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Regulation of human monocyte functional properties by phosphatidylinositide 3-kinase

University of British Columbia

Mononuclear phagocytes are important regulators and effectors of both the innate and acquired immune responses. Extensive research has highlighted an important role for phosphoinositides in monocyte cell regulation. The 3’-phosphoinositide metabolites produced by phosphatidylinositide 3-kinase (PI3K) family of lipid kinases are known to be involved in regulating numerous monocyte activities including phagocytosis, adherence, oxidative burst, and cytokine secretion. An important research objective is to develop an understanding of how specificity is achieved in PI3K signaling [i.e. PI3K signalling] for these diverse biological functions. The goal of this thesis was to determine when particular monocyte functional properties are governed by PI3K in an isoform-specific manner versus situations in which PI3K family members have redundant functions. Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110α isoform of PI3K was silenced using a lentiviral vector expressing short hairpin RNA. This resulted in the generation of stable THP-1 and U-937 human monocytic cell lines deficient in p110α. The role of p110α in regulating cell adherence, phagocytosis, the phagocyte oxidative burst, and LPS-induced cytokine secretion was examined. Monocyte adherence induced in response to either LPS or vitamin D₃ was blocked by PI3K inhibitor LY294002. However, while adherence induced in response to D₃ was sensitive to silencing of p110α. LPS-induced adherence was not. These findings demonstrate that LPS and vitamin D₃ use distinct isoforms of class IA PI3K to induce functional responses. We also observed that p110α was required for phagocytosis of IgG and serum opsonized particles in differentiated U-937 cells. The phagocyte oxidative burst induced in response to either PMA or opsonized zymosan in differentiated THP-1 cells was also found to be dependent on p110α. Furthermore, p110α was observed to exert selective and bi-directional effects on the secretion of pivotal cytokines apparently independently of other PI3K isoforms. LPS stimulation of p110α deficient THP-1 cells demonstrated that p110α was required for inducing IL-12 and IL-6 production, whereas this isoform of PI3K appeared to negatively regulate the production of TNF-α and IL-10. The results reported in this thesis demonstrate that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology. Furthermore, taken together, the data suggest that p110α PI3K is involved in regulating important monocyte effector functions independently of other PI3K family members.

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2010-01-16

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http://hdl.handle.net/2429/18217

Pathology

University of British Columbia

UBCV

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