In vitro study of pro-inflammatory and anti-tumour properties of microvesicles from bacterial cell wall of Pantoea agglomerans.

In the environment, Gram-negative bacteria are capable of producing large amounts of endotoxin-containing microvesicles - spherical structures measuring 30-50 nm in diameter, emerging by fragmentation of the outer membrane of the bacterial cell wall. Microvesicles are suspected of inducing inflammatory lung diseases, but possibly also of stimulating anti-tumour defence mechanisms. The present study was aimed at assessing the pro-inflammatory and anti-tumour properties of microvesicles in vitro. Peripheral blood mononuclear cells of 5 healthy volunteers were cultured for 6 h, 24 h, 3 days, and 5 days with microvesicles (MV) of Pantoea agglomerans at concentrations ranging from 0.48-1500 microg/ml. The following outcomes were measured: secretion of IFN-gamma and TNF-alpha (by ELISA and ELISpot), intensity of cell proliferation (LPT), expression of surface markers CD8, CD14, CD16, CD25, CD69, CD80, CD83, HLA-DR, and apoptosis markers (by flow cytometry). After 24 hours, a clear dose-dependent response to microvesicles was seen for IFN-gamma production, starting already at the lowest concentration of 0.48 microg/ml (p=0.04). A 2-fold increase in TNF-alpha production was seen after 3 days at the concentration of 1,500 microg/ml (p=0.05). A clear and significant dose-dependent increase in cell proliferation in response to MV was detectable after 5 days (p=0.001). A decrease in the percentage of CD14(+)CD83(+) monocytes was observed after 1 day of culture. We conclude that IFN-gamma and TNF-alpha are triggered at different concentrations of microvesicles: at lower concentrations only IFN-gamma is upregulated, whereas at higher concentrations both IFN-gamma and TNF-alpha are secreted.