Effector mechanisms of human antimycobacterial immunity

Fazal, Nadeem (1994). Effector mechanisms of human antimycobacterial immunity. University of Birmingham. Ph.D.

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Abstract

A number of effector mechanisms thought to be involved in immunity to mycobacterial infections in humans were examined in this thesis. The details of the studies performed in each chapter can be summarised as follows:

Chapter 2 .

Different methods of determining Bacillus Calmette Guerin (BCG) and Mycobacterium avium intracellulare (MAI) viability based on colony forming unit (CFU) counting and radio-isotope labelling were comparatively assessed. These included radio-isotope labelling with [3H]uracil, [3H]uridine, [3H]glycerol, and CFU counting, by both agar plate dilution, and microcolony counting in broth. The sensitivity ranges of the different techniques were determined in both macrophage-free and macrophage-treated systems and used to assess the anti-mycobacterial potential of human monocyte-derived macrophages following BCG/MAI infection. A combination of radiolabelling with [3H]uridine, confirmed by CFU assessment using microcolony counting, was found to be the most sensitive and reproducible means of determining mycobacterial growth and survival respectively. These techniques were then utilised in subsequent studies to assess various effector mechanisms thought to be involved in immunity to mycobacterial infections in human and murine cells.

Chapter 3
The role of reactive oxygen intermediates (ROI) and cytokine activation in the killing or inhibition of growth of mycobacteria within human phagocytes, were separately examined in this study. To look initially at ROI effects, peripheral blood-derived neutrophils and monocytes were isolated from normal healthy control individuals and from Chronic Granulomatous Disease (CGD) patients. Such patients are known to have a defect in their cellular membrane NADPH oxidase pathway, resulting in a failure of their phagocytes to generate oxygen radicals. Neutrophils and monocyte-derived macrophages from these two groups were then infected with BCG/MAI, and bacterial growth rates in each cell type from both groups were determined. No difference in intracellular BCG/MAI growth was demonstrable within neutrophils or macrophages, derived from either CGD patients or normal healthy controls. The results suggest that ROI are not important mediators in the intracellular control of mycobacterial infections within human phagocytes. Further studies were then undertaken to examine the effect of various cytokine treatments, known to activate mycobactericidal activity in murine cells, to affect BCG growth/survival within infected human monocyte-derived macrophages. The results revealed that macrophage activation with either, recombinant human GM-CSF, IFN-y, TNF-oc, or IFN-y and TNF-a together, for between 4 to 7 days post-infection with BCG, had no demonstrable effect on bacterial growth or survival, as compared to non-stimulated control cells. The results then suggest that other activation signals may be required to stimulate mycobactericidal activity in human macrophages.

Chapter 4
The role of tumour necrosis factor alpha (TNF-a) in macrophage-mediated killing of mycobacteria was also investigated in human cells. Four agents, thalidomide, oxpentifylline, dexamethasone and a polyclonal anti-TNF-a antibody, were all shown by specific ELISA to block endogenous TNF-a production by BCG-infected human monocyte-derived macrophages in vitro culture. There was however no significant enhancement of intracellular BCG growth, over a 7-day incubation period, in infected human cells, in the presence of any of the four TNF-a blocking agents, as determined by both radiometric and CFU counting methods of assessing bacterial growth and viability. The results suggest that the action of TNF-a alone is unlikely to be an important effector mechanism in antimycobacterial immunity within human cells.

Chapter 5
Five strains of M. smegmatis with different catalase expression/activity, were used to investigate the role of nitric oxide (NO) as an anti-mycobacterial mediator within murine and human macrophages. The strains: mc2155 (wild type parental), BH1 (spontaneous, INHresistant mutant of M. smegmatis me2155 which is deficient for catalase-peroxidase production), BHl/pBH4 (original shuttle plasmid), BH1/T35 (katG+), & BH1/T586 (partially overlaps katG but does not carry the catalase-peroxidase gene from M. tuberculosis), were used to infect both the murine macrophage cell line J-774 and human peripheral blood mononuclear cells (PBMC)-derived macrophages. The levels of nitrite (NO2") in the cell culture supernatants, and bacterial survival/growth, following infection were then determined. None of the five strains of M. smegmatis were observed to be killed within activated murine or human macrophages, as assessed by colony forming unit (CFU) determination and radiometric labelling, although high levels of nitrite (40-70nMol/ml) were detected in murine but not human cell culture supernatants. Nitrite titres in J774 supernatants moreover were not found to differ significantly following infection with the different catalase expressing strains. Possible catalase-mediated resistance to NO killing was also evaluated by assessing strain survival within cell-free cultures, containing differing concentrations of sodium nitrite (NaNO2‘) or sodium nitroprusside (SNP), as in vitro generators of NO. The growth of the different strains of M. smegmatis were not affected by up to lOmM of either sodium nitrite or nitroprusside. The results suggest that mycobacterial catalase activity does not appear to affect either bacterial survival within murine or human macrophages, nor NO synthesis by infected cells. Additional results are also detailed, showing that BCG are similarly unaffected by physiological levels of NaNO2" in vitro cell free culture, and that L-NMMA, an analogue of L- rginine, which has been shown to block NO-synthase (NOS), and hence NO production in murine cell systems, had no effect on the growth of BCG in human macrophage cultures. Overall therefore, the lack of detectable mycobacterial inhibition or killing in the presence of high levels of NO in murine systems, either synthesised intracellularly or generated in vitro, or in human cells following blocking of NOS, suggest that the NO pathway is unlikely be involved in controlling mycobacterial infections within either murine or human macrophages.

Chapter 6
Human CD4+, mycobacteria-specific, cytolytic T-cell clones were used to lyse BCG-infected macrophages, and the effect on the subsequent growth and viability of the organisms was examined. The survival of released bacteria following cell lysis was assessed by both [3H] uridine labelling and colony forming unit (CFU) estimation. The results indicate that even when effective antigen-specific or lectin-mediated cytolysis of the infected macrophages was achieved, there was no evidence for a direct mycobactericidal effect on the intracellular bacteria. This remained the case even if the period of co-culture of T cells and macrophages was extended up to 48 hours. Pre-treatment of the macrophages with IFN-y was not able to act together with T cell-mediated lysis to produce inhibition of mycobacterial growth. Furthermore, no demonstrable effect on bacterial growth or viability was observed following re-phagocytosis of released BCG, by fresh human monocytes.

Chapter 7
A number of TB patients were identified in 7 family clusters with more than one individual with clinical TB. These families were considered as good candidate subjects to look for expression of cellular dysfunctions which might correlate with genetic markers of susceptibility. Monocyte-derived macrophage cultures were prepared from infected and noninfected family members and normal healthy controls. Mycobacterial growth rates, following a standard infection with BCG were then assessed for each subject’s macrophages, and the levels of TNFa produced, on stimulation of uninfected cells with IFN-y and/or LPS were also determined. The results show that in one Caucasian family of 14 in which 8 members in three generations developed tuberculosis, macrophages from 7 out of 8 diseased subjects, permitted high rates of intracellular growth, while 5 out of 6 asymtomatic family members exhibited low rates, comparable to those in unrelated healthy controls subjects. In a further 45 individuals from 6 Asian multicase families however, no such correlation was demonstrable between individuals with putative susceptibility to TB, and higher bacterial growth rates. Furthermore, no correlations were observed in any of the multicase families between the disease status of the subjects and TNFa production by their macrophages following IFN-y and/or LPS stimulation in vitro.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):