Puan, Kia Joo; (1999) Mycobacterial inositol phosphoglycans and their effects on macrophages. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ligand binding of a number of growth factors and cytokines can result in the release of mediators known as inositol phosphoglycans (IPG) catalyzed by a glycosyl phosphatidylinositol specific phospholipase D. Two distinct subtractions of IPG derived from M. vaccae were able to mimic metabolic effects of mammalian IPG such as activating pyruvate dehydrogenase phosphatase and stimulating lipogenesis in adipocytes. In contrast, the IPG isolated from M. tuberculosis H37Rv were inhibitory in the mammalian assay systems, leading to the speculation that they might exert pathogenic roles in infected cells. Therefore, the effects of the IPG from M. vaccae and M. tuberculosis were evaluated on the functions of a murine macrophage cell line, J774A.1. Mycobacterial IPG of both fractions were not able to stimulate nitrite production in J774A.1 cells. However, they can synergise with IFN-[gamma] to produce high levels of nitrite. Western blotting analysis of the J774A.1 cell lysates treated with mycobacterial IPG plus IFN-[gamma] showed that the nitrite produced was attributed to the induction of inducible nitric oxide synthase protein. Unlike lipopolysaccharides (LPS), mycobacterial IPG-induced nitrite production was not inhibited by polymyxin B. However, LPS and mycobacterial IPG-induced nitrite production were inhibited by the L-arginine analogue, NG-monomethyl-L-arginine (L-NMMA) but not by its inert enantiomer, NG-monomethyl-D-arginine (D-NMMA). As IPG are released by GPI-PLD, an enzyme assay system using [3H] myristate-labelled membrane-form variant surface glycoprotein (mfVSG) was set up to monitor the purification of GPI-PLD. It was found mannosylated lipoarabinomannan (ManLAM) inhibits GPI-PLD, raising the possibility that in mammalian cells the mycobacterial LAM inhibits release of endogenous IPG, while the mycobacterium substitutes its own IPG, resulting in subtly disordered regulation of macrophage function.

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