Two Loci on 15 Control Experimentally Induced Arthritis through the Differential Regulation of IL-6 and Lymphocyte Proliferation This information is current as of September 24, 2021. Tibor T. Glant, Sándor Szántó, Aniko Vegvari, Zoltan Szabo, Katalin Kis-Toth, Katalin Mikecz and Vyacheslav A. Adarichev J Immunol 2008; 181:1307-1314; ; doi: 10.4049/jimmunol.181.2.1307 Downloaded from http://www.jimmunol.org/content/181/2/1307

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Two Loci on Chromosome 15 Control Experimentally Induced Arthritis through the Differential Regulation of IL-6 and Lymphocyte Proliferation1

Tibor T. Glant,* Sa´ndor Sza´nto´,*‡ Aniko Vegvari,*‡ Zoltan Szabo,*‡ Katalin Kis-Toth,*‡ Katalin Mikecz,*† and Vyacheslav A. Adarichev2*§

Using genetic linkage analysis of proteoglycan-induced arthritis (PGIA), a murine model for rheumatoid arthritis, we iden- tified two loci, Pgia8 and Pgia9, on chromosome 15 (chr15) that appear to be implicated in disease susceptibility. Immuni- zation of congenic strains carrying the entire chr15 and separately each of the two loci of DBA/2 arthritis-resistant origin in susceptible BALB/c background confirmed locations of two loci on chr15: the major Pgia9 and lesser Pgia8 locus. Distal part of chr15 (Pgia9) showed a major suppressive effect on PGIA susceptibility in females (40%, p < 0.001), whereas the Downloaded from effect of this locus in congenic males was still significant but weaker. Proximal part of chr15 (Pgia8) demonstrated mild and transient effect upon arthritis; this effect was PGIA-promoting in males and suppressive in females. Pgia8 and Pgia9 loci demonstrated an additive mode of inheritance, since when they were both incorporated in consomic chr15 strain, the total effect was a sum of the two loci. Using F2 population of the intercross of wild-type and chr15 consomic strain, we confirmed and refined quantitative trait locus positions and identified a strong correlation between disease susceptibility and lympho- cyte-producing cytokines of TNF-␣ and IL-6. Both Pgia8 and Pgia9 loci on chr15 appear to control IL-6 production in spleen http://www.jimmunol.org/ cultures of arthritic mice, providing an important link to the mechanism of autoimmune inflammation. The Journal of Immunology, 2008, 181: 1307–1314.

heumatoid arthritis (RA)3 is a chronic inflammatory au- ical assessments, biochemical and immunologic tests, and histo- toimmune disease influenced by both genetic and envi- pathology studies of diarthrodial joints (12). Like RA, PGIA is an R ronmental factors (1, 2). The genetic background of RA autoimmune disease, which is critically dependent on MHC (H-2 is complex and heterogeneous, being linked to different sets of complex in mice); however, multiple chromosome loci were found chromosome regions in different ethnic populations (3–9). Meta- to control disease in different crosses of murine strains (12). Ge- by guest on September 24, 2021 analysis of genome scans of RA-affected families has located the nome-wide linkage analysis of MHC-matched (BALB/c ϫ DBA/ most common susceptibility loci on human 1p, 2q, 2)F2 mice revealed that different sets of non-MHC loci were linked 6p, 6q, 8p, 12p, 16p, and 18q (10). to PGIA susceptibility, severity, or disease onset, while gender was Progressive polyarthritis induced in susceptible BALB/c mice a major modulator of the quantitative trait loci (QTLs) affecting via systemic immunization with human cartilage proteoglycan either male- or female-restricted penetrance (14). (PG) aggrecan is a murine model for RA (11–13). PG-induced Women are affected with RA two to three times more frequently arthritis (PGIA) resembles RA in many ways, as indicated by clin- than men, and joint deformities are also more pronounced in women (15, 16); however, factors that account for the increased susceptibility of females are still not completely understood. Dis- *Section of Molecular Medicine, Department of Orthopedic Surgery, and †Depart- parity of XY vs XX chromosomes in males vs females could be ment of Biochemistry, Rush University Medical Center, Chicago, IL 60612; ‡Depart- ment of Internal Medicine 3, Health Science Center, University of Debrecen, Deb- directly linked to higher female preponderance. Therefore, a direct recen, Hungary; and §Albert Einstein College of Medicine of Yeshiva University, effect of sex chromosomes upon arthritis phenotype is a compel- Department of Medicine, Department of Microbiology and Immunology, Bronx, NY ling hypothesis. Indeed, several linkage studies have confirmed the 10461 involvement of chromosome X in patients with RA (3–5, 7), and Received for publication September 15, 2006. Accepted for publication May 5, 2008. of both chromosomes X and Y in a number of animal models of 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 arthritis (14, 17–24). Apparently gender is an important risk factor with 18 U.S.C. Section 1734 solely to indicate this fact. for arthritis, and sex chromosome loci may indeed influence ar- 1 This work was supported in part by National Institutes of Health/National Institute thritis susceptibility. of Arthritis and Musculoskeletal and Skin Diseases Grants AR051101, AR045652 In addition to X and Y chromosomes, genetic loci on somatic and AR040310. V.A.A. is a recipient of the Sontag Foundation and the Arthritis National Research Foundation Awards. chromosomes may also affect sex-related penetrance of the disease 2 Address correspondence and reprint requests to Dr. Vyacheslav A. Adarichev, Al- (20). For example, in PGIA, a number of QTLs showed asso- bert Einstein College of Medicine of Yeshiva University, Department of Medicine, ciation with gender when the MHC effect was excluded in 1300 Morris Park Avenue, Forchheimer, Room 405, Bronx, NY 10461. E-mail ad- ϫ dress: [email protected] BALB/c DBA/2 intercross (14). Remarkably, chromosome 15 (chr15) carries two gender-effected loci: Pgia8 and Pgia9. 3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis; chr15, chromosome 15; LRS, likelihood ratio statistic; Ncf, neutrophil cy- To gain insight into the mechanisms by which these two QTLs tosolic factor; PG, proteoglycan; PGIA, PG-induced arthritis; PIA, pristane-induced on chr15 can differentially affect arthritis in males and females, arthritis; QTL, quantitative trait locus (loci). we transferred the entire chr15, and each locus separately, from Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 the arthritis-resistant DBA/2 strain to the PGIA-susceptible www.jimmunol.org 1308 CHROMOSOME 15 IN MURINE ARTHRITIS

(25). N2 males were genotyped with 130 genomic markers selected for detectable length polymorphism between the parental strains using in- formation from the Mouse Genome Informatics database (http:// www.informatics.jax.org). N2 males bearing DBA/2-type heterozygous QTLs on chr15 and the highest number of BALB/c alleles in the rest of the genome were selected for the next backcross. Approximately forty offspring males after each backcross were genotyped with ten chr15- specific markers, and additional markers were used for the genomic regions found to be heterozygous in the previous backcross. At back- cross level N6, when the entire genome was BALB/c homozygous ex-

cept chr15, N6 males and females were intercrossed, generating N6F1 population of the consomic strain B-15D (Fig. 1). Chromosome Y of DBA/2 origin was replaced with BALB/c through the mating of B-15D females with BALB/c males. Simultaneously, we selected mice with recombinations carrying either proximal (B-15pD) or distal (B-15dD) parts of chr15 from DBA/2 origin, thus, establishing either Pgia8-or Pgia9-specific congenic strains (Figs. 1 and 2C).

Chr15-specific genetic linkage analysis For chr15-specific genetic linkage analysis, wild-type BALB/c females D ϫ D were mated with B-15 males; (BALB/c B-15 )F1 hybrids were then

ϭ Downloaded from intercrossed to produce F2 hybrid mice (total n 195; 114 females and 81 males) (Table I), which were all genotyped with chr15-specific

genomic markers listed in Fig. 3. All chromosomes in these F2 hybrids were wild-type BALB/c type except for chr15, which was either ho- mozygous BALB/c, homozygous DBA/2, or heterozygous. Genetic linkage analysis, marker regression, and interval mapping for putative QTLs within chr15 were performed with Map Manager QTX software (26). Significant thresholds for linkage were calculated http://www.jimmunol.org/ for each trait (arthritis and Ag-specific IL-6 production) using empirical 5000 permutations at 1-cM interval (26). Permutation test for arthritis score revealed threshold for suggestive linkage at likelihood ratio sta- tistics (LRS) 4.2 and for significant linkage at LRS 11.5. Permutations for level of IL-6 production in lymphocytes trait established suggestive threshold at LRS 4.0 and significant threshold at LRS 11.3. QTL po- sitions were confirmed using bootstrap re-sampling method incorpo- rated in Map Manager QTX (26).

Ag and immunization by guest on September 24, 2021 Articular cartilage from knee joints was obtained from osteoarthritic FIGURE 1. Chr15 loci of DBA/2 origin (filled chromosome symbols) patients undergoing joint replacement surgery. The use of human car- were transferred to BALB/c genetic background (open chromosome sym- tilage for PG isolation was approved by the Institutional Review Board bols) using marker-assisted speed congenic backcrossing breeding protocol of Rush University Medical Center, Chicago, IL. PG isolation has been described in detail (27, 28). Mice were immunized with human PG at 12 for six generations (N1 through N6). Inheritance of chr15 (shorter chro- wk of age using a standard immunization protocol (27, 28). Emulsion mosome symbols) and sex chromosomes X (long symbols) and Y (shortest containing 100 ␮g of PG and 2 mg of dimethyldioctadecylam- D symbols) is shown. First, a consomic strain B-15 was generated. Then monium bromide adjuvant in 200 ␮l of PBS (pH 7.4) was injected i.p. consomic females B-15D were crossed with BALB/c males to produce on days 0, 21, 42, and 63. Arthritis severity was determined using a recombinations within chr15 and select subcongenic strains B-15pD and visual scoring system based on the extent of swelling and redness of the B-15dD. front and hind paws (11, 27). We performed earlier visual scoring under control of histopathological examination and found a strong correlation between visually observed parameters of arthritis and microscopy pic- ture of joint tissues upon H&E staining (12). Animals were examined at BALB/c background, then analyzed clinical phenotypes of least two times a week and inflammation was scored from 0 to 4 for these PG-immunized consomic and congenic mice. In addition, each paw, thus, resulting in a cumulative arthritis score ranging from 0 to 16 for each animal (11, 27). Susceptibility to disease for each mouse we produced an F2 population in which chr15 carried BALB/c, was considered as a binary qualitative trait; i.e., susceptibility has only DBA/2, or both alleles, while all other chromosomes were two values: either “1” for positive (arthritis-susceptible) or “0” for non- BALB/c homozygous. Using this two-way approach, we were arthritic (resistant) animals. Incidence of disease was expressed as per- able to determine how the Pgia8 and Pgia9 loci affect arthritis cent of arthritic mice to the total number of PG-immunized mice. Dis- susceptibility and severity in males and females via a T cell- ease severity score (ranging from 1 to 16) was applied only to arthritic mice. Day of disease onset was used to characterize how quickly a mediated cytokine-controlled mechanism. mouse developed arthritis. Mice were sacrificed two weeks after the fourth injection.

Materials and Methods Lymphocyte responses and cytokine production Marker-assisted congenic animal breeding Ag-specific lymphocyte responses were measured in spleen cell cultures All animal experiments were approved by the Institutional Animal Care in the presence of 25 ␮g/ml human PG Ag. IL-1␤, IL-4, IL-6, IFN-␥, and Use Committee (Rush University Medical Center, Chicago, IL). and TNF-␣ production were measured in cell culture supernatants of Ag Animals were maintained in a pathogen-free environment. PGIA-sus- (PG)-stimulated spleen cell cultures on day 4 using ELISA (BD Bio- ceptible BALB/c female and PGIA-resistant DBA/2 male mice (Na- sciences). Secreted cytokine concentrations were normalized and ex- tional Cancer Institute, Kingston colony, NY) were mated, and the F1 pressed as ng/million cells (12, 27, 29). Ag-specific IL-2 production males were backcrossed to parental BALB/c strain to obtain N2 gen- was measured as a proliferation response of CTLL-2 cells to IL-2 in eration (n ϭ 150) (Fig. 1). Marker-assisted selection protocol for speed 48-h spleen cell supernatants (CTLL-2 bioassay) (27). Lymphocyte pro- congenic breeding was used to generate QTL-specific congenic strains liferation was assessed on day 5 by incorporation of [3H]thymidine The Journal of Immunology 1309

Table I. Incidence and severity of PGIA in chr15-specific F2 hybridsa

n Incidence Severity Ϯ SEM

Females 114 83.33 5.37 Ϯ 0.38a Males 81 87.65 6.85 Ϯ 0.42 Total 195 85.13 6.01 Ϯ 0.29

a Males developed more severe arthritis than females according to non-paired two-tails Student’s t test, p Ͻ 0.01.

Statistical analysis Statistical analysis was performed using SPSS statistical software package (SPSS). Since the incidence of disease demonstrated a non-parametric dis- tribution, we used the Mann-Whitney U test to examine differences be- tween populations. To compare immunization curves, comparison between a group of wild-type and a group of congenic mice was performed for every day of scoring. Two-sample non-paired Student’s t test was used for comparison of means of two groups, where the data showed normal dis- tribution (severity of the disease, cytokines). To determine associations Downloaded from between cytokine levels and arthritis, we used Spearman’s rank correlation coefficient ␳. Significance level for all statistical tests was routinely set at p Ͻ 0.05. Results Arthritis in chr15 congenic mice http://www.jimmunol.org/ Previously, we performed genome-wide linkage analysis of PG- ϫ immunized (BALB/c DBA/2)F2 hybrid mice and identified two gender-dependent loci on chr15 (14). To confirm positions of loci and their genetic effects upon arthritis phenotypes, we transferred the entire chr15D from arthritis-resistant DBA/2 to arthritis-sus- ceptible BALB/c strain in a series of marker-assisted speed con- genic backcrosses, thus, generating B-15D consomic females (Fig. 1). Additionally, we selected B-15pD and B-15dD congenic strains that carried either proximal or distal parts of chr15 corresponding by guest on September 24, 2021 to Pgia8 and Pgia9 loci (Figs. 1 and 2C). We immunized males and females of consomic B-15D, congenic B-15dD, and congenic B-15pD strains, and of wild-type BALB/c littermates (Fig. 2). The strongest effect upon PGIA was observed in congenic B-15dD mice carrying the distal part of chr15 covering FIGURE 2. Incidence of arthritis in chr15-specific congenic males (A) Pgia9 locus (Fig. 2). The onset of arthritis in congenic males (Fig. and females (B). Congenic B-15pD (blue circles, 26 females and 23 males), D 2A) and females (Fig. 2B) was delayed by at least 2 wk, and dis- congenic B-15d strain (red diamonds, 27 females and 17 males), con- D somic B-15D (green triangles, 20 females and 24 males), and wild-type ease incidence in B-15d congenic females was as little as 50% BALB/c mice (gray squares, 21 females and 19 males) were immunized that in wild-type BALB/c female littermates ( p Ͻ 0.001, Mann- D with PG. The third and fourth immunizations were given at days 42 and 63, Whitney U test) (Fig. 2B). In B-15d congenic males, suppression shown with arrows below post immunization day-axis. Arthritis-suppres- of arthritis was also evident, although the significance was weaker sive effect of the distal part of chr15 was most significant in B-15dD fe- ( p Ͻ 0.05). males (p Ͻ 0.001). Effect of the proximal part of chr15 (Pgia8 in B-15pD) Immunization of B-15pD congenic females carrying the proxi- was transient in both males and females (p Ͻ 0.05). The distal part of chr15 mal part of the chr15 also revealed a reduced PGIA susceptibility, D (Pgia9 in B-15d ) suppressed arthritis in both males and females. Effect of albeit much weaker than in B-15dD females (Fig. 2B). The prox- the proximal part of chr15 (Pgia8 in B-15pD) was transient in both males imal part of chr15, which incorporates Pgia8 locus, did not show and females (p Ͻ 0.05). Color-coded brackets above the immunization curves show the time windows for the statistically significant difference any effect on arthritis severity in either males or females when between wild-type BALB/c and congenic mice according to Mann-Whit- compared with wild-type littermates (data not shown). We ob- ney U test. C, Pgia8 and Pgia9 loci; genomic markers and genotypes of the served higher disease susceptibility and a more rapidly developing D congenic strains are shown. Congenic mice were either DBA/2-type ho- disease after the third immunization in B-15p congenic males mozygous (filled areas within chr15) or BALB/c-type homozygous (open than in wild-type control male littermates, although this effect was areas). transient and was sustained for only 10–14 days (Fig. 2A). There- fore, proximal chr15 loci demonstrated opposing effects upon PGIA, exacerbating disease in males and suppressing it in females (Fig. 2, A vs B), although the effect was not very strong ( p Ͻ 0.05) (30). Ag-specific lymphocyte proliferation was expressed as stimulation and could be detected only during a period of less than two weeks. index. Similarly, we expressed cytokine responses using “normalized Consomic mice carry the entire chr15D; thus, both Pgia8 and change,” which was calculated as follow: [(cytokine concentration mea- Ϫ Pgia9 loci are present. As one might expect, the immunization sured in PG-stimulated spleen cultures) (cytokine concentration mea- D sured in non-stimulated cultures)]/(cytokine concentration measured in curve of B-15 mice is close to the arithmetic average of immu- non-stimulated cultures). nizations of B-15pD and B-15dD congenics. In B-15D consomic 1310 CHROMOSOME 15 IN MURINE ARTHRITIS

In a summary of results for congenic strains immunization, the major suppressive effect upon arthritis susceptibility was identified for Pgia9 locus on the distal part of chr15, while the effect of Pgia8 locus upon disease susceptibility was transient.

Genetic control of arthritis in chr15-specific F2 hybrid mice Using congenic strains, we confirmed the genetic effects of located both within the proximal/centromeric and distal/middle parts of chr15 upon clinical and immunologic arthritis phenotypes (Fig. 2). The size of either Pgia8 or Pgia9 locus, however, was too large, and the identification of causative genes within these loci required a dramatic reduction in size of these chromosome inter- vals. For this purpose, we used one of the modification of selective phenotyping methods (31) and generated a chr15-specific (BALB/ ϫ D c B-15 )F2 hybrid population. In these F2 mice, all genetic loci are BALB/c homozygous, except for chr15 alleles. Therefore, the overall genetic variance in this cross is dramatically reduced, and linkage analysis is focused for chr15 loci only.

ϫ D Downloaded from We immunized (BALB/c B-15 )F2 males and females (total n ϭ 195) and scored them for PGIA (Table I). Males were slightly more susceptible to PGIA (incidence 87.7 vs 83.3%) and devel- oped more severe arthritis than littermate females; the difference between genders was significant for arthritis severity ( p Ͻ 0.01)

(Table I). The entire F2 population was genotyped for chr15 al-

leles, and genetic linkage analysis identified two major loci linked http://www.jimmunol.org/ to the clinical score of PGIA (Fig. 3A). Interval mapping also found two loci controlling IL-6 production by PG-stimulated lym- phocytes. Positions of these two IL-6-controlling loci (Fig. 3B) were identical with clinical loci (Fig. 3A); furthermore, they matched previously identified Pgia8 and Pgia9 intervals (14). Lo- cus positions were further confirmed with bootstrap re-sampling. Pgia8 locus controlled arthritis in females and lymphocyte IL-6 response in males. Pgia9 locus controlled arthritis in both males and females and was equally responsible for IL-6 production in by guest on September 24, 2021 both genders. ϫ D Arthritis in (BALB/c B-15 )F2 mice is associated with IL-6 and TNF-␣ production

Lymphocytes from F2 hybrid mice were assayed for Ag-induced ␣ ␥ ␤ FIGURE 3. Genetic linkage analysis for arthritis susceptibility (A) production of TNF- , IFN- , IL-1 , IL-2, IL-4, and IL-6. Pair- and IL-6 normalized change production by PG-stimulated splenocytes wise correlations between all measured cytokines and arthritis ϫ D (B) in chr15-specific (BALB/c B-15 )F2 hybrid population (114 fe- were calculated for all mice (Table II and Fig. 4). In basal condi- males and 81 males, total n ϭ 195 mice). All chromosomes in this cross tions, when no PG-stimulation was applied to lymphocytes, arthri- were of BALB/c origin, except chr15, which loci were from either tis score did not correlate with in vitro production of any cytokine BALB/c or DBA/2 or both. Interval mapping for both phenotypes was (Table II “Basal”). However, the existence of an associative cyto- performed using LRS (on y-axis). Peak positions of Pgia8 and Pgia9 kine network is expected (12–14, 32), even though that seems to be loci were confirmed with bootstrapping test (26) relative to genomic irrelevant to the clinical phenotype, because cytokine levels cor- markers, which are shown with vertical ticks on x-axis. The size of related with each other. For example, analysis of association be- chr15 is illustrated with 10 million base pairs scale bar. Calculation of linkage was performed separately for females (circles) and males (tri- tween lymphocyte proliferation and cytokine production by non- angles), as well as for the combined population (solid line). Thresholds stimulated splenocytes indicated that the most potent growth for suggestive (dotted lines) and significant (dashed lines) linkage lev- factors were IL-2, IL-4, and especially IL-6. This is indicated by els were calculated for each trait empirically using 5000 permutations the strength of their coefficient of correlation (␳) with cell prolif- with Map Manager QTX. eration index, which was highest for IL-6 (␳ 0.703) (Table II). However, when lymphocytes were stimulated with Ag, correla- tion between arthritis severity and cytokine levels became obvious (Table II “PG-stimulated” and Fig. 4). Surprisingly, PGIA corre- females, the arthritis-suppressive effect is significant ( p Ͻ 0.01), lated significantly with only two cytokines, TNF-␣ and IL-6; but weaker than in B-15pD congenic females ( p Ͻ 0.001) (Fig. moreover, these correlations were negative. Apparently, at the next 2B). In B-15D consomic males, the PGIA-suppressive effect of the level, TNF-␣ and IL-6 concentrations affected all other measured proximal part of chr15 is offset by the arthritis-promoting effect of cytokines (Fig. 4). For PG-stimulated lymphocytes, again, IL-6 the distal part of chr15, resulting in an insignificant genetic effect was the dominant cell growth inducer (␳ 0.48, p Ͻ 0.01), having (Fig. 2A). Severity of paw inflammation in B-15pD or B-15dD mice even greater effect than TNF-␣ and IL-2. Thus, IL-6 and TNF-␣ was similar to that assessed in wild-type BALB/c mice during the appear to provide a link between the disease and the underlying entire observation period (data not shown). immune mechanisms of inflammation. The Journal of Immunology 1311

Table II. Spearman’s coefficients of correlation between cytokine levels produced by lymphocytes in vivo in basal conditions (no stimulation) and ϫ D a upon stimulation with arthritogenic PG Ag in (BALB/c B-15 )F2 hybrid mice

IL-1␤ TNF-␣ IL-6 IL-4 IFN-␥ IL-2 Proliferation

Basal Arthritis 0.069 0.097 0.095 0.073 0.079 0.133 0.101 Proliferation 0.410** 0.307** 0.703** 0.581** 0.374** 0.584** IL-2 0.154* -0.141 0.442** 0.336** 0.171* IFN-␥ 0.405** 0.550** 0.368** 0.649** IL-4 0.473** 0.470** 0.489** IL-6 0.350** 0.293** TNF-␣ 0.356** PG-stimulated Arthritis 0.011 -0.198* -0.155* -0.119 -0.109 -0.088 -0.101 Proliferation 0.202** 0.394** 0.480** 0.001 0.172* 0.422** IL-2 0.123 0.018 0.376** 0.173* 0.101 IFN-␥ 0.037 0.234** 0.239** 0.433** IL-4 -0.128 -0.080 0.271** IL-6 0.094 0.211** TNF-␣ 0.362**

a Basal pair-wise Spearman’s coefficients of correlation rho between arthritis susceptibility and cytokines in basal conditions, when no lymphocyte stimulation was applied, Downloaded from are shown. PG-stimulated, Ag-specific correlations between arthritis susceptibility and cytokines produced by lymphocyte cultures stimulated with cartilage PG. Both positive .p Ͻ 0.01 ,ءء p Ͻ 0.05 and ,ء :and negative significant coefficients are boldfaced

To further understand the mechanism of disease, particularly in relation to Pgia8 and Pgia9 genetic loci and gender, we deter- mined the allele-specificity of lymphocyte responses in (BALB/ http://www.jimmunol.org/ ϫ D c B-15 )F2 hybrid population. For each of two loci, normalized changes were calculated separately for mice carrying either ho- mozygous (BALB/c or DBA/2) or heterozygous alleles at locus peak genomic marker (Fig. 5). DBA/2 alleles of both Pgia8 and Pgia9 loci increased Ag-specific production of TNF-␣ by 1.5- to 2.0-fold. In contrast, the effect of these DBA/2 alleles upon IL-6 production was opposing: Pgia9D increased IL-6 secretion 2-fold, D but Pgia8 suppressed IL-6 level almost 3-fold. Since IL-6 and by guest on September 24, 2021 ␣ TNF- were the two major proinflammatory cytokines in this F2 population (Table II), when they worked in the same direction, as happened for Pgia9D, the total effect upon T cell proliferation be- came significant (Fig. 5). In the case of Pgia8D, growth stimulation effects of IL-6 and TNF-␣ were mixed, and the overall result upon proliferation was not significant.

ϫ D FIGURE 4. PGIA susceptibility in (BALB/c B-15 )F2 hybrid pop- ulation significantly and negatively correlated with TNF-␣ and IL-6. Cytokines IL-1␤, IL-2, IL-4, IL-6, and IFN-␥ production (normalized FIGURE 5. Allele-specificity of lymphocyte responses. Proliferation, change) correlated with either TNF-␣ or IL-6 or both major immune stimulation index for lymphocyte proliferation; IL-6, TNF-␣, normalized biomarkers. Associations between arthritis and cytokines produced in change for cytokine production upon stimulation with PG immunizing Ag. vitro by PG-stimulated spleen lymphocytes were estimated using pair- For each locus, Pgia8 or Pgia9, stimulation indices were calculated sepa- wise non-parametric Spearman’s correlation. Whenever correlation was rately for BALB/c homozygous (B), DBA/2 homozygous (D), and het- significant, the line connects two parameters; solid single line shows erozygous (H) mice segregating by locus-specific peak markers. Mice car- negative correlation coefficients; double line shows positive correlation. rying DBA/2 allele of the Pgia9 locus showed greater lymphocyte Only significant (italic, p Ͻ 0.05) and highly significant (bold and italic, proliferation, accompanied by greater production of TNF-␣ and IL-6 in p Ͻ 0.01) biases are shown. Note that IL-6 shows the largest number of both males and females. Pgia8 locus exhibited a heterogeneous profile for connections (n ϭ 5) and, therefore, appears to be the most significant the presented three immune parameters. Data are combined males and link between the disease and cytokine network. females. 1312 CHROMOSOME 15 IN MURINE ARTHRITIS

Discussion Two QTLs that control murine arthritis, Pgia8 and Pgia9 on chr15, were identified in genome screening studies of (BALB/c ϫ ϫ C3H)F2 and (BALB/c DBA/2)F2 hybrid populations (13, 14, 30). In a most recent analysis, we found that these two loci affected PGIA severity in a gender-specific mode (14). To further analyze the genetic effect of these two chr15 loci, we transferred the prox- imal part of chr15 containing Pgia8 locus and the distal part of chr15 bearing Pgia9 locus to the BALB/c genetic background. Immunization of congenic strains confirmed the presence of ar- thritis-controlling genes on chr15, although the effects of these loci were of different magnitude and gender specificity. The major lo- cus was found on a distal part of chr15, Pgia9. In females, this locus suppressed arthritis susceptibility by ϳ50% (Fig. 2), and it seemed to have no effect upon severity of inflammation (data not shown). In males, the Pgia9 locus demonstrated a lesser but still significant arthritis suppression (ϳ10%). The genetic effect of the Pgia8 locus was smaller than that of Pgia9 and either suppressed Downloaded from or promoted arthritis susceptibility, depending on gender. B-15pD females were less susceptible to PGIA; however, arthritis inci- D dence in B-15p males was higher, albeit for only a limited period FIGURE 6. Chr15 loci are homologous to multiple loci in rat and hu- D of time (Fig. 2). The two loci together in B-15 consomic mice man genome. In the mouse, Pgia8 locus clusters with QTLs controlling exhibited an additive mode of inheritance for Pgia8 and Pgia9 CIA (Cia30, Cia31, and Cia32), experimental allergic encephalomyelitis loci, since PGIA susceptibility in these consomic mice appeared to (Eae2), progression of autoimmune arthritis (Paam1), Mycobacterium bo- be an average of disease susceptibilities of congenic mice bearing vis-induced systemic lupus erythematosus (Mbis3), and Borrelia burgdor- http://www.jimmunol.org/ proximal and distal regions of chr15. feri-associated arthritis (Bbaa14). Pgia9 locus clusters with Cia4, Eea32, Genetic analysis of chr15-specific F hybrid population not only Eae38, and Bbaa22 in mouse genome. In the rat genome, chr15 loci cor- 2 responds to Pia17 and Cia7 QTLs. Human chromosomes 5, 8, and 22 carry confirmed the presence of two loci on chr15, but also refined loci loci for multiple autoimmune disorders. Whenever the causative is position. However, the major finding of this study was that the believed to be known, its name is given in brackets: Ankh (progressive locus controlling murine arthritis (Pgia9) occupies the same ankylosis), C6, C7, C9 (complement cascade components 6, 7, and 9), genomic position as the locus governing production of IL-6 by IL-7R (IL-7 receptor), Opg (osteoprotegerin), Mif (macrophage migration spleen cells. IL-6 cytokine has a pleiotropic effect (33), but it is inhibitory factor), and Rac2 (Ras-related C3 botulinum toxin substrate 2).

probably best known for its strong stimulation of growth of murine To build the chromosome homology map in this figure, we used the En- by guest on September 24, 2021 plasmacytomas and myelomas (34). More relevant to our data on sembl Genome Database (http://www.ensembl.org), the Rat Genome Da- the control of IL-6 production by chr15 loci, IL-6 has been dem- tabase (http://rgd.mcw.edu/), the On-line Mendelian Inheritance in Man ϭ onstrated to decrease aggrecan and collagen type II production by database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db OMIM), and NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Genes chondrocytes; therefore, cytokine is associated with cartilage de- effecting up- or down-regulation in inflamed joints of arthritic mice (50) terioration (35). Moreover, IL-6 supports osteoclast formation, were located on chr15 and are shown with arrows next to the murine loci: which leads to irreversible cartilage and bone erosion in inflamed C1qtnf3 and C1qtnf6 (C1q and TNF-related 3 and 6), Gcsf2rb1 joints (36). and Gcsf2rb2 (granulocyte-macrophage CSF 2 receptor b1 and b2), Dab2 The hypothesis explaining Pgia9 etiology could be based on the (disabled homologue 2), Ext1 (multiple exostoses 1), Fam49b (family with neutrophil cytosolic factor (Ncf4) gene located within the locus. sequence similarity 49 member B), Kdelr3 (KDEL endoplasmic reticulum This gene is also part of rat pristane-induced arthritis locus 17 protein retention receptor 3), Mal2 (T cell differentiation protein mal2), (Pia17), which overlaps with Pgia9 (Fig. 6). Several lines of ev- Mapk12 (MAPK 12), Mgb (myoglobin), Ncf4, Osmr (oncostatin M recep- tor), Pdzk3 (PDZ domain containing 3 isoform A), Rai14 (retinoic acid idence point to the Ncf4 gene. First, our data indicate that this gene induced protein 14), Sla (Src-like adaptor), Wisp1 (WNT1 inducible pro- is up-regulated in arthritis paws (37) (Fig. 6). Second, a recent tein 1), and Zfp7 (zinc finger protein 7). case-control study identifies an association of the NCF4 encod- ing gene with RA (38) and Crohn’s disease (39). Third, an earlier study of PIA in rats identified Ncf1 as a causative ar- thritis factor within Pia4 locus on rat chr12 (40). Ncf4 is a close translocation to IgH chain locus on murine chr12, thereby inducing homologue to Ncf1; both proteins are part of NADPH-oxidase T(12;15) plasmacytoma formation (45). The major growth factor complex. We hypothesize that enhanced expression of Ncf4 in promoting survival and proliferation of such plasmacytomas is macrophages of B-15dD congenic females leads to increased IL-6 (45). As for another type of arthritis, PIA, the formation of production of reactive oxygen species by macrophages, which plasmacytomas in BALB/c peritoneum, lymph nodes, lamina pro- have been shown to suppresses T cell responses and arthritis pria, and Payer’s patches has been demonstrated earlier (45). In- development in mice (41). terestingly, that correlation between PIA and plasmacytoma for- Interestingly, reactive oxygen is one of the mechanisms for mation in immunized BALB/c females was negative (46), like the chromosome aberrations (42, 43). Moreover, TNF-␣, which was negative correlation between PGIA and lymphocyte IL-6 produc- another cytokine associated with arthritis in this study, is also an tion in this study. These similarities further support our hypothesis inducer of chromosome abnormalities independently of the reac- about the mechanism of murine inflammation. tive oxygen species pathway (44). From this point of view, local- The Pgia8 and Pgia9 loci on chr15 correspond to multiple lo- ization of c-myc encoding gene right in the middle of the locus is cations on rat and human genomes. The Pgia8 locus clusters with rather remarkable, since c-myc is a well-known site for illegitimate multiple murine loci associated with type II CIA (Cia30, Cia31, The Journal of Immunology 1313 and Cia32), experimental allergic encephalomyelitis (Eae2), pro- the gene loci that predispose to rheumatoid arthritis. Int. Immunol. 10: gression of autoimmune arthritis (Paam1), Borrelia burgdorferi- 1891–1895. 8. Barton, A., S. Eyre, A. Myerscough, B. Brintnell, D. Ward, W. E. Ollier, associated arthritis (Bbaa14), and Mycobacterium bovis-induced J. C. Lorentzen, L. Klareskog, A. Silman, S. John, and J. Worthington. 2001. systemic lupus erythematosus (Mbis3) (Fig. 5). The murine Pgia8 High resolution linkage and association mapping identifies a novel rheumatoid arthritis susceptibility locus homologous to one linked to two rat models of in- locus corresponds to CIA-controlling locus Cia7 on rat chromo- flammatory arthritis. Hum. Mol. Genet. 10: 1901–1906. some 2. The Pgia8-syntenic region is located on the short arm of 9. Osorio, Y., J. Fortea, H. Bukulmez, E. Petit-Teixeira, L. Michou, C. Pierlot, human , a segment linked to several immune defi- S. Cailleau-Moindrault, I. Lemaire, S. Lasbleiz, O. Alibert, et al. 2004. Dense genome-wide linkage analysis of rheumatoid arthritis, including covariates. Ar- ciencies such as complement cascade-related C6, C7, and C9 de- thritis Rheum. 50: 2757–2765. ficiencies and severe combined immunodeficiency associated with 10. Choi, S. J., Y. H. Rho, J. D. Ji, G. G. Song, and Y. H. Lee. 2006. Genome scan a mutant IL-7 receptor (Fig. 5). The mouse Pgia9 locus clustered meta-analysis of rheumatoid arthritis. Rheumatology 45: 166–170. 11. Glant, T. T., K. Mikecz, A. Arzoumanian, and A. R. Poole. 1987. Proteoglycan- with murine Cia4, Eea32, Eae38 and Bbaa22 loci, and with rat induced arthritis in BALB/c mice. Clinical features and histopathology. Arthritis Pia17 locus in PIA (Fig. 5). Homologous human loci include Rheum. 30: 201–212. genes associated with Hashimoto’s thyroiditis and juvenile Paget’s 12. Glant, T. T., A. Finnegan, and K. Mikecz. 2003. Proteoglycan-induced arthritis: immune regulation, cellular mechanisms and genetics. Crit. Rev. Immunol. 23: disease on human chromosome 8q, and with juvenile RA and neu- 199–250. trophil immunodeficiency syndrome loci on human chromosome 13. Otto, J. M., R. Chandrasekaran, C. Vermes, K. Mikecz, A. Finnegan, 22 (Fig. 5). S. E. Rickert, J. T. Enders, and T. T. Glant. 2000. A genome scan using a novel genetic cross identifies new susceptibility loci and traits in a mouse model of Clustering Pgia8 and Pgia9 loci with a dozen loci that control rheumatoid arthritis. J. Immunol. 165: 5278–5286. numerous autoimmune diseases in mice and rats might indicate a 14. Adarichev, V. A., A. B. Nesterovitch, T. Bardos, D. Biesczat, R. Chandrasekaran, complex genetic structure within each locus, i.e., the presence of C. Vermes, K. Mikecz, A. Finnegan, and T. T. Glant. 2003. Sex effect on clinical Downloaded from and immunological quantitative trait loci in a murine model of rheumatoid ar- multiple genes that govern disease. Indeed, we detected an inter- thritis. Arthritis Rheum. 48: 1708–1720. action between Pgia26, the major locus on mouse chromosome 3 15. Linos, A., J. W. Worthington, W. M. O’Fallon, and L. T. Kurland. 1980. The (14), with Pgia8 on chr15 (47). Both Pgia26 and Pgia8 governed epidemiology of rheumatoid arthritis in Rochester, Minnesota: a study of inci- dence, prevalence, and mortality. Am.J. Epidemiol. 111: 87–98. arthritis susceptibility, and both loci were female-specific (14). 16. Weyand, C. M., D. Schmidt, U. Wagner, and J. J. Goronzy. 1998. The influence When locus-locus interaction was considered, Pgia26 and Pgia8 of sex on the phenotype of rheumatoid arthritis. Arthritis Rheum. 41: 817–822. 17. Gulko, P. S., Y. Kawahito, E. F. Remmers, V. R. Reese, J. P. Wang, each appeared to be a complex multi-chromosomal locus. This http://www.jimmunol.org/ S. V. Dracheva, L. Ge, R. E. Longman, J. S. Shepard, G. W. Cannon, et al. 1998. phenomenon of locus-locus interaction in PG-immunized F2 hy- Identification of a new non-major histocompatibility complex genetic locus on brids (47) was similar to recent findings reported for CIA (48, 49). chromosome 2 that controls disease severity in collagen-induced arthritis in rats. The Cia5/Eae3 locus on mouse chromosome 3 has recently been Arthritis Rheum. 41: 2122–2131. 18. Joe, B., E. F. Remmers, D. E. Dobbins, J. L. Salstrom, T. Furuya, S. Dracheva, separated into three loci (48), and the Eae2 locus on chr15 into P. S. Gulko, G. W. Cannon, M. M. Griffiths, and R. L. Wilder. 2000. Genetic four (Cia26, Cia30, Cia31, and Cia32), when these genomic re- dissection of collagen-induced arthritis in chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influ- gions were analyzed simultaneously on chromosomes 3 and 15 ences. Immunogenetics 51: 930–944. (49). QTL locations were confirmed with congenic strains and ad- 19. Holm, B. C., X. Wei, L. Jacobsson, A. Larsson, H. Luthman, and J. C. Lorentzen. vanced intercross techniques (48, 49). 2001. Rats made congenic for Oia3 on chromosome 10 become susceptible to

squalene-induced arthritis. Hum. Mol. Genet. 10: 565–572. by guest on September 24, 2021 In this study, we addressed a mechanism whereby somatic chro- 20. Furuya, T., J. L. Salstrom, S. McCall-Vining, G. W. Cannon, B. Joe, mosome regions Pgia8 and Pgia9 loci on chr15 function in a gen- E. F. Remmers, M. M. Griffiths, and R. L. Wilder. 2000. Genetic dissection of a der-specific way and affect the susceptibility and progression of rat model for rheumatoid arthritis: significant gender influences on autosomal modifier loci. Hum. Mol. Genet. 9: 2241–2250. PG-induced autoimmune arthritis. Despite the absence of major 21. Joe, B., G. W. Cannon, M. M. Griffiths, D. E. Dobbins, P. S. Gulko, R. L. Wilder, PGIA loci on chr15, the fact is that Pgia8 and Pgia9 loci account and E. F. Remmers. 2002. Evaluation of quantitative trait loci regulating severity for ϳ15% of arthritis variance in BALB/c ϫ DBA/2 cross, con- of mycobacterial adjuvant-induced arthritis in monocongenic and polycongenic rats: identification of a new regulatory locus on rat chromosome 10 and evidence genic strains and chromosome-specific genetic linkage analyses of overlap with rheumatoid arthritis susceptibility loci. Arthritis Rheum. 46: helped us to confirm that these genomic regions indeed carry genes 1075–1085. affecting arthritis susceptibility. 22. Remmers, E. F., B. Joe, M. M. Griffiths, D. E. Dobbins, S. V. Dracheva, A. Hashiramoto, T. Furuya, J. L. Salstrom, J. P. Wang, P. S. Gulko, et al. 2002. Modulation of multiple experimental arthritis models by collagen-induced arthri- Disclosures tis quantitative trait loci isolated in congenic rat lines. Arthritis Rheum. 46: 2225–2234. The authors have no financial conflict of interest. 23. Wilder, R. L., M. M. Griffiths, E. F. Remmers, G. W. Cannon, R. R. 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