Chin J, Kostura M J
Department of Cellular and Molecular Pharmacology, Merck Research Laboratories, Rahway, NJ 07065.
J Immunol. 1993 Nov 15;151(10):5574-85.
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] (SK&F 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 SK&F 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.
人血单核细胞在低剂量细菌细胞壁产物刺激下可合成但不分泌白细胞介素-1β（IL-1β）。基于这一观察结果，我们开发了一种两步分期释放测定法，该方法将IL-1β的合成与分泌区分开来。此测定法可用于鉴定IL-1β分泌的促分泌剂或抑制剂以及导致IL-1β释放的生化事件。首先用低剂量（<50 pg/ml）的脂多糖（LPS）处理人血单核细胞，这会导致细胞内前体IL-1β的合成。用100 ng/ml的LPS或1×10⁶CFU/ml的热灭活金黄色葡萄球菌进一步处理可诱导细胞内IL-1β的释放。分期释放测定法中IL-1β分泌的量和效率与用单剂量100 ng/ml LPS处理血单核细胞的标准方法相当。IL-1β的分泌不需要持续的蛋白质合成，因为在存在蛋白质合成抑制剂放线菌酮的情况下仍会发生成熟IL-1β的释放。我们在标准和分期释放测定法中比较了四种不同的细胞因子合成抑制剂对IL-1β产生的影响。我们发现，地塞米松或IL-10与100 ng/ml LPS一起添加时，分别以0.2 μM和2.0 ng/ml的IC50水平抑制IL-1β的产生。当针对用50 pg/ml LPS预处理的单核细胞进行测试时，IC50水平增加超过50倍。这些数据表明地塞米松和IL-10对IL-1β的分泌影响很小。相反，IL-1β转化酶抑制剂Ac-Tyr-Val-Ala-Asp-CHO（L-709,049）和抗炎剂[5-(4-吡啶基)6(4-氟苯基)-2,3-二氢咪唑并(2,1-b)噻唑]（SK&F 86002）在标准和分期释放测定法中均以1 μM的IC50抑制IL-1β的释放。数据表明L-709,049和SK&F 86002干扰了IL-1β分泌所涉及的步骤，而非合成步骤。结果还证明了一种用于描绘IL-1β生物合成所需途径中不同事件的新方法，并进一步区分了两类能够调节IL-1β分泌的化合物。
