Student Research: Lynn Bekris

MS, , 2001
Faculty Advisor: Elaine M. Faustman

Glutamate Cystein Ligase Levels in Human Peripheral Blood Lymphocytes


Abstract

Glutathione (GSH) plays a critical role in the antigen mediated immune response associated with inflammation. Abnormal GSH homeostasis has been implicated in inflammatory lung diseases such as idiopathic pulmonary fibrosis, cystic fibrosis and chronic beryllium disease. Glutamate cysteine ligase (GCL) is the enzyme catalyzing the rate-limiting step in the formation of GSH. The aim of this investigation was to determine the levels of GCL catalytic subunit (GCLC) in human peripheral blood lymphocyte (HPBL) subtypes according to cell cycle in normal healthy individuals. A method was developed which utilizes ethanol or paraformaldahyde fixation, saponin, anti-GCLC antibody, lymphocyte subtype specific antibodies and DNA stains to analyze the level of GCLC in resting an phytohemmaglutinin (PHA) stimulated HPBL by flow cytometry. Multiparameter analyses were performed utilizing Multiplus software. Without lymphocyte immunophenotyping PHA stimulated lymphocytes were found to have similar GCLC levels as non-stimulated lymphocytes. Upon immunostaining for lymphocyte subtype, CD19 PHA stimulated B-cells were found to have intermediate levels of GCLC that increase upon PHA stimulation. CD14 monocytes were found to have low GCLC levels that decrease further upon PHA stimulation. CD8 T-cells have the highest levels of GCLC. Cell cycle analyses suggest GCLC levels are highest in CD4 and CD8 T-cells in G1 phase, whereas, G1 and S/G2 phase GCLC levels are similar in CD14 monocytes. GCLC levels in G1 phase CD19 B-cells are higher in PHA stimulated B-cells than in non-stimulated B-cells. This information will contribute to the understanding of the role of GSH and GCL in the proliferation of lymphocyte subtypes and in defining potential immunotoxicity within sensitive populations.