BALB/c mice genetically susceptible to proteoglycan-induced arthritis and spondylitis show colony-dependent differences in disease penetrance.

Abstract

The major histocompatibility complex (H-2d) and non-major histocompatibility complex genetic backgrounds make the BALB/c strain highly susceptible to inflammatory arthritis and spondylitis. Although different BALB/c colonies develop proteoglycan-induced arthritis and proteoglycan-induced spondylitis in response to immunization with human cartilage proteoglycan, they show significant differences in disease penetrance despite being maintained by the same vendor at either the same or a different location.
BALB/c female mice (24 to 26 weeks old after 4 weeks of acclimatization) were immunized with a suboptimal dose of cartilage proteoglycan to explore even minute differences among 11 subcolonies purchased from five different vendors. In vitro-measured T-cell responses, and serum cytokines and (auto)antibodies were correlated with arthritis (and spondylitis) phenotypic scores. cDNA microarrays were also performed using spleen cells of naïve and immunized BALB/cJ and BALB/cByJ mice (both colonies from The Jackson Laboratory, Bar Harbor, ME, USA), which represent the two major BALB/c sublines.
The 11 BALB/c colonies could be separated into high (n = 3), average (n = 6), and low (n = 2) responder groups based upon their arthritis scores. While the clinical phenotypes showed significant differences, only a few immune parameters correlated with clinical or histopathological abnormalities, and seemingly none of them affected differences found in altered clinical phenotypes (onset time, severity or incidence of arthritis, or severity and progression of spondylitis). Affymetrix assay (Affymetrix, Santa Clara, CA, USA) explored 77 differentially expressed genes (at a significant level, P < 0.05) between The Jackson Laboratory's BALB/cJ (original) and BALB/cByJ (transferred from the National Institutes of Health, Bethesda, MD, USA). Fourteen of the 77 differentially expressed genes had unknown function; 24 of 77 genes showed over twofold differences, and only 8 genes were induced by immunization, some in both colonies.
Using different subcolonies of the BALB/c strain, we can detect significant differences in arthritis phenotypes, single-nucleotide polymorphisms (SNPs), and a large number of differentially expressed genes, even in non-immunized animals. A number of the known genes (and SNPs) are associated with immune responses and/or arthritis in this genetically arthritis-prone murine strain, and a number of genes of as-yet-unknown function may affect or modify clinical phenotypes of arthritis and/or spondylitis.

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Vol 11 No 1Research article
BALB/c mice genetically susceptible to proteoglycan-induced
arthritis and spondylitis show colony-dependent differences in
disease penetrance
Balint Farkas1, Ferenc Boldizsar1,2, Oktavia Tarjanyi1, Anna Laszlo1, Simon M Lin3, Gabor Hutas1,
Beata Tryniszewska1, Aaron Mangold1, Gyorgy Nagyeri1, Holly L Rosenzweig4, Alison Finnegan5,6,
Katalin Mikecz1,6,7 and Tibor T Glant1,5,7
1Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, 1735 W. Harrison Street, Cohn Research
Building, Chicago, IL 60612, USA
2Department of Immunology and Biotechnology, University of Pecs, Ifjusag u. 13, Pecs, Hungary
3Biomedical Informatics Center, Northwestern University, 750 N. Lake Shore Drive, Chicago, IL 60611, USA
4Department of Ophthalmology, Portland, Oregon Health Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA
5Department of Internal Medicine (Section of Rheumatology), Rush University Medical Center, 1730 W. Harrison Street, Cohn Research Building,
Chicago, IL 60612, USA
6Department of Immunology/Microbiology, Rush University Medical Center, 1730 W. Harrison Street, Cohn Research Building, Chicago, IL 60612,
USA
7Department of Biochemistry, Rush University Medical Center, 1730 W. Harrison Street, Cohn Research Building, Chicago, IL 60612, USA
Corresponding author: Tibor T Glant, tglant@rush.edu
Received: 5 Dec 2008 Revisions requested: 14 Jan 2009 Revisions received: 31 Jan 2009 Accepted: 16 Feb 2009 Published: 16 Feb 2009
Arthritis Research & Therapy 2009, 11:R21 (doi:10.1186/ar2613)
This article is online at: http://arthritis-research.com/content/11/1/R21
© 2009 Farkas et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction The major histocompatibility complex (H-2d) and
non-major histocompatibility complex genetic backgrounds
make the BALB/c strain highly susceptible to inflammatory
arthritis and spondylitis. Although different BALB/c colonies
develop proteoglycan-induced arthritis and proteoglycan-
induced spondylitis in response to immunization with human
cartilage proteoglycan, they show significant differences in
disease penetrance despite being maintained by the same
vendor at either the same or a different location.
Methods BALB/c female mice (24 to 26 weeks old after 4
weeks of acclimatization) were immunized with a suboptimal
dose of cartilage proteoglycan to explore even minute
differences among 11 subcolonies purchased from five different
vendors. In vitro-measured T-cell responses, and serum
cytokines and (auto)antibodies were correlated with arthritis
(and spondylitis) phenotypic scores. cDNA microarrays were
also performed using spleen cells of naïve and immunized
BALB/cJ and BALB/cByJ mice (both colonies from The Jackson
Laboratory, Bar Harbor, ME, USA), which represent the two
major BALB/c sublines.
Results The 11 BALB/c colonies could be separated into high
(n = 3), average (n = 6), and low (n = 2) responder groups
based upon their arthritis scores. While the clinical phenotypes
showed significant differences, only a few immune parameters
correlated with clinical or histopathological abnormalities, and
seemingly none of them affected differences found in altered
clinical phenotypes (onset time, severity or incidence of arthritis,
or severity and progression of spondylitis). Affymetrix assay
(Affymetrix, Santa Clara, CA, USA) explored 77 differentially
expressed genes (at a significant level, P < 0.05) between The
Jackson Laboratory's BALB/cJ (original) and BALB/cByJ
(transferred from the National Institutes of Health, Bethesda,
MD, USA). Fourteen of the 77 differentially expressed genes
had unknown function; 24 of 77 genes showed over twofold
differences, and only 8 genes were induced by immunization,
some in both colonies.
Conclusions Using different subcolonies of the BALB/c strain,
we can detect significant differences in arthritis phenotypes,
single-nucleotide polymorphisms (SNPs), and a large number of
differentially expressed genes, even in non-immunized animals.
A number of the known genes (and SNPs) are associated with
immune responses and/or arthritis in this genetically arthritis-
DDA: dimethyldioctadecyl-ammonium bromide; ELISA: enzyme-linked immunosorbent assay; IFN-γ: interferon-gamma; IL: interleukin; IVD: interverte-
bral disc; MHC: major histocompatibility complex; NCI: National Cancer Institute (Bethesda, MD, USA); NIH: National Institutes of Health (Bethesda,
MD, USA); PG: proteoglycan; PGIA: proteoglycan-induced arthritis; PGIS: proteoglycan-induced spondylitis; QTL: quantitative trait locus; RA: rheu-
matoid arthritis; RUMC: Rush University Medical Center (Chicago, IL, USA); SNP: single-nucleotide polymorphism.

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prone murine strain, and a number of genes of as-yet-unknown
function may affect or modify clinical phenotypes of arthritis and/
or spondylitis.
Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease
that leads to inflammatory cartilage destruction and bone ero-
sion in synovial joints. Although the pathological mechanism of
RA is unknown, both environmental and genetic factors are
thought to be involved in the etiology and pathogenesis of the
disease [1]. Animal models, especially those that involve joint
pathology in genetically altered rodents, are invaluable aids in
the research of human autoimmune diseases [2-6]. Among the
systemic animal models of RA, cartilage proteoglycan (PG)
aggrecan-induced arthritis (PGIA) is a T cell-dependent and
autoantibody/B cell-mediated disease in BALB/c mice which
is frequently accompanied by spondylitis [7-10]. In addition to
the major histocompatibility complex (MHC), PGIA and PG-
induced spondylitis (PGIS) are controlled by multiple genetic
loci [9,11]. Although various non-MHC genetic loci (quantita-
tive trait loci; QTLs) may contribute to disease, different com-
binations of these QTLs may result in a remarkably uniform
clinical phenotype of arthritis [12].
Due to a specific genetic background, the BALB/c strain
shows a strong predisposition toward arthritis. In addition to
PGIA, immunization with cartilage link protein [13] or human
cartilage glycoprotein-39 (HC-gp39) [14] can induce arthritis,
but only in BALB/c mice. Moreover, interleukin-1 (IL-1) recep-
tor antagonist protein-deficient mice [15] and SKG mice, in
which a spontaneous point mutation occurred in ZAP-70,
develop spontaneous arthritis [16], both only in the BALB/c
background.
Despite the efforts of companies to maintain genetically
homogenous inbred colonies, there are differences among
BALB/c colonies/substrains (for example, in body weight, size
of littermates, and the composition of intestinal bacterial flora)
maintained at different locations by the same vendor. Accord-
ing to the online public database of The Jackson Laboratory
(Bar Harbor, ME, USA) [17], there are at least 492 single-
nucleotide polymorphism (SNP) differences between their two
inbred BALB/cJ and BALB/cByJ colonies; of these, at least 59
SNPs are present in 33 immune-regulatory genes in the
mouse genome (F. Boldizsar and T.T. Glant, unpublished in
silico analysis data). Some of these known, or as-yet-unknown,
mutations may significantly influence the pathogenesis and
progression of PGIA or PGIS.
Since we have 'simplified' the model by replacing the highly
purified human fetal cartilage PG [7,18] with PG isolated from
human osteoarthritic cartilage [19,20] and changed the Fre-
und's adjuvants to a synthetic adjuvant [21], the PGIA/PGIS
model became available to a wide range of applications,
including the testing of new pharmacological agents. How-
ever, we and others observed differences in the onset, inci-
dence, and severity of arthritis, even when the source of
antigen (for example, our laboratory) and immunization proto-
cols were the same. Therefore, either local environmental com-
ponents or the source of BALB/c colony might account for the
different levels of susceptibility to, or severity of, PGIA.
Because environmental factors also play critical roles in RA
susceptibility [22] and different BALB/c colonies may have dif-
ferent panels of spontaneous mutations, it has become neces-
sary to test these components under uniform conditions. In the
present study, we investigated the disease parameters (onset
time, susceptibility, severity, and progression) simultaneously
in various colonies of BALB/c mice in the same experimental
setup. Because the BALB/c strain is highly susceptible to
PGIA (and PGIS) and sooner or later all immunized animals
develop arthritis independently of the colony source, we used
a suboptimal dose of PG antigen to be able to monitor even
minute differences among the colonies.
Materials and methods
Chemicals, antigen, animals, and immunization of mice
with cartilage proteoglycan aggrecan
All chemicals, unless otherwise indicated, were purchased
from Sigma-Aldrich (St. Louis, MO, USA) or Fischer Scientific
Co. (Chicago, IL, USA). Mouse-specific cytokine enzyme-
linked immunosorbent assay (ELISA) kits were purchased
from R&D Systems (Minneapolis, MN, USA) or BD Bio-
sciences (San Jose, CA, USA). Cartilage specimens from
knee joints were obtained from osteoarthritic patients under-
going joint replacement surgery. The use of human cartilage
for PG isolation was approved by the Institutional Review
Board of Rush University Medical Center (RUMC) (Chicago,
IL, USA). PG isolation has been described in detail [19,20]. All
animal experiments were approved by the Institutional Animal
Care and Use Committee of RUMC. Animals were maintained
in a pathogen-free environment in the same room and rack. A
total of 178 (retired breeder) female BALB/c mice of 11 colo-
nies (Table 1) were ear-tagged, and registered mice (all 24 to
26 weeks old) were randomly mixed and left for acclimatization
to the local environment for 4 weeks prior to the first immuni-
zation.
Mice were injected intraperitoneally with a 'suboptimal' dose
of human cartilage PG aggrecan (equivalent to 75 μg, instead
of the 'standard' dose of 100 μg of core protein of PG) emul-
sified with 2 mg of dimethyldioctadecyl-ammonium bromide
(DDA) adjuvant in 200 μL of phosphate-buffered saline (pH

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7.4). DDA is a synthetic adjuvant with positively charged
micelle-forming hydrophobic-hydrophilic (detergent-like) prop-
erties and does not contain mineral oil or mycobacterial com-
ponents as Freund's adjuvants do [21]. Intraperitoneal
injections were given on days 0, 21, and 42 of the experiment,
and mice were sacrificed on days 63 and 64. The goal of using
a suboptimal dose of cartilage PG aggrecan was necessary;
otherwise, all animals develop arthritis with a high arthritis
score after the third PG aggrecan (100 μg) injection in DDA
[20,21]. About half a year later, these experiments were
repeated using BALB/cJ and BALB/cByJ strains (The Jackson
Laboratory) and Portage P08 and Hollister H42 (Charles River
Laboratories, Inc., Wilmington, MA, USA) as well as National
Cancer Institute (NCI) (Bethesda, MD, USA) (Kingston)
BALB/c colonies (10 to 15 animals per group).
Clinical and histological assessment of arthritis and
spondylitis
Arthritis severity was determined using a visual scoring system
based on the degree of swelling and redness of the front and
hind paws [7,18,20]. Animals were examined at least three
times a week and inflammation was scored from 0 to 4 for
each paw, resulting in a cumulative arthritis score ranging from
0 to 16 for each animal [7,20]. To monitor early inflammatory
reactions as well, in this particular study, an acute arthritis
score of 0.5 was given if at least two interphalangeal, meta-
carpo-phalangeal, or metatarso-phalangeal joints were swol-
len but the paw inflammation (swelling and redness) did not
reach the 'standard' level of an arthritis score of 1 [20]. Ani-
mals were scored alternatively by two investigators in a blind
manner. Incidence of the disease was expressed as the per-
centage of arthritic mice to the total number of PG-immunized
mice per colony. Acute arthritis (severity) score was applied
only to arthritic animals. In addition, an arbitrary score (from 5
to 0) from the earliest onset of arthritis (onset score of 5) to
negative (onset score of 0) was established for each mouse
[23,24]. This onset score represents how quickly a mouse
developed arthritis.
Upon sacrifice, limbs and spines were removed, fixed in 10%
formalin, acid-decalcified, and processed in accordance with
standard histological procedures [7-9,20]. A total of 2,298
intervertebral discs (IVDs) of 127 spines (7 to 15 per colony)
were examined and scored. A modified histological scoring
system of the spine [10] was established by assessment of the
severity of spine involvement, which may achieve a score for
each IVD, ranging from 0 to 8. No inflammation was scored as
0, inflammatory (leukocyte) cell accumulation (peridiscitis and
Table 1
Arthritis susceptibility, severity, and onset of different BALB/c colonies
Colonya
VendorsArthritic/total number of animalsArthritis score (acute)b
Onset scorec
Number SymbolIncidence Percentage
1
●
Portage P08; Charles River Laboratories, Inc.
(Wilmington, MA, USA)
16/16100% 11.0 ± 1.1 2.3 ± 0.3
2
●
Canada II; Charles River Laboratories, Inc.15/15100% 10.3 ± 1.32.5 ± 0.4
3
●
Harlan Laboratories, Inc. (Indianapolis, IN, USA) 12/1392% 9.7 ± 1.5 2.8 ± 0.5
4
■
Kingston K72; Charles River Laboratories, Inc.13/1493% 8.8 ± 1.2 2.1 ± 0.4
5
■
Raleigh R02; Charles River Laboratories, Inc.15/15 100%7.6 ± 1.01.9 ± 0.3
6
■
Taconic Farms, Inc. (Hudson, NY, USA); Charles
River Laboratories, Inc.
15/18 83%7.5 ± 1.41.6 ± 0.3
7
■
NCI/Kingston (Charles River Laboratories, Inc.)17/20 85% 6.8 ± 0.91.5 ± 0.2
8
■
BALB/cJ; The Jackson Laboratory
(Bar Harbor, ME, USA)
16/1984%6.6 ± 1.12.1 ± 0.3
9
■
Raleigh R12; Charles River Laboratories, Inc.10/1377%6.4 ± 1.51.2 ± 0.2
10
▲
Hollister H42; Charles River Laboratories, Inc.10/1567% 3.8 ± 0.81.1 ± 0.2
11
▲
Bailey's BALB/c ByJ; The Jackson Laboratory15/2075%2.4 ± 0.71.0 ± 0.2
All animals were immunized with human cartilage proteoglycan aggrecan in dimethyldioctadecyl-ammonium bromide. Values represent mean ±
standard error of the mean. aColony numbers indicate the different BALB/c colonies. bHighest arthritis score measured at any time point of the
experiment. cOnset score was calculated at the end of the experiment (days 63 and 64) and ranged from the earliest onset of inflammation (5) to
no arthritis (0). Circles (●) represent the most arthritis-prone colonies. Designation was based on the statistical analysis showing no significant
differences between these three colonies (1 to 3) comparing two major clinical variables: onset and severity. Therefore, these three colonies were
combined and designated as group I (Figure 1a). Squares (■) indicate the average clinical phenotype of arthritis (colonies 4 to 9) with no
significant differences using onset and severity scores as clinical phenotype markers. This combined group is designated as group II (Figure 1a).
Triangles (▲) represent the two least arthritic or least susceptible colonies, 10 and 11. Data of these two colonies were combined, and the two
colonies together were designated as group III (Figure 1a). NCI, National Cancer Institute (Bethesda, MD, USA).

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enthesitis) was scored as 1 or 2, progression of IVD resorption
was scored as 3 to 6, fibrotic or fibro-cartilaginous ankylosis
(with complete disc resorption) received a score of 7, and
complete ankylosis due to chondrophyte/osteophyte forma-
tion was scored as 8. A cumulative spodyloarthropathy score
(the sum of spondylitis scores per spine) was calculated for
each animal.
Measurements of serum cytokines and anti-
proteoglycan antibodies and the lymphocyte responses
Serum cytokines IL-1β, IL-4, IL-6, interferon-gamma (IFN-γ),
and tumor necrosis factor-alpha were measured by ELISA.
Antigen-specific lymphocyte responses were measured in
spleen cell cultures in the presence or absence of 50 μg/mL
human PG antigen. Antigen-specific IL-2 production was
measured as a proliferative response of CTLL-2 cells to IL-2 in
48-hour spleen cell supernatants (CTLL-2 bioassay) [20].
Lymphocyte proliferation was assessed on day 5 of culture by
measuring [3H]-thymidine incorporation [18,20], and antigen-
specific T-cell proliferation was expressed as stimulation index
[7,18,20]. In vitro production of the above-listed cytokines
was also measured in supernatants of antigen (PG)-stimulated
(50 μg/mL) spleen cell cultures on day 5 using ELISA.
Secreted cytokine concentrations were normalized to nano-
grams per million cells [9,11].
PG-specific serum antibodies were measured by ELISA using
at least three different serum dilutions. Highly purified human
or mouse cartilage PG [25] was immobilized onto the surface
of Nunc-Maxisorp 96-well plates (Nalge Nunc, Naperville, IL,
USA) [20]. For PG-specific IgG isotype assays, peroxidase-
labeled goat anti-mouse IgG1 (Zymed Laboratories, Inc., now
part of Invitrogen Corporation, Carlsbad, CA, USA) and IgG2a
(BD Biosciences) were employed. Serum PG-specific anti-
body levels were calculated using serial dilutions of pooled
and standardized sera of mice with PGIA [20].
Affymetrix hybridization and related statistical analysis
RNA samples from spleen cells of naïve and immunized (12
days after the intraperitoneal PG injection) mice were
extracted with TriReagent (Sigma-Aldrich) in accordance with
the instructions of the manufacturer. Affymetrix hybridization
(Affymetrix, Santa Clara, CA, USA) was performed using
'Mouse Genome 430 2.0' gene chips. Biotinylation of cRNA,
labeling, and hybridization were processed by the Genomic
Core Facility of the University of Illinois at Chicago. Data were
analyzed using the GeneSpring GX 10.0 software package
(Agilent Technologies, Inc., Santa Clara, CA, USA). Robust
multi-array average [26] summarization algorithm (with quan-
tile normalization and median polish probe summarization pro-
cedures) and baseline transformation (that is, per gene
normalization; baseline to median of all 12 samples) were run
on data using a logarithmic scale. All sample replications
passed quality control. For pairwise comparisons, the Stu-
dent-Newman-Keuls post hoc test was performed after one-
way analysis of variance on four groups (naïve BALB/cJ, naïve
BALB/cByJ, immunized BALB/cJ, and immunized BALB/cByJ)
to identify statistically significant (P < 0.05) differentially
expressed transcripts and statistical differences between
naïve and immunized mice of the two colonies. Asymptotic P
value computation and Benjamini-Hochberg false discovery
rate multiple testing correction were applied [27]. Hierarchical
clustering was applied to significantly differentially expressed
genes, based on the Pearson centered distance metric and
centroid linkage rule. Differentially expressed transcripts were
annotated with the GeneSpring software. The bivariate linear
correlation (Pearson) test was performed to identify statistical
correlations among spine and arthritis parameters. The Fisher
exact chi-square test was applied when normal and diseased
animals were compared. These statistical analyses were per-
formed using SPSS version 16.0 statistical software (SPSS
Inc., Chicago, IL, USA).
Results
Susceptibility, severity, and onset of arthritis in different
BALB/c colonies
Based on the visual scoring system [20] and later confirmed
by histology, we could sort the 11 BALB/c colonies into three
major groups. There were no statistical differences in arthritis
severity and onset time within any of the three groups (Table 1
and Figure 1a). Overall, arthritis scores ranged from 2.4 ± 0.7
to 11.0 ± 1.1 and the onset score of arthritis ranged from 1.0
± 0.2 to 2.8 ± 0.5 (Table 1). Most of the BALB/c colonies (col-
ony numbers 4 to 9, henceforth called group II) developed
arthritis at an average severity of 7.2 ± 0.5 and at onset scores
of 1.8 ± 0.1 (Table 1 and Figure 1a). Compared with group II,
group I (colonies 1 to 3, Table 1) comprised the most suscep-
tible substrains, which developed arthritis earlier and with
greater severity than any other colonies. In contrast, group III
(colonies 10 and 11, Table 1) showed the least severe arthritis
(mean arthritis score of 3.0 ± 0.6) with delayed onset time (1.0
± 0.2), and approximately 30% of the immunized animals did
not have arthritis at the end of the experiment (Table 1 and Fig-
ure 1a). In arthritic animals, the histopathological abnormalities
(cellular infiltration, synovitis, pannus formation, and cartilage
and bone erosions) were similar to those (data not shown)
described earlier in numerous papers [7-9,20,28], and there
were no differences in the histopathology when peripheral
joints of any subcolonies with the same clinical scores [20]
were compared (data not shown).
Histopathology of the spine
A total of 127 spines were formalin-fixed, x-ray-imaged, and
then processed for histological analysis. Following the earlier
scoring system [10], IVD involvement was analyzed using
three parameters: (a) the cumulative spondyloarthropathy
score of each animal (Figure 1c), (b) the mean (IVD) inflamma-
tory score per animal (Figure 1d), and (c) the ratio of the
number of the inflamed IVDs per total number of IVDs
(expressed as a percentage) (Figure 1e). In the scoring of the

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127 spine sections, spondylitis was diagnosed in 62.2% of
BALB/c mice, which was significantly lower (P < 0.05) than
the mean of arthritis incidence (86.5%; n = 178). This obser-
vation confirmed that arthritis and spondylitis could occur
either together or separately in BALB/c mice [9] and that,
most likely, different genes of different QTLs control PGIA and
PGIS [9,29]. However, the most susceptible and most
severely arthritic BALB/c colonies (Table 1) showed the most
extensive spine involvement (Figure 1c–e) as assessed by
using any of the three spondylitis parameters listed above.
Similarly, animals that developed arthritis sooner exhibited
more progressive spondylitis (Figure 1b). Although no PGIS-
resistant BALB/c colony was found, there were large individ-
ual variations in the spine involvement. In addition, neighboring
IVDs of the same animal frequently showed different stages of
inflammation. Typically, the most affected spine segments
were the distal lumbar and distal cervical regions, whereas the
IVDs in the thoracic and proximal lumbar regions remained
less affected.
T cell- and B cell-mediated immune responses
Despite screening a wide spectrum of immunological parame-
ters, we could not identify 'colony-specific' cytokine, T-cell, or
B-cell responses. In vitro tests (T-cell proliferation and
cytokine production) showed evidence of T-cell activation in
response to PG stimulation, but T cell responses did not cor-
relate significantly with either arthritis or spondylitis scores.
Interestingly, female BALB/c mice of the Hollister and ByJ col-
onies (Table 1, colonies 10 and 11, group III), which were the
animals least susceptible to PGIA and PGIS, produced the
highest levels of anti-inflammatory IL-4, proinflammatory IL-6
and IFN-γ cytokines when assayed in spleen cultures. How-
ever, there was no evidence that any of these cytokine genes
(Figure 2) were expressed differentially in BALB/cJ versus
BALB/cByJ colonies (data not shown). We hypothesized that
BALB/c mice of the Hollister and ByJ 'low-susceptibility' colo-
nies (with delayed onset and less severe arthritis) still might be
in the initiative (proinflammatory) phase of arthritis at the end
of the experiments (days 63 and 64). This was supported by
the serum levels of autoantibodies to mouse PG (either IgG1
or IgG2a), which were significantly lower in animals of group
III than in any other colonies (data not shown). Indeed, in sup-
plemental experiments using age-matched females of The
Jackson Laboratory's BALB/cJ and BALB/cByJ colonies or of
Kingston and Hollister colonies of Charles River Laboratories,
Inc. (average versus low-susceptibility animals) injected with
the standard dose of 100 μg of PG protein on day 42 (third
Figure 1
Progression and severity of arthritis in 11 BALB/c colonies sorted into three different groups (listed in Table 1), correlation between the onset of arthritis and spine involvement, and comparison of the three arthritic groups with different spine inflammation scoresProgression and severity of arthritis in 11 BALB/c colonies sorted into three different groups (listed in Table 1), correlation between the onset of
arthritis and spine involvement, and comparison of the three arthritic groups with different spine inflammation scores. (a) Each animal was scored for
arthritis three times a week, and scores are shown as mean ± standard error of the mean. Arrow indicates the third injection, administrated on day
42. Significant differences (P < 0.01), calculated by one-way analysis of variance, were found from day 32 between groups I, II, and III. (b) The ratio
of the number of inflamed intervertebral discs (IVDs) per the total number of IVDs showed positive significant correlation (Pearson correlation coeffi-
cient ρ = 0.485; P < 0.0005) with the onset of arthritis. (c-e) Significant differences were found among the three arthritic groups when compared
with three spine representative scores: cumulative spondylitis score (c), the mean spondylitis score (d), and the ratio of the number of inflamed IVDs
per total number of IVDs (e). Asterisks indicate the level of significance between groups (*P < 0.05 and **P < 0.01) using Tamhane (c, e) (n = 127)
and least significant difference (d) (n = 79) post hoc tests.