Title

The effects of intracellular and extracellular metallothionein on the immune response

Date of Completion

January 2003

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Metallothionein (MT), a stress response protein, has been shown to play numerous roles in the cell: it serves as a reservoir of trace metals, scavenges free radicals, and can sequester toxic heavy metals. These various functions suggest that MT can participate in modulating immune responses. This thesis was designed to evaluate the effects of intracellular and extracellular MT on humoral immunity. First, the humoral immune function of animals with either a targeted disruption of Mt1 and Mt2 genes (MTKO) or containing 56 extra copies of Mt1 (MT-TGN) per haploid were compared with wild-type counterparts. MTKO mice displayed a significantly higher humoral response to ovalbumin (OVA) challenge compared to wild-type controls. Immune responses in MT-TGN animals were not significantly different from controls. The changes in immune capacity in MTKO mice correlated with increased B cell differentiation following OVA challenge and enhanced lymphoproliferative response to mitogens. Finally, we have found that splenocytes from MTKO animals displayed significantly elevated NF-κB activity compared to controls. These findings suggest that endogenous metallothionein may provide a mechanism to downregulate in vivo immune function after antigenic challenge. ^ Given the potential of metallothionein to regulate the immune response, the mechanism of regulation was investigated as a function of interactions with plasma membrane proteins. Antigen presentation assays were used to investigate the possibility that MT may be interacting with proteins of the immune synapse. This thesis shows that MT alters T cell epitope recognition after presentation by antigen presenting cells (APCs). In the presence of MT, OVA-stimulated DO-11.10 T cells (OVA-specific T cell line) secreted 20–30% less cytokines than controls. DO-11.10 responses were also suppressed in the presence of MT when antigen was presented by paraformaldehyde-fixed APCs, suggesting that suppression does not depend on alterations in processing of OVA. T cell proliferation induced by immobilized anti-CD3ϵ antibodies was similarly decreased in the presence of MT. The interpretation that MT interferes with antigen specific T cell responses was corroborated by co-localization studies using confocal microscopy. MT associated with aggregated CD3ϵ on the surface of DO-11.10 T cells; suggesting MT may be altering recognition of OVA epitopes through direct interaction with proteins of the immune synapse. ^ Taken together, these observations suggest MT plays an integral role within the cell and outside of the cell in regulating the immune response. ^