TOXICOLOGICAL HIGHLIGHT |
Metallothionein and Anti-Metallothionein, Complementary Elements of Cadmium-Induced Renal Disease
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125
1 To whom correspondence should be addressed at Department of Molecular and Cell Biology, 91 N. Eagleville Road, University of Connecticut, Storrs, CT 06269-3125. E-mail: michael.lynes{at}uconn.edu.
Received March 1, 2006; accepted March 2, 2006
Key Words: metallothionein; nephropathy; immune complex; inflammation; cadmium.
Metallothionein (MT) is a low–molecular weight, cysteine-rich stress response protein with a high affinity for divalent heavy metals. MT was originally identified as a cadmium-rich protein in the equine renal cortex. A large amount of subsequent work has shown MT to serve in many cell types in the management of essential divalent metal cations, to interfere with the toxic effects of xenobiotics, heavy metals, and free radicals, and to serve as a regulator of specific transcription factors. As a consequence of these various activities, MT synthesis has an impact on developmental processes and on the cellular responses to stressful conditions (reviewed in (Klaassen et al., 1999
; Vasak, 2005)).
MT is primarily synthesized on free polysomes (Shapiro et al., 1980). Based on these observations, MT has often been considered to function exclusively as an intracellular protein. While MT lacks the signal peptide sequences or other protein trafficking signals that would result in the protein's entering the traditional secretory pathway, it is nevertheless detected in serum, urine, pancreatic secretions, and other biological fluids as well as in bronchoalveolar spaces, liver sinusoids, and other extracellular spaces. This pool of extracellular MT may originate from necrotic cell death that may accompany some forms of stress, but it is also possible that MT may be selectively released from stressed cells by a nontraditional secretory pathway (Lynes et al., in press). Regardless of its origin, there is compelling evidence that this pool of extracellular MT has interesting roles of its own to play.
One possible role of extracellular MT is as a distribution mechanism for both essential and toxic heavy metals. Another aspect of MT's extracellular influences is its influence on immune capacity. Exogenous extracellular MT suppresses the adaptive immune response in vivo. This immunosuppression can be countered by the simultaneous injection of a monoclonal anti-MT antibody. The observation that injection of the monoclonal anti-MT antibody in the absence of exogenous MT can enhance the humoral immune response to T-dependent antigen challenge suggests that endogenous extracellular MT is capable of affecting immune regulation in a manner similar to that observed with exogenous sources of MT. This conclusion is supported by the observation that MT can be detected on the surface of splenic leukocytes from animals immunized in the presence of adjuvant and on the surface of leukocytes harvested from congenitally autoimmune animals. The nature of the binding of MT to these plasma membranes is as yet undefined: binding could be nonspecific via formation of mixed disulfides with cysteine-rich surface proteins such as CD45, it might be via interactions with pattern recognition receptors such as the Toll-like receptor family, or it might be a specific interaction with an MT-selective receptor as has been described on astrocytes (El Refaey et al., 1997). In light of the reports of anti-MT antibodies produced as a consequence of metal exposure, it may also be possible that MT interactions with some leukocyte plasma membranes are also mediated by Fc receptor capture of the immune complex on these cells.
Injected exogenous MT can also suppress the progression of experimental autoimmune disease. Both experimental autoimmune encephalomyelitis (Penkowa and Hidalgo, 2003) and collagen-induced arthritis (Youn et al., 2002) are diminished in severity following treatment with exogenous MT. Conversely, targeted disruption of the Mt1 and Mt2 genes exacerbates the severity of viable moth-eaten congenital murine autoimmune disease (Lynes et al., 1999).
From a biochemical standpoint, MT is a novel protein. Roughly 33 mol% (20 of 61 amino acids) of the MT sequence is cysteine. These cysteines play central roles in the capture of the divalent heavy metals that MT holds, and they also serve as targets for oxidant attack. The amino-terminal beta domain of the protein has been shown to contain the two immunodominant epitopes. Winge and Garvey (1983) showed that the principle antigenic determinants are to be found in residues 1–5 and 25–30 within MT's beta domain. In contrast, the COOH-terminal domain (residues 30–61) is thought to exhibit only modest immunoreactivity. Because of its size (
7 kDa), antisera and monoclonal antibodies have generally been experimentally produced by immunizing animals with chemically cross-linked MT: either cross-linked to itself or as a hapten covalently coupled to a carrier protein. MT can naturally form mixed disulfides with other cysteine-containing peptides and proteins, which (within the cell) may serve to regulate the availability of Zn (Ghezzi, 2005). If interactions that form disulfides occur in the oxidative environment that exists outside the cell, these interactions may serve to produce MT-associated complexes that are sufficiently large and complex to provoke naturally occurring anti-MT antibodies. In particularly stressful and oxidative cellular environments (e.g., sites of inflammation, tissue wounding, or toxicant accumulation), MT cross-linking may be at its greatest. In light of the presence of significant amounts of MT in circulation following some forms of stress, the cross-linking of MT may result in the production of naturally occurring antibodies to this stress protein.
It has previously been reported that anti-MT antibody is present in the circulation of some healthy adults, in both normal and autistic children, and in atopic dermatitis patients (Jin et al., 2003; Singh and Hanson, 2005). In this highlighted paper (Chen et al., 2005), the authors measure the levels of anti-MT antibody in individuals that were occupationally exposed to elevated cadmium levels in the eastern section of Hunan Province of Central China. The authors report that renal dysfunction correlates with urinary cadmium levels and that anti-MT antibody levels correlated with two biomarkers of renal dysfunction: urinary ß2-microglobulin and N-acetyl-ß-D-glucosaminidase. Furthermore, high levels of serum anti-MT were associated more frequently with nephrotoxicity than lower levels of anti-MT antibodies. They note that these observations suggest that anti-MT antibody in circulation may prove to be a useful biomarker of the susceptibility to renal damage that is often associated with cadmium exposure. It seems reasonable to extend this conjecture even further: elevated levels of circulating MT may provoke anti-MT antibodies, and these two elements may accumulate as immune complexes in the kidney. Simultaneously, free anti-MT may enhance the immune response to antigen challenge by blocking the suppressive effects of free MT. Thus, the production of anti-MT antibodies may enhance the production of other autoantibodies made under toxic or otherwise stressful conditions. As Chen et al. (2005) note, a variety of autoantibodies have been associated with exposure to cadmium. Autoantigen-containing immune complexes may provoke renal nephropathy by activation of the complement cascade or by activation of resident or inflammatory macrophages and neutrophils. Previous work has suggested that MT that originates in the liver upon cadmium exposure may deliver metals to the kidney via the circulation (Klaassen et al., 1999). Intriguingly, in vivo Cd-MT is more toxic to the kidney than CdCl2, yet in vitro the reverse is true (Liu et al., 1994). This could suggest that the in vivo Cd or Cd-MT exposure is responsible for the production of anti-MT antibody that subsequently facilitates accumulation of the immune complexes in the kidney.
If the accumulation of anti-MT autoantibodies does play a role in the development of Cd-associated renal disease, then other stimuli that provoke elevated levels of MT may similarly be associated with immune complex disease and glomerulonephritis as a consequence of MT-related immune complexes. In cadmium-exposed individuals, other observed effects of cadmium such as vasculitis, uveitis, and arthritis might also reflect the formation of these immune complexes. Moreover, treatments of immune complex disease (e.g., plasmapheresis or complement depletion) could serve to diminish the severity of the renal damage if it is related to cadmium-induced MT–anti-MT immune complexes. One way to test this experimentally would be to cadmium-treat animals that have a congenital complement system defect (e.g., B10.D2/o mice), or mice treated with cobra venom factor to deplete the complement cascade.
In contrast to the consequences of circulating anti-MT on the immune response, both free MT and Cd-MT have been found to be immunosuppressive. These observations suggest a mutually antagonistic relationship: MT suppresses the immune response and also can induce formation of anti-MT antibodies that are immunoenhancing. Under normal circumstances, the small amounts of MT that are ordinarily present in circulation do not interfere with the normal functioning of the immune response. When MT appears outside the cell in a normal immune response, this appearance may represent one form of "danger signal" used to initiate or sustain an effective immune response. We have recently shown that leukocytes respond to an MT gradient with a chemotactic response that has many of the hallmarks of a traditional response to chemokines (Yin et al., 2005). When MT levels are high in circulation (stimulated, for example, by toxic levels of cadmium), the normal chemotactic response may be impossible because the normal gradients have been overwhelmed. Alternatively, inappropriately high levels of MT that originate from tissues where heavy metal may accumulate (e.g., lung, liver, or kidney) may produce alternative gradients for cells that should be responding to an antigenic challenge. Such misdirection of the relevant immune cells could ultimately result both in the diminished adaptive immunity and in a concordant increase in inflammatory damage to tissues that are the source of the chemotactic MT gradient.
While little work has been done to quantify the population variance in MT synthesis capacity, it seems reasonable to hypothesize that any variation will be reflected both in the propensity to subsequently produce anti-MT antibodies and in the resulting dimensions of any MT immune complex formation. One way to further explore this issue would be to harvest blood leukocytes from the cadmium-exposed population and determine an individual's in vitro MT synthesis level in the presence of defined concentrations of cadmium. Thus, each individual's risk might be related both to the relative amount of MT synthesis that their cells could produce and the subsequent amount of anti-MT synthesis that the synthesized stress protein might provoke. Similarly, the propensity to produce other autoantibodies in the presence of cadmium may also vary between individuals. Thus, it would be useful to assess the production of a set of autoantibodies (e.g., anti-DNA, anti-histone, and anti-erythrocyte) to determine whether the correlations that the authors report for anti-MT are uniquely associated with MT or are more generally associated with a broader set of autoantigens.
The presence of MT and anti-MT in the serum of these individuals produces a challenging technical quandary. Since an immunoassay was used to measure the anti-MT levels, the assessment of antibody in the serum is complicated by the presence of MT in the same serum and the immune complexes that should be present in equilibrium in those sera. This makes the interpretation of the results more challenging unless the individuals have been removed from the cadmium-contaminated environment for a time sufficient to clear the autoantigen. While the biological half-life of MT has been reported to be brief, the lengthy half-life of cadmium makes this approach difficult. Since MT is so small, it is possible that any immune complexes formed in the serum could be disassembled in high-salt or low-pH buffer and removed by dialysis of the serum with a 12- to 14-kDa molecular weight cutoff dialysis membrane. The 7-kDa form of MT would be removed from the serum, leaving free anti-MT behind. One would thus predict that the detectable amounts of serum anti-MT would increase following this manipulation if circulating immune complexes represent an appreciable percentage of the total anti-MT in the patient serum samples.
This highlighted paper provides intriguing evidence that may suggest immunological changes linking this remarkable stress response protein to the renal disease that is commonly associated with exposure to heavy metals. Ultimately, it may point the way to a wider understanding of the role played by the stress response in a variety of inflammatory diseases and suggest novel therapeutic approaches to these diseases.
ACKNOWLEDGMENTS
This work was supported in part by an award from the National Institutes of Health: ES007408.
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