Semester

Spring

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

John B. Barnett.

Abstract

Approximately 5.3 billion pounds of pesticides are applied annually across the United States and 15 of the top 25 most used pesticides are herbicides. Total herbicide use the in the United States represents 28% of all worldwide herbicide use. DCPA (3,4 dichloropropionanilide, common name propanil) is a post emergent herbicide that is used extensively for control against several broadleaf plants and grasses. It is the 17th most common herbicide in the United States and 6--9 million pounds are applied annually to 2 million acres of rice fields. In mammals, DCPA is metabolized in the liver and produces 3,4-dichloroaniline (DCA) and propionic acid as its major metabolites. DCA is further biotransformed leading to the production of 2 hydroxylated metabolites; 6-hydroxy-3,4-dichloroaniline (6OH-DCA) and N-hydroxy-3,4-dichloroaniline (NOH-DCA). The immunomodulatory effects of DCPA are well documented but only limited data is available on the effects of its metabolites. Previous studies have shown that DCPA alters transcription factors involved in the expression of IL-2 and decreases mRNA and IL-2 protein in human and mouse T cells. IL-2 is an early cytokine that is secreted by activated T cells and plays an important role in the activation, proliferation and differentiation of several immune cells. Expression of IL-2 relies on activation and influx of calcium through channels in the plasma membrane. This study was conducted to examine the effects of DCPA and its metabolites on T cell activation and function and to propose a mechanism for the observed effects. Human Jurkat T cells, a model cell line for T cell signaling, were exposed to increasing concentrations of DCPA or its metabolites and T cell function was assessed by measuring IL-2 secretion. DCPA and its metabolites all inhibit IL-2 secretion in a concentration-dependent manner, however, NOH-DCA is the most potent inhibitor, followed by DCPA. To better understand the mechanism by which they suppress IL-2, NFAT activity and calcium influx were investigated. Interestingly, DCPA and DCA inhibited IL-2 in a calcium-dependent manner whereas the hydroxylated metabolites inhibited IL-2 in a calcium-independent manner. The calcium-dependent alterations in IL-2, NFAT and calcium influx are influenced by the presence of chlorines, as substitution with fluorines abrogated all effects. Further studies investigating the role of DCPA in calcium release-activated calcium (CRAC) channels revealed that activation of a key protein, Stromal interaction molecule-1 (Stim1), is inhibited by DCPA. Collectively, this data supports the conclusion that DCPA suppresses IL-2 production by inhibiting Stim1. This mechanism describes a novel pathway for immunosuppression.

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