Title

Metallothionein gene effects on metal-mediated immunomodulation: Traditional and microarray-based assessments

Date of Completion

January 2007

Keywords

Biology, Cell|Engineering, Biomedical

Degree

Ph.D.

Abstract

Metallothionein (MT) is a cysteine rich stress response protein found within the cell. The oxido-reductive power of the high cysteine content provides MT with the capacity to perform numerous functions in the cell. MT can act as a reservoir and donor of essential metals, provide protection against oxidative stress, and sequester toxic metals such as cadmium. The Lynes research group has found that MT can modulate immune function through these unique abilities. To better understand the complex roles and interactions of MT and cadmium in cellular activation and immune response, two MT mutant mouse strains on the C57BL6/J background were developed: C57BT/6J-TgN(Mt1)174Bri (denoted B6-MTTN) and C57B1/6J-Mt1tm1BriMt2 tm1Bri (denoted B6-MTKO). B6-MTKO mice have insertions in the Mt1 and Mt2 genes resulting in an incomplete transcript; B6-MTTN mice have a total of 116 Mt1/Mt2 genes as opposed to just 4 in the wildtype control. Along with the three strains of mice, we have also developed a novel protein microarray platform based on the surface plasmon resonance phenomenon to aid in our studies. Grating-coupled surface plasmon resonance imaging (GCSPRI) uses a grated sensor chip which allows us to analyze hundreds of regions of interest (ROIs) simultaneously. These ROIs can consist of antibodies specific to analytes of interest, such as secreted cytokines and cells expressing membrane-associated targets.^ Our results suggest that both MT gene dose and cadmium, alone and in concert, influence immune function. MT gene dose and cadmium influence in vitro lymphocyte cytokine profiles when stimulated with PMA/ionomycin. B6-MTKO mice secreted more IL-4 and less IFN-γ compared to B6-WT and B6-MTTN. B6-MTTN exhibited suppressed cytokine levels compared to B6-WT. In general, the in vitro cadmium exposure at the highest concentration (25μM) had an immunosuppressive effect on all strains and cytokines, except IFN-γ and IL-6, while not influencing viability. The low concentration of cadmium (6μM) had minimal effect. Taken together, this suggests that above a certain threshold, cadmium influences only specific signaling pathways in lymphocytes, leading to immune suppression. A lack of functional MT tips the balance of immune function towards a Th2 response, while too much MT has an immunosuppressive effect on T-cell dependent adaptive immunity. ^