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Human blood monocytes synthesize but do not secrete IL-1 beta in response to low doses of bacterial cell-wall products. With this observation, we have developed a two-step staged release assay that separates IL-1 beta synthesis from secretion. This assay can be used to identify secretagogues or inhibitors of IL-1 beta secretion as well as the biochemical events leading to IL-1 beta release. Human blood monocytes are first treated with low (50 pg/ml) doses of LPS, which causes the synthesis of intracellular proIL-1 beta. Release of intracellular IL-1 beta can be induced by further treatment with 100 ng/ml of LPS or 1 x 10(6) CFU/ml of heat-killed Staphylococcus aureus. The amount and the efficiency of IL-1 beta secretion in the staged release assay was comparable with that of a standard method of treating blood monocytes with a single dose of 100 ng/ml LPS. Ongoing protein synthesis was not required for IL-1 beta secretion because mature IL-1 beta release occurred in the presence of the protein synthesis inhibitor cycloheximide. We have compared the effects of four different inhibitors of cytokine synthesis on IL-1 beta production in the standard and staged release assays. We find that dexamethasone or IL-10, when added together with 100 ng/ml LPS, inhibits IL-1 beta production with IC50 levels of 0.2 microM and 2.0 ng/ml, respectively. The IC50 levels increase greater than 50-fold when tested against monocytes pretreated with 50 pg/ml of LPS. These data suggest that dexamethasone and IL-10 have little effect on IL-1 beta secretion. Conversely, the IL-1 beta converting enzyme inhibitor, Ac-Tyr-Val-Ala-Asp-CHO (L-709,049) and the anti-inflammatory agent [5-(4-pyridyl)6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b)thiazole] (SKF 86002) inhibited IL-1 beta release in both the standard and staged release assays with IC50 of 1 microM. The data suggest that L-709,049 and SKF 86002 interfere with steps involved in IL-1 beta secretion, as opposed to synthesis. The results also document a novel method for delineating separate events in the pathway required for IL-1 beta biosynthesis and further distinguish two classes of compounds capable of modulating IL-1 beta secretion. |
