Materials and methods

Mice
Inbred NFR/N mice were originally obtained from the National Institute of Health (Bethesda, MD, USA) and the B10.Q mice were originally bought from The Jackson Laboratory (Bar Harbor, ME, USA). (B10Q × NFR/N)F1 hybrids and (B10.Q × NFR/N) × B10.Q N2 mice were bred in individual ventilated cages in the BMC Barrier animal facility and at the animal house of the Department of Pathology, Lund University, Sweden. The animals were fed ad libitum with standard rodent chow (LAB FOR R36, irradiated breeding food for rats and mice; Lactamin AB, Stockholm, Sweden) and water in a photoperiod of 12 hours:12 hours light:dark. The mice used in the present study had clean health monitoring protocols according to the Federation of European Laboratory Animal Sciences Association recommendations. Ethical permissions were M125-04 (embryo transfer) and M290-03 (reproduction and arthritis).

Experimental design
A total of 200 female mice (approximately ten months old) of N2 backcross (B10.Q × NFR/N) × B10.Q were used. The mice had earlier undergone a reproductive study where each animal had four mating opportunities. First, the female mice were allowed to mate twice with B10.RIII males (allogeneic mating by means of MHC (major histocompatibility complex) and finally to mate twice with B10.Q males (syngeneic mating by means of MHC). After four possible pregnancies CIA was induced to the female mice.

Induction and evaluation of CIA
To induce CIA, the mice were immunized subcutaneously at the base of the tail with 100 μg rat collagen type II (CII) emulsified in 0.1 M acetic acid combined with an equal amount of complete Freud's adjuvant (Difco Laboratories, Detroit, MI, USA). A booster injection containing 50 μg CII emulsified in 0.1 M acetic acid combined with an equal amount of Freud's incomplete adjuvant (Difco Laboratories) was given after 30 days. The clinical scoring of arthritis commenced 25 days after the first immunization. Animals were examined for clinical signs of CIA three times per week and were graded on a 12-point scale. The arthritis index was assigned to each mouse using the following criteria: 0 = no visible sign of arthritis, 1 = redness and swelling in a single joint, 2 = inflammation in multiple joints, and 3 = severe inflammation in the entire paw and/or anklebone. Each paw was given the scores 0–3, with the index being the sum of all four paws. The severity trait is the maximum score observed in each individual female. The onset is the number of days calculated from the first immunization to the first clinical signs of arthritis excluding unaffected animals.

Microsatellite genotyping and linkage analysis
Tail biopsies were collected from all N2 females and the F0 generation. DNA was isolated according to a previously described protocol [12]. After screening of parental DNA with approximately 450 mouse fluorescence-labeled microsatellite markers (INTERACTIVA, Ulm, Germany), 125 informative markers were selected covering the genome. Two hundred and thirty-seven N2 mice were genotyped with markers covering all chromosomes except for the Y chromosome. PCR amplification for the markers was performed in a final volume of 10 μl in a 96-well V-bottom microtiter plate using 20 ng DNA, 10 mM KCl, 20 mM Tris–HCl, 10 mM (NH4)2SO4, 2 mM MgCl2, 0.1% Triton X-100, pH 8.8 (New England BioLabs Inc., Ipswich, MA, USA), 3 μM (10 pmol) each primer, 2 mM dNTPs (Advanced Biotechnologies, Epsom, Surrey, UK) and 0.25 U Taq DNA Polymerase (New England BioLabs Inc.). The following program was used to amplify the DNA: denaturation at 95°C for 3 minutes, annealing at 56°C for 45 seconds, polymerization at 72°C for 1 minute, 30 cycles of 95°C for 30 seconds, 56°C for 45 seconds and 72°C for 1 minute, and a final extension step of 7 minutes at 72°C. The PCR products were analyzed on a MegaBACE™ 1000 (Amersham Pharmacia Biotech Inc Piscataway, NJ, USA) according to the manufacturer's protocol. Data were analyzed with Genetic Profiler 1.1 (Amersham Pharmacia Biotech Inc).
Map Manager QTXb20 free software [13] was used to perform linkage analysis and the permutation test. Ninety percent of the mouse genome was within a 20 cM intermarker distance. The marker map was generated using the Kosambis map function and 1,000 permutations were performed for every phenotype (P < 0.05). The permutation tests were carried out to establish empirical significance thresholds for the interactions. A threshold equal to or above the 37th percentile (P = 0.63) was considered suggestively significant, and the level for the significant threshold was set to the 95th percentile (P = 0.05). Interval mapping was made with a 2 cM increase under the additive regression model in order to calculate the test statistics.

Enzyme-linked immunosorbent assay
The adult female mice were sacrificed at 15 months of age and sera were collected. Anti-CII antibody titers in sera were analyzed by a sandwich ELISA technique [14]. In brief, CII (10 μg/ml) was coupled to immunosorbent plates overnight at 4°C. Bovine serum albumin (Sigma Chemical St Louis, MO, USA) was used for blocking, and thereafter different dilutions of control sera (purified mouse anti-CII antibodies), test sera, and positive and negative controls were added. The presence of CII-specific IgG was visualized by means of peroxidase-conjugated goat antimouse IgG.

Statistical analysis
Statistical comparison between the different experimental groups was performed using the Mann-Whitney U test or Student's unpaired t test.