Immunosuppressive and autoimmune effects of thimerosal in mice

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Abstract

The possible health effects of the organic mercury compound thimerosal (ethylmercurithiosalicylate), which is rapidly metabolized to ethylmercury (EtHg), have recently been much debated and the effect of this compound on the immune system is largely unknown. We therefore studied the effect of thimerosal by treating A.SW (H-2s) mice, susceptible to induction of autoimmunity by heavy metals, with 10 mg thimerosal/L drinking water (internal dose ca 590 μg Hg/kg body weight/day) for up to 30 days. The lymph node expression of IL-2 and IL-15 mRNA was increased after 2 days, and of IL-4 and IFN-γ mRNA after 6 and 14 days. During the first 14 days treatment, the number of splenocytes, including T and B cells as well as Ig-secreting cells decreased. A strong immunostimulation superseded after 30 days treatment with increase in splenic weight, number of splenocytes including T and B cells and Ig-secreting cells, and Th2- as well as Th-1-dependent serum immunoglobulins. Antinucleolar antibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin, and systemic immune-complex deposits developed. The H-2s strains SJL and B10.S also responded to thimerosal treatment with ANoA. The A.TL and B10.TL strain, sharing background genes with the A.SW and B10.S strain, respectively, but with a different H-2 haplotype (t1), did not develop ANoA, linking the susceptibility to H-2. Thimerosal-treated H-2s mice homozygous for the nu mutation (SJL-nu/nu), or lacking the T-cell co-stimulatory molecule CD28 (B10.S–CD28−/−), did not develop ANoA, which showed that the autoimmune response is T-cell dependent. Using H-2s strains with targeted mutations, we found that IFN-γ and IL-6, but not IL-4, is important for induction of ANoA by thimerosal. The maximum added renal concentration of thimerosal (EtHg) and inorganic mercury occurred after 14 days treatment and was 81 μg Hg/g. EtHg made up 59% and inorganic mercury 41% of the renal mercury. In conclusion, the organic mercury compound thimerosal (EtHg) has initial immunosuppressive effects similar to those of MeHg. However, in contrast to MeHg, thimerosal treatment leads in genetically susceptible mice to a second phase with strong immunostimulation and autoimmunity, which is T-cell dependent, H-2 linked and may at least partly be due to the inorganic mercury derived from the metabolism of ethyl mercury.

Introduction

Thimerosal has for a long time been used as a wound disinfectant and a preservative in medical preparations, not least human vaccines (Magos, 2001). However, more extensive childhood immunization schedules and increased concern regarding the potential effect of low level exposure of organic mercurials on neurodevelopment, recently raised the question of thimerosal in vaccines as a public health concern (Stratton et al., 2001a). As a precautionary measure, the use of thimerosal in vaccines has now been largely abandoned in the US (Ball et al., 2001).

Knowledge on the toxicokinetics and toxicology of thimerosal is limited (Clarkson, 2002), and to a large extent based on comparisons with methyl mercury (MeHg), which due to its presence as a common environmental contaminant has been more intensely studied (Stratton et al., 2001b). Thimerosal consists of an organic radical, ethylmercury (EtHg), bound to the sulfur atom of the thiol group of salicylic acid. Thimerosal contains 49.6% mercury by weight, and following tissue adsorption, EtHg rapidly dissociates from the thiolsalicylic acid moiety and binds to the thiol ligands in tissue proteins (Magos, 2003). Identified effects of EtHg on the immune system are sparse. Thimerosal is a frequent skin sensitizer according to the patch test performed in patients with suspected contact allergy (Goncalo et al., 1996). However, the clinical relevance of sensitization is low (Suneja and Belsito, 2001), and thimerosal rarely causes systemic hypersensitivity (Maibach, 1975, Zenarola et al., 1995).

Based on the similarities between EtHg and MeHg with regard to chemistry, initial distribution in organisms and tissue (brain) damage (Clarkson, 2002), it is a plausible hypothesis that the effect of EtHg on the immune system is similar to that of MeHg. MeHg is a well-known immunotoxic substance (reviewed in (Descotes, 1986)). In vitro MeHg reduces T- and B-cell responses (Brown et al., 1988, Nakatsuru et al., 1985, Shenker et al., 1992, Shenker et al., 1993). In vivo, immunosuppression has been found after exposure to sufficient doses of MeHg. Short-term treatment (up to 1 week) with very high doses (corresponding to 3000–9000 μg Hg/kg bw/day) reduces primary and secondary immune responses in rodents (Brown et al., 1988, Hirokawa and Hayashi, 1980, Ohi et al., 1976) and may even cause atrophy of the immune system (Klein et al., 1972, Hirokawa and Hayashi, 1980).

More modest doses of MeHg (130–600 μg Hg/kg bw/day), comparable to those given in the present study, caused in mice after 3 weeks treatment reduced primary and secondary immune responses (Blakley et al., 1980), and after 12 weeks treatment thymic atrophy, reduced NK cell activity (Ilbäck, 1991) and impaired ability to handle viral infections (Ilbäck et al., 2000, Koller, 1975).

We recently reported (Havarinasab et al., 2004) that sufficient doses of thimerosal induce an autoimmune condition in genetically susceptible mice. This condition shared many characteristics with the autoimmune syndrome induced in such mice after exposure to inorganic mercury in the form of metallic mercury vapor (Warfvinge et al., 1995) or mercuric chloride via the oral (Hultman and Eneström, 1992) and the subcutaneous (Robinson et al., 1986) route. The autoimmune syndrome is characterized by lymphoproliferation with polyclonal B-cell activation and hypergammaglobulinemia (Pietsch et al., 1989, Pollard and Hultman, 1997), production of autoantibodies targeting the 34-kDa nucleolar protein fibrillarin (Hultman and Eneström, 1989, Reuter et al., 1989), and development of immune-complex deposits (Hultman and Eneström, 1988, Robinson et al., 1997). In the mouse, susceptibility to induction of antinucleolar/antifibrillarin antibodies (ANoA/AFA) with inorganic mercury is linked to the mouse MHC (H-2) haplotypes s and q, while most other haplotype are resistant to induction of ANoA/AFA (Hultman et al., 1992).

In this study, we assessed if thimerosal has immunosuppressive properties, and examined the relationship between the immunosuppressive and autoimmune effects. Secondly, we studied cellular and molecular requirements for the autoimmune effect of thimerosal, including cytokine expression. Finally, we tried to link the effects on the immune system to the toxicokinetics of thimerosal.

Section snippets

Mice

A.SW, B10.S (H-2s) mice were obtained from Taconic M & B (Ry, Denmark). SJL/N mice (H-2s) heterozygous (nu/+) or homozygous (nu/nu) for the nude mutation (Hultman et al., 1995a) were obtained from National Institute of Health (Bethesda, MD, USA) and bred in the animal facilities of the Faculty of Health Sciences, Linköping. Breeding pairs of A.TL and B10.TL mice (H-2t1) mice were obtained from Harlan Ltd. (Oxon, UK) and Department of Immunogenetics, University of Tübingen Germany, respectively,

Mesenterial lymph node cytokine mRNA expression

A.SW mice treated with thimerosal for 2.5 days showed a significant increase in IL-2 and IL-15 mRNA expression compared with the controls (Fig. 1). After 6 days treatment, the expression of these two cytokines had declined and was not any longer significantly different from the controls. However, at this time the IFN-γ and IL-4 mRNA expression showed a 2- and 7-fold increase, respectively, which was significantly different from the controls (Fig. 1). The expression of IFN-γ and IL-4 mRNA then

Discussion

In a previous study (Havarinasab et al., 2004), we showed that a sufficient dose of thimerosal could induce all features of the mercury-induced autoimmune disease described after treatment with inorganic mercury in genetically susceptible mice (Pollard and Hultman, 1997). The present study concerns effects of thimerosal on cellular and humoral immunity, including cytokine expression, during development of autoimmune disease in such mice.

We found a reduced number of splenic T- and B-cells,

Acknowledgments

This study was supported by a grant from the Swedish Research Council, Branch of Medicine (project no. 09453). The technical assistance of Elham Nikookhesal and Marie-Louise Eskilsson is gratefully acknowledged.

References (62)

  • P. Hultman et al.

    Murine susceptibility to mercury. I. Autoantibody profiles and systemic immune deposits in inbred, congenic, and intra-H-2 recombinant strains

    Clin. Immunol. Immunopathol.

    (1992)
  • P. Hultman et al.

    Genetic susceptibility to silver-induced anti-fibrillarin autoantibodies in mice

    Clin. Immunol. Immunopathol.

    (1995)
  • P. Hultman et al.

    Murine mercury-induced autoimmunity: the role of T-helper cells

    J. Autoimmun.

    (1995)
  • N.G. Ilbäck

    Effects of methyl mercury exposure on spleen and blood natural killer (NK) cell activity in the mouse

    Toxicology

    (1991)
  • U. Johansson et al.

    Effects of the murine genotype on T cell activation and cytokine production in murine mercury-induced autoimmunity

    J. Autoimmun.

    (1997)
  • D.S. Matheson et al.

    Mercury toxicity (acrodynia) induced by long-term injection of gammaglobulin

    J. Pediatr.

    (1980)
  • C.J. Robinson et al.

    Mercuric chloride-, gold sodium thiomalate-, and D-penicillamine-induced antinuclear antibodies in mice

    Toxicol. Appl. Pharmacol.

    (1986)
  • C.J. Robinson et al.

    Murine strain differences in response to mercuric chloride: antinucleolar antibodies production does not correlate with renal immune complex deposition

    Clin. Immunol. Immunopathol.

    (1997)
  • T. Suneja et al.

    Thimerosal in the detection of clinically relevant allergic contact reactions

    J. Am. Acad. Dermatol.

    (2001)
  • K. Warfvinge et al.

    Systemic autoimmunity due to mercury vapor exposure in genetically susceptible mice: dose–response studies

    Toxicol. Appl. Pharmacol.

    (1995)
  • L.K. Ball et al.

    An assessment of thimerosal use in childhood vaccines

    Pediatrics

    (2001)
  • U. Boehm et al.

    Cellular responses to interferon-gamma

    Annu. Rev. Immunol.

    (1997)
  • T.W. Clarkson

    The three modern faces of mercury

    Environ. Health Perspect.

    (2002)
  • J. Descotes

    Immunotoxicology of drugs and chemicals

    Elsevier, Amsterdam

    (1986)
  • M. Goncalo et al.

    Hypersensitivity to thimerosal: the sensitizing moiety

    Contact Dermatitis

    (1996)
  • B. Häggqvist et al.

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

    Clin. Exp. Immunol.

    (2001)
  • K. Hirokawa et al.

    Acute methyl mercury intoxication in mice—Effect on the immune system

    Acta Pathol. Jpn.

    (1980)
  • P. Hultman et al.

    Mercury induced antinuclear antibodies in mice: characterization and correlation with renal immune complex deposits

    Clin. Exp. Immunol.

    (1988)
  • P. Hultman et al.

    Murine mercury-induced immune-complex disease: effect of cyclophosphamide treatment and importance of T-cells

    Br. J. Exp. Pathol.

    (1989)
  • N.G. Ilbäck et al.

    Trace element distribution in heart tissue sections studied by nuclear microscopy is changed in Coxsackie virus B3 myocarditis in methyl mercury-exposed mice

    Biol. Trace Elem. Res.

    (2000)
  • U. Johansson et al.

    Murine silver-induced autoimmunity: silver shares induction of antinucleolar antibodies with mercury, but causes less activation of the immune system

    Int. Arch. Allergy Immunol.

    (1997)
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