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The Journal of Immunology, 2005, 175: 2475-2483.
Copyright © 2005 by The American Association of Immunologists

Two Major Interacting Chromosome Loci Control Disease Susceptibility in Murine Model of Spondyloarthropathy1

Anikó Végvári*,{dagger}, Zoltán Szabó*,{dagger}, Sándor Szántó{dagger}, Andrew B. Nesterovitch*, Katalin Mikecz*, Tibor T. Glant* and Vyacheslav A. Adarichev2,*

* Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL 60612; and {dagger} Department of Internal Medicine 3, University of Debrecen, Debrecen, Hungary


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Autoimmune spondylitis was induced in BALB/c mice and their MHC-matched (BALB/c x DBA/2)F1 and F2 hybrids by systemic immunization with cartilage/intervertebral disk proteoglycan (PG). As in human ankylosing spondylitis, the MHC was the major permissive genetic locus in murine PG-induced spondylitis (PGIS). Two major non-MHC chromosome loci with highly significant linkage were found on chromosomes 2 (Pgis2) and 18 (Pgis1) accounting for 40% of the entire F2 trait variance. The dominant spondylitis-susceptibility allele for Pgis2 locus is derived from the BALB/c strain, whereas the Pgis1 recessive allele was present in the disease-resistant DBA/2 strain. The Pgis1 locus significantly affected the disease-controlling Pgis2 locus, inducing as high incidence of spondylitis in F2 hybrids as was found in the spondylitis-susceptible parent BALB/c strain. Additional disease-controlling loci with suggestive linkage were mapped to the chromosomes 12, 15, and 19. Severity of spondylitis in F2 mice positively correlated with serum levels of amyloid A, IL-6, and Pg-specific Abs, and showed negative correlation with Ag-induced T cell proliferation, IFN-{gamma}, IL-4, and TNF-{alpha} production. A major locus controlling serum IL-6 was found on chromosome 14 near osteoclast differentiation factor Tnfsf11. Locus on chromosome 11 near the Stat3 and Stat5 genes controlled serum level of the Ig IgG2a isotype. The two major genetic loci Pgis1 and Pgis2 of murine spondylitis were homologous to chromosome regions in human genome, which control ankylosing spondylitis in human patients. Thus, this animal model of experimentally induced spondylitis might facilitate the identification of spondylitis-susceptibility genes in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chronic inflammation of the axial skeleton and sacroiliac joints is the hallmark of ankylosing spondylitis (AS)3 in human patients. The incidence of AS is 0.1–0.3% in the Caucasian population; it is higher in Native Americans and lower in African-Americans (1, 2, 3, 4). Although the etiology of the disease is unknown, environmental and genetic components have been implicated as predisposing factors. To date, the strongest genetic risk factor is the HLA-B27 allele among the MHC class I genes (5). Although 92% of Caucasian patients with AS carry HLA-B27, the risk associated with this allele alone is rather low: <10% of HLA-B27 individuals develop AS (1, 3, 4, 6). Therefore, extensive studies have been undertaken to identify other, non-MHC genetic factors (6, 7, 8, 9, 10) and, indeed, approximately a dozen chromosome regions have been linked to AS. Two major loci were consistently mapped to chromosomes 2q and 16q in Oxford scans of British pedigrees (7, 8, 9), although a broader European ancestry pedigree could not confirm the locus on chromosome 2q, and only a weak linkage was found on chromosome 16q (10). The genetic basis of AS appeared to be distinct in these two pedigrees, and the MHC seemed to be the only shared genetic locus (10).

Although mapping of disease susceptibility genes in the human genome is the ultimate goal of genetic studies, animal models offer a plausible alternative. Animal models for spondyloarthropathy, however, are limited to a few HLA-B27 transgenic murine lines (11, 12), and a transgenic mouse expressing truncated TNF-{alpha} (13, 14). In contrast to these animal models with artificially altered genetic background, spondylitis, sacroiliitis, and enthesitis can be induced in unmodified susceptible mouse strains (BALB/c and C3H) using systemic immunization with certain constituents of cartilage and/or intervertebral disks (IVDs), such as proteoglycan (PG), link protein, or versican (15, 16, 17, 18). Clinical symptoms, histopathological and x-ray abnormalities, and the progression of the disease in PG-immunized mice, are very similar to those in human AS. As in human patients, inflammation in mice occurs first in sacroiliac joints occupied with enthesitis, and then expands upwards involving multiple IVDs (15, 19).

We have mapped non-MHC genetic loci associated with clinical or immunological parameters of the disease in F2 hybrid mice from spondylitis-susceptible BALB/c and a virtually spondylitis-resistant DBA/2 cross, and found two major quantitative trait loci (QTL) with significant linkage to genes on mouse chromosomes 2 and 18. The first murine locus is homologous to human chromosomes 5q and 18q, both of which have significant linkage with AS found in British and European kindreds (6, 9, 20). The second locus overlaps with the cluster of IL-1 genes and is homologous to an AS locus described in human studies (8).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, Ag, and immunization

Inbred female BALB/c and male DBA/2 mice were purchased from the National Cancer Institute and mated to generate F1 and then F2 offspring. All animal experiments were approved by the Institutional Animal Care and Use Committee of Rush University Medical Center. All mice were maintained in a pathogen-free environment. At 12 wk of age, mice were immunized i.p. with 100 µg of PG (measured as protein) emulsified with 2 mg of dimethyldioctadecylammonium bromide (DDA) adjuvant in 100 µl of 1x PBS (0.15 M NaCl, 0.01 M potassium phosphate, pH 7.4) as described (21, 22). Mice received total of four injections every 3 wk (days 0, 21, 42, and 63) and were sacrificed 3–4 wk after the fourth PG injection, i.e., on days 84–91 of the experiment.

Clinical and immunological phenotypes

The axial skeleton spanning the spine from the midcervical to distal lumbar region was dissected, fixed in neutral buffered 10% formalin, decalcified, and embedded in paraffin. Spine sections were stained with H&E and individual IVDs scored as described earlier (15). In brief, enthesitis, inflammatory cell accumulation around the IVD (discitis), and/or infiltration of the annulus fibrosus with or without evident tissue damage was recorded as severity score 1; massive but <50% resorption/erosion of the IVD received a score of 2; nearly complete resorption (>50%) of the IVD was recorded as score 3; and cartilaginous/bony ankylosis was given a score of 4. An average of 18 IVDs per mouse was scored. Finally, a spondylitis index (SPI) for each animal was calculated by dividing the sum of scores (all IVD scores) with the number of IVDs examined histologically. In addition, a specific index for mice exhibiting late onset of spondylitis (SPILS) was also calculated. A mouse was assigned SPILS"1" if at least one of the IVDs had a mild severity score of 1. Otherwise a mouse with progressive spondylitis was assigned SPILS"0", even if IVDs had a more advanced form of the disease indicated by severity scores 2, 3, or 4.

Ag (PG)-specific T cell proliferation was measured by [3H]thymidine incorporation in response to in vitro PG treatment as described (22). Ag-induced cytokine production (IL-2, IFN-{gamma}, IL-4, and TNF-{alpha}), and serum levels of Abs, amyloid A, and cytokines (IL-1, IL-4, IL-6, and TNF-{alpha}) were determined using ELISA as described previously (21, 22, 23).

Genomic markers

Markers were selected for detectable simple sequence length polymorphism between the parent BALB/c and DBA/2 strains from the mouse genome database (<www.informatics.jax.org>) or alternatively were designed using primers flanking regions of short, usually <100 bp, tandem repeats in the mouse genome. Differences in length between PCR fragments of BALB/c and DBA/2 alleles were >3%. Polymorphism between the strains was detected in 3.5% high resolution Aquapore agarose gel (National Diagnostics) upon staining with ethidium bromide and UV illumination. All 20 mouse chromosomes, except chromosome Y, were covered with a total of 224 polymorphic markers at an average spacing of 6.2 cM (10.8 Mbp).

Genome screening and statistical analysis

Genomic DNA was isolated from mouse kidney using proteinase K and sodium lauryl sulfate (23, 24). DNA was genotyped with simple sequence length polymorphic markers (MWG Biotech) using conventional PCR and gel-electrophoresis as described previously (23, 24). Initial linkage map was generated with Map Manager QTX (25) using the Kosambi mapping function. The order of markers was further adjusted using the "ripple" command and then confirmed according to physical positions of oligonucleotide primers in the National Center for Biotechnology Information mouse genome assembly (<www.ensembl.org/Mus_musculus/>) and the Celera Discovery System genome database <www.celeradiscoverysystem.com/>). Single marker effect was estimated using marker regression in Map Manager QTX. For traits that demonstrated association with genomic markers stronger than {chi}2 > 10 (p < 0.01), both simple and complex interval mappings were performed on the entire genome using Windows QTL Cartographer (26). Experiment-specific empirical thresholds for likelihood ratio statistic (LRS) were established for each trait with a permutation test (n = 2000, 1 cM walk speed) according to an algorithm proposed by Churchill and Doerge (27) and implemented in Map Manager QTX and Windows QTL Cartographer (25, 26). Levels for genome-wide highly significant ({alpha} < 0.001) and significant ({alpha} < 0.05) linkage were used (28). For a suggestive linkage, we used a p < 0.05 chromosome-wise significance level ({alpha} < 0.63), which corresponds to one false-positive QTL for the entire genome (28).

Statistical analysis was performed using the SPSS statistical software package. As spondylitis indices demonstrated nonparametric distribution in the F2 hybrid population, we used the Mann-Whitney U test to examine differences between groups and the Spearman’s correlation coefficient (rs) to evaluate biases between traits. {chi}2 statistics and Kruskal-Wallis H tests were used to determine the difference between distributions of traits in the genotypes. The two-sample Student’s t test was used for comparison of means of two groups, when data showed normal distribution. The significance level was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Clinical and immunological parameters of spondylitis in parental BALB/c and DBA/2 strains

Upon systemic immunization with cartilage Pg, BALB/c mice develop spondylitis with an incidence of 61.5% (Table I and Fig. 1). Using the same immunization protocol, DBA/2 mice demonstrated much lower susceptibility (4%) with notably less severe IVD damage than BALB/c mice, and spine ankylosis was never detected in the DBA/2 strain (19).


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  		Table I. Comparison of clinical and immunological parameters between BALB/c and DBA/2 progenitor mouse strains upon systemic immunization with PG for induction of PGISa

 


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  		FIGURE 1. SPI (y-axis values are shown in a logarithmic scale) in parental BALB/c and DBA/2 strains, and their F1 and F2 hybrids. Each triangle represents one animal. Symbols placed below the horizontal line at SPI = 0 represent spondylitis-resistant mice with no histological abnormalities in the spine. Severity was calculated as average SPI in the affected mice and is shown as a short horizontal line for each animal group.

 
The major immunological characteristics were measured in progenitor mice and correlated with disease susceptibility (Table I). The DBA/2 strain had almost three times higher serum levels of Igs as compared with BALB/c mice (17.8 vs 6.5 mg total Ab per milliliter of serum with IgG1 being the major Ig isotype in both strains). The serum level of IgG2a isotype showed an opposite distribution between the strains: it was three times more abundant in BALB/c. Based on the IgG1/IgG2a Ig isotype ratio, both BALB/c and DBA/2 strains demonstrated a Th2-type immune response; however, in Pg-immunized DBA/2 mice, the Th2-type response was more prominent. Interstrain differences found in histological scores were highly correlated with the elevated concentration of proinflammatory cytokines IL-1{beta} and IL-6 in sera of spondylitis-susceptible BALB/c mice, although serum levels of IL-4 and TNF-{alpha} were comparable.

Ag (PG)-stimulated lymphocytes of BALB/c mice demonstrated significantly higher production of IFN-{gamma} and IL-4 when compared with lymphocytes from DBA/2, and the IFN-{gamma}/IL-4 ratio was 2.2-times higher in BALB/c (5.1) than in DBA/2 mice (2.3). This observation also indicates that BALB/c mice with PG-induced spondylitis (PGIS) exhibit a more significant shift to Th1 dominance, whereas the virtually resistant DBA/2 strain does not.

Correlations between clinical and immunological traits in F1 and F2 hybrid populations

The incidence of spondylitis in F1 hybrids of BALB/c and DBA/2 mice was 35.5%, approximately the median value between the progenitor strains, although the severity of the disease was relatively low (SPI 0.16) (Fig. 1). Unexpectedly, the incidence of spondylitis in F2 hybrid mice was the same as in the susceptible parent BALB/c strain with a severity score nearly twice higher than in BALB/c mice (Fig. 1).

Because mice of the F1 generation are genetically homogeneous, only nongenetic environmental factors might be responsible for trait variance. In contrast, F2 hybrid mice are genetically heterogeneous and both genetic and environmental factors might be in action. Thus, the correlation between spondylitis and immunological parameters in F1 and F2 populations must come from different sources. One of the best examples is the SPI, which very tightly correlated with SPILS in the F1 population (rs 0.97), but this correlation was notably weaker in F2 hybrid mice (rs 0.54) because the heterozygous combination of genes differently affected the SPI and SPILS in (BALB/c x DBA/2) F2 hybrids (Table II).


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  		Table II. Cross-correlation of clinical and immunological traits in the genetically homogenous F1 and the segregating F2 hybrid populations of the BALB/c x DBA/2 cross upon immunization with PGa

 
To determine the most important immunological parameters that might be associated with spondylitis in F1 and F2 hybrid populations, we calculated correlation coefficient rs (Table II). The strongest positive correlation of PGIS was found with serum concentrations of amyloid A (SAA; an acute phase protein in mice) and IL-6, rs 0.28 and 0.43, respectively (Table II), which is consistent with an even stronger correlation between SAA and IL-6 (rs 0.74). Spondylitis correlated positively with Ab production (both for IgG1 and IgG2a isotypes) in the F2 hybrid population. Surprisingly, spondylitis correlated negatively with all measured T cell responses (T cell proliferation, IFN-{gamma}, IL-4, and TNF-{alpha} production) but only in the F2 mice (Table II). There was no correlation between spondylitis and Ag-specific T cell responses (or the serum levels of most Abs) in homozygous F1 hybrid mice (Table II). In contrast, these correlations are strong in the segregating F2 population, suggesting that spondylitis is controlled by immune response-related genes and/or allele combinations in this animal model. In summary, enhanced serum levels of IL-6, SAA, and Ab, together with decreased production of IFN-{gamma}, IL-4, and TNF-{alpha} by PG-stimulated T cells, could be considered the strongest predictors of spondylitis in this MHC-matched cross of PGIS-susceptible and resistant mice.

Linkage analysis for spondylitis in (BALB/c x DBA/2)F2 hybrids

To map non-MHC spondylitis-susceptibility genes, we used a BALB/c x DBA/2 cross, where both progenitor strains carry the same H-2d haplotype, thus deliberately excluding the effects of the MHC genes from linkage analysis. To perform an effective scan for murine genes regulating SPI and SPILS clinical traits, F2 hybrid male and female mice were immunized with PG, scored for spondylitis, and a set of immune-related parameters was measured. Accordingly, all mice were genotyped for the entire mouse genome (20 chromosomes) with 224 markers. Interval mapping and single marker effect analysis indicated the presence of a very limited number of chromosome loci controlling spondylitis (SPI, SPILS), serum level of Ab (IgG2a isotype to mouse PG), and IL-6 in the sera of F2 mice, whereas other traits did not show significant linkage (Fig. 2).



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  		FIGURE 2. Genome-wide interval mapping for clinical and immunological parameters of spondylitis in (BALB/c x DBA/2)F2 hybrid population. LRS thresholds were established with 2000 genome-wide permutations at {alpha} < 0.63 (suggestive; long-dashed line), {alpha} < 0.05 (significant; short-dashed line), and {alpha} < 0.001 (highly significant; dotted line). Chromosome numbers are shown at the bottom of each plot.

 
The major genetic locus controlling spondylitis was identified on the telomeric part of chromosome 18 (Pgis1). The LRS reached a value of 31, which exceeds the highly significant cut-off level introduced by Lander and Kruglyak (28) and empirically established a highly significant threshold at {alpha} < 0.001 (Fig. 3A). Composite interval mapping (Windows QTL Cartographer, standard model 6 with control of five markers inside of a 10 cM window) confirmed the QTL peak position for markers D18Mit51 and D18Mit142 (Fig. 3A).



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  		FIGURE 3. Simple and composite interval mapping for all genome-wide significant QTLs ({alpha} < 0.05) shown in Fig. 2. LRS thresholds were suggestive (long-dashed line), significant (short-dashed line), or highly significant (dotted line). Composite interval mapping (thick solid curves) confirms and narrows the QTL position given by simple interval mapping (light solid curves). Chromosomes 2 and 18 carry the major QTLs for spondylitis (A–C). Chromosome 11 carries a locus controlling the production of IgG2a to mouse Pg (D), while chromosome 14 contains a locus controlling serum IL-6 level (E). Association of spondylitis with genotypes of QTL peak markers is shown on F. Genotypes: B, BALB/c homozygous, {square}; D, DBA/2 homozygous, {blacksquare}; H, heterozygous, {cjs2108}. Student’s t test p values for the difference in SPI between BALB/c- and DBA/2-homozygous mice are presented for each QTL. The dominant spondylitis-susceptibility Pgis2 allele of chromosome 2 was originated from the BALB/c strain, and a recessive spondylitis-susceptible allele, located inside the Pgis1 locus of chromosome 18, was from the DBA/2 strain.

 
The second major spondylitis QTL was identified on chromosome 2 (Pgis2). This locus, however, was fairly vague, when simple interval mapping was used, and occupied up to one-third of the chromosome (Fig. 3B). Using composite interval mapping, we found the peak for this QTL (LRS 16.9, {alpha}< 0.05) located near marker D2Mit241 (Fig. 3B). When the integrated effects of each chromosome upon spondylitis susceptibility was calculated, chromosomes 2 and 18 appeared to jointly control 40.5% of the entire SPI trait variance in the F2 population (Fig. 2).

The additional spondylitis phenotype index, SPILS, was introduced in this study. This is a binary index with score "1" for mice with late onset of spondylitis, and it was tightly correlated with low PGIS severity. Despite the high correlation between SPI and SPILS, the genetic basis of these two clinical traits seems to be different. For example, there is no QTL for SPILS on chromosome 18, whereas the major QTL controlling late/weak spine inflammation was found at the same position as the Pgis2 locus (Figs. 2 and 3C). However, the Pgis2 locus was more prominent and narrower for SPILS than for SPI. Pgis2 alone contributed to 28.7% of the entire SPILS trait variance in the F2 population (Fig. 2). The Pgis2 peak was near the D2Mit296 marker (LRS 21.1, {alpha} < 0.05, simple interval mapping), and the position was confirmed by composite interval mapping (LRS 25.9, {alpha} < 0.001) (Fig. 3C).

Because spondylitis in F2 hybrid mice correlated significantly with certain immunological phenotypes (T and B cell responses, serum cytokines), we performed linkage analysis for intermediate immunological traits as well. Serum concentration of IgG2a isotype autoantibodies seemed to be under control of several QTLs (Fig. 2). Two major loci were mapped close to each other on mouse chromosome 11 (LRS 17.7 and 19.4, {alpha} < 0.001), and two weaker QTLs were found on chromosomes 1 and 5 (LRS 11.8 and 10.8, {alpha} < 0.63) (Figs. 2 and 3D). Serum concentration of the proinflammatory cytokine IL-6 was under the control of a single QTL located in the central part of chromosome 14 (LRS 14.9, {alpha} < 0.05), and composite interval mapping confirmed the peak position (Figs. 2 and 3E).

Interaction between the two major clinical QTLs on chromosomes 2 and 18

Although both BALB/c and DBA/2 strains carry genes for susceptibility to spondylitis, these strains responded very differently to PG immunization. To determine the source of disease-controlling alleles in the F2 population, which might come either from the BALB/c or the DBA/2 strain, we calculated the average SPI separately for BALB/c homozygous (PgisB), DBA/2 homozygous (PgisD), and BALB/c-DBA/2 heterozygous mice (PgisH), and for each QTL (Fig. 3F). Unexpectedly, we have found that F2 mice, which were homozygous for the DBA/2 allele of Pgis1 (Pgis1D), developed spondylitis at a significantly higher incidence and severity rate than BALB/c homozygous animals (Pgis1B vs Pgis1D, p < 0.000003), which suggested that the major disease susceptibility allele was derived from a resistant DBA/2 strain. Another QTL, Pgis2 on chromosome 2, showed "normal" phenotype-genotype relationship, and mice with BALB/c homozygosity for this region were significantly more spondylitis-susceptible than F2 hybrid mice with DBA/2 homozygosity (Pgis2B vs Pgis2D, p < 0.0005), thus indicating that the disease allele was derived from the spondylitis-susceptible BALB/c strain. For both Pgis1 and Pgis2 QTLs, the BALB/c alleles were dominant over the DBA/2 allele, since BALB/c-DBA/2 heterozygous mice had as high SPI as BALB/c homozygous animals (Fig. 3F).

Thus, in F2 hybrid mice the highest susceptibility to spondylitis was observed in DBA/2 homozygous animals (Pgis1D); that seems to contradict the virtual resistance of the parental DBA/2 mice having the same genotype of the Pgis1 locus (Table I and Fig. 1). As we found only two major loci controlling spondylitis in the BALB/c x DBA/2 cross, to explain the silence of the Pgis1D allele in the parental DBA/2 strain, we examined the influence of one locus to the other. The SPI and incidence of the disease were calculated based on the hypothesis of cooperation between Pgis1 and Pgis2 loci; average values for phenotypes were calculated separately for each of the nine allele combinations (Figs. 4). When the effects of Pgis1 genotypes upon clinical traits were analyzed independently of the Pgis2 locus, Pgis1D homozygous mice were most spondylitis-susceptible, Pgis1B homozygous mice demonstrated the lowest susceptibility to the disease, and Pgis1H heterozygous animals were in-between (Fig. 3F). Surprisingly, the relationship between Pgis1 alleles was found critically dependent upon the genetic composition of the Pgis2 locus. It seemed that at least one copy of the Pgis2 of BALB/c origin (Pgis2B) was necessary for the high penetrance of the Pgis1D allele and its function as a spondylitis-permissive gene (Fig. 4). Thus, mice bearing the Pgis1D allele were the most spondylitic (average SPI = 0.96) and mice with the Pgis1B allele were the least affected (SPI = 0.13) (Fig. 4, block A). However, the phenotypic difference between Pgis1D and Pgis1B allele-carrying mice vanished on a pure Pgis2D genetic background (Fig. 4, block C). Thus, the spondylitis-promoting Pgis1D allele needs the Pgis2B allele for the complete trait penetrance and full action. F2 hybrid mice that carry the most malicious Pgis1D–Pgis2B allele combination (Fig. 4, the bar marked with black arrow) demonstrated the highest spondylitis severity and 100% incidence of the disease, further confirming the importance of both loci for disease development.



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  		FIGURE 4. The effect of the Pgis1 locus upon spondylitis depends on the allelic composition of Pgis2. Pairwise Mann-Whitney U test p values indicate difference in spondylitis severity (SPI) and incidence (%) between mice carrying different combinations of Pgis1 and Pgis2 alleles. Genotype description is the same as in Fig. 3. Table presents mean SPI, SEM, and number of mice for all nine haplotype combinations. All F2 hybrid mice were included into analysis; no selection was applied. Black arrow indicates combination of alleles controlling complete 100% spondylitis susceptibility. Note, that there is no difference in spondylitis severity and incidence of mice carrying Pgis1D vs Pgis1B alleles on the Pgis2D background (block C), however the difference is obvious on the Pgis2B and PgisH backgrounds (blocks A and B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we present for the first time a complete genome scan for genetic loci controlling spondylitis susceptibility in mice. We determined the genetic basis of trait variance in a complex phenotype that developed from natural genetic variation instead of artificial modification, i.e., gene deficiency. Despite a number of murine models for spondyloarthropathy established and studied earlier, the genomic basis of disease susceptibility is not known, except for the contribution of HLA-B27 and a few other genes to disease pathology (11, 12, 13, 14, 29, 30).

Earlier we demonstrated that spondylitis susceptibility of mice of different parental strains and genetic crosses was associated with certain H-2-permissive haplotypes, which indicates the leading role of the MHC in murine arthritis and spondylitis (19). The major goal of this study was finding non-MHC genes. We have found only two major genetic loci regulating spondylitis in the BALB/c x DBA/2 cross: the Pgis1 disease-controlling allele originating from the DBA/2 strain (although the gene remained silent in this strain), and the Pgis2 allele which is derived from the BALB/c strain. Obviously, the combined effects of these two loci/genes resulted in high disease incidence in BALB/c x DBA/2 F1 hybrids (35.5%), and even higher susceptibility in F2 hybrid mice (61.7%) when two spondylitis-promoting alleles of two genes supplemented each other. Similarly, disease severity, which was defined only in SPI-positive mice, was even higher in F2 hybrids than in BALB/c progenitor mice (Fig. 1) due to a cumulative effect of two permissive genes in the F2 population.

Despite the differences between spondylitis-susceptible and resistant mice in immune function-related traits (T and B cell responses, and serum cytokines), and the known pattern of interaction among SPI-linked genes inside the Pgis1 and Pgis2 loci, at this stage of investigation it is not possible to identify spondylitis-contributing primary causative genes within these loci. Comparison of genomic maps of disease-controlling loci, as shown in Fig. 5, in human patients and in mice and syntenic mapping, might aid in identification of gene candidates in future studies.



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  		FIGURE 5. Murine spondylitis loci overlap with chromosome loci controlling different autoimmune diseases in mice and humans. Black vertical boxes show murine spondylitis-linked chromosome segments, where numbers on the right represent the exact QTL location at a distance of million base pairs from the chromosome centromere. Murine QTLs are shown on the left in italics. Abh, bronchial hyperresponsiveness; Pcfm, periosteal circumference and femur length; Fcsa, femoral cross-sectional area; Sbmd, spinal bone mineral density; Stia, serum transfer-induced arthritis; Aig, autoimmune gastritis; Cia, collagen-induced arthritis QTLs 2 and 4; Eae, experimental allergic encephalomyelitis; Heal, wound healing; Lbw, murine lupus; Cmo, chronic multifocal osteomyelitis; Pgia11, Pg-induced arthritis QTL 11. Gene candidates within these loci include IL-1rn, IL-1R antagonist inhibitor; IL-1f5, IL-1f6, IL-1f7, IL-1f8, IL-1 family members 5, 6, 7, and 8; C8g, complement component 8 {gamma}; C5, hemolytic complement 5; Adamts19, a metalloproteinase 19 of the ADAMTS family; IL17b, IL 17 {beta}-chain; Cd74, MHC class II associated invariant chain; Csf1r, macrophage CSF 1 receptor; and Nfatc1, NF 1 of activated T cells. Syntenic with mouse, human chromosome loci are presented as gray bars on the right. The murine Pgis1 locus is homologous to loci in human chromosomes 5 and 18; the Pgis2 locus is syntenic with human chromosomes 2 and 9. Base pair positions are indicated on the right of the human chromosome regions. Gray arrows point to identified human ankylosing spondylitis loci; AS(Ox), loci found in the Oxford pedigree (9 ); AS(Eu), loci in pedigree of European origin (10 ).

 
The major SPI locus in the BALB/c x DBA/2 cross was found on the telomeric region of mouse chromosome 18 (Figs. 2 and 3A). The Pgis1 locus is flanked by D18Mit55 and D18Mit80 markers and occupies the region from 54 Mbp to the chromosome telomere. The Pgis1 locus overlaps with murine QTLs for a number of autoimmune diseases such as murine lupus (Lbw6), Pg- and collagen-induced arthritis (Pgia11 and Cia18), experimental allergic encephalomyelitis (Eae25), wound healing (Heal9), and chronic multifocal osteomyelitis (Cmo) (Fig. 5).

The murine Pgis1 locus (54–81 Mbp) is homologous with human chromosomes 5q (segments 110–129 Mbp and 137–150 Mbp) and 18q (segments 17–52 Mbp and 64–76 Mbp) (<www.ncbi.nlm.nih.gov/mapview/>). These human chromosome segments contain two loci for AS; one on chromosome 18q was found in Oxford pedigree AS(Ox) (9), and the AS(Eu) locus was found in the kindred of European origin on chromosome 5q (10) (Fig. 5). The list of gene candidates in the locus includes metalloproteinase Adamts19, IL IL-17b, MHC class II-associated invariant chain Cd74, macrophage CSF I receptor Csf1r, and others. The region also contains the Nfatc1 gene encoding calcineurin-dependent NF 1 of activated T cells. This gene regulates IL-2 and IL-4 gene transcription, differentiation, proliferation, and activation-induced cell death in T lymphocytes (<http://harvester.embl.de/harvester/>). Because we have found a significant correlation between SPI and T cell responses, this gene inside the Pgis1 locus is a plausible candidate as a primary causative factor.

The size of the second major locus, Pgis2, is larger than Pgis1. The Pgis2 locus is flanked by the D2Mit293 and D2Mit156 markers located at 26.4 and 57.0 Mbp, respectively. Simple interval mapping suggested slightly different positions for SPI and SPILS on mouse chromosome 2 (Fig. 3, B and C). However, we maintain the hypothesis that a single gene/locus in this region controls disease susceptibility based on the following considerations. First, the mode of inheritance for SPI and SPILS peak position markers is identical, and demonstrated a dominant action of the BALB/c allele. Second, we did not succeed in splitting Pgis2 into two loci by varying the number of markers in composite interval mapping. Third, both SPI and SPILS clinical traits were closely correlated both in the F1 and F2 hybrids of the BALB/c x DBA/2 cross.

The murine Pgis2 locus (24–57 Mbp) contains numerous autoimmune loci linked to Ag-induced bronchial hyperresponsiveness (Abhr1 and Abhr2), experimental autoimmune gastritis (Aig), collagen-induced arthritis (Cia2 and Cia4), serum transfer-induced arthritis (Stia2), and experimental allergic encephalomyelitis (Eae21) (Fig. 5). Interestingly, Pgis2 also overlaps with QTLs controlling the anatomical development of the skeleton such as femoral cross-sectional area (Fcsa5) and periosteal circumference and femur length (Pcfm1).

Combining data from publicly available online genome resources and analysis of published literature (<www.ncbi.nlm.nih.gov/mapview/>), we found that the Pgis2 locus is syntenic with two segments in the human genome: one located on chromosome 2q between 143 and 161 Mbp, and the second on chromosome 9 between 120 and 137 Mbp. Both intervals on chromosomes 2 and 9 contain QTLs for AS identified in British pedigree (6, 7, 8, 9) (Fig. 5). Besides this, linkage with C5 deficiency monogenic syndrome resulting in recurrent local and systemic infections and systemic lupus erythematosus (Mendelian inheritance in man 120900) was also found in the syntenic human loci.

Gene candidates in the locus include complement component 8 {gamma} (C8{gamma}) and an IL-1 gene cluster, namely IL-1R antagonist and five members of the IL-1 family. The latter gene cluster is considered the major gene candidate for AS on human chromosome 2 in the Oxford study (8). After initial genome scan and identification of positive linkage on chromosome 2 (7, 9, 31), the Oxford group found significant association between single polymorphisms and IL-1-related haplotypes (8). In our study, IL-1 itself did not show any significant linkage, probably because the difference in serum concentrations of IL-1 was not significantly different in BALB/c and DBA/2 mice (Table I). Any of the gene candidates located between the telomeric end of Pgis2 locus and the IL-1 cluster might also be functionally associated with the spondylitis phenotype.

The best-known gene proposed to have a crucial role in ankylosis in mice is the Ank gene. Spontaneous mutation(s) on chromosome 15, resulting in progressive ankylosis, was described in 1981 (29) and 1988 (32, 33). Later, the mutation was mapped inside the Ank gene (30). At present it is believed that mutation of this gene is responsible for craniometaphyseal dysplasia (34) and crystal deposition arthropathy (Mendelian inheritance in man 118600). In our genome scan we found only a suggestive QTL on mouse chromosome 15, on which the Ank gene is located (Fig. 2). However, this locus was linked with PgIA in our earlier studies (35, 36), suggesting that chromosome 15 loci might be partially involved in spondylitis in this murine model.

In addition to the two major SPI loci on chromosomes 2 and 18, and a suggestive locus on chromosome 15, four more loci demonstrated suggestive linkage (Fig. 2). We found QTLs on chromosomes 11, 12, and 19 (LRS 11.0, 12.5 and 12.1, respectively). Suggestive spondylitis QTL on the telomeric part of chromosome 12 contains a cluster of Ig H chain genes (<www.ensembl.org/Mus_musculus/>). This locus has been found in several crosses produced in our laboratory to control PgIA or serum concentration of IgG2a, or both traits, although this QTL reached a significant level of linkage with arthritis only in the C3H x C57BL/6 cross (37).

Major QTLs for IgG2a and IL-6 did not fall into either Pgis1 or Pgis2 regions on chromosomes 2 and 18. However, there is an overlap between spondylitis and immunological QTLs when a suggestive threshold for linkage is considered. The telomere part of chromosome 11 carries suggestive a spondylitis-susceptibility locus, which coincides with the major peak for IgG2a (Fig. 2). Additionally, our earlier studies showed two arthritis-controlling QTLs on chromosome 11 (Pgia7 and Pgia28) (35, 36, 37), which occupy the same regions as newly discovered QTLs for IgG2a in this murine cross. Chromosome 14 carried the arthritis Pgia29 locus which shares a position with IL-6 QTL.

The pivotal role of both T and B cell populations in the induction of murine arthritis and spondylitis was shown in the experiments with adoptive disease transfer (38). Depletion of donor lymphocytes with Abs specific to Th and T suppressor populations, similar to depletion of B cells, prevented the successful disease transfer. Neither anti-Pg Abs nor Pg-specific B cells alone were able to transfer disease, but the cooperation between T and B cell subpopulations is necessary (38, 39, 40). Progenitor BALB/c mice upon immunization with Pg produced twice more IL-6 than DBA/2 mice (57.8 pg/ml vs 30.1 pg/ml, p < 0.013); and the IgG2a Ig isotype concentration was three times higher whereas IgG1 concentration was three times lower in BALB/c than in DBA/2 mice (Table I). The correlations between disease severity and Th2-supported Ag-specific Ig levels were significant in (BALB/c x DBA/2)F2 hybrid mice as well. The strongest correlations with spondylitis were found for the IL-6 and IgG1 Ab isotype: the SPI vs IL-6 coefficient of correlation rs was 0.43, and for SPI vs IgG1 was 0.23. Th1-type associated IFN-{gamma} and TNF-{alpha} production by Pg-stimulated lymphocytes in vitro were found to be negatively correlated with spondylitis in the F2 hybrid mice further supporting the leading role of Th2-type cells in disease pathogenesis. Therefore, we conclude that a strong genetic predisposition toward a Th2 response and susceptibility to arthritis and spondylitis is similar in progenitor strains and in genetically mixed F2 hybrid mice, and the F2 population is modeling similar pathology as seen in the parental BALB/c strain.

Colocalization of QTLs for clinical and immunological traits, as was demonstrated for chromosomes 11 and 12, is a powerful approach to the effective mapping of spondylitis-susceptibility genes and better understanding of the involvement of these loci, thus shedding light upon the mechanisms of spondylitis.


   Acknowledgments
 
We thank Drs. Alison Finnegan and Katalin Ludági for valuable discussions and Sonja Velins for assistance in preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (AR 40310 and AR 45652). Back

2 Address correspondence and reprint requests to Dr. Vyacheslav A. Adarichev, Section of Molecular Medicine, MC 101, Department of Orthopedic Surgery CRB011, Rush University Medical Center, 1735 West Harrison, Room 724, Chicago, IL 60612. E-mail address: vadarich{at}rush.edu Back

3 Abbreviations used in this paper: AS, ankylosing spondylitis; IVD, intervertebral disk; PG, proteoglycan; QTL, quantitative trait locus; SPI, spondylitis index; SPILS, late-onset SPI; LRS, likelihood ratio statistic; PGIS, PG-induced spondylitis; SAA, serum amyloid A; rs, Spearman’s correlation coefficient; PGIA, PG-induced arthritis. Back

Received for publication March 2, 2005. Accepted for publication May 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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