Per Hultman

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

Inbred, congenic, and intra-H-2-recombinant mouse strains were given subcutaneous injections of either 1.6 mg HgCl2/kg body wt or 0.1 ml NaCl thrice weekly for 5-6 weeks. Mercury-treated mice from strains carrying the H-2s haplotype developed antinucleolar antibodies (ANoA), which targeted the 34-kDa nucleolar protein fibrillarin, and in some instances also nucleolar proteins of 60-70 and 10-15 kDa, the latter corresponding to histones. Strains with H-2b and H-2d haplotypes were resistant to induction of ANoA. The susceptibility to development of AnoA/antifibrillarin antibodies (AFA) was mapped to the H-2A-region using intra-H-2-recombinant strains. We were not able to confirm earlier reports that expression of H-2E genes dampens the development of ANoA. Mercury treatment caused a substantial increase in the titer of antichromatin (ACA) and/or antihistone (AHA) antibodies in a fraction of SJL/J, A.SW, A.TH, B10.S, and B10.HTT mice (H-2s), and in A/J (H-2k) mice, whereas mice from the C57BL/6J and C57BL/10J (H-2b), and the DBA and BALB/c (H-2d) strains were low or nonresponders. The development of AHA and ACA could not be linked to the H-2 complex. A significant, substantial increase of granular mesangial and systemic vessel wall IgG deposits occurred in mice with serum ANoA/AFA. However, the B10.S(9R) and B10.HTT strains, which express the H-2E genes, developed only an intermediately increased titer of mesangial IgG deposits. Systemic vessel wall IgG deposits occurred in only 60-80% of the B10.S(9R) mice and in none of the B10.HTT mice. This contrasted with the high titer of mesangial IgG deposits and uniform development of systemic vessel wall IgG deposits observed in B10.S mice not expressing H-2E. Mice with mesangial IgG deposits showed a mild glomerulonephritis. There was no systemic vasculitis. The susceptibility to development of ANoA, AHA, ACA, and systemic, granular IgG deposits in the B10.S strain was influenced by the sex, since males showed less uniform development of these immunopathologic features than females.

Does Amalgam Affect the Immune System? A Controversial Issue (Part 1 of 2)

Although in use for more than 150 years, dental amalgam has been questioned more or less vigorously as a dental restoration material due to its alleged health hazard. Humans are exposed to mercury and the other main dental amalgam metals (Ag, Sn, Cu, Zn) via vapour, corrosion products in swallowed saliva, and direct absorption into the blood from the oral cavity. Dental amalgam fillings are the most important source of mercury exposure in the general population. Local, and in some instances, systemic hypersensitivity reactions to dental amalgam metals, especially mercury, occur at a low frequency among amalgam bearers. Experimental and clinical data strongly indicate that these and other subclinical systemic adverse immunological reactions to dental amalgam metals in humans will be linked to certain MHC genotypes, and affect only a small number of the exposed individuals. These individuals will be very difficult to detect in a mixed population of susceptible and resistant individuals, including persons with alleged symptoms due to dental amalgam fillings, where many of the individuals are likely to suffer from conditions with no proven immunological background such as multiple chemical sensitivity syndrome. Intensified studies should be performed to identify such susceptible MHC genotypes, taking advantage of the reported cases of more heavily metal-exposed humans with systemic autoimmune reactions. Further studies will also be needed to ascertain whether the combined exposure to the metals in dental amalgam may lower the threshold for adverse immunological reactions, since recent studies have shown that the metals in alloy, especially silver, may induce autoimmunity in genetically susceptible mice.

Dose-response studies in murine mercury-induced autoimmunity and immune-complex disease

Female SJL/N mice were given either 5.0, 2.5, 1.25, or 0.625 mg mercuric chloride per liter drinking water (ppm HgCl2). Serum antinucleolar antibodies (ANuA) of the IgG class were seen in mice given at least 1.25 ppm HgCl2 for 10 weeks, a dose which corresponded to a mean renal mercury concentration, as measured with atomic absorption spectrophotometry, of 2.4 +/- 0.43 microgram Hg/g wet weight (ppm Hg; means +/- 1 SD). At a dose of 5.0 ppm HgCl2 all mice showed IgG ANuA with a mean titer of 1:846 and a mean renal mercury concentration of 14.8 +/- 3.9 ppm. Significantly increased titers of granular IgG deposits, corresponding to immune-complex (IC) deposits, developed in the renal mesangium of mice given 5.0 ppm HgCl2. Mice with heavy mesangial IgG deposits showed a mild glomerular endocapillary cell proliferation and widening of the mesangium. Renal vessel wall IgG deposits were found only in mice given 5.0 ppm HgCl2, whereas such deposits were seen in splenic and cardiac arteries of mice receiving 1.25 ppm or more of HgCl2. The renal and splenic mercury concentration was significantly increased in all groups of mercuric chloride-exposed mice and correlated with the dose. We conclude that 10 weeks peroral treatment with mercuric chloride in drinking water is able to elicit autoimmunity and IC disease in genetically homogeneous, mercury-sensitive mice at a body burden similar to that reported in some occupationally exposed humans.

In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel

BACKGROUND A conventional lymphocyte transformation test (LTT) was compared to the commercially available MELISA (memory lymphocyte immunostimulation assay), a lymphoproliferative assay that has been suggested to be a valuable instrument for the diagnosis of metal allergy. Sensitivity and specificity of the two assays were calculated using a patch test as a reference method. METHODS 34 patients were patch-tested for gold sodium thiosulfate, palladium chloride and nickel sulfate, and the lymphocyte proliferation to these metals was tested in vitro using mononuclear cells from peripheral blood. RESULTS No significant differences regarding sensitivity and specificity were found between MELISA and conventional LTT. The sensitivity varied between 55 and 95% and the specificity between 17 and 79%. CONCLUSIONS The low specificity of the two in vitro assays suggests that they are not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since a large number of false-positive results will be obtained.

Adverse immunological effects and autoimmunity induced by dental amalgam and alloy in mice.

Dental amalgam fillings are the most important source of mercury exposure in the general population, but their potential to cause systemic health consequences is disputed. In this study, inbred mice genetically susceptible to mercury‐induced immune aberrations were used to examine whether dental amalgam may interfere with the immune system and cause autoimmunity. Female SJL/N mice were implanted in the peritoneal cavity with 8‐100 mg silver amalgam or silver alloy for 10 weeks or 6 months. Chronic hyperimmunoglobulinemia, serum IgG autoantibodies targeting the nucleolar protein fibrillarin, and systemic immune‐complex deposits developed in a time‐ and dose‐dependent manner after implantation of amalgam or alloy. Splenocytes from mice implanted with amalgam or alloy showed an increased expression of class II molecules. The functional capacity of splenic T and B cells was affected in a dose‐dependent way: 10 weeks of low‐dose and 6 months of high‐dose amalgam implantation strongly increased mitogen‐induced T and B cell proliferation, whereas 10 weeks of high‐dose implantation decreased the proliferation. Not only mercury but also silver accumulated in the spleen and kidneys after amalgam implantation. In conclusion, dental amalgam implantation in a physiological body milieu causes chronic stimulation of the immune system with induction of systemic autoimmunity in genetically sensitive mice. Implantation of silver alloy not containing mercury also induced autoimmunity, suggesting that other elements, especially silver, have the potential to induce autoimmunity in genetically susceptible vertebrates. Accumulation of heavy metals, from dental amalgam and other sources, may lower the threshold of an individual metal to elicit immunological aberrations. We hypothesize that under appropriate conditions of genetic susceptibility and adequate body burden, heavy metal exposure from dental amalgam may contribute to immunological aberrations, which could lead to overt autoimmunity.—Hultman, P., Johansson, U., Turley, S. J., Lindh, U., Enestrom, S., Pollard, K. M. Adverse immunological effects and autoimmunity induced by dental amalgam and alloy in mice. FASEB J. 8, 1183‐1190 (1994)

Toxicology of autoimmune diseases.

Susceptibility to most autoimmune diseases is dependent on polygenic inheritance, environmental factors, and poorly defined stochastic events. One of the significant challenges facing autoimmune disease research is in identifying the specific events that trigger loss of tolerance and autoimmunity. Although many intrinsic factors, including age, sex, and genetics, contribute to autoimmunity, extrinsic factors such as drugs, chemicals, microbes, or other environmental factors can also act as important initiators. This review explores how certain extrinsic factors, namely, drugs and chemicals, can promote the development of autoimmunity, focusing on a few better characterized agents that, in most instances, have been shown to produce autoimmune manifestations in human populations. Mechanisms of autoimmune disease induction are discussed in terms of research obtained using specific animal models. Although a number of different pathways have been delineated for drug/chemical-induced autoimmunity, some similarities do exist, and a working model is proposed.

Resistance to Xenobiotic-Induced Autoimmunity Maps to Chromosome 1

Although evidence indicates that environmental factors play a major role in precipitating systemic autoimmunity in genetically susceptible individuals, little is known about the mechanisms involved. Certain heavy metals, such as mercury, are potent environmental immunostimulants that produce a number of immunopathologic sequelae, including lymphoproliferation, hypergammaglobulinemia, and overt systemic autoimmunity. Predisposition to such metal-induced immunopathology has been shown to be influenced by both MHC and non-MHC genes, as well as susceptibility to spontaneous lupus, in mice and other experimental animals. Among the various mouse strains examined to date, the DBA/2 appears to uniquely lack susceptibility to mercury-induced autoimmunity (HgIA), despite expressing a susceptible H-2 haplotype (H-2d). To define the genetic basis for this trait, two genome-wide scans were conducted using F2 intercrosses of the DBA/2 strain with either the SJL or NZB strains, both of which are highly susceptible to HgIA. A single major quantitative trait locus on chromosome 1, designated Hmr1, was shown to be common to both crosses and encompassed a region containing several lupus susceptibility loci. Hmr1 was linked to glomerular immune complex deposits and not autoantibody production, suggesting that DBA/2 resistance to HgIA may primarily involve the later stages of disease pathogenesis. Identification and characterization of susceptibility/resistance genes and mechanisms relevant to the immunopathogenesis of mercury-induced autoimmunity should provide important insights into the pathogenesis of autoimmunity and may reveal novel targets for intervention.

The Genotype Determines the B Cell Response in Mercury–Treated Mice

Background: Mercury causes in mouse strains of the H–2s haplotype an autoimmune syndrome with antibodies to the nucleolar protein fibrillarin and systemic immune complex (IC) deposits. Other strains, like BALB/C (H–2d), develop only IC deposits, and most strains are resistant. However, mercury activates the murine immune system and causes lymphoproliferation in most strains: H–2s strains are high–responders also in this respect, while the relation between lymphoproliferation and autoimmune manifestations is unclear for other strains. We examined the B cell response to mercury in order to better understand the relation between lymphoproliferation and systemic autoimmunity, using the high–responder H–2s strains (A.SW and SJL), the intermediate responder strain BALB/C (H–2d), and the A.TL (H–2tl) and DBA/2 (H–2d) strains which are resistant to systemic autoimmunity. Methods: During 4–7 weeks of subcutaneous mercuric chloride injections, the number of B cells and the expression of cell surface activation and proliferation markers was monitored by flow cytometry. The number of cytoplasmic Ig+ splenocytes was determined by direct immunofluorescence technique on slides, and serum Ig isotype levels as well anti–ssDNA and anti–DNP antibodies were determined by ELISA. Serum ANA were monitored weekly by indirect immunofluorescence technique. Results: Mercury–treated A.SW and SJL mice (H–2s) developed an increased expression of the proliferation marker CD71 on B cells, an increased number of B cells in the spleen, and an early, strong, but transient increase in serum Ig concentrations of Th1– as well as Th2–regulated Ig isotypes. Mercury–treated H–2s mice rapidly developed a polyclonal B cell response including the IgM isotype, but also antinucleolar antibodies (ANoA) of the IgG isotype with a clumpy pattern, characteristic for antifibrillarin antibodies. The IgG ANoA response was of a long duration and high titer. The A.TL strain (H–2tl) showed only a slight, restricted B cell activation. The BALB/C strain developed a slight, transient B cell activation dominated by IgG1 and IgE, and antinuclear antibodies (ANA). The DBA/2 strain showed only a minimal B cell response without ANA. Conclusion: Mercury induces an early, transient, polyclonal B cell activation linked to the H–2A or H–2K locus in H–2s strains on the A background. This polyclonal response differs from the long–lasting, high–titered IgG autoantibody response to a nucleolar antigen with characteristics of fibrillarin in H–2s strains, which indicates that these responses arise from separate mechanisms. Another group of strains, exemplified by BALB/C (H–2d), responds to mercury with a slight, transient, Th2–dominated B cell response, a restricted antibody specificity targeting the cell nucleus, and systemic IC deposits. Another H–2d strain, DBA, is essentially resistant to mercury, illustrating the importance of non–H–2 genes for regulating the response to mercury.

Costimulation Requirements of Induced Murine Systemic Autoimmune Disease

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L−/− mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.

Mercury toxicokinetics—dependency on strain and gender

Mercury (Hg) exposure from dental amalgam fillings and thimerosal in vaccines is not a major health hazard, but adverse health effects cannot be ruled out in a small and more susceptible part of the exposed population. Individual differences in toxicokinetics may explain susceptibility to mercury. Inbred, H-2-congenic A.SW and B10.S mice and their F1- and F2-hybrids were given HgCl2 with 2.0 mg Hg/L drinking water and traces of (203)Hg. Whole-body retention (WBR) was monitored until steady state after 5 weeks, when the organ Hg content was assessed. Despite similar Hg intake, A.SW males attained a 20-30% significantly higher WBR and 2- to 5-fold higher total renal Hg retention/concentration than A.SW females and B10.S mice. A selective renal Hg accumulation but of lower magnitude was seen also in B10.S males compared with females. Differences in WBR and organ Hg accumulation are therefore regulated by non-H-2 genes and gender. Lymph nodes lacked the strain- and gender-dependent Hg accumulation profile of kidney, liver and spleen. After 15 days without Hg A.SW mice showed a 4-fold higher WBR and liver Hg concentration, but 11-fold higher renal Hg concentration, showing the key role for the kidneys in explaining the slower Hg elimination in A.SW mice. The trait causing higher mercury accumulation was not dominantly inherited in the F1 hybrids. F2 mice showed a large inter-individual variation in Hg accumulation, showing that multiple genetic factors influence the Hg toxicokinetics in the mouse. The genetically heterogeneous human population may therefore show a large variation in mercury toxicokinetics.