published_or_final_version / Pathology / Master / Master of Philosophy

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No description available.

Investigation of the molecular alterations of macrophage function during intracellular infection by Leishmania donovani revealed both adverse and positive influences of this protozoan on host cell function. Chapter I delineates a negative effect which this parasite has on signal transduction pathways in its host cell. In macrophages put in contact, or infected with L. donovani, c-fos gene expression mediated through protein kinase A was unaffected under conditions where there was an impairment of protein kinase C-mediated c-fos gene expression. Selective impairment of protein kinase C-, or, calmodulin-dependent protein kinase-mediated signal transduction in the macrophage was found to influence the establishment of infection. Chapters two and three describe a positive enhancement of macrophage function by L. donovani. Intramacrophage infection with L. donovani was shown to enhance host cell viability in the absence of growth factor. This was attributable to the elaboration of a soluble factor(s) by infected macrophages into the cell culture supernatant, which enhanced macrophage viability in a manner independent of cell replication. Further characterization of the mechanism of this enhancement revealed that L. donovani infection, and lipophosphoglycan, inhibited macrophage death by apoptosis. Cell supernatants derived from L. donovani infected cells were also capable of inhibiting macrophage apoptosis. To identify the active factor in infected cell supernatants, the cytokine gene expression profile of L. donovani infected macrophages was delineated and possible candidate cytokines were further investigated. Levels of TNF-$ alpha$ capable of causing an abrogation of apoptosis were found to be produced by infected macrophages. However, antibody neutralization of TNF in infected cell cultures could not reverse the inhibition of apoptosis by L. donovani, implicating the involvement of multiple factors in the abrogation of apoptosis by L. donovani.

Infections with Entamoeba histolytica are associated with impaired cell mediated immunity by an unknown mechanism. Macrophages are the most important cells in host defense against invasive amebiasis. The present study investigated the effect of E. histolytica on macrophage functions. Macrophages isolated from gerbils with amebic liver abscess and naive macrophages exposed to soluble amebic proteins induced profound alteration of eicosanoid formation both in cyclooxygenase and lipoxygenase pathways by enhanced PGE$ sb2$ and LTC$ sb4$ production. TNF-$ alpha$ production by macrophages was altered locally in amebic granulomas and at systemic sites during the infection and in response to amebic proteins stimulation in vitro. PGE$ sb2$ produced by macrophages in response to amebic proteins was involved in the down-regulation of TNF-$ alpha$ production. E. histolytica-induced dysfunction of macrophage cytotoxicity against amebae and tumor cells occurred by suppressing NO and by enhancing PGE$ sb2$ production. Amebic proteins suppressed the induction. of IFN-$ gamma$-induced bone marrow macrophage class II MHC Ia molecule synthesis and I-A$ beta$ mRNA expression by stimulating PGE$ sb2$ production. Taken together, these results demonstrate that E. histolytica induced PGE$ sb2$ production plays a central role in the suppression of macrophage effector and accessory cell functions.

Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. Activated macrophages are the main host effector cells in host defence against E. histolytica, through the production of nitric oxide (NO) which is cytotoxic for the parasite. NO is upregulated by tumor necrosis factor-alpha (TNF-$ alpha$) produced by macrophages. The objective of this study was to determine the effect of amebic components on TNF-$ alpha$ and NO production by macrophages. Soluble E. histolytica proteins stimulated naive macrophages for enhanced TNF-$ alpha$ mRNA expression through PKC signal transduction. E. histolytica-induced TNF-$ alpha$ mRNA expression was unstable, and macrophages pretreated with E. histolytica proteins expressed reduced levels of TNF-$ alpha$ mRNA in response to LPS or IFN-$ gamma$ + LPS. In contrast, the purified galactose-adherence lectin (Gal-lectin) of E. histolytica stimulated naive macrophages for stable TNF-$ alpha$ mRNA expression and protein production. Furthermore, IFN-$ gamma$ primed macrophages produced TNF-$ alpha$ and NO in response to the Gal-lectin. Naive macrophages exposed to Gal-lectin + IFN-$ gamma$ were activated to kill E. histolytica trophozoites in vitro by NO. Anti-lectin monoclonal antibodies that recognize non-overlapping epitopes of the kDa heavy subunit of the Gal-lectin identified amino acids 596-1082 as important in mediating amebic adherence to target cells and TNF-$ alpha$ mRNA induction in macrophages. Likewise, a region between amino acids 596-818 of the 170 kDa Gal-lectin, in conjunction with IFN-$ gamma$, activated macrophages for TNF-$ alpha$ and NO production and amebicidal activity. This research demonstrates the immunogenic potential of the E. histolytica Gal-lectin and the critical regions that could be used as a subunit vaccine candidate against amebiasis.

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A general metabolic profile of macrophages was established by measurement of maximum catalytic activities of enzymes in energy-producing pathways and rates of utilisation of glucose, glutamine, fatty acids and ketone bodies under various conditions. It was found that glucose, glutamine and fatty acids can be used to satisfy the energy requirement of the cell. Although a significant proportion of utilised glutamine or fatty acid was converted to C0₂ by the macrophage, most glucose was not oxidised and was converted, almost stoichiometrically, to lactate. Utilised fatty acids were not only oxidised by the macrophage, but were incorporated into cellular lipid (mainly triacylglycerol and phospholipid). The triacylglycerol rich macrophage was shown to be able to release fatty acids into the culture medium. The importance of glutamine in macrophages was indicated from the high activity of phosphate-dependent glutaminase. Glutamine is probably metabolised by the following enzymes in macrophages: phosphate-dependent glutaminase, aspartate aminotransferase (or other amino acid aminotransferases), oxoglutarate dehydrogenase followed by enzymes of the TCA cycle and metabolism of oxaloacetate by phosphoenolpyruvate carboxykinase. Pyruvate derived via this pathway may be metabolised via pyruvate dehydrogenase or pyruvate carboxylase. A study of the sub-cellular distribution of some of these enzymes suggested that phosphate-dependent glutaminase has a cytosolic as well as a mitochondrial localisation. Further characterisation suggested that the non-mitochondrial activity could be associated with the plasma membrane. To the author's knowledge, this is the first report of a non-mitochondrial localisation for phosphate-dependent glutaminase. Glutaminase was shown to be activated by phosphate and inhibited by glutamate and 2-oxoglutarate. Significant inhibition of glutaminase occurred only at high concentrations of these compounds. Glucose and glutamine were utilised at very high rates by the macrophage, but were not fully oxidised even though the cells were incubated in aerobic conditions. The significance of these high rates of utilisation to the macrophage is discussed.