Toxicology. 2007 Jan 5;229(1-2):23-32. Epub 2006 Sep 24.
Dose and Hg species determine the T-helper cell activation in murine autoimmunity.
Havarinasab S, Björn E, Ekstrand J, Hultman P.
Inorganic mercury (mercuric chloride—HgCl2) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. The organic mercury species methylmercury (MeHg) and ethylmercury (EtHg—in the form of thimerosal) induce AFA, while the other manifestations of HgIA seen after treatment with HgCl2 are present to varying extent. Since these organic Hg species are converted to the autoimmunogen Hg2+ in the body, their primary autoimmunogen potential is uncertain and the subject of this study.
A moderate dose of HgCl2 (8 mg/L drinking water−internal dose 148 g Hg/kg body weight [bw]/day) caused the fastest AFA response, while the induction was delayed after higher (25 mg/L) and lower (1.5 and 3 mg/L) doses. The lowest dose of HgCl2 inducing AFA was 1.5 mg/L drinking water which corresponded to a renal Hg2+ concentration of 0.53 g/g. Using a dose of 8mg HgCl2/L this threshold concentration was reached within 24 h, and a consistent AFA response developed after 8–10 days. The time lag for the immunological part of the reaction leading to a consistent AFA response was therefore 7–9 days. A dose of thimerosal close to the threshold dose for induction of AFA (2 mg/L drinking water—internal dose 118 g Hg/kg bw per day), caused a renal Hg2+ concentration of 1.8 g/g. The autoimmunogen effect of EtHg might therefore be entirely due to Hg2+ formed from EtHg in the body.
The effect of organic and inorganic Hg species on T-helper type 1 and type 2 cells during induction of AFA was assessed as the presence and titre of AFA of the IgG1 and IgG2a isotype, respectively. EtHg induced a persistent Th1-skewed response irrespectively of the dose and time used. A low daily dose of HgCl2 (1.5–3 mg/L) caused a Th1-skewed AFA response, while a moderate dose (8 mg/L) after 2 weeks resulted in a balanced or even Th2-skewed response. Higher daily doses of HgCl2 (25 mg/L) caused a balanced Th2–Th1 response already from onset. In conclusion, while metabolically formed Hg2+ might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose.
A moderate dose of HgCl2 (8 mg/L) caused the earliest onset of AFA (4–8 days), while the response was delayed following lower (1.5–3 mg/L) or higher (25 mg/L) doses. Although the exact mechanism for induction of AFA by Hg remains unknown, two major possibilities have been indicated. First, Hg may interact directly with fibrillarin/fibrillarin peptides causing a physically altered molecule which is immunogenic (Pollard et al., 1997). Secondly, Hg-induced cell death (necrosis) might modify the cleavage pattern for fibrillarin, resulting in neo-peptides of fibrillarin (Pollard et al., 1997), which expose immunogenic epitopes (Pollard et al., 2000) to T cells (Kubicka-Muranyi et al., 1995).
These mechanisms will all require a certain concentration of Hg in the tissues for the initiating cellular–molecular event to develop, which is also supported by the dose–response relationship observed for induction of AFA with HgCl2 (Nielsen and Hultman, 2002). Therefore, the longer time needed for development of AFA using lower doses of Hg (present study) might be explained by a delay in reaching the threshold concentration of Hg2+. In contrast, the delayed AFA response using a high dose of HgCl2 (25 mg/L) might be due to the dose-dependent suppression of Hg on T and B cells (Shenker et al., 1992; Daum et al., 1993; Shenker et al., 1993), which are all vital for induction of AFA (van Vliet et al., 1993; Hultman et al., 1995).
However, even using the optimal dose of HgCl2 for a rapid AFA response (8 mg/L), 8–10 days were needed for a consistent AFA response (response in a majority of the mice) to develop. This time lag is mainly caused by the specific immune response, since the renal Hg2+concentration needed for induction of AFA with HgCl2 (0.53g Hg/g), was reached already within 24 h after onset of treatment with 8 mg/L. A lag phase of 7–9 days for the AFA immune response tallies with the lag phase for induction of an antigen-specific response (Janeway et al., 2005). Using low doses of HgCl2 the Th1-associated IgG2a isotype of ANoA dominated. In contrast, a moderate dose of HgCl2 first caused a dominance of the IgG2a isotype followed by an increase of the Th2-related IgG1 isotype, leading to a balance between Th1/Th2 (IgG2a–IgG1 index nil) after 14 days.
A higher dose of HgCl2 caused already from the onset of ANoA development a balanced IgG2a–IgG1 index (nil), which developed into a modestly negative index (IgG1/Th2-dominance) after 12 days treatment. The distinct dose– esponse relationship of the IgG2a–IgG1 index, with a dominating Th2 phenotype (IgG1) after higher doses, tallies with the predominant Th2 phenotype of HgIA (Goldman et al., 1991). However, the Th1 (IgG2a) dominance after lower doses of HgCl2 tallies with the observation that Th1-linked IFN- is vital for the initial induction of AFA (Kono et al., 1998), while IL-4 is superfluous, but confers a Th2 phenotype to the HgIA response.
How might these observations from mice given different doses of HgCl2 be used to discern between a primary and secondary autoimmunogen effect of EtHg? A dose of 2mg thimerosal L−1 drinking water, which is close to the threshold for induction of AFA with thimerosal (Havarinasab et al., 2004), corresponded to a renal tissue Hg2+ concentration of 1.8g Hg/g after 8 days treatment. This concentration is at least three-fold higher than theHg2+ concentration needed for induction ofAFAwith HgCl2, showing that the induction of AFA by thimerosal might have been caused entirely by the Hg2+ formed from EtHg in the body.
However, although organic EtHg may lack or only have a weak primary autoimmunogen effect, it affects the quality of the autoimmune response, since the IgG2a–IgG1 index in EtHg-treated mice consistently showed a Th1-dominated pattern, also after 14 days treatment with a high dose of EtHg.
The observation of a shift in the balance in favor of a Th1 phenotype during treatment with organic Hg (EtHg), tallies with previous observations (Hultman and Hansson-Georgiadis, 1999; Haggqvist and Hultman, 2001; Havarinasab et al., 2004). The IL-2 and IFN- mRNA expression was higher in EtHg-treated mice (Havarinasab et al., 2005) compared with mice given HgCl2 (Haggqvist and Hultman, 2001).
EtHg caused a seven-fold increase of IL-4 mRNA and 30-fold increase of IgE (Havarinasab et al., 2005), compared with a 15-fold increase of IL-4 mRNA and a 40–160-fold increase of IgE in HgCl2-treated mice (Hultman and Hansson-Georgiadis, 1999; Haggqvist and Hultman, 2001). This indicates that the Th1-dominance in EtHgtreated mice might be due to a strong and/or more protracted increase of the Th1-associated cytokines IL-2 and IFN- (Havarinasab et al., 2005), which should also lead to a reciprocal inhibition of Th2 cells.
The effect of Hg on activation of Th1 or Th2 cells has previously been studied. For example, HgCl2 was shown to cause a Th2 skewing in vitro (Heo et al., 1997).
Another observation indicates that intracellular levels of glutathione might be involved, since in rats glutathione (GSH) is required for Con A-mediated IFN- induction in vitro, which tallies with the induction of IFN- by Hg in vivo (van der Meide et al., 1993). Depletion of GSH in mice causes a decreased IFN- production and an increased IL-4 production upon in vitro stimulation of T cells (Peterson et al., 1998). Furthermore treatment of human T cells in vitro with aGSHprecursor inhibited IL-4 production (Jeannin et al., 1995).
These observations indicate that a low level of GSH favors a Th2 response and a high level of GSH a Th1 response. Organic as well as inorganic Hg species cause a reduction of intracellular GSH, which has been associated with the cytotoxicity of Hg (Naganuma et al., 1990; Sanfeliu et al., 2003; James et al., 2005), but it is unknown if the organic Hg species affect GSH levels differently than inorganic Hg. While EtHg causes an initial, transient immunosuppression with reduction of CD3+ and CD4+ cells (Haggqvist et al., 2005; Havarinasab et al., 2005), further studies are needed in order to try to establish the effect of different doses and Hg species directly on the T- helper cell subsets.
Another possibility is that other cells than T cells affecting the Th1/Th2-ratio, such as mast cells (Wu et al., 2001) and possibly dendritic cells (Agrawal et al., 2003), may react differently to inorganic and organic Hg.