at day 90 after immunization. One
possible explanation could be the dynamic of the CD4:
CD8 ratio during the immunization as we described
above. Therefore, the gene in this locus regulating the
CD4:CD8 ratio is probably affected by the immunization
with CII.
Another new locus that we identified in this
study, Lp1, is located on chromosome 16 and linked to
lymphocyte cell proliferation determined by XTT assay.
This locus showed colocalization with Eae11, which is
also associated with spleen cell proliferation as well as
onset and incidence in EAE (41,42).
The performed 2-locus analysis showed that several of the previously defined loci, e.g., Cia2, Cia4, Cia8,
and Cia9, were involved in controlling the disease and
antibody responses. The disease traits of onset and
severity all involved the chromosome 2 marker C5 (Cia2
or Cia4). The antibody responses were controlled by
Cia9 and Cia22 (IgG1) and Cia27 and Cia29 (IgG2a).
When we analyzed the gene region of interest for the
QTLs Cia27, Cia28, and Cia29, we could not reveal any
explanation as to why there was an association with
antibody levels.
Our present analysis has identified 5 unique loci
and confirmed 2 common loci for CIA susceptibility,
collagen-specific antibody responses, the CD4:CD8 ratio, and cell proliferation (measured by XTT test). Using
a combination of congenic mapping, positional cloning,
and gene expression profiling, we are currently pursuing
identification of the candidate genes within a few of
these loci.
ACKNOWLEDGMENTS
The authors wish to thank Dr. G. Brockmann for
advice on linkage analysis, as well as Ilona Klamfuss and Eva
Lorbeer for help with the animals.
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