Investigation into a role for metallothionein in cadmium-induced immunotoxicity and immunocompetence of hemocytes from the eastern oyster, Crassostrea virginica

Abstract

Metallothioneins (MT) are a class of low molecular weight, sulfhydryl-rich, metal binding proteins that function in metal tolerance, essential metal metabolism and radical scavenging. Mammalian research suggests MT plays a role in immune function, including the activities of macrophages/monocytes. MT is also found in the macrophage-like cells, hemocytes, of the bivalve, Crassostrea virginica. Hemocytes are known to accumulate high levels of several metals including cadmium (Cd). We hypothesize that MT expression influences hemocyte immune function: modulation of MT expression alters hemocyte resistance to Cd toxicity and alters cellular immune response to invading microorganisms. In vitro exposure of hemocytes to sublethal Cd concentrations had marked effects on MT expression, as measured by a quantitative RT-PCR assay, inducing MT in a dose-dependent manner to a maximum, above which greater Cd exposure inhibited induction. Cadmium also suppressed reactive oxygen species (ROS) levels and lysosomal stability. Conference of protection by oyster MT against Cd-induced toxicity was demonstrated in hemocytes through disruption of MT expression by phosphorothioate oligodeoxynucleotides antisense to a portion of the MT mRNA. Results from down regulation by antisense also suggest a role for MT in ROS generation by hemocytes, consistent with observations from higher organisms. The mechanism by which MT is involved in macrophage and hemocyte ROS generation remains to be elucidated. Biological significance of the Cd and antisense induced changes in immune activities were assessed in a functional assay for microbial killing. Immunocompetence was tested towards two different microbes: the non-pathogenic (to oysters) marine bacterium Bacillus megaterium and the oyster protozoan parasite, Perkinsus marinus. Despite the treatment induced suppression of ROS and decreased stability of the lysosomal compartment, no significant changes were observed in hemocyte ability to destroy either of the test microorganisms. Thus, MT expression does not appear to be critical for hemocytes to mount an effective immune response against at least certain invading microorganisms.