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

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-prone murine strain, and a number of genes of as-yet-unknown function may affect or modify clinical phenotypes of arthritis and/or spondylitis.

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 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 coefficient 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.

Figure 2

Hierarchical clusterization comparing the 77 genes expressed differently at significant levels in spleen cells of naive and proteoglycan-immunized (not-yet-arthritic) mice of BALB/cJ and BALB/cByJ colonies (n = 3 of each, four-group cross-comparison: naïve BALB/cJ versus immunized BALB/cJ, naïve BALB/cByJ versus immunized BALB/cByJ, naïve BALB/cJ versus naïve BALB/cByJ, and immunized BALB/cJ versus immunized BALB/cByJ). Color code indicates the normalized intensity expression values (with baseline transformation) on a logarithmic scale. Twenty-three genes showing over twofold differences in any of the four comparisons are labeled with asterisks. Whenever a gene name was not identified (n = 14), the original probe set ID (number_at), the Riken ID (numberRik), or the expressed sequence tag clone number is used. Those genes that showed significant differences only in response to immunization (n = 8) are labeled with the '†' symbol. Original data files are available via Gene Expression Omnibus (accession number [GEO:GSE13730] and National Center for Biotechnology Information tracking system number 15549466).