Exposure to mercuric chloride during the induction phase and after the onset of collagen-induced arthritis enhances immune/autoimmune responses and exacerbates the disease in DBA/1 mice

Abstract

In susceptible mice, mercuric chloride induces a systemic autoimmune response that is characterized by elevated serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of anti-nucleolar antibodies (ANolAs) and the formation of renal IgG deposits. We have previously shown that mercury can also enhance immune/autoimmune responses in mouse strains genetically prone to develop spontaneous autoimmune disease. Here, we investigated whether mercury can enhance the severity of murine collagen-induced arthritis (CIA), an inducible (acquired) autoimmune disease that cannot be induced by mercury itself. While mercury administered prior to the induction phase of CIA exerted little, if any, influence, administration of mercury during the induction phase and following onset aggravated the symptoms of this disease and increased the serum levels of IgE and IgG2a antibodies directed against collagen type II (CII). Furthermore, while animals injected with mercury alone exhibited a significant decrease in the ratio of the levels of interferon-γ (IFN-γ) to interleukin-4 (IL-4) mRNA in their spleens, this ratio was increased in mice with CIA, with or without administration of mercury. Finally, the production of anti-nuclear antibodies, a hallmark of autoimmunity in response to mercury, was observed in all mice with CIA treated with this heavy metal. Our findings suggest that exposure to mercury during the development of CIA may influence immunological factors in such a way as to synergistically promote disease development.

Introduction

Exposure of various mammalian species – including humans, rabbits, rats and mice – to mercurial compounds can give rise to immunosuppression and/or autoimmunity.^1–4 The autoimmune effects of, in particular, inorganic mercury salts have been extensively characterized in susceptible rodent strains.^3–6 In susceptible mice, for example, such autoimmunity involves the polyclonal activation of B cells by CD4⁺ T cells, increased serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), the production of antibodies with different specificities [in particular, anti-nucleolar autoantibodies (ANolA)] and the formation of renal deposits of IgG.^7–10

Although the mechanisms underlying mercury-induced autoimmunity are poorly understood, genetic predisposition and immunological parameters appear to be important factors.^{11, 11–13} Studies on mice have revealed that both H-2 and other, background genes are involved in determining the susceptibility to such autoimmunity.^{11, 11–14} Moreover, the exposure of susceptible mice to mercury has been reported to result in the aberrant production of various cytokines – i.e. the early up-regulation of interleukin (IL)-2 and interferon-γ (IFN-γ), followed by pronounced expression of IL-4 mRNA, ¹⁵ thereby giving rise to a T helper 2 (Th2) type of autoimmune response.⁵

As the spontaneous development of autoimmune disease in humans and animal models is also thought to involve genetic predisposition and immune dysregulation, ^16–18 our hypothesis was that the immunotoxicity of mercury might be more pronounced in individuals predisposed to spontaneous development of autoimmune disease.¹⁹ Upon exposing young (NZB × NZW) F1 hybrid and tight skin 1 (Tsk1) mice, which are genetically prone to develop diseases resembling systemic lupus erythematosus and scleroderma, respectively, to mercury, we observed potent and characteristic immune/autoimmune activation in both strains.^{19, 20} Furthermore, other investigators have demonstrated that exposure of lupus-prone BXSB mice to mercury enhances both the cellular and humoral features of their systemic autoimmunity.²¹

Although consistent with our hypothesis, these studies do not reveal whether mercury interacts with genetic and/or immunological parameters to either increase susceptibility to and/or exacerbate the manifestation of autoimmune disease. The present investigation focuses on this question. To this end, normal mice, in which collagen-induced arthritis (CIA) was induced by intradermal injection of this protein, ²² were exposed simultaneously to mercuric chloride.

Characteristic for CIA is symmetric swelling of the peripheral joints, involving synovial membrane hyperplasia, massive infiltration of mononuclear cells, pannus formation, and erosion of cartilage and bone.^23–25 These changes are accompanied by an elevated production of proinflammatory cytokines and the production of specific, pathogenic antibodies towards collagen.^23–25 Mercuric chloride, by itself, does not cause such alterations. Therefore, by injecting this salt at various time-points during the induction phase and onset of CIA, we were able to characterize the influence of mercury on the development of this disease.

Materials and methods

Mice

Female DBA/1 (H-2^q) mice (6–8 weeks of age at the beginning of each experiment; M & B A/S, Ry, Denmark) were housed in the animal facilities of the Department of Immunology, Stockholm University (Stockholm, Sweden), under conditions of 12-hr dark/12-hr light and with free access to standard chow and tap water.

Induction of CIA and treatment with HgCl₂

Chicken collagen type II (CII) (Sigma Chemical Co., St Louis, MO) was dissolved at a concentration of 2 mg/ml by stirring overnight in 0·05 m acetic acid at 4°. Complete Freund's adjuvant (pulverized, heat-killed Mycobacterium tuberculosis H37RA; Difco Laboratories, Detroit, MI) was emulsified in incomplete Freund's adjuvant (Sigma) at a concentration of 4 mg/ml. Under light isofluorane anaesthesia, mice were injected intradermally (i.d.) at the base of the tail with 0·1 ml of a 1 : 1 mixture of these two preparations. Twenty-one days later, these same animals were injected intraperitoneally (i.p.) with 100 µg of CII alone dissolved in 0·1 ml of 0·05 m acetic acid.

The mice were divided randomly into six groups of six animals each, and certain of these groups were injected subcutaneously (s.c.) with HgCl₂ (0·1 ml dissolved in physiological saline; 1·6 mg/kg body weight; Merck, Darmstadt, Germany) as follows:

1. Group 1 received HgCl₂ once every third day for a period of 4 weeks prior to immunization with CII (referred to as Hg treatment prior to the induction of CIA).

2. Group 2 (included in the first, but not in the second experiment) received mercury at the same time as the first immunization with CII (Hg treatment during induction of CIA).

3. Group 3 received mercury at the time of appearance of CII-induced joint swelling (Hg treatment after development of CIA).

4. Group 4 was not exposed to mercury (CIA without Hg treatment).

5. Group 5 received HgCl₂ according to the same schedule as Group 3, but no CII (Hg treatment alone).

6. Group 6 were controls, receiving only physiological saline (0·1 ml, s.c., once every third day).

This protocol is summarized in Fig. 1. These experiments were approved by the Northern Ethical Committee for Animal Experimentation in Sweden.

Figure 1.

Experimental protocol for administration of mercury to DBA/1 mice with collagen-induced arthritis (CIA).

Monitoring the development of arthritis

Following secondary administration with CII, the mice were assessed (at the time-points indicated in the figures) for the appearance of arthritis by examination of their forepaws and hindpaws. The severity of arthritis was scored by using an established system: 0, normal joint; 1, mild, but apparent erythema and swelling of the ankle or wrist or swelling limited to individual digits (regardless of the number affected); 2, moderate erythema and swelling of the ankle and wrist; 3, severe erythema, with swelling of the entire paw, including the digits; and 4, maximally inflamed limb with the involvement of multiple joints. The sum of the scores for all four paws in each mouse (maximally 16) was employed as an index of the overall severity and progression of arthritis in each animal. The severity of arthritis was scored only on the animals that developed the disease; the incidence of arthritis in each experiment was 90–95%.

Collection and preparation of blood, spleen and limb samples

In separate experiments, 34 and/or 62 days after secondary immunization with CII, the mice were bled by retro-orbital puncture under light isofluorane anaesthesia. Thereafter, the animals were killed by cervical dislocation, their spleens were carefully removed and the limbs most affected by arthritis were removed.

The blood was allowed to clot at 4°, centrifuged and the serum thus obtained stored at −20° until assayed for antibody/autoantibody levels. About 75% of each spleen was teased apart with forceps in Earle's balanced salt solution and the single-cell suspensions thus obtained were washed three times and resuspended in 5 ml of this same solution for performing the protein A plaque assay.

The protein A plaque assay

The numbers of spleen cells secreting antibodies of different immunoglobulin classes and subclasses were quantified by utilizing the protein A plaque assay.²⁶ Rabbit anti-mouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC) and IgG2b (Nordic Immunological Laboratories, Tillburg, the Netherlands) were employed as developing agents.

Detection of IgG1-type and IgG2a-type ANolAs in serum by indirect immunofluorescence

Hep-2 cells grown as monolayers on slides (Immuno Concepts, Sacramento, CA) were exposed to fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotechnology, Birmingham, AL) and the pattern and titres of different ANolAs were subsequently assessed by using a Reichard-Jung Plyvar microscope (Vienna, Austria). The highest dilution of serum at which specific green nucleolar fluorescence was detected was defined as the titre of the IgG1 and/or IgG2a ANolA (with serum exhibiting no such fluorescence at a dilution of 1 : 50, being assigned a value of zero).

ELISA quantification of mouse IgE

Total serum IgE levels were determined with a sandwich enzyme-linked immunosorbent assay (ELISA), described previously, ¹⁹ by using a rat anti-mouse IgE monoclonal antibody (mAb) (E-35-72; Pharmingen, San Diego, CA) for capture and a biotinylated rat anti-mouse IgE mAb (R35-92; Pharmingen) for detection.

Detection of anti-CII by ELISA

Serum IgG1 and IgG2a antibodies against CII were assayed by using a standard ELISA procedure. Briefly, each well of 96-well, flat-bottom micro ELISA plates (Corning Costar, Cambridge, MA) was coated with chicken and/or mouse CII (Chondrex; MD Biosciences, Zurich, Switzerland) overnight at 4°, after which unbound sites were blocked by incubation (for 2 hr at room temperature) with phosphate-buffered saline (PBS) containing 1% (v/v) bovine serum albumin (BSA). Then, 50 µl of sera serially diluted in PBS containing 1% (v/v) Tween-20 [beginning with dilutions of 1 : 1000 (for IgG1) and/or 1 : 2000 (IgG2a)] was added to the wells and the plates were incubated overnight at 4°, washed four times with PBS-Tween, and incubated with alkaline phosphatase-labelled goat anti-mouse IgG1 and/or IgG2a (Southern Biotechnology) for 2 hr at room temperature, followed by three further washes. Subsequently, the plates were incubated with p-nitrophenyl phosphate (Sigma), as a phosphatase substrate, for 30 min at room temperature, followed by determination of the absorbance (A) at 405 nm. Serum from an arthritic mouse was used as a positive control.

RNA extraction, cDNA synthesis and quantification of IL-4 and IFN-γ mRNA

RNA was prepared from the remaining 25% of each spleen by using a commercial extraction kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. cDNA was synthesized by using 5 µg of this total RNA as template. The 20-µl reaction mixture, containing 4 µl of 5× reaction buffer, 2 µl of 10 mm dNTP, 1·5 µl of 100 µm dithiothreitol (DTT), 1 µl of Random hexamer Pd (N6; 10 pmol/ml) and 1 µl of reverse transcriptase (all purchased from Gibco/BRL, Life Technologies, Gaithersburg, MD), was incubated at 42° for 45 min.

To determine the relative expressions of IL-4 and IFN-γ mRNAs in the spleens of our mice, the real-time polymerase chain reaction (PCR)²⁷ was performed on an ABI Prism 7700 Sequence Detector (Perkin-Elmer/Applied Biosystems, Foster City, CA) with primers and probes spanning an exon junction, to avoid amplification of any contaminating genomic DNA.²⁸ Following initial activation of AmpErase UNG (2 min at 50°) and Ampli Taq Gold^® (10 min at 95°), each run consisted of 40 cycles of two-step PCR amplification (15 seconds at 95°; 1 min at 60°) on MicroAmp Optical 96-well reaction plates with optical caps (Perkin-Elmer/Applied Biosystems). An equal volume of DNA was used in all assays, PCR conditions were optimized with respect to the concentrations of primers, probes and MgCl₂, and the level of mRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for standardization.

Defining the mRNA levels for IL-4 and IFN-γ in group 6 (no treatment with Hg, no CIA) as 1, the relative increase or decrease in these levels was determined from the expression 2^–ΔCT, according to the Perkin-Elmer instruction manual (User Bulletin #2, December 11, 1997).

Histopathological examination

The limbs removed were fixed in 4% (v/v) formaldehyde in PBS, placed in 70% (v/v) ethanol, decalcified using 5% (v/v) formic acid and then embedded in paraffin. Sections of 5 µm were stained with haematoxylin and eosin and examined for histological evidence of inflammation, pannus formation and damage to cartilage and bone.

Expression of data and statistical analysis

Serum levels of IgE, IgG1 and IgG2a anti-CII, and titres of IgG1 and IgG2a ANolAs are all expressed as mean values + standard error (SE), with the medians also being given. The ratios of IFN-γ/IL-4 mRNA are also presented as mean values + SE. Differences between these parameters for the control group 6 and the other groups were analysed for statistical significance by using analysis of variance (anova). This same test was used to analyse the differences between CIA mice injected with mercury at different time-points (groups 1–3) and the control CIA group 4 with respect to severity of arthritis (determined in mice which developed the disease only). A P-value of < 0·05 was considered statistically significant. All statistical analyses were performed utilizing winstat software (R. Fitch Software; Medina AB, Vänerborg, Sweden).

Results

Administration of mercuric chloride either during induction or following the onset of arthritis exacerbates the development of CIA in DBA/1 mice

As expected, CII induced polyarthritis in DBA/1 mice, with maximal symptoms developing 17–22 days after secondary immunization with CII (Fig. 2). Thereafter, inflammation (swelling) in the joints decreased gradually and led to low joint mobility (ankylosis) by day 62 following CII administration (Fig. 2b). Development of CIA after injection with mercury prior to the induction phase (Group 1) was very similar to that observed in the absence of mercury (Group 4, Exp. 1; Group 3, Exp. 2) (Fig. 2).

Figure 2.

Exposure of DBA/1 mice to mercury during the induction phase and after disease onset aggravates the development of collagen-induced arthritis (CIA). The animals were immunized, on day 1, with chicken collagen type II (CII) emulsified in complete Freund's adjuvant and then boosted with this same antigen on day 21. In two separate experiments (a and b), mercury was administered to different groups of mice (five per group) every third day for a period of 4 weeks, as follows: Group 1 (bef; black triangles) began receiving mercury prior to the induction phase; Group 2 (dur; black circles; not included in experiment 2), mercury administered during the induction phase; and Group 3 (aft; black pentagons), mercury administered after disease onset. Group 4 (black squares) was not treated with mercury; while two control groups, of three mice each, received either mercury (Group 5, open squares) or physiological saline (Group 6, open circles) alone. The severity of arthritis was scored as described in the Materials and methods and the values shown are the means + standard error (SE) of these scores. Experiments 1 and 2 are representative of four and two independent experiments, respectively. *P < 0·05, **P < 0·01.

In contrast, compared to the control group 4, the severity of CIA was enhanced in mice that received mercury during the induction phase (Group 2, Exp. 1) or after development of the disease (Group 3, Exp. 1; Group 2, Exp. 2) (Fig. 2). Moreover, disease symptoms were seen 3 days earlier in mice administered mercury during induction (Group 2), although the development of CIA in these animals resembled that in mice not receiving mercury 17 days after secondary immunization (Fig. 2a). In contrast, the severity scores for animals which received mercury after developing CIA (Group 3) remained relatively elevated throughout the experimental period (Fig. 2a). Control mice injected with mercury (Group 5) or saline (Group 6) alone demonstrated no signs of arthritis at any time (Fig. 2).

To further characterize the disease in our experimental animals, representative tissue samples from joints with the highest severity scores 34 days after secondary immunization with CII were subjected to histopathological examination. All such samples from these mice, regardless of whether or not they were also injected with mercury, exhibited a similar appearance, with massive infiltration of inflammatory cells and destruction of cartilage (Fig. 3a, 1, 2, 3), in contrast to the animals administered mercury or saline alone (Fig. 3e, 3f, respectively).

Figure 3.

Influence of mercury treatment on the histopathology of collagen-induced arthritis (CIA). Histological examination of the paw joints (staining with haematoxylin and eosin) was performed 34 days after secondary immunization with chicken collagen type II (CII) (a–f). A representative slide from a mouse with CIA, not treated with mercury (d), reveals massive infiltration of inflammatory cells (indicated with white arrows), as also seen in the joints of mice with CIA treated with mercury at different time-points, i.e.(a)prior to the induction phase;(b)during the induction; and(c)after the onset of CIA. Representative joints from animals administered mercury(e)or saline(f)alone show no such infiltration. Note that in all mice with CIA, with or without mercury treatment, the most severely affected limb was chosen for histopathological examination.

Together, these findings demonstrate that treatment with mercury during either the induction phase or after the effector phase of CIA potentiates the clinical manifestations of this syndrome.

Administration of mercuric chloride following the onset of CIA enhances the production of IgE

This potentiation of CIA by mercury motivated examination of the major characteristic immunological parameters associated with this disease, including specific and non-specific activation of B cells.²³ Accordingly, the spleens and sera documented in Fig. 2 were tested for the presence of antibody-secreting cells and IgE, respectively. Thirty-four and 62 days after secondary immunization with CII, all CIA mice, with or without mercury treatment, as well as the animals receiving mercury alone, exhibited elevated numbers of spleen cells secreting IgG1 and IgG2b antibodies and enhanced serum levels of IgE (compared to mice receiving saline alone; data not shown). At these time-points the numbers of cells secreting IgM, IgG1, IgG2b and IgG3 antibodies were similar in CIA mice treated or not treated with mercury and in animals receiving mercury alone. However, at both time-points, mice treated with mercury after the onset of CIA exhibited serum levels of IgE that were markedly higher than in the other groups (Fig. 4a,4b).

Figure 4.

Treatment with mercury after the onset of collagen-induced arthritis (CIA) enhances and prolongs the elevation of serum immunoglobulin E (IgE) levels. Thirty-four(a)and 62(b)days after secondary immunization with chicken collagen type II (CII), serum IgE levels for the same animals documented in Fig. 2 (see the legend to this figure for an explanation of the symbols and abbreviations employed) were determined employing a sandwich enzyme-linked immunosorbent assay (ELISA), as described in the Materials and methods. Each symbol represents a single animal. The mean values + standard error (SE) are presented as thin vertical lines, while the median values are represented by small bold horizontal lines. *P < 0·05, **P < 0·01.

Treatment with mercuric chloride augments the production of IgG2a anti-CII by mice with CIA

Production of collagen-specific IgG antibodies is considered to be the hallmark of humoral immune responses associated with CIA.^{23, 25} Thirty-four days after secondary immunization with CII, all of the CIA mice documented in Fig. 2, regardless of whether they were treated with mercury, demonstrated levels of IgG1 antibodies against chicken CII that were similar and much higher than those in control animals administered mercury or saline alone (Fig. 5a). By day 62, these IgG1 antibody levels in CIA mice had decreased somewhat, but still remained higher than in the control animals (Fig. 5c). In addition, 34 days after immunization, CIA animals treated with mercury, either during the induction phase or after the onset of disease, possessed higher serum levels of IgG2a anti-chicken CII than untreated CIA mice (Fig. 5b) and in the case of treatment with mercury after the onset of CIA, these elevated levels persisted at least until day 62 (Fig. 5d).

Figure 5.

Treatment with mercuric chloride augments the production of immunoglobulin G2a (IgG2a) antibodies against chicken collagen type II (CII) by mice with collagen-induced arthritis (CIA). Thirty-four (a and b) and 62 (c and d) days after secondary immunization with chicken CII, the serum levels of IgG1 (a and c) and IgG2a (b and d) antibodies against chicken CII for the same animals documented in Fig. 2 (see the legend to this figure for an explanation of the symbols and abbreviations employed) were determined employing a sandwich enzyme-linked immunosorbent assay (ELISA), as described in the Materials and methods. In all cases a serum dilution of 1 : 4000 was used and the values obtained were within the linear range of the dilution curve. Each symbol represents a single animal. The mean values + standard error (SE) are presented as thin vertical lines, while the median values are represented by small bold horizontal lines. *P < 0·05, **P < 0·01. In all cases the mean values for all groups of mice CIA were significantly higher (P < 0·05) than those for the control animals treated with mercury or saline alone.

In order to test, in greater detail, the specificity of the IgG2a antibodies in our experimental animals, we also measured the serum levels of these antibodies directed towards murine CII. Again, we found that at both time-points, CIA mice treated with mercury, either during the induction phase or after the onset of disease, exhibited higher serum levels of IgG2a anti-mouse CII than did untreated mice with CIA (Fig. 6a,6b).

Figure 6.

Treatment with mercuric chloride augments the production of immunoglobulin G2a (IgG2a) antibodies against mouse collagen type II (CII) by mice with collagen-induced arthritis (CIA). Thirty-four (a) and 62 (b) days after secondary immunization with chicken CII, serum levels of IgG2a (b and d) antibodies against mouse CII for the same animals documented in Fig. 2(see the legend to this figure for an explanation of the symbols and abbreviations employed) were determined employing a sandwich enzymelinked immunosorbent assay (ELISA), as descrbed in the Materials and methods. In all cases a serum dilution of 1 : 2000 was used and the values obtained were within the linear range of the dilution curve. Each symbol represents a single animal. The mean values + standard error (SE) are presented as thin vertical lines, while the median values are represented by small bold horizontal lines. *P < 0·05. In all cases the mean values for all groups of CIA mice were significantly higher (P < 0·05) than those for the control animals treated with mercury or saline alone.

Mercury-induced expression of IL-4 and IFN-γ mRNAs in the spleen of mice with CIA

As mentioned above, dysregulation in cytokine production also plays an important role in the pathogenesis of CIA. Thus, IL-2, IFN-γ and tumour necrosis factor-α (TNF-α), produced mainly by T-helper type 1 (Th1) cells, appear to exacerbate autoimmunity, whereas products of Th2 cells, such as IL-4, have been suggested to be protective.^23–25 In the present study, real-time PCR revealed that 34 and 62 days after secondary immunization with CII, the ratios of the splenic levels of mRNA encoding IFN-γ (a representative Th1 cytokine) and IL-4 (a representative Th2 cytokine) were significantly higher in untreated and mercury-treated CIA mice than in mice that received mercury alone. CIA mice administered mercury after the onset of the disease exhibited somewhat (albeit not statistically significant) higher ratios than did untreated CIA mice (Fig. 7a,7b). This finding suggests that mercury may exert a small synergistic effect on the cytokine imbalance associated with CIA.

Figure 7.

Ratios of interferon-γ (IFN-γ)/interleukin (IL)-4 mRNAs in the spleens of mice with collagen-induced arthritis (CIA), treated or not treated with mercury. Thirty-four (a)and 62(b)days after secondary immunization with chicken collagen type II (CII), the levels of IFN-γ and IL-4 mRNAs in the spleens of the same animals documented in Fig. 2 (see the legend to this figure for an explanation of the abbreviations employed) were determined by employing the real-time polymerase chain reaction (PCR) procedure described in the Materials and methods. The data presented represent the mean values + standard error (SE) of the IFN-γ/IL-4 ratios. In all cases the mean values for the groups of mice with CIA were significantly higher (P < 0·05) than those for the mice administered mercury or saline alone.

Mercury-induced production of ANolAs is largely preserved during the development of CIA

Production of ANolA is one of and possibly the major characteristic of mercury-induced autoimmunity in mice, ^{3, 4, 7, 9} including DBA/1 mice.^{11, 11–13} When serum levels of IgG1 and IgG2a ANolAs in the mice presented in Fig. 2(b) were measured by indirect immunofluorescence 62 days after secondary immunization with CII, untreated CIA animals and mice receiving saline alone did not exhibit any detectable amounts of these antibodies (Fig. 8a,8b). In contrast, mice treated with mercury alone and all of the CIA animals receiving mercury, irrespective of when, demonstrated high levels of both isotypes of ANolAs (Fig. 8a,8b). Similar observations were made on day 34, except that in this case CIA mice treated with mercury after onset of the disease exhibited lower serum levels than mice in the other mercury-treated groups (data not shown). Thus, mercury-induced autoimmune responses are maintained during the development of CIA.

Figure 8.

The production of immunoglobulin G1 (IgG1) and immunoglobulin G2a (IgG2a) anti-nucleolar antibodies (ANolAs) induced by mercury is maintained during the course of collagen-induced arthritis (CIA). Sixty-two days after secondary immunization with chicken collagen type II (CII), serum IgG1 (a)and IgG2a(b), ANolA levels for the same animals documented in Fig. 2 (see the legend to this figure for an explanation of the symbols and abbreviations employed) were determined employing an indirect immunofluorescence technique, as described in the Materials and methods. Each symbol represents a single animal. The mean values + standard error (SE) are presented as thin vertical lines, while the median values are represented by small bold horizontal lines. In all cases, the mean values for the groups treated with mercury were significantly higher (P < 0·05) than those for untreated mice with CIA or for animals receiving saline alone.

Discussion

We^{19, 20} and others²¹ have previously shown that chronic exposure of mice which spontaneously develop autoimmune disease to mercury exacerbates the disease. In the present study we have examined the influence of chronic exposure to subtoxic doses of mercuric chloride on the development of collagen-induced (acquired) autoimmunity in DBA/1 mice. Such treatment, prior to the induction phase, is without effect. In contrast, administration of mercury either during the induction phase or after onset of disease enhances the severity of CIA, as well as causing the symptoms to appear earlier, in the former case.

These findings are consistent with our hypothesis that mercury influences immunological processes in such a way as to potentiate the development of autoimmune diseases. Further evidence for this idea comes from the demonstration that as little as 2 weeks of exposure to low doses of mercury is sufficient to enhance disease severity in a murine model of chronic graft-versus-host disease (GVHD).²⁹ Our present results also indicate that the initial stage, but not the final stage, of mercury-induced immunoactivation interacts synergistically with both the induction and effector phases of CIA, thereby enhancing disease severity. This conclusion is also supported by reports that during the period soon after the initial administration of mercury (2–8 days), the production of proinflammatory cytokines which play important roles in the induction and development of CIA, such as IL-1, ³⁰ IL-2 and IFN-γ, ^{15, 31} is potently induced. Thus, characterization of the entire pattern of inflammatory cytokine production in CIA mice treated with mercury is clearly warranted.

In the present study we also observed that administration of mercury to mice after the onset of CIA results in maintenance of the elevated serum IgE levels associated with this disease.³² No such phenomenon is seen when mercury is injected alone or at other stages of CIA. The increase in the total serum IgE level associated with the effector and remission phases of CIA includes an increase in collagen-specific IgE, ³² and the potentiation caused by mercury may thus also involve collagen-specific antibodies. Consistent with this proposal is the report that co-treatment of susceptible BN rats immunized with ovalbumin and mercury potentiates both the total and ovalbumin-specific IgE responses.³³ However, this hypothesis must be tested further, e.g. by characterization of the IgE antibodies produced by mercury-treated CIA mice.

It is well established that collagen-specific IgG antibodies of different isotypes are produced during the development of CIA²³ and that these antibodies might be involved in the pathogenesis, as indicated by the fact that mice lacking B cells do not develop CIA.³⁴ Here, we found that treatment with mercury, especially after the development of CIA, enhances primarily the production of IgG2a anti-chicken and murine CII antibodies, which might then exacerbate the disease. Although the exact mechanism underlying this effect is unknown, it seems likely that under the inflammatory conditions associated with exposure to mercury and collagen together, mercury acts as an additional adjuvant that preferentially enhances the production of anti-collagen II IgG2a. This suggestion is supported by findings that mercury exerts adjuvant effects on the specific humoral (IgG) immune responses exhibited by rats immunized with ovalbumin³⁵ and by mice immunized with horse erythrocytes.³⁶

The cytokine profiles associated with both CIA and mercury-induced autoimmunity are complex and heterogeneous and vary with the disease stage.^{5, 23–25} Our analyses of the splenic production of IFN-γ (a characteristic Th1 cytokine) and IL-4 (a prototype Th2 cytokine) revealed that at all time-points tested, the ratio of IFN-γ/IL-4 mRNA is increased in all animals with CIA, but reduced in mice treated with mercury alone, suggesting that the cytokine profiles associated with the late stages of mercury-induced autoimmunity and CIA differ.

Furthermore, we found that treatment with mercury prior to or during the induction phase does not alter the ratio of IFN-γ/IL-4 mRNA in mice with CIA, while such treatment after disease onset even slightly enhances this ratio. Together with the observation that the development of mercury-induced autoimmunity is totally dependent on the expression of IFN-γ, ³⁷ our finding implies that mercury may induce the production of this cytokine in a manner synergistic with CIA, resulting in a stable Th1 phenotype that cannot be altered to the Th2 pattern. Indeed, this proposal is supported by the fact that differentiated Th1 cells do not retain the ability to produce IL-4 when restimulated under Th2-polarizing conditions.³⁸

High titres of IgG ANolAs, the hallmark of mercury-induced autoimmunity in susceptible mice, ^{4, 5, 7, 9, 11, 11–13, 19} can be detected 2 weeks after treatment¹⁹ and persist in certain strains for approximately 12 months, i.e. several months after the cessation of mercury treatment.³⁹ In the present study, production of both IgG1 and IgG2a isotypes of ANolAs in mercury-treated CIA mice was seen to persist, indicating that CIA and mercury-induced autoimmunity can co-exist and that ANolAs can be expressed as additional autoantibodies during CIA. Whether such expression is involved in the aggravation of CIA by mercury remains to be elucidated.

Considered together, our present findings reveal that mercury can both potentiate the clinical symptoms of and enhance the immune/autoimmune responses associated with CIA. This conclusion supports the hypothesis that environmental risk factors, such as infections and immunotoxic agents, can accelerate ongoing autoimmune processes. Accordingly, we suggest that although subtoxic doses of mercury cannot, in themselves, induce arthritis, this heavy metal might trigger an autoimmune disease, similar to CIA, by potentiating otherwise appropriate immune responses against certain infectious agents. We are currently testing this hypothesis.

Abbreviations

BSA

bovine serum albumin

CIA

collagen-induced arthritis

CII

collagen type II

DTT

dithiothreitol

EKISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate

i.d.

intrademal

IFN-γ

interferon-γ

IgE

immunoglobulin E

IgG

immunoglobulin G

IgM

immunoglobulin M

IL

interleukin

i.p.

intraperitoneal

mAb

monoclonal antibody

PBS

phosphate-buffered saline

s.c.

subcutaneous

Th1

T helper 1

Th2

T helper 2

TNF-a

tumour necrosis factor-a