Hodge S, Hodge G, Flower R, Han P
University of South Australia; Haematology Department, Women's and Children's Hospital, Adelaide, South Australia.
Scand J Immunol. 1999 May;49(5):548-53. doi: 10.1046/j.1365-3083.1999.00538.x.
Glucocorticosteroids (GCS) have been used successfully in the treatment of inflammatory conditions such as asthma and acute graft-vs-host disease, but their mode of action remains unclear. There have been numerous reports of the in-vitro suppression of cytokine production by GCS based on quantitation of cytokines by ELISA on bulk supernatants from isolated cell culture systems. We report the use of a whole-blood intracellular cytokine assay which is more representative of an in-vivo environment. We examined the effects of GCS, prednisolone and dexamethasone, on cytokine production by individual cells (monocytes, T lymphocytes and natural killer or NK cells) in heterogenous cell populations. Cells in whole blood were activated with various stimuli: phorbol ester and calcium ionophore for T cells, Escherichia coli lipopolysaccharide (LPS) for monocytes, and phytohaemagglutinin (PHA) plus interleukin (IL)-12 for NK cells. Brefeldin A was used as an intracellular transport inhibitor to enhance the detection of intracellular cytokine production. The effects of various concentrations (10-5, 10-7, 10-9 and 10-11 m) of GCS on cytokine production were studied using multiparameter flow cytometry. After surface staining with fluorescently-conjugated monoclonal antibodies (MoAbs) to identify cell type, cells were fixed and permeabilised. Intracellular cytokines interferon (IFN)-gamma, IL-10, IL-1alpha and beta, IL-2, tumour necrosis factor (TNF)-alpha, and IL-12 were stained with their respective conjugated MoAbs. The GCS both caused a dose-dependent modulation of cytokine production by T cells, monocytes and NK cells. After 4 h, a decrease in the MFI (amount of cytokine produced per cell) was noted for all cell types. After 24 h a decrease in both MFI and the percentage of cells producing cytokine was observed for all cell types. The exception was monocyte production of IL-10 which was enhanced at low concentrations of GCS (10-9 and 10-11 m). Our findings thus suggest that one anti-inflammatory mechanism of GCS action may be through inhibition of the release of pro-inflammatory cytokines IL-1alpha and beta, IL-2, IFN-gamma and TNF-alpha, and up-regulation of the anti-inflammatory cytokine IL-10.
糖皮质激素（GCS）已成功用于治疗哮喘和急性移植物抗宿主病等炎症性疾病，但其作用方式仍不清楚。基于酶联免疫吸附测定（ELISA）对分离细胞培养系统的大量上清液中的细胞因子进行定量分析，已有大量关于GCS体外抑制细胞因子产生的报道。我们报告了一种全血细胞内细胞因子检测方法的应用，该方法更能代表体内环境。我们研究了GCS、泼尼松龙和地塞米松对异质细胞群体中单个细胞（单核细胞、T淋巴细胞和自然杀伤细胞或NK细胞）产生细胞因子的影响。全血中的细胞用各种刺激物激活：佛波酯和钙离子载体用于T细胞，大肠杆菌脂多糖（LPS）用于单核细胞，植物血凝素（PHA）加白细胞介素（IL）-12用于NK细胞。布雷菲德菌素A用作细胞内转运抑制剂，以增强细胞内细胞因子产生的检测。使用多参数流式细胞术研究了不同浓度（10-5、10-7、10-9和10-11 m）的GCS对细胞因子产生的影响。在用荧光共轭单克隆抗体（MoAbs）进行表面染色以识别细胞类型后，将细胞固定并通透化。细胞内细胞因子干扰素（IFN）-γ、IL-10、IL-1α和β、IL-2、肿瘤坏死因子（TNF）-α和IL-12用各自的共轭MoAbs进行染色。GCS对T细胞、单核细胞和NK细胞产生细胞因子均有剂量依赖性调节作用。4小时后，所有细胞类型的平均荧光强度（MFI，即每个细胞产生的细胞因子量）均下降。2小时后，所有细胞类型的MFI和产生细胞因子的细胞百分比均下降。例外的是单核细胞产生IL-10的情况，在低浓度GCS（10-9和10-11 m）时会增强。因此，我们的研究结果表明，GCS作用的一种抗炎机制可能是通过抑制促炎细胞因子IL-1α和β、IL-2、IFN-γ和TNF-α的释放，以及上调抗炎细胞因子IL-10。
