Mercury and silver induce B cell activation and anti-nucleolar autoantibody production in outbred mouse stocks: are environmental factors more important than the susceptibility genes in connection with autoimmunity?

Abstract

Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury- and silver-induced B cell activation and anti-nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody-secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H-2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long-term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.

Introduction

Systemic autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, systemic sclerosis and Sjögren's disease occur in up to 1–1·5% of the general population and cause considerable morbidity and disabilities [1,2]. The underlying aetiological factors involved in the initiation and development of these diseases are mainly unknown. However, it has been proposed that predisposing genetic factors in combination with environmental risk factors contribute to the development of systemic autoimmune diseases [3,4]. Although it is not known how and to what extent these risk factors influence the development of these diseases, it has been suggested that both genetic and environmental factors affect the susceptibility to autoimmune diseases at three levels, including general reactivity of the immune system, specific antigen presentation and recognition and target tissue restriction [4]. This suggestion is indeed supported by the results of experimental studies including my own. For instance, it has been shown that chronic exposure to subtoxic doses of widely spread environmental pollutants, mercurial and silver compounds [5–7] induces a systemic type of autoimmunity in rodents [8–16]. In susceptible mice, mercury-induced autoimmunity is characterized by a potent CD4⁺ T cell-dependent polyclonal activation of B cells, increased serum levels of immunoglobulin (Ig)G1 and IgE, production of autoantibodies of different specificities, particularly anti-nucleolar autoantibodies (ANolA) and the formation of immune complex deposits in the kidney [5,9,10]. Similar to mercury, silver also induces a CD4⁺ T cell-dependent B cell activation, which leads to the production of high levels of ANolA in susceptible mice [8,11,13,16]. However, despite being as effective as mercury in induction of ANolA production, silver induces a weak activation of the immune system [13,16].

The results from the studies in the inbred mouse strains have demonstrated clearly that susceptibility to both mercury- and silver-induced autoimmune manifestations is controlled genetically [11,13–20]. For instance, genes within H-2 loci are shown to control the susceptibility to ANolA production, i.e. only mouse strains of H-2^s and H-2^q genotypes, irrespective of their background genes, produced ANolA after treatment with mercury or silver [11,14,16,18]. By using intra-H-2 recombinant mouse strains, susceptibility to mercury-induced ANolA production could be mapped to the I-A loci of H-2 class II genes [19]. Furthermore, non-H-2 genes could largely influence the severity of B cell activation and the magnitude of the production of ANolA caused by exposure to mercury and silver [11,14,18,20]. In connection with this statement, results of my own study have shown that among 15 inbred mouse strains harbouring seven different H-2 genotypes, only the DBA/2 (H-2^d) strain was fully resistant to mercury-induced autoimmunity [14]. In contrast, all other strains were found to be mercury-susceptible, i.e. each of them was able to develop at least one characteristic of mercury-induced autoimmune manifestations [14]. On the other hand, it has been demonstrated that, in a non-H-2 dependent manner, exposure to mercury exacerbates the disease process in mouse strains, which are genetically are prone to develop SLE-like disease [12,21,22]. On the basis of these findings and by considering the concept that environmental factors play a crucial role in the initiation and exacerbation of autoimmune diseases, it is assumable that intensive and prolonged exposure of genetically heterogeneous populations to potent environmental insults can activate the immune system to produce autoimmunity without requiring any specific autoimmune susceptible genes. To test this assumption, in the present study I employed three different outbred mouse stocks and assessed the induction of B cell activation and ANolA production by mercury and silver in these animals. I found that a large proportion of the tested animals exhibited a strong B cell activation and ANolA production in response to these xenobiotic metals. These results suggest that certain environmental factors do not necessarily require the contribution of the susceptibility genes for the activation of the immune system to produce autoimmune reactions.

Materials and methods

Animals

Female outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice (6–8 weeks old at the beginning of each experiment) were obtained from Olac Harlan (Bicester, UK), Scanbur (Sollentuna, Sweden) and Taconic (Ry, Denmark) respectively. The animals were housed in the animal facilities at the Wenner-Gren Institute, Stockholm University, with a 12-h dark/12-h light cycle and access to tap water and standard chow (R70 containing 4·5% fat, 14·5% protein and 60·1% carbohydrate (Lantmännen, Stockholm, Sweden) ad libitum. The experiments described here were pre-approved by the Northern Stockholm Ethical Committee for Animal Experimentation (N138/98).

HgCl₂ and AgNO₃ treatment

A solution of 0·4 mg/ml HgCl₂ (analytical grade; Merck, Darmstadt, Germany) was prepared in sterile 0·9% NaCl solution. A solution of 0·573 mg/ml AgNO₃ (analytical grade; Merck) was prepared in sterile water. Groups of ICR, NMRI and Black Swiss outbred mice (five to 20 mice/group as indicated in the figures or figure legends) were injected subcutaneously (s.c.) with either 0·1 ml of HgCl₂ (1·6 mg/kg body weight) or AgNO₃ (2·5 mg/kg body weight) solutions every third day for 4 weeks. Control groups received 0·1 ml of sterile 0·9% NaCl by s.c. route.

Collection of blood, spleens and preparation of serum and cell suspensions

Following the 4-week treatment period, the mice were bled by retro-orbital puncture under light isofluorane anaesthesia and thereafter killed by cervical dislocation and their spleens dissected out aseptically. The blood samples were allowed to clot at 4°C and then centrifuged to obtain the serum, which was stored at −20°C until being assayed for antibody/autoantibody levels. The spleens were teased apart with forceps in Earle's balanced salt solution in order to obtain single cell suspensions, which were washed three times and resuspended in 5 ml of this same solution for performance of the protein A plaque assay.

The protein A plaque assay

The numbers of splenic cells secreting antibodies belonging to different Ig classes and subclasses were quantified utilizing the protein A plaque assay described by Gronowicz et al.[23], employing rabbit anti-mouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC, USA) and IgG2b (Nordic Immunological Laboratories, Tillburg, the Netherlands) as the developing reagents.

Quantification of mouse IgE by enzyme-linked immunosorbent assay

Total serum levels of IgE were determined with a sandwich enzyme-linked immunosorbent assay (ELISA) procedure, as described previously [12]. A rat anti-mouse IgE monoclonal antibody (mAb) (R35–72; Pharmingen, San Diego, CA, USA) was utilized as the ‘capture’ antibody and a biotinylated rat anti-mouse IgE mAb (R35–92; Pharmingen) as the ‘detection’ antibody. The concentrations of IgE in the sera were determined by comparison with a calibration curve generated using an internal standard (Pharmingen).

Detection of ANolA by indirect immunofluorescence

The levels of IgG1- and IgG2a-type ANolAs in serum samples were determined employing indirect immunofluorescence. For this purpose, HEp-2 cells grown as monolayers on slides (Immuno Concepts, Sacramento, CA, USA) served as the substrate and fluorescein isothiocyanate-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotechnology, Birmingham, AL, USA) as the visualizing antibodies. The pattern and titres of different ANolAs were assessed subsequently under a Reichard–Jung Polyvar microscope (Vienna, Austria), with serum samples that exhibited no specific green fluorescence at a dilution of 1:50 being assigned a value of zero. The highest dilution at which nucleolar fluorescence could still be detected was defined as the titre of the IgG1 and/or IgG2a ANolA.

Expression and statistical analysis of the data

The numbers of splenic cells secreting IgM, IgG1, IgG2b and IgG3 antibodies, serum levels of IgE and the titres of IgG1 and IgG2a ANolA are all expressed as means ± standard error. Differences between these parameters for the control and treated groups were analysed for statistical significance using analysis of variance. Differences between IgG1 and IgG2a ANolA in mercury- and silver-treated animals were analysed for statistical significance employing the Mann–Whitney U-test. All statistical analyses were performed utilizing WinSTAT software (R. Fitch Software, Medina AB, Vänerborg, Sweden).

Results

Mercury and silver induce a potent B cell activation and antibody formation in outbred ICR, NMRI and Black Swiss mice

I first evaluated whether xenobiotic heavy metals, mercury and silver were able to activate the immune system in genetically heterogeneous mouse populations. Groups of young ICR, NMRI and Black Swiss mice (five to 35 mice per group, as indicated Table 1) were treated with subtoxic doses of HgCl₂ and AgNO₃ for 4 weeks. Control mice received sterile saline. As a parameter for mercury- and silver-induced immune activation, I first determined the Ig production of different isotypes in the treated and control animals by employing the protein A plaque assay. As shown in Table 1, the three tested outbred mouse stocks were able to respond to mercury and/or silver by exhibiting an increase in the numbers of splenic antibody-secreting cells of at least one or more Ig isotype(s). For instance, in ICR mice, while treatment with mercury induced a significant increase in the numbers of splenic IgG1, IgG2b and IgG3 antibody-secreting cells, treatment with silver enhanced the production of IgG1 in these animals (Table 1). In NMRI mice, treatment with mercury increased significantly the numbers of splenic IgM and IgG1 antibody-secreting cells, whereas these animals produced elevated numbers of IgG1, IgG2b and IgG3 antibody-secreting cells in response to silver (Table 1). On the other hand, Black Swiss mice responded to mercury by producing high numbers of IgM, IgG1 and IgG2b antibody-secreting cells, and in response to silver they showed a significant increase in the production of IgG1 and IgG3 (Table 1). Of particular note is that all the tested outbred mouse stocks, irrespective of their origins, exhibited an increase in IgG1 production in response to both mercury and silver (Table 1). However, in all cases the magnitude of IgG1 synthesis induced by silver was lower than that caused by mercury (Table 1). Unfortunately, although it was highly informative to determine if mercury and silver can also affect the production of IgG2a in the tested outbred mice, due to unavailability of a suitable developing antibody (anti-mouse IgG2a) for the protein A plaque assay, I was not able to enumerate the numbers of splenic IgG2a antibody-secreting cells in these animals. However, in order to verify the role of IgG2a response in heavy metal-induced autoimmunity in outbred mice, I determined instead the serum levels of IgG2a ANolA measured in mercury- and silver-treated animals (see below).

Table 1.

Mercury and silver induce B cell activation of different isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice.^a

Mouse stock	No. of mice	Treatment	IgM PFC	IgG1 PFC	IgG2b PFC	IgG3 PFC
ICR
	10 (6)b	Saline	64 100 ± 7 900	410 ± 50	380 ± 100	560 ± 190
	35	HgCl2	85 700 ± 8 200	12 000 ± 3 700**	900 ± 160**	1 850 ± 520*
	5	AgNO3	73 900 ± 11 100	750 ± 80*	250 ± 110	204 ± 70
NMRI
	10 (7)	Saline	50 800 ± 6 400	1 400 ± 360	1830 ± 490	2 880 ± 560
	30	HgCl2	90 000 ± 1 100**	8 800 ± 2 600**	2430 ± 620	2 260 ± 610
	12	AgNO3	46 500 ± 5 600	8 400 ± 1 700**	8230 ± 1830***	10 100 ± 3 940*
Black Swiss
	5 (5)	Saline	48 800 ± 3 900	1 600 ± 340	920 ± 340	960 ± 200
	11	HgCl2	89 200 ± 5 500***	13 900 ± 2 100***	2130 ± 430*	640 ± 150
	12	AgNO3	64 800 ± 6 200	4 900 ± 530*	1580 ± 310	4 150 ± 1 600*

^*P < 0·05

^**P < 0·01 and

^***P < 0·001.

^aIn separate experiments, groups of female ICR, NMRI and Black Swiss outbred mice were injected repeatedly subcutaneously (s.c.) with HgCl₂ or AgNO₃ and/or NaCl (saline) for 4 weeks. At the end of each experiment, the spleens were tested for immunoglobulin (Ig)M, IgG1, IgG2b and IgG3 antibody-secreting cells (plaque-forming cells) by employing a protein A plaque assay. Significant differences between the parameters in mercury-, silver- and saline-injected mice were calculated by analysis of variance test.

^bThe number of control animals used along with AgNO₃-treated animals.

Mercury, but not silver enhances the production of IgE in outbred ICR, NMRI and Black Swiss mice

The enhanced total serum IgE levels have been considered as one of the major characteristics of mercury-induced autoimmunity in genetically susceptible mice [24,25]. Treatment with silver has also been shown to increase the serum levels of IgE in some, but not all mercury susceptible mouse strains [13]. Thus, in order to elucidate if treatment with these xenobiotic metals can also increase the production of IgE in outbred mice, I next measured the levels of IgE in the sera of mercury- and silver-treated and untreated ICR, NMRI and Black Swiss mice employing an ELISA technique. As shown in Fig. 1a–c, in response to mercury all the tested outbred mouse stocks exhibited a marked increase in the serum IgE levels. In contrast, none of the silver-treated stocks showed any significant change in the serum IgE levels (Fig. 1a–c).

Fig. 1.

Mercury, but not silver, induces an increase in IgE production in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice. Groups of female ICR (a), NMRI (b) and Black Swiss (c) outbred mice were injected repeatedly subcutaneously (s.c.) with HgCl₂ (solid bars) or AgNO₃ (grey bars) and/or NaCl (saline) (open bars) for 4 weeks, as described in the footnote for Table 1. At the end of each experiment the mice were bled and killed. The sera were tested for total immunoglobulin (Ig)E concentration using an enzyme-linked imunosorbent assay method. The data are shown as the mean values for the serum IgE concentrations + 1 standard error. Significant differences between the serum IgE concentrations in mercury-, silver- and saline-injected mice were calculated by analysis of variance test. ***P < 0·001.

Induction of ANolA production in outbred ICR, NMRI and Black Swiss mice by mercury and silver

The main hallmark of mercury- and silver-induced autoimmunity in genetically susceptible mice is the production of ANolA [8,11,13,14,16]. ANolA react with fibrillarin, a 34 kDa protein, which is associated with the U3, U8, U13, U14, X and Y small nucleolar RNAs in vertebrates [26]. Interestingly, ANolA with anti-fibrillarin specificity have also been detected in a subset of patients with systemic scleroderma [27]. Because several studies have demonstrated that only mouse strains with certain H-2 genotypes produced ANolA after treatment with mercury and silver [11,14,18,20], I next performed experiments to test if these xenobiotic metals could induce ANolA production in apparently H-2 heterozygous mouse populations. Sera of control, mercury- and silver-treated ICR, NMRI and Swiss Black mice were analysed for the presence of IgG1 and IgG2a ANolA, which could detect the nucleoli in a clumpy pattern, and with several nuclear dots in the nucleoplasm by employing indirect immunofluorescence. As demonstrated in Fig. 2a and b, 28 days‘ treatment with mercury induced the production of both IgG1 and IgG2a ANolA of variable titres in 88% (31 of 35) of ICR, 96% (29 of 30) of NMRI and 100% (11 of 11) of Black Swiss mice. The production of mercury-induced ANolA in ICR was dominated by the IgG2a isotype (P < 0·05), whereas NMRI and Black Swiss mice did not show any isotype preferences (P = 0·09 and P = 0·06 respectively) for the production of ANolA in response to mercury (Fig. 2a and b).

Fig. 2.

Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with mercuric chloride (HgCl₂). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with HgCl₂ (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of IgG1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of mercury-treated animals, which produce ANolA.

Similar to mercury treatment, 28 days‘ treatment with silver also induced ANolA production of both IgG1 and IgG2a isotypes in 60% (three of five) of ICR, 75% (nine of 12) of NMRI and 100% (12 of 12) of Black Swiss mice (Fig. 3a and b). However, in all tested outbred stocks, the magnitudes of silver-induced ANolA responses (Fig. 3a and b) were lower than those induced by the treatment with mercury (Fig. 2a and b). Moreover, while the magnitudes of silver-induced IgG1 and IgG2a ANolA responses were comparable in ICR and NMRI mice (P = 0·45 and P = 0·18 respectively), in Black Swiss mice silver induced a dominant IgG1 ANolA response (P < 0·01) (Fig. 3a and b).

Fig. 3.

Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with silver nitrate (AgNO₃). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with AgNO₃ (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of immunoglobulin (Ig)G1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of silver-treated animals, which produce ANolA.

Discussion

The present study was designed to test the assumption that potent environmental insults can activate the immune system to produce autoimmunity per se without requiring any specific autoimmune susceptible genes. Thus, I assessed the induction of autoimmunity by xenobiotic metals, mercury and silver (as representative of environmental factors) in different genetically heterogeneous outbred mouse stocks, including ICR, NMRI and Black Swiss. Like many other outbred mouse stocks, these stocks are originated from two male and seven female mice known as ‘Swiss mice’, which were imported to the United States and bred by Clara J. Lynch at the Rockefeller Institute for Medical Research in New York in 1926 [28]. Thus, ICR, NMRI and Black Swiss mice are related, but they were separated during the late 1930s [28]. Moreover, analysis of genetic variation in outbred Swiss mice of different stocks has demonstrated that the heterozygocity of each stock is very similar to estimates for feral mouse and human populations [29,30]. Hence, ICR, NMRI and Black Swiss mouse stocks can indeed be considered as genetically heterogeneous populations.

My first observation was that all tested outbred mouse stocks, irrespective of their origins, were able to respond to both mercury and silver by exhibiting a significant increase in the splenic antibody-secreting cells of different isotypes. This finding suggests that, at least, the initial activation of the immune system, which is a prerequisite for the development of autoimmunity, is determined to a large extent by the nature (e.g. biological, biochemical and immunochemical properties) of the environmental factor, but not the genetic make-up of the host. In fact, this suggestion is supported by my previous finding that mercury induced immune/autoimmune activation in 14 of 15 inbred mouse strains of different H-2 genotypes [14]. This suggestion is also consistent with the observations that treatment with mercury, but not silver, induced a substantial increase in the production of IgE in all tested outbred mouse stocks (this study) and that silver is a weaker activator of the immune system compared with mercury [this study and 13,16].

As mentioned previously, studies on the genetic susceptibility of inbred mouse strains to mercury- and silver-induced autoimmunity have revealed that susceptibility to the development of ANolA production is controlled strictly by the class II of H-2 genes, i.e. only certain mouse strains with specific H-2 genotypes (e.g. H-2^s and H-2^q) produce ANolA upon exposure to mercury or silver [11,14,16,18,19]. Moreover, by employing F₁ hybrid crosses between the highly mercury-resistant DBA/2 (H-2^d) and mercury-susceptible SJL (H-2^s), (DBA/2 × SJL)F₁ and also back-cross hybrids between (DBA/2 × SJL)F₁ and SJL, it was demonstrated that mice carrying the heterozygous H-2 genotype (H-2^d/s) were highly resistant to mercury-induced ANolA production [15]. Based on these results, I expected to observe that in genetically heterogeneous mouse populations only a very small number of animals produce ANolA upon exposure to mercury and silver. In contrast to my expectation, I found that treatment with these xenobiotics resulted in the development of high serum titres of both IgG1 and IgG2a ANolA in a large number of outbred ICR, NMRI and Black Swiss mice. In line with this finding, Robinson et al. [31] and Saegusa et al. [32] have also shown that chronic treatment of male and female ICR mice with mercury at a concentration of 1 mg/kg body weight induces the production of ANolA of the IgG class in 62% of these animals. These findings clearly imply that unlike in inbred mouse strains, H-2 heterozygosity does not confer resistance to mercury- and silver-induced ANolA production. This observation also suggests that certain environmental factors, without requiring the presence of specific susceptibility genes, can induce autoimmune manifestations in members of a genetically heterogeneous population.

The underlying mechanisms by which mercury and silver induce ANolA production in outbred mice remain to be elucidated. However, this phenomenon can, hypothetically, be elucidated by two explanations. First, it is likely that in a genetically heterozygous mouse population, a broad range of MHC (here H-2) class II genes confer susceptibility to mercury- and silver-induced ANolA production. Secondly, it is also possible that in resistant inbred mouse strains, H-2-associated resistance effects are unnaturally amplified, because the background genes in these strains are extremely homogeneous, whereas in outbred mouse stocks the resistance effects of H-2 are diluted out owing to their diverse genetic backgrounds. Clearly, further studies are required to verify these possibilities.

Results from initial studies based on the immune characteristics and cytokine profiles have suggested that mercury-induced autoimmunity in rodents is mediated by T helper type 2 (Th2) responses, i.e. there is a polyclonal B cell activation with hyper-IgE and -IgG1 as well as an up-regulation of interleukin (IL)-4, a prototype cytokine produced by activated Th2 cells, which induces IgG1 and IgE responses in the mouse [25,33–35]. However, further studies have revealed that the development of mercury-induced autoimmune manifestations cannot be explained simply by Th2-biased immune responses. For instance, by employing IL-4 and interferon (IFN)-γ gene knock-out mice, it was demonstrated that not only both Th1- and Th2-type responses contribute to the development of mercury-induced autoimmunity [36]; also IFN-γ, which is a principal cytokine produced by activated Th1 cells and induces IgG2a response in the mouse, is absolutely required for the induction of ANolA production in this autoimmune condition [37]. Morover, the results from my own study have shown that treatment with mercury and silver can induce IgG ANolA responses dominated by either IgG1 or IgG2a or both isotypes in a strain-dependent manner [16]. In the present study, I also found that treatment with either mercury or silver induced IgG1 and IgG2a ANolA responses differentially in outbred ICR, NIMRI and Black Swiss mice, and that the magnitude of ANolA responses in Black Swiss was higher than those in NMRI and ICR mice. These findings again confirm that the Th1/Th2 dichotomy cannot be used to define the production of ANolA in heavy metal-induced induced autoimmunity and suggest further that not only the individual genetic makeup [16]), but also the genetic makeup of the mouse population can play an important role in the severity and outcome of this autoimunity.

One of the major characteristics of mercury-, but not silver-induced autoimmune manifestations in susceptible inbred mice is the development of renal immune complex deposits, which is secondary to mercury-induced B cell activation and autoantibody production, and appears in a mesangial granular pattern 3 weeks after initiation with mercury treatment [8–16]. Interestingly, studies have demonstrated that the deposition of mercury-immune complexes in the kidney does not cause severe glomerulonephritis [38–40]. Regarding this issue, and the findings that both mercury and silver are capable of inducing strong immune/autoimmune responses in outbred mice, it is of high importance to elucidate whether these animals are also able to develop renal immune complex deposits and even glomerulonephritis upon exposure to mercury and silver. I am currently performing experiments to answer this question.

Considered together, results of the present study provide a solid experimental support for the hypothesis that in a genetically heterozygous population, chronic exposure to certain environmental factors can activate the immune system per se to produce autoimmunity in the absence of specific susceptible genes. Indeed, this hypothesis is also supported by the human studies in which it has been shown that 40% of a human population chronically exposed to relatively high levels of mercury (a gold-mining community) exhibit different levels of ANolA in their sera [41,42]. Thus, based on the above-mentioned hypothesis, one can suggest that environmental factors might play a more important role than the autoimmune susceptible genes in triggering the autoimmune processes and that susceptibility genes can function as exacerbating factors, potentiating the risks and severity of clinical manifestations. I believe that heavy metal-induced autoimmunity in outbred mouse stocks provides a suitable model to test this suggestion.