Resistance to Xenobiotic-Induced Autoimmunity Maps to 1 Dwight H. Kono, Miyo S. Park, Agnieszka Szydlik, Katarina M. Haraldsson, Jason D. Kuan, Deborah L. Pearson, Per This information is current as Hultman and K. Michael Pollard of September 27, 2021. J Immunol 2001; 167:2396-2403; ; doi: 10.4049/jimmunol.167.4.2396 http://www.jimmunol.org/content/167/4/2396 Downloaded from

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Resistance to Xenobiotic-Induced Autoimmunity Maps to

Dwight H. Kono,2* Miyo S. Park,* Agnieszka Szydlik,* Katarina M. Haraldsson,* Jason D. Kuan,* Deborah L. Pearson,† Per Hultman,‡ and K. Michael Pollard†

Although evidence indicates that environmental factors play a major role in precipitating systemic autoimmunity in genetically susceptible individuals, little is known about the mechanisms involved. Certain heavy metals, such as mercury, are potent envi- ronmental immunostimulants that produce a number of immunopathologic sequelae, including lymphoproliferation, hypergam- maglobulinemia, and overt systemic autoimmunity. Predisposition to such metal-induced immunopathology has been shown to be influenced by both MHC and non-MHC , as well as susceptibility to spontaneous lupus, in mice and other experimental animals. Among the various mouse strains examined to date, the DBA/2 appears to uniquely lack susceptibility to mercury-induced autoimmunity (HgIA), despite expressing a susceptible H-2 haplotype (H-2d). To define the genetic basis for this trait, two Downloaded from -wide scans were conducted using F2 intercrosses of the DBA/2 strain with either the SJL or NZB strains, both of which are highly susceptible to HgIA. A single major quantitative trait on , designated Hmr1, was shown to be common to both crosses and encompassed a region containing several lupus susceptibility loci. Hmr1 was linked to glomerular immune complex deposits and not autoantibody production, suggesting that DBA/2 resistance to HgIA may primarily involve the later stages of disease pathogenesis. Identification and characterization of susceptibility/resistance genes and mechanisms relevant to the immunopathogenesis of mercury-induced autoimmunity should provide important insights into the pathogenesis of auto- http://www.jimmunol.org/ immunity and may reveal novel targets for intervention. The Journal of Immunology, 2001, 167: 2396–2403.

ercury is a widely distributed environmental and in- and susceptibility to immune complex disease have been shown to dustrial pollutant (1). Exposure to large doses results depend on H-2 haplotype, with H-2s and H-2d haplotypes associ- in acute renal tubular lesions and immunosuppression, ated with the greatest susceptibility, H-2k intermediate suscepti- M b whereas chronic administration of smaller doses can lead to the bility, and H-2 the lowest susceptibility (16, 17). However, be- development of systemic autoimmunity (2–4). The characteristic cause many of these comparisons are among different strains of 3

features of mercury-induced autoimmunity (HgIA) are very sim- mice, this classification may have been influenced by background by guest on September 27, 2021 ilar to many of the manifestations of systemic lupus erythemato- genes. The anti-nucleolar Ab (ANoA) response, which is largely sus, and include lymphocyte proliferation (5), increased levels of directed against fibrillarin (18), is one of the best-characterized class II MHC expression (6, 7), hypergammaglobulinemia (8, 9), manifestations of HgIA. It has been linked to H-2s (19) and, more polyclonal Abs to self Ags, including anti-nuclear Abs (8, 10), specifically, to the class II molecule I-As, by analysis of H-2 con- immune complex glomerulonephritis (GN) (10), and necrotizing genic mice (16, 17). The ANoA response is absent in F1 hybrids vasculitis (11). HgIA is also similar to lupus in that the disease s/b ϩ (H-2 ), and recent adoptive bone marrow transfer studies suggest process requires CD4 T cells (12, 13), certain B and T cell co- that this is due to an intrinsic resistance of H-2s/b heterozygous B ␥ stimulatory molecules (14), and IFN- (15), which strongly sup- cells to produce ANoA rather than differences in thymic education ports the possibility of related or identical pathogenic mechanisms. or a lack of antifibrillarin-specific T cell help (20). The development of HgIA depends not only on the amount and There is also considerable evidence implicating non-MHC duration of heavy metal exposure, but also on the genetic back- genes in susceptibility to HgIA. Among H-2d haplotype strains, for ground of the exposed animal. In mice, autoantibody specificities example, BALB/c mice are highly susceptible to both lymphopro- liferation and immune complex GN, B10.D2 mice are susceptible Departments of *Immunology and †Molecular and Experimental Medicine, The to lymphoproliferation, but develop less severe immune complex Scripps Research Institute, La Jolla, CA 92037; and ‡Division of Molecular and GN than BALB/c mice, while DBA/2 mice are reportedly resistant Immunological Pathology, Department of Health and Environment, Linko¨ping Uni- versity, Linko¨ping, Sweden to both lymphoproliferation and GN (16, 17, 21). Other studies Received for publication February 12, 2001. Accepted for publication June 4, 2001. have also recently demonstrated that lupus-prone strains are par- ticularly sensitive to the induction of systemic autoimmunity fol- 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 lowing exposure to mercury (22, 23). Among the various strains, with 18 U.S.C. Section 1734 solely to indicate this fact. the DBA/2 background appears to be the most resistant to HgIA, 1 This is The Scripps Research Institute Publication 13915-IMM. This work was because it is the only background expressing a susceptible H-2 that supported in part by National Institutes of Health Grants ES08666, AR42242, ES07511, and ES08080, and Grant 9543 from the Swedish Medical Research does not develop autoimmunity and, of 22 strains, it alone did not Council. develop hypertrophy of popliteal lymph nodes following HgCl2 2 Address correspondence and reprint requests to Dr. Dwight H. Kono, Department of injection (21). Although the basis for this resistance is not known, Immunology/IMM3, The Scripps Research Institute, 10550 North Torrey Pines Road, DBA/2 mice have reduced in vitro mercury-induced activation of La Jolla, CA 92037. E-mail address: [email protected] CD4ϩ, but not CD8ϩ T cells compared with BALB/c mice (12), 3 Abbreviations used in this paper: HgIA, mercury-induced autoimmunity; ANoA, anti-nucleolar Ab; GN, glomerulonephritis; LR, likelihood ratio; QTL, quantitative suggesting that the initiation of the autoimmune response may be trait locus/loci. compromised. However, this lack of susceptibility to HgIA does

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 2397

not appear to be due to a generalized resistance to autoimmunity, Ͻ1:40 were considered background. Vessel wall IgG deposits were graded ϩ because the DBA/2 strain is highly susceptible to syngeneic graft- ona0–4 scale (15). Slides were examined under blind conditions. vs-host disease induced by cyclosporin A (24) and adoptive trans- Mapping. Construction of linkage maps spanning all autosomal chromo- fer of DBA/2 spleen cells into (C57BL/6 ϫ DBA/2)F mice is a somes and PCR typing of mice were performed as previously described 1 (29). A complete list of polymorphic markers used in this study can be commonly studied model of chronic graft-vs-host disease that in- obtained from the corresponding author. QTL were identified using QTL cludes the development of immune-mediated GN (25, 26). Defi- Cartographer version 1.14 (http://statgen.ncsu.edu/qtlcart) with maps con- nition of the basis for HgIA resistance will undoubtedly provide structed by Mapmaker3 (http://waldo.wi.mit.edu/ftp/distribution/software/ insights into the pathogenesis of HgIA, which will also most likely mapmaker3) (30). Likelihood ratios (LR) were calculated using the LR- mapqtl program. Composite interval mapping was performed using model be relevant to spontaneous lupus. Furthermore, because the DBA/2 6 of the Zmapqtl program with options set at 2-cM intervals, 10-cM win- strain is not known to be immune deficient, this information may dow size, and five background parameters. The experimentwise signifi- also suggest disease interventions that will have minimal effects on cance level for each trait was determined by analyzing 1000 random shuf- the immune response to pathogens. fling permutations of the actual phenotype data. Given the complexity of heavy metal interaction with cellular Statistics and subcellular components of the immune system and the large Data were analyzed by unpaired t test, Fisher’s exact test, or ␹2 (StatView; number of molecules that may be directly or indirectly affected, we Abacus Concepts, Berkeley, CA), as indicated in the legends. Values of p initiated genetic studies to define the DBA/2 genes responsible for Ͻ0.05 were considered significant. resistance to HgIA through a genetics approach. This strategy has the advantage of not requiring previous knowledge of the genes or Results mechanisms, and direct examination of the relationship of genetic Susceptibility of SJL, DBA/2, SDF1, and SDF2 mice to HgIA Downloaded from alterations predisposing to HgIA and spontaneous lupus by com- To identify loci that influence susceptibility to HgIA, the SJL (sus- paring the locations of quantitative trait loci (QTL) and eventually ceptible) and DBA/2 (resistant) strains were selected for analysis. the specific genetic alterations. Genome-wide searches were per- Of particular interest was the identification of DBA/2 genes that formed using two F2 intercrosses involving the resistant DBA/2 to confer resistance to mercury-induced autoimmune disease. Fig. 1 either the SJL or NZB strains, both HgIA susceptible. These stud- and Table I summarize the major autoimmune manifestations elic- http://www.jimmunol.org/ ies identified a single locus on chromosome 1 present in both sets ited in the parental strains, F1 hybrid, and F2 intercrosses following of crosses. Hmr1 is particularly interesting because it confers re- a 4-wk exposure to HgCl2. Consistent with previous observations sistance to HgIA in nonautoimmune (SJL) and autoimmune (NZB) (31, 32), both autoantibody responses and end organ deposits of backgrounds and is derived from a background strain (DBA/2) that immune complexes were markedly reduced or not detectable in the is resistant to HgIA, but otherwise not immune compromised. DBA/2 mice, whereas SJL mice developed typical features of HgIA ( p Ͻ 0.05, Table I).

Materials and Methods The hybrid SDF1 responses were also assessed (Table I and Fig. Mice 1). From the autoantibody profiles, it appeared that both dominant and recessive modes of inheritance were applicable, depending on ϫ by guest on September 27, 2021 DBA/2, SJL/J, NZB/B1Scr, (SJL DBA/2)F1 (SDF1), and SDF2 mice were obtained from The Scripps Research Institute animal facility and were specificity. The lack of ANoA response in SDF1 mice is consistent maintained under specific pathogen-free conditions. Exposure to mercury with dominant transmission of resistance by the DBA/2 back- ␮ consisted of twice per week injections from 6 wk of age with 40 g HgCl2 ground, while the anti- Ab response of equal magnitude in 100 ␮l PBS s.c. for 4 wk, as previously described (15). H-2 haplotypes to the SJL mice suggests recessive transmission. Alternatively, the were determined by the D17Mit16 or Tnf microsatellite markers (Research anti-chromatin Ab and ANoA responses may indicate recessive Genetics, Huntsville, AL); both are located ϳ0.5 cM from the I-A ␤ . and additive dominant transmission of susceptibility genes from Serology the SJL background. Based on these findings, F2 intercrosses were used to map QTLs, because this approach permits analysis of all Antinuclear Abs. Indirect immunofluorescence was performed as de- scribed previously (18). Briefly, HEp-2 cells on slides (Bion Enterprises, combinations of alleles from both parental strains. Fig. 1 and Table Park Ridge, IL) were incubated with 2-fold serial dilutions of serum start- I show the incidence and severity of disease manifestations among ing from 1/100, followed by a 1/100 dilution of FITC-conjugated goat the SDF mice. Average SDF responses were generally between ϩ 2 2 anti-mouse IgG IgM Abs (Caltag Laboratories, Burlingame, CA). Anti- the two parental strains. The incidence of kidney vessel IgG (15 nuclear Ab patterns and titers were assessed under blinded conditions. An- ti-chromatin Abs were measured by ELISA, as previously described (15). mice) and C3 (4 mice) deposits in the SDF2 mice was insufficient Ig levels. Serum IgG1 levels were quantitated as previously described (15, for performing mapping studies. ␬ 27). Briefly, ELISA plate wells were coated with goat anti-mouse Ab Loci linked to HgIA traits in SDF mice (Caltag Laboratories), blocked with 0.1% gelatin in PBS, then incubated 2 with 200 ␮l diluted sera in duplicates. Total IgG1 mouse Abs were detected Genome-wide mapping for autoantibody production and glomer- by the appropriate HRP-conjugated goat anti-mouse IgG or IgG1 Abs ular deposits was performed on 211 SDF2 mice using 96 markers (Caltag Laboratories) and substrate solution. A standard curve consisting of ϳ serial dilutions (400 ng/ml to 3.125 ng/ml) of a polyclonal mouse reference spanning roughly 90% of the autosomal genome ( 1300 cM). The sera (The Binding Site, Birmingham, U.K.) was included on each plate. incidence of vessel deposits was too low in the SDF2 mice for Serum IgG2a levels were similarly quantitated with the following modifi- analysis. Three QTL linked to mercury-induced traits were iden- cations. Plates were coated with rat anti-mouse Ig ␬ (PharMingen, San tified on 1, 7, and 17. The locus on , Diego, CA), and reagents that detected both the a and b allotypes of IgG2a defined by the Tnf marker within the H-2 complex, was linked very were used throughout the assay, because some mice had the IgHa allotype from 129 strain (embryonic stem cell derived). Standard curves for IgG2a tightly to ANoA with a LR of 96, a value substantially higher than were generated by serial dilutions of purified mouse IgG2a of both allo- the genome-wide calculated significance level of 12.4, for ␣ ϭ types (PharMingen). 0.05 (experimentwise significance level was determined by ana- Immunohistology. Sections of kidney and spleen were stained for direct lyzing, for each trait, 1000 permutations of the actual data after immunofluorescence, as previously described (28). Briefly, 4-␮m cryostat random shuffling). In fact, none of the H-2d, and only a small sections were fixed with ethanol, then incubated with serial dilutions of s/d FITC-conjugated goat anti-mouse IgG Abs (Southern Biotechnology, Bir- fraction (5.6%) of H-2 heterozygous SDF2 mice had ANoAs mingham, AL). Glomerular deposits were graded by the highest dilution of (Table II), consistent with previous studies suggesting that anti- Ab that could detect a specific fluorescence (end-point titer). Positive titers fibrillarin Ab production requires the I-As haplotype (17, 20), or 2398 QTL CONFERRING RESISTANCE TO HgIA

FIGURE 1. Manifestations of HgIA among

SJL, DBA/2, SDF1, and SDF2 mice. Column scat- tergraphs of anti-nucleolar and anti-chromatin Ab levels, and glomerular IgG, IgM, or C3 deposits Downloaded from are shown. See Table I for a summary of the re- sults and additional details. http://www.jimmunol.org/ by guest on September 27, 2021

possibly, that the H-2d haplotype suppresses the response. In ad- Hmr1 using composite interval mapping did not quite reach ge- dition, the tight linkage of ANoA to the H-2s haplotype despite the nome-wide significance (16.1 and 16.7, respectively); therefore, a diverse F2 backgrounds indicates that production of ANoA is un- second set of mice was analyzed in an attempt to verify this locus. likely to be greatly influenced by non-MHC DBA/2 genes. Fur- thermore, the H-2 (Tnf) region was not linked to glomerular de- posits ( p Ͼ 0.05), nor were there significant differences in Mercury susceptibility of NZB mice and crosses glomerular deposits of IgG and C3 in mice with or without ANoAs For the second mapping study, DBA/2 mice were crossed with or anti-chromatin Abs ( p Ͼ 0.05, data not shown). The H-2 region NZB mice partly based on our recent finding that lupus-susceptible was also linked to IgG anti-chromatin Ab production (LR 12.8, strains are more sensitive to HgIA (22, 23), and the possibility that experimentwise significance level ␣ ϭ 0.05 at LR of 12.4). if Hmr1 or other DBA/2 gene (or genes) is capable of suppressing In contrast to the chromosome 17 locus, the other two QTL disease in these strains, they are more likely to be significant and identified were linked solely to later stages of disease pathogene- possibly relevant to the pathogenesis of spontaneous disease. NZB sis. The chromosome 1 locus was linked to glomerular deposits of mice were specifically selected because they are the only lupus- both IgG (D1 Mit15, LR 10.4, genome-wide permutation signifi- prone strain expressing the same H-2d haplotype as the DBA/2. cance level ␣ ϭ 0.05 at LR of 13.1, nominal p value ϭ 0.001) and Furthermore, NZB susceptibility loci promoting spontaneous dis- C3 (D1Nds1, LR 16.2, ␣ ϭ 0.05 at LR of 12.5), whereas the ease have been mapped by us (29) and others (33–36). acromeric QTL was weakly linked only to IgG In preliminary experiments, female NZB, DBA/2, and (NZB ϫ ␣ ϭ glomerular deposits (D7 Mit57, LR 9.4, 0.05 at LR of 13.1, DBA/2)F1 (BDF1) mice were exposed to the standard regimen of ϭ nominal p value 0.002). In all instances, resistance to disease HgCl2 at an age before the development of spontaneous disease (6 mapped to the DBA/2 genome. To better define the location of wk), and then examined for immunopathology (Table III and Fig. these QTL, composite interval mapping was then performed, as 3). NZB mice developed significantly greater amounts of IgG and shown in Fig. 2. This method combines interval mapping and mul- C3 deposits in glomeruli and vessels ( p Ͻ 0.05) than both the tiple regression to adjust for the presence of other QTL. The chro- DBA/2 mice and control NZB mice given PBS alone (NZB-PBS). mosome 1 locus, designated Hmr1 (heavy metal resistance 1), was Thus, similar to other autoimmune strains, NZB mice appear mapped to the middle-distal portion of the chromosome and the highly susceptible to HgIA. In contrast, BDF1 hybrids exhibited chromosome 7 locus to the proximal region. However, the LR of low susceptibility to mercury with little or no vessel deposits and The Journal of Immunology 2399

Table I. Manifestations of HgIA in SJL, DBA/2, SDF1, and SDF2 micea

Mice ANoA ANoA score Anti-Chromatin Glom IgG Glom IgM Glom C3 Kid Ves IgG

DBA/2 0% 0 0.005 Ϯ 0.002 0 320 Ϯ 56 80 Ϯ 14 0 SJL 90% 1.7 Ϯ 0.3 0.264 Ϯ 0.148 180 Ϯ 87 620 Ϯ 166 190 Ϯ 32 160 Ϯ 84 SDF1 0 0 0.296 Ϯ 0.116 ND ND ND ND SDF2 23% 0.5 Ϯ 0.1 0.035 Ϯ 0.010 69 Ϯ 13 501 Ϯ 23 226 Ϯ 13 33 Ϯ 12 pb 0.0002 0.0001 0.04 0.02 0.04 0.002 0.03

a Phenotypes examined were ANoA positivity (ANoA titers Ն ϩ1) and score, anti-chromatin Abs, and immunofluorescence examination of kidney (Kid) for glomerular (Glom) and vessel (Ves) deposits. Serum anti-nuclear Abs were detected on HEp-2 cells by indirect immunofluorescence, autoAb levels were determined by ELISA, and endpoint titers for deposits were obtained by scoring immunofluorescence-stained cryostat sections (15). Mean Ϯ SE are shown except for ANoA (percent positive). Deposit scores are inverse titers. b Values of p for comparisons between DBA/2 and SJL mice are shown. The unpaired t test was used for all phenotypes except for ANoA (Fisher’s exact test). Number of mice: 8 DBA/2, 8–20 SJL, 26 SDF1, 210–211 SDF2. Among SDF2 mice, there was only low incidence of IgG (15 mice) and C3 (4 mice) deposits in kidney vessels. intermediate amounts of glomerular IgG material (Table III and mosome 7, but also to . Thus, resistance of the Fig. 3), suggesting dominant transmission of DBA/2 resistance. DBA/2 appears to be multigenic and largely due to the effects of Hmr1 and, to a lesser extent, to a locus on chromosome 7. Loci modifying susceptibility/resistance to HgIA in BDF2 Interestingly, Hmr1 overlaps a previously identified locus on the Downloaded from intercross mice NZB background, Lbw7 or nba2, that has been linked to the spon-

For mapping of mercury-related QTLs, 282 BDF2 mice were ex- taneous development of autoantibodies, splenomegaly, and/or GN amined for susceptibility to HgIA. The distributions of individual in several different crosses of the NZB with nonautoimmune and mice for glomerular and vessel deposits, and levels of autoanti- autoimmune background strains (29, 34, 35, 38). However, map- bodies are shown in Fig. 3, and the results are summarized in Table ping studies have suggested that the major contribution of Lbw7 or III. Genome-wide analysis was performed with 98 markers encom- nba2 is enhancement of autoantibody production, while Hmr1 was passing all autosomal chromosomes and ϳ90% coverage, includ- not linked to this trait in either cross. Nevertheless, in the case of http://www.jimmunol.org/ ing all regions previously linked to spontaneous lupus in the NZB the BDF2 cross, linkage to chromosome 1 may be partly due to the strain (29, 37). Glomerular IgG deposits mapped very strongly to presence of the NZB Lbw7 allele if, in fact, this allele can promote chromosome 1 (D1 Mit111, LR 33.7, genome-wide permutation susceptibility to HgIA. Further dissection of this region with in- significance level ␣ ϭ 0.05 at LR of 17.1). Similar to Hmr1, re- terval-specific congenic sublines will address this issue. sistance to autoimmunity mapped to the DBA/2 genome (mean Ϯ The Hmr1 interval on chromosome 1 is particularly interesting, Ϯ Ϯ Ϯ SE for DBA/2 384 58, NZB 1093 95, BDF1 695 60). No because it overlaps with other lupus-predisposing loci in addition other QTLs were identified that were at the ␣ ϭ 0.05 level for to the NZB locus mentioned previously, as well as with several

genome-wide significance. Composite interval mapping demon- genes that, when altered, either enhance or reduce susceptibility to by guest on September 27, 2021 strated that the NZB chromosome 1 locus (ϳ80–101.2 cM, Mouse lupus-like disease. The other predisposing loci include Sle1a-c Genome Informatics; The Jackson Laboratory, Bar Harbor, ME) from the NZM/Aeg2410 and NZW backgrounds (39–41) and overlapped with Hmr1 (ϳ66.8–93.2 cM) (Fig. 4). Composite in- Bxs3 from the BXSB strain (42). Strikingly, all of the highly sus- terval mapping also revealed an additional locus on chromosome 4 ceptible lupus-prone strains except for the MRL-Faslpr have loci (LR 18.2, Fig. 4) linked to HgIA resistance in the DBA/2 back- that overlap with the Hmr1 interval. Genes associated with pre- Ϯ Ϯ Ϯ ground (DBA/2 483 124, NZB 862 111, BDF1 623 68). disposition to the development of lupus include Fasl () The proximal chromosome 7 region was also weakly linked to glo- (43), Sap (serum amyloid P-component) (44), Fcgr2b (Fc␥RIIB) merular IgG deposits (D7 Mit178, LR 8.1, nominal p ϭ 0.005, Fig. 4). (45), Cr2 (CD21/CD35) (46, 47), and Ptprc (CD45) (48), whereas Mapping of the other traits did not identify any other QTL that deficiency of Fcer1g (FcR ␥-chain) (49) results in resistance to reached genome-wide significance by composite interval mapping. Furthermore, autoantibody responses were not linked to the above identified loci on chromosomes 1, 4, and 7 (nominal p Ͼ 0.05).

Discussion In this study, genome-wide mapping of two independent crosses was used to define the genetic resistance of DBA/2 mice to HgIA.

In the SDF2 cross, glomerular deposits of IgG and/or complement mapped to chromosome 1 (designated Hmr1), and more weakly to proximal chromosome 7. In the BDF2 cross, glomerular deposits of IgG similarly mapped to chromosome 1 and weakly to chro-

Table II. Influence of H-2 haplotype on HgIA FIGURE 2. Loci linked to glomerular deposits in SDF2 mice. Compos- ite interval maps of LR test (LRT) results are shown for glomerular IgG (solid line) and C3 (dashed line) deposits. Horizontal lines showing ex- MHC No. Mice ANoAa Glom. IgG Glom. C3 perimentwise significance levels for ␣ ϭ 0.1 and 0.05 were 15.3 (thin, H-2s 51 82.4% 29.4% 35.3% dashed) and 17.7 (thin, solid) for IgG deposits, and 14.6 (thick, dashed) and H-2d 52 0% 19.2% 44.2% 16.7 for C3 deposits. Markers for chromosome 1 (from acromere: D1 H-2s/d 108 5.6% 23.1% 38.0% Mit66, D1 Mit212, D1 Mit306, D1Nds1, D1 Mit265, D1 Mit15, D1 Mit36, D1 Mit17) and chromosome 7 (D7 Mit57, D7 Mit23, D7 Mit227, D7 a Percentage of mice with ANoA scores Ն 1ϩ are shown. Three H-2s, three H-2s/d, and no H-2d haplotype mice had trace positive ANoA scores. p Ͻ 0.0001 for Mit211, D7Nds1, D7 Mit323, D7 Mit134) are indicated by solid triangles. ANoA; p Ͼ 0.05 for glomerular IgG and C3 deposits (␹2 test). D1 Mit15 and D1 Mit36 are closely situated and appear as a single triangle. 2400 QTL CONFERRING RESISTANCE TO HgIA

Table III. Immunopathology of NZB, DBA/2, BDF1, and BDF2 micea

Mice Glom IgG Glom C3 Kid Ves IgG Kid Ves C3

DBA/2 0 80 Ϯ 14 0 0 NZB 1760 Ϯ 234 660 Ϯ 183 2.5 Ϯ 0.19 2.6 Ϯ 0.18 BDF1 560 Ϯ 193 93 Ϯ 41 0 0 BDF2 603 Ϯ 40 951 Ϯ 41 0.52 Ϯ 0.06 0.14 Ϯ 0.03 NZBpbs 120 Ϯ 56 194 Ϯ 46 0 0 pb Ͻ0.001 0.0068 Ͻ0.001 Ͻ0.001

a Mean Ϯ SE are shown. Same set of DBA/2 mice from Table I are included. Deposit results for glomeruli are inverse titers and vessel deposits are scored by intensity and degree of involvement (0–3 scale). b Values of p for unpaired t test comparisons of the DBA/2 and SJL mice are shown. Number of mice: 7–8 DBA/2, 8 NZB (HgCl2-exposed), 7 NZBpbs (PBS controls), 12 BDF1, 237–282 BDF2. Eight-week-old female mice were injected s.c. with 40 ␮ ␮ g HgCl2 in 100 l sterile PBS twice a week. After 1 mo, mice were sacrificed, and kidney sections were examined for glomerular and vessel deposits for IgG and C3 by direct immunofluorescence.

autoimmunity. Although the relationship of Hmr1 to these loci and overlaps the NZW susceptibility locus, Sle2 or Lbw5 (29, 39) and genes remains to be determined, this study appears to add yet the CD22 gene. Gene knockout of CD22 has been associated with another gene to a region that is particularly rich in lupus-affecting lupus-like disease (54–56), and it has been recently suggested that genes. The concentration of such genes in this interval may ac- the CD22a allele may predispose to autoimmunity because of an Downloaded from count for some of the linkage of systemic lupus erythematosus insertion of a B1 repetitive element in the second intron that leads traits to homologous regions on human chromosome 1 in diverse to alternative splicing of the 5Ј untranslated region (exons 1–3) and populations (50–53). reduced levels of CD22 in unstimulated and LPS-stimulated B The other possible DBA/2 resistance locus on chromosome 7, cells (57). However, it is unlikely that the CD22a allele is respon- which was identified in both crosses, encompasses a region that sible for the DBA/2 resistance, because both DBA/2 and NZB http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Manifestations of HgIA among

NZB, DBA/2, BDF1, and BDF2 mice. Column scattergraphs of glomerular and kidney vessel deposits of IgG and C3 are shown in the top four panels. See Table III for a summary of results and additional details. Autoantibody levels (bot- tom panel) show the distribution of individual

animal responses for the DBF2 mice only. CG, IgG anti-chromatin Ab; CM, IgM anti-chromatin Ab; SG, IgG ssDNA Ab; SM, IgM ssDNA Ab (OD units are in log scale). The Journal of Immunology 2401

FIGURE 4. Loci linked to glomerular deposits in BDF2 mice. Composite interval maps for glomerular IgG are shown. Experimentwise significance levels were 15.2 for ␣ ϭ 0.1 (dashed horizontal line), 17.1 for ␣ ϭ 0.05 (solid horizontal line), and 23.6 for ␣ ϭ 0.01. Markers for chromosome 1 (from acromere: D1 Mit296, D1 Mit236, D1 Mit132, D1 Mit387, D1 Mit200, D1 Mit105, D1 Mit507, D1 Mit15, D1 Mit111, D1 Mit36, D1 Mit406, D1 Mit17) and chromosome 4 (D4 Mit18, D4 Mit272, D4 Mit142, D4 Mit175, D4 Mit203, D4 Mit70, D4 Mit54, D4 Mit251, D4 Mit256) are indicated by solid triangles. Downloaded from mice express the CD22a allele, while SJL mice express the normal tion of autoantibodies, which would be consistent with our map- CD22b allele (58). ping studies. This study demonstrates that NZB mice are highly sensitive to Mercury is only one of a number of immunostimulatory heavy HgIA, similar to observations in most lupus strains or crosses, metal xenobiotics that can induce adverse immunotoxicity. Several ϩ/ϩ including BXSB, MRL , and BWF1 (22, 23). Furthermore, the of these, such as silver (31, 63, 64) or gold (65, 66), also promote s two significant loci identified in the BDF2 cross-mapped to previ- the production of anti-fibrillarin autoantibodies in H-2 haplotype http://www.jimmunol.org/ ously identified NZB lupus susceptibility loci on chromosomes 1 mice. Interestingly, the DBA/2 strain is also resistant to both sil- (Lbw7 and nba2)and4(nba1, Sle2, and Lbw2) (29, 39, 59–61), ver- and gold-induced autoimmunity. Thus, findings related to although no linkage was observed for three other NZB loci linked Hmr1 resistance to mercury-induced disease may be more broadly to lupus traits on chromosomes 6 (29), 11 (29, 35), and 13 (34). applicable to other xenobiotic agents. This suggests that mercury may synergize with some, but not all, Predisposition to the adverse effects of environmental agents loci predisposing to spontaneous lupus. In contrast to the chromo- may be influenced by genetic variations in the population that af- some 1 locus (Hmr1), none of the traits in the SDF2 cross-mapped fect both susceptibility and resistance. Herein, we have identified to the chromosome 4 interval (nominal p Ͼ 0.05). Thus, linkage to a locus, Hmr1, that appears to contribute to the resistance of by guest on September 27, 2021 chromosome 4 in the BDF2 cross is more likely due to the NZB DBA/2 mice to HgIA and that maps to a region on chromosome 1 susceptibility locus (nba1, Sle2, and Lbw2) rather than a resistance implicated in susceptibility to spontaneous lupus. Definition of ge- locus from the DBA/2 strain. Another interesting finding was the netic alteration and mechanisms responsible for the Hmr1 pheno- much stronger linkage of the chromosome 1 region (Lbw7)to type should provide new insights into the relationship of environ- HgIA than the chromosome 4 interval (Lbw2), which contrasts mental and genetic susceptibility in autoimmune diseases. with our previous observation in BWF2 crosses that the Lbw2 lo- cus was much more strongly linked to GN (29). A possible expla- nation for the major effect of the chromosome 1 interval in the Acknowledgments BDF cross may be a combined effect of the Hmr1 resistance locus We thank Aiko Lawson for technical help and Kat Occhipinti for editorial 2 assistance. from the DBA/2 and the NZB susceptibility locus.

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