CD14-DEPENDENT INTERNALIZATION OF BACTERIAL LIPOPOLYSACCHARIDE (LPS) IS STRONGLY INFLUENCED BY LPS AGGREGATION BUT NOT BY CELLULAR-RESPONSES TO LPS

We analyzed the impact of ligand aggregation and LPS-induced signaling on CD14-dependent LPS internalization kinetics in human monocytic THP -1 cells and murine macrophages. Using two independent methods, we fou nd that the initial rate and extent of LPS internalization increased w ith LPS aggregate size. In the presence of LPS binding protein (LBP), large LPS aggregates were internalized extremely rapidly (70% of the t ell-associated LPS was internalized in 1 min). Smaller LPS aggregates were internalized more slowly than the larger aggregates, and LPS mono mers, complexed with soluble CD14 in the absence of LBP, were internal ized very slowly after binding to membrane CD14 (5% of the cell-associ ated LPS was internalized in 1 mini. In contrast, the initial aggregat ion state had little or no effect on the stimulatory potency of the LP S, Previous studies suggest that LPS-induced signal responses may infl uence the intracellular traffic and processing of LPS, We found that e licited peritoneal macrophages from LPS-responsive (C3H/HeN) and LPS-h yporesponsive (C3H/HeJ) mice internalized LPS with similar kinetics. I n addition, pre-exposure of THP-1 cells to LPS had no effect on their ability to internalize subsequently added LPS, and pre-exposure of the cells to the LPS-specific inhibitor, LA-IJ-PPI inhibited stimulation of the cells without inhibiting LPS internalization. In these cells, L PS is thus internalized by a constitutive cellular mechanism(s) with k inetics that depend importantly upon the physical state in which the L PS is presented to the cell.