Immunosuppressive and autoimmune effects of thimerosal in mice
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.
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