CD14 and tolerance to lipopolysaccharide: biochemical and functional analysis

To evaluate the role of the high-affinity monocyte receptor for lipopolysaccharide (LPS), CD14, in the process of tolerance to LPS, the human monocytic cell line Mono-Mac-6 was cultured in the absence or presence of different amounts of LPS. The kinetics of CD14 modulation in these cells showed an initial 4-day period characterized by increased cell-surface expression, rate of biosynthesis (peaking at 48 hr) and release of its soluble forms (sCD14) which correlated with the amount of LPS in the culture. At this time, tolerance to LPS was already established, as measured by tumour necrosis factor-alpha (TNF-alpha) induction, it was LPS dose dependent and persisted up to 15 days. LPS also reduced the cell proliferation rate in a dose-dependent manner. After 8 days and up to 15 days, the CD14 biosynthesis, cell-surface expression and release of sCD14 inversely correlated with the level of LPS in the culture. The 48-hr LPS-pretreated cells showed a slightly decreased CD14 affinity for LPS, a relative high number of CD14 molecules per cells, and desensitization also to a phorbol 12-myristate 13-acetate (PMA) challenge. An anti-CD14 monoclonal antibody (mAb) protected the cells from tolerization when added at the beginning of culture, as revealed by challenge with LPS and PMA. The data indicate that in this model tolerization to LPS (1) precedes CD14 down-modulation, (2) operates by alteration of the receptor affinity for LPS and by a mechanism which affects a protein kinase C (PKC)-dependent signalling pathway, and (3) that CD14 plays a critical role in the establishment of tolerance to LPS. In addition, analysis of the data suggests the existence of a PKC-independent signalling pathway for LPS tolerization and a CD14-independent mechanism for establishing tolerance.