The effect of lipopolysaccharide on the CD14 expression of human monocytic cell-lines.
Tamplin, Gareth Paul
Cardiff Metropolitan University
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Introduction: LPS is an essential component in the cell wall of gram-negative bacteria. The lipid-A moiety is the major determinant of LPS endotoxicity, whereas variation in the O-antigen accounts for numerous different LPS serotypes and phenotypes. Activation of TLR-4 receptors on human monocytic cells is essential in mediating the immune response to infection such as during meningitis. CDl4, found in both membrane-bound and soluble form, is an important component of the TLR-4 receptor complex involved in recognition of LPS and its subsequent presentation to TLR-4. Materials & Methods: THP1 and MM6 cell stocks were grown to 80% confluence in media containing FBS. A quantity of THPl cells were also differentiated using PMA. Cells were incubated for four hours or overnight with five strains of LPS from E.coli, S.typhimurium, or S.typhosa, four of which were smooth-type LPS, and one E.coli rough-type. Following incubation supernatants were removed and analysed for sCDl4 using ELISA. To the remaining cells Mouse IgGl isotype control-FITC was added to controls, and Mouse anti-human CDl4-FITC was added to samples prior to mCDl4 analysis via flow cytometry. Results: In terms of mCDl4 expression, non-differentiated THPl exhibited a time-dependent increase (P<0.05 four hours, p<0.01 overnight), PMA-differentiated THPl exhibited a time-dependent decrease (p<0.001 both time-points), and MM6 exhibited no significant difference after four hours (P=0.420), but increased overnight (p<0.001). In terms of sCDl4 non-differentiated THPl exhibited a time-dependent increase, PMA-differentiated THPI exhibited massively increased baseline levels which decreased overnight, and MM6 exhibited more sensitivity to Salmonella than E.coli at four hours which was not observed overnight. In all samples E.coli smooth-type produced higher CDl4 levels than rough-type. Discussion: Each cell type responded differently to LPS despite similar morphology, and these responses were similarly varied between strains. Non-differentiated THPl increased expression, as did MM6 albeit after longer incubation. It also appears that PMA-treatment reduces expression in THPI through cleavage of mCDl4. Some bacterial strains may cause early sCDl4 release in MM6 to remove LPS from the system and stimulate inflammatory responses in surrounding tissues in the interim period between exposure and macrophage activation. Further research should focus on the variable expression caused by rough- and smooth-type LPS, the differences between expression in THPI/MM6 cell-lines compared with whole-blood monocytes, discrimination between cleaved and intracellular sCD14, and the relationship between CDl4 expression and cytokine production.
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