FIG. 8.

LBP and sCD14 enhance monocyte stimulation by bacterial membranes; PLTP does not. (A) To prepare complexes of sCD14 with bleb or pLPS, 100 ng of pLPS or blebs containing 100 ng of LPS were incubated at 37°C for 30 min with 0.06 μg of LBP and 0.5 μg of sCD14. The mixtures were centrifuged onto 4 to 24% sucrose gradients, and aliquots of the top halves of the gradients were counted to measure the sCD14-associated [³H]LPS. As a control, blebs were added to a sucrose gradient without LBP and sCD14. Equal amounts of [³H]LPS from the three preparations were then diluted in serum-free medium and added to vitamin D₃-differentiated THP-1 cells (7 × 10⁵), followed by incubation for 2 h at 37°C. The cells were pelleted by centrifugation, and IL-8 was measured in the culture medium. (B) Blebs containing 100 ng of LPS were incubated at 37°C for 30 min with or without 0.03 μg of LBP, 0.5 μg of sCD14, or 0.5 μg of PLTP, and the mixtures were incubated with THP-1 cells as described above. IL-8 was measured in the culture supernatants. Symbols show the means of duplicate determinations. Each of these experiments was repeated with similar results.