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. 2001 Oct;126(1):157–164. doi: 10.1046/j.1365-2249.2001.01636.x

Murine metal-induced systemic autoimmunity: baseline and stimulated cytokine mRNA expression in genetically susceptible and resistant strains

B Häggqvist 1, P Hultman 1
PMCID: PMC1906178  PMID: 11678913

Abstract

Cytokines play an important and complex role in the pathogenesis of systemic autoimmune diseases. In susceptible H-2s mice, inorganic mercury (Hg) induces lymphoproliferation, antinucleolar antibodies against the 34-kDa-protein fibrillarin, and systemic immune-complex (IC) deposits. Here, we report extensive analysis of cytokine mRNA levels in susceptible A.SW (H-2s) and resistant A.TL (H-2tl) mice under unstimulated conditions and during oral treatment with Hg and/or silver nitrate (Ag). Cytokine mRNA expression in lymphoid tissues was assessed using the ribonuclease protection assay and phosphorimaging. Baseline expression of IL-2 and IFN-γ mRNA was higher in A.SW than in A.TL mice. In A.SW mice, Hg treatment caused early up-regulation of IL-2 and IFN-γ levels, followed by substantial expression of IL-4 mRNA, which was significant compared to control A.SW and Hg-treated A.TL mice. Hg-exposed A.TL mice exhibited unchanged IFN-γ, reduced IL-2 and greatly increased IL-10 mRNA expression. Ag-treated A.SW mice, which develop antifibrillarin antibodies (AFA) but exhibit minimal immune activation and no IC deposits, showed an early increase in IL-2 and IFN-γ mRNA, but only a small and delayed rise in IL-4 mRNA. In conclusion, H-2-linked resistance to Hg-induced AFA is characterized by low constitutive expression of IL-2 and IFN-γ mRNA, which is not increased by Hg, and a marked increase in IL-10 expression. Conversely, the key features of H-2-linked susceptibility to Hg- and Ag-induced AFA are up-regulation of IL-2, IFN-γ and IL-4 mRNA expression, and down-regulation of IL-10 expression.

Keywords: mercury, mice, autoimmunity, cytokines, mRNA

Introduction

Cytokines play an important role in the pathogenesis of systemic autoimmune diseases, but this is difficult to elucidate due to the large number of cytokines and their pleiotropic effects [1]. Some rodent strains show rapid responses to treatment with inorganic mercury (Hg), which include activation of the immune system and induction of a systemic autoimmune disease referred to as Hg-induced autoimmunity (HgIA) [2]. In mice with HgIA, the dominating autoantibody targets a 34-kDa nucleolar protein called fibrillarin. The same type of autoantibody occurs in human scleroderma patients [3,4]. Considerable evidence suggests that the antifibrillarin antibodies (AFA) that are associated with HgIA in mice are the result of an antigen-specific reaction [5–9]. In light of that, and considering the fact that Hg also can induce systemic IC-deposits, HgIA in mice (M-HgIA) constitutes a suitable model for studying the role of cytokines in systemic autoimmunity [10].

Activated CD4+ cells become polarized into two main subsets designated T-helper (Th) 1 and Th2-type cells, which are associated with expression of certain patterns of cytokines [1]. In 1991, Goldman et al. [11] proposed that there is a skewing towards a Th2 response in animals that are susceptible to HgIA and a bias towards a Th1 response in HgIA-resistant animals. There is ample support for this hypothesis in the rat [12–14]. Also, it was initially considered plausible for M-HgIA [15], although this was contradicted by results showing that susceptible (H-2s) mice displaying inherited [16] or induced [17] IL-4 deficiency still develop AFA in response to Hg. Using IFN-γ−/– and IL-4−/– knock-out (KO) mice, Kono et al. [18] showed that induction of AFA is critically dependent on Th1-associated IFN-γ but not Th2-associated IL-4, and the latter finding was confirmed by Bagenstose et al. [19].

Several investigators have examined the expression of cytokines in murine lymphocytes treated with Hg in vitro. For instance, Hg-treated cells from C57BL/6 and BALB/C mice were found to show an increased production of IL-2 [20] and IFN [21], respectively, and lymphocytes from Brown Norway rats that were exposed to Hg exhibited raised levels of IL-4 and IFN-γ mRNA [22]. Furthermore, other studies in vitro have indicated that cytokine mRNA expression in rats [23] and cytokine production in mice [24] may be directly triggered by Hg. However, a comprehensive study of the cytokine profile that occurs during development of AFA in vivo in the M-HgIA model has not yet been reported.

In the present study we used the ribonuclease protection assay (RPA) to simultaneously assess the expression of mRNA coding for several different cytokines. We employed three main approaches to identify cytokines invoked in induction of M-HgIA and AFA. First, we assessed the unstimulated, baseline level of cytokine mRNA expression in lymphoid tissues of susceptible and resistant strains of mice. Second, we studied the cytokine mRNA profile in lymphoid tissues from Hg-treated A.SW and A.TL mice; these two strains are, respectively, susceptible and resistant to Hg-induced AFA [6], and they have the same non-H-2 genetic background (A), thus any dissimilarities in the cytokine patterns they exhibit must be due to differences in their H-2 haplotype (H-2s for A.SW and H-2t1 for A.TL). The third approach was to find the minimal cytokine requirements for induction of AFA by studying the cytokine mRNA profile after treating mice with silver (Ag). In susceptible strains such as A.SW, Ag is known to induce AFA but only slight immune activation and no other M-HgIA manifestations [25].

Materials and methods

Mice

A.SW (H-2s) and A.TL (H-2tl) mice were obtained from M & B A/S (Ry, Denmark) and Harlan UK Ltd. (Bicester, Oxon, England), respectively. The animals were housed in steel-wire cages under a 12-h dark−12- h light cycle, and they had access to pellets (Type R 36, Lactamin, Vadstena, Sweden) ad libitum. The mice were 8–12 weeks old at the onset of experiments.

Treatment with mercury and silver

The animals were exposed to mercury and silver by dissolving HgCl2 (6 mg/l) or AgNO3 (0·5 g/l) in the drinking water, which was available ad libitum. Fresh solutions were prepared once a week, and control mice were given water without added metals.

Experimental design

Previous experiments had shown that cytokine responses to mercury are rapidly induced and down-regulated [26]. Therefore, we initially monitored expression of cytokine mRNA in the lymph nodes (LN) and spleens of animals sacrificed every 24 h, starting 36 h after the onset of treatment with Hg or Ag and continuing for the next 15 days. The results of the screening experiment using two animals at every time point formed the basis for selecting the critical times to compare the cytokine mRNA expression in larger groups of metal-treated and control mice (described in the Results section). We also compared the baseline cytokine mRNA expression in untreated A.SW and A.TL mice.

Preparation of tissues

The spleens and mesenteric LN of the experimental animals were aseptically removed and transferred to tubes placed on ice and containing denaturing Ultraspec™ RNA reagent (Biotecx Laboratories, Inc., Houston, TX). The RNA isolation procedure was either performed directly after removal of the organs or after storage at − 70°C. Fresh or frozen tissue (20–100 µg) was homogenized in 1 ml of Ultraspec™ RNA reagent with an Omnitron 17106 homogenizer (Omni International, Waterbury, CT). Before each session, the homogenizer was sterilized and RNase was removed using RNase Zap™ (Sigma, St Louis, MO). The homogenizer was thoroughly cleaned with RNase-free water between homogenizations to avoid any cross contamination between samples.

Isolation and purification of total mRNA

We employed a single-step RNA isolation method using acid guanidinum thiocyanate [27], according to the protocol provided by the manufacturer of the Ultraspec™-II RNA isolation system (Biotecx Bulletin no. 28, 1993). In short, after homogenization of tissue and dissociation of the nucleoprotein complex, 0·2 ml of chloroform was added, and the sample was centrifuged for 15 min at 12000 g and 4°C. The resulting RNA-containing upper aqueous phase was mixed with 0·5 vol. of isopropanol and 0·05 vol. of RNA-binding RNATack™ resin (Biotecx Laboratories, Inc.). The binding resin was collected by centrifugation, and the pellet was washed several times with 75% ethanol. Thereafter, the ethanol was removed, and RNase-free water was used to elute the RNA from the resin. The A260/A280 ratio was always > 1·8, which indicates highly pure RNA.

Assessment of cytokine mRNA

Expression of mRNA for IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IFN-γ and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was assessed using the RPA according to the instructions of the manufacturer (Riboquant® Instruction Manual, 5th edn, Pharmingen, San Diego, CA). Briefly, multiprobes incorporating [α32P]UTP were transcribed from the template set mCK-1 (Cat. #45001P, Pharmingen) using T7 polymerase from an in vitro transcription kit (Cat. #45004K, Pharmingen).

Hybridization and digestion of unprotected probes was done with an RPA kit (Cat# 45004K, Pharmingen). In short, 15–20 µg of purified total RNA from a spleen or LN was hybridized overnight at 56°C with an excess of the multiprobe set in hybridization buffer.

Unprotected RNA probes were digested with RNase A + T1 mixture, and the protected probes were extracted with phenol-chloroform and precipitated with ethanol. Protected probes were dissolved in loading buffer and separated by electrophoresis on a 40-cm-long, 0·4-mm-thick 5% polyacrylamide sequencing gel (19 : 1 acrylamide/bis) containing 9 m urea. The gel was subsequently absorbed on Whatman no. 3 filter paper (Whatman, Clifton, NJ) and dried in a gel dryer for 1 h at 80°C.

A phosphorimaging plate was exposed to the dried gels, and the photo-stimulated luminescence [28] was assessed using a BAS-1000 instrument (Fuji Photo Film Co. Ltd, Japan). Science Laboratory 97, Image Gauge 3·01 software (Fuji Photo Film Co. Ltd) was employed to evaluate the gel images that displayed bands representing cytokines. The housekeeping gene GAPDH was measured and used as a normalizing factor.

Statistics

Statistical differences between mercury- and silver-treated animals and controls were calculated using the nonparametric Kruskal–Wallis test followed by Dunn's post test, and differences in basal mRNA expression were analysed with the Mann–Whitney test. Results were considered statistically significant at a level of P < 0·05.

Results

General observations

The ratio of cytokine to GAPDH expression was lower in the spleens than in the LN (data not shown). The lymphoid tissues showed higher constitutive expression of mRNA for IL-10, IFN-γ, and particularly IL-15, compared to the other cytokines we assessed (Figs 1 and 2).

Fig. 1.

Fig. 1

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Cytokine expression in A.SW mice treated with mercury. The RPA was performed using 18 µg of total RNA from mesenteric lymph nodes of groups of A.SW mice (five animals in each) that were not treated (Cont.) or were exposed to mercury (6 mg/l HgCl2) in drinking water for 2·5 and 5·5 days. A phosphorimaging plate is shown.

Fig. 2.

Fig. 2

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Cytokine expression in A.SW mice treated with silver. The RPA was performed using 18 µg of total RNA from mesenteric lymph nodes of A.SW mice that were not treated (Cont.) or were exposed to silver (0·5 g/l AgNO3) in drinking water for 2·5 and 5·5 days. A phosphorimaging plate is shown.

Cytokine mRNA expression in untreated animals

Comparing untreated A.SW and A.TL mice, we found that the LN of the former showed higher mean expression of IFN-γ mRNA, a significantly higher level of IL-2 mRNA, and also higher mean expression of IL-10 mRNA (Fig. 3). However, the spleens of these two groups of mice contained similar levels of IL-10 mRNA (data not shown). Moreover, baseline expression of IL-4 mRNA was very low in these untreated animals, with no significant difference between the two strains (Fig. 3).

Fig. 3.

Fig. 3

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Baseline IFN-γ, IL-2, IL-10 and IL-4 mRNA expression in relation to GAPDH levels in the lymph nodes of untreated female A.TL (□) and A.SW (Inline graphic) mice. Bars indicate + 1 s.d., and ** denotes significant difference (P < 0·01) between the strains according to the Mann–Whitney test.

IL-4 mRNA

Our initial screening experiments (see Experimental design under Materials and Methods) revealed that, already after 3·5 days, the LN of A.SW mice treated with Hg showed higher mean expression of IL-4 mRNA compared to controls (data not shown). Peak expression of IL-4 occurred after 5·5 days and, at that time, was 16-fold higher than in the non-Hg-treated control group (Figs 1 and 4a) and also significantly higher than in Hg-treated A.TL and Ag-treated A.SW mice (P < 0·01). IL-4 expression decreased but was still sevenfold higher than in the controls on day 8, and continued to slowly decline until day 16. In the A.TL mice exposed to Hg, compared to controls, the mean expression of IL-4 in the LN decreased after treatment for 2·5 days, but showed a significant fourfold increase on days 8 and 12, and had declined to the control level on day 16 (Fig. 4a). We found a similar pattern of IL-4 mRNA expression in the spleens of the Hg-treated A.SW and A.TL mice, although the ratio of IL-4 to GAPDH was lower (data not shown). IL-4 expression was slightly increased in the LN of A.SW mice exposed to Ag (Fig. 2), and this difference was significant compared to controls after 7 days of treatment (Fig. 4a).

Fig. 4.

Fig. 4

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Expression of cytokine mRNA in mesenteric lymph nodes of A.SW (□) and A.TL mice (▵) exposed to mercury (6 mg/l HgCl2 in drinking water) and A.SW mice (○) treated with silver (0·5 g/l AgNO3 in drinking water). The four diagrams represent (a) IL-4, (b) IL-2, (c) IFN-γ and (d) IL-10. The mean cytokine expression in four or five metal-treated mice at the indicated time points is given as percentage of the mean cytokine expression in control animals (100%). The bars denote mean + 1 s.d., and * indicates significant difference (P < 0·05) from values for control mice of the same strain, calculated using the Kruskal–Wallis test followed by Dunn's post test.

IL-2 mRNA

In Hg- and Ag-treated A.SW mice, levels of IL-2 mRNA increased more rapidly than levels of IL-4 mRNA. Considering the LN, the mean expression of IL-2 mRNA in the Hg-exposed A.SW mice was 48% higher than in controls after 2·5 days and 58% higher after 5·5 days (Fig. 4b) Also, the mean expression of IL-2 mRNA in the Hg-exposed A.SW mice was significantly higher than in Hg-treated A.TL mice after 2·5 days and 5·5 days (P < 0·05 and P < 0·01, respectively). Mean IL-2 expression in the LN of A.TL mice began to decrease at the onset of exposure to Hg, reached a nadir after 5·5 days, and returned to control levels after 8 days (Fig. 4). Moreover, levels of IL-2 mRNA in the the LN were significantly higher in the Ag-treated A.SW mice than in the Hg-treated A.TL mice after 2·5 days (P < 0·05) and showed a significant, almost twofold higher peak compared to controls after 4 days. Mean expression of IL-2 mRNA in the LN was higher in Ag-treated A.SW mice than in the control animals until 5·5 days after treatment was begun (Fig. 4), at which time there was a rapid decline back to control levels.

IFN-γ mRNA

Mean expression of IFN-γ mRNA in the LN was 25% higher in A.SW mice exposed to Hg than in the controls 2·5 and 5·5 days after the onset of treatment (Fig. 4c), and significantly higher than in A.TL animals after 2·5 days of Hg treatment (P < 0·01). During exposure to Hg, IFN-γ expression in the LN declined to the control level on day 8 in A.SW mice, and, in the A.TL mice, was comparable to the control group for the first 8 days, but increased slightly after 12 days. The mean expression of IFN-γ in the LN was higher in A.SW mice 2·5, 4 and 8 days after the onset of treatment with Ag, as compared to controls (Fig. 4c). The IFN-γ mRNA level was much lower in the spleens than in the LN, and the mean expression of IFN-γ increased slightly in the spleens of Hg- and Ag-treated A.SW and Hg-treated A.TL mice after 6·5 days of exposure (data not shown).

IL-10 mRNA

Considering levels of IL-10 mRNA in the LN after treatment with Hg, the A.SW mice showed no major difference compared to controls during the first 8 days of exposure, but displayed a significant decrease after 12 days (Fig. 4d). In contrast, the mean expression levels in the LN of the A.TL mice increased after only 5·5 days of Hg treatment, and peaked at a threefold higher level after 8 days; this rise was significant compared to both controls and Hg-treated A.SW mice (P < 0·05), and persisted after 12 days of treatment. A similar increase in IL-10 mRNA expression was seen in the spleens of the A.TL but not the A.SW mice after 8 and 12 days of exposure to Hg (Fig. 5). Treatment with Ag caused no significant changes in IL-10 mRNA expression in the LN of the A.SW mice, compared to controls( Figs 2 and 4d).

Fig. 5.

Fig. 5

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IL-10 mRNA expression in relation to GAPDH levels in the spleens of female and male A.TL mice and female A.SW mice that were not treated (C, ○) or were exposed to mercury (6 mg/l HgCl2 in drinking water, •). Significant difference (P < 0·05) from controls is denoted * and was calculated using the Kruskal–Wallis test followed by Dunn's post test.

IL-13 mRNA

Expression of IL-13 mRNA was generally low in both of the murine strains we studied, regardless of metal treatment. However, a significant rise in IL-13 did accompany the increase in IL-10 mRNA in the LN but not in the spleens of Hg-treated A.TL mice (not shown).

IL-15 mRNA

There was substantial expression of IL-15 mRNA in the spleens and the LN of both control and metal-treated A.SW and A.TL mice (Figs 1 and 2). However, after 5·5 days, such expression was significantly lower in the LN of Hg- and Ag-treated A.SW mice, as well as Hg-treated A.TL mice (data not shown).

IL-5, IL-6 and IL-9 mRNA

Levels of IL-5, IL-6 and IL-9 mRNA were close to the detection limits in both the spleens and the LN of metal-treated as well as control animals. The bands representing these cytokines were very faint, indicating that mercury and silver had no major impact on expression of the mRNA coding for these cytokines (Figs 1 and 2). However, there was a slight increase in mean IL-5 expression in parallel with marked expression of IL-4 in the LN of Hg-treated A.SW mice (data not shown).

Discussion

We found that Hg treatment of susceptible A.SW mice significantly increased the expression of IL-4 mRNA in the LN, which agrees with findings in B10.S mice carrying the H-2s haplotype [15], and with results showing an increase in the number of IL-4-producing cells in Hg-treated A.SW mice [26]. However, we also detected a much smaller but significant increase in IL-4 mRNA in resistant A.TL mice exposed to Hg. This finding is in accordance with Northern blot data reported by van Vliet et al. [15], which demonstrated that the expression of IL-4 mRNA was augmented in resistant B10.D2 mice treated with Hg, although this increase was slight compared to the response seen in susceptible B10.S mice. IL-4 is not necessary for production of IgG1, but it is the dominant factor regulating this immunoglobulin [29]. We have previously observed that A.TL mice that were treated with Hg exhibited a slight rise in serum IgG1 but no increase in serum IgE [26], which indicates that this metal does not induce sufficient expression of IL-4 mRNA to generate the amount of IL-4 needed to cause B-cells to switch to production of IgE.

In the present study, we found that the LN of susceptible A.SW mice exposed to Hg showed an early increase in mean expression of IL-2 mRNA, which was significant compared to the controls after 5·5 days of treatment. This finding agrees with our previous results [26] demonstrating an early rise in both levels of splenic IL-2-receptor markers and numbers of IL-2-producing LN cells in Hg-treated A.SW mice. In sharp contrast to this, the resistant A.TL mice in the present experiments exhibited reduced mean IL-2 mRNA expression in response to Hg, and this reached a nadir after 5·5 days of treatment, which conforms with the previously observed reduction in levels of the very early activation marker CD69 on T-cells in Hg-treated A.TL mice [26]. Furthermore, we observed significantly lower baseline expression of IL-2 mRNA in A.TL than in A.SW mice. This suggests that low baseline levels of IL-2, which are further reduced by exposure to Hg, play an important role in resistance to M-HgIA in the A.TL strain. Indeed, Jiang and Möller [30] recently proposed that IL-2 may be a limiting factor that prevents the lymphocytes in resistant DBA/2 mice from responding to Hg in vitro. Interestingly, it has been reported that self-tolerance in mice is maintained by a CD25+CD4+ subset of T-cells that exert their suppressive effect by reducing expression of IL-2 [31].

We found that the LN of A.SW mice, as compared to controls, showed a modest but significant increase in expression of IFN-γ after 2·5–5·5 days of treatment with Hg. Moreover, IFN-γ levels in the LN were significantly higher in A.SW than in A.TL mice after 2·5 days of exposure to Hg. It is possible that the early rise in IFN-γ in Hg-treated A.SW mice is rapidly nullified by the reciprocal suppression, which the forceful increase in IL-4 may be expected to exert on IFN-γ [32,33]. In support of that, we have previously observed that a single injection of mercury caused an early and marked increase in the number of IFN-γ+ LN cells and, to a lesser extent, IL-4+ cells; a second injection 3 days later suppressed the rise in number of IFN-γ+ cells and instead markedly increased the number of IL-4 producing cells [26].

Although it is known that M-HgIA is dependent on IFN-γ [18], it is not clear what effects of this pleiotropic cytokine [34] are necessary to induce such autoimmunity. Inasmuch as IFN-γ−/– KO (H-2s) mice are resistant to HgIA [18], susceptibility to HgIA in H-2s mice may require either constitutive or increased levels of IFN-γ. It has been found that heterozygous IFN-γ−/+ KO (H-2s) mice are partially responsive to Hg and development of AFA [18], which indicates that a subnormal level of IFN-γ is sufficient to confer such susceptibility. We observed higher baseline IFN-γ mRNA levels in A.SW than in A.TL mice, and Charles et al. [35] have also recently reported strain-dependent differences in baseline cytokine mRNA expression in mice. Notably, we found that baseline mRNA expression is linked to H-2, as indicated by our results showing that higher constitutive IFN-γ and IL-2 levels were conferred by the H-2s haplotype than by the H-2t1 haplotype. Hypothetically, the H-2t1 haplotype may mediate low baseline levels of Th1 cytokines and thereby lead to anergy, a state in which the growth of autoreactive T cells is arrested, which, in turn, could efficiently prevent HgIA. It has been shown that an efficient Th1 response against foreign antigens, such as the E. coli MalE protein [36], is linked to certain H-2 haplotypes. In that context, we observed an early Th1 cytokine response in the susceptible A.SW mice but not in the resistant A.TL mice, which indicates a similar link between the H-2 haplotype and the immune response to Hg-induced self-antigens.

In our experiments, resistant A.TL mice responded to mercury by expressing increased levels of IL-10 mRNA in both the spleen and LN, starting 5·5 days after treatment and reaching a maximum after 8 days, which is in contrast to the decline in IL-10 mRNA that occurred in the susceptible A.SW mice exposed to Hg. It is known that IL-10 reduces the expression of IL-12 [37,38], and that effect inhibits secretion of IFN-γ and subsequently prevents development of Th 1 cells and cell-mediated immune responses [39]. Considering these findings, together with other results in the literature showing that IL-10 blocks antigen-presenting cell function by down-regulating surface expression of MHC class II molecules [40] and also directly inhibits secretion of IL-2 from CD4+ lymphocytes [41], it seems likely that IL-10 is responsible for the suppression of IL-2 expression (see above) and therefore also the resistance to HgIA in A.TL mice. Indeed, it has previously been suggested that IL-10 is involved in resistance to HgIA in Lewis rats [12], and a suppressive effect of IL-10 has recently been implicated in a number of other autoimmune conditions as well [42–44].

We concluded our study by examining the differential effects of Hg and Ag on the immune system in order to elucidate the mechanisms that are necessary for development of AFA. Both of these metals can induce AFA, but silver causes minimal immune activation without increase in IgE [25]. We found only a delayed and small, albeit significant, increase in IL-4 mRNA in Ag-treated A.SW mice, which supports that this cytokine is of little importance for induction of AFA. Conversely, the LN of Ag-treated A.SW mice showed an early significant increase in IL-2 mRNA expression, as well as a slight increase in mean expression of IFN-γ mRNA, which is analogous to what was seen after treatment with Hg. The rapid return to normal levels of mRNA for these cytokines after 5·5 days of exposure to Ag may indicate that such expression is cyclic in nature, or that it involves two separate immunological events. In conclusion, our results support the hypothesis that early expression of IL-2 and IFN-γ may be necessary for induction of AFA by mercury and silver.

Acknowledgments

This study was supported by a grant from the Swedish Medical Research Council (Project 9453).

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