Variability of TNF-α secretion by peripheral blood mononuclear cells from healthy males

Payne, Shawn (1994) Variability of TNF-α secretion by peripheral blood mononuclear cells from healthy males. Masters thesis, Memorial University of Newfoundland.

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Abstract

Tumor necrosis factor-α (TNF-α) is a cytokine with inflammatory and regulatory properties produced by monocytes and activated T-cells. Levels of TNF-α secretion in vitro by peripheral blood mononuclear cells (PBMC) stimulated with bacterial lipopolysaccharide (LPS) orphorbol 12-myristate 13-acetate (PMA) plus concanavalin A (ConA) have been reported to vary among individuals and to be associated with HLA-DR alleles, namely HLA-DR2 with low levels of TNF-α secretion and HLA-DR3 and DR4 with high levels. -- In this study we have measured levels of TNF-α secreted by LPS-stimulated PBMC from 72 healthy young males. There was a large degree of variation in the level of secreted TNF-α and this interindividual variation was relatively stable in four individuals tested 2 to 4 times. The study panel was also typed for HLA-DR, -A, -B and -C antigens. A weak association was shown between HLA-DR3 and low levels of TNF-α secretion (p = 0.014, uncorrected). No association was shown between levels of TNF-α secretion and HLA-DR2, -DR4 or HLA-B antigens. -- We further explored the possibility that differences in levels of TNF-α secretion might be due to (1) individual differences in the kinetics of TNF-α secretion and/or (2) differences in the relative number of TNF-α secreting cells when different stimulants were used. The kinetics of 20μg/ml LPS-induced and 1ng/ml LPS-induced TNF-α secretion by PBMC were very similar but the total amounts secreted were higher with 20 μg/ml LPS. The kinetics of TNF-α secretion were quite different when PBMC were stimulated by LPS or ConA plus PMA. LPS-induced TNF-α peaked at 3 to 6 hours while ConA/PMA-induced TNF-α secretion did not reach an obvious peak in our experiments but was low at 3 hours and continued to rise at 53 hours. Five individuals were classified as high to low secretors using the 3-hour, 20 μg/ml LPS, stimulation protocol. This classification changed dramatically when the ConA/PMA, 53-hour protocol was used and three individuals classified high became low or vice versa. This is an important observation and must be considered when investigating associations between levels of TNF-α secretion and HLA antigens. -- To investigate if differences in secretion might be due to individual differences in the number of TNF-α secreting cells, a two-color immunofluorescence assay was developed to monitor cell phenotype and cytoplasmic TNF-α by flow cytometry. There was no consistent relationship between the proportion of CD14 monocytes in the samples staining positive for cytoplasmic TNF-α and the levels of secreted TNF-α by LPS-stimulated PBMC from two individuals tested, but this may have been due to technical difficulties. However, differential counts of the male study panel PBMC samples by flow cytometry for the subsets CD14 monocytes, CD4 T-cells, CD8 T-cells and CD45RO T-cells showed no correlation between the relative numbers of cells in each subset and secreted TNF-α levels.

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