DISSOCIATION OF IL-1-BETA SYNTHESIS AND SECRETION IN HUMAN BLOOD MONOCYTES STIMULATED WITH BACTERIAL-CELL WALL PRODUCTS

Human blood monocytes synthesize but do not secrete IL-1beta in respon se to low doses of bacterial cell-wall products. With this observation , we have developed a two-step staged release assay that separates IL- 1beta synthesis from secretion. This assay can be used to identify sec retagogues or inhibitors of IL-1beta secretion as well as the biochemi cal events leading to IL-1beta release. Human blood monocytes are firs t treated with low (<50 pg/ml) doses of LPS, which causes the synthesi s of intracellular proIL-1beta. Release of intracellular IL-1beta 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 efficienc y of IL-1beta secretion in the staged release assay was comparable wit h 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-1beta secretion because mature IL-1beta release occurred in the pre sence of the protein synthesis inhibitor cycloheximide. We have compar ed the effects of four different inhibitors of cytokine synthesis on I L-1beta production in the standard and staged release assays. We find that dexamethasone or IL-10, when added together with 100 ng/ml LPS, i nhibits IL-1beta production with IC50 levels of 0.2 muM and 2.0 ng/ml, respectively. The IC50 levels increase greater than 50-fold when test ed against monocytes pretreated with 50 pg/ml of LPS. These data sugge st that dexamethasone and IL-10 have littre effect on IL-1beta secreti on. Conversely, the IL-1beta converting enzyme inhibitor, Ac-Tyr-Val-A la-Asp-CHO (L-709,049) and the anti-inflammatory agent [5-(4-pyridyl)6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b)thiazole] (SK&F 86002) inhi bited IL-1beta release in both the standard and staged release assays with IC50 Of 1 muM. The data suggest that L-709,049 and SK&F 86002 int erfere with steps involved in IL-1beta secretion, as opposed to synthe sis. The results also document a novel method for delineating separate events in the pathway required for IL-1beta biosynthesis and further distinguish two classes of compounds capable of modulating IL-1beta se cretion.