Henjakovic M, Sewald K, Switalla S, Kaiser D, Müller M, Veres T Z, Martin C, Uhlig S, Krug N, Braun A
Fraunhofer Institute of Toxicology and Experimental Medicine, Department of Immunology, Allergology and Immunotoxicology, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany.
Toxicol Appl Pharmacol. 2008 Aug 15;231(1):68-76. doi: 10.1016/j.taap.2008.04.003. Epub 2008 Apr 11.
The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon gamma (IFNgamma), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1alpha, TNFalpha, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNgamma resulting in increased levels of TNFalpha, IL-12(p40), RANTES, and IL-1alpha. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.
本研究的目的是建立精密肺切片（PCLS），作为一种合适的体外替代方法，用于替代动物实验来研究免疫调节作用。为此，我们对PCLS与免疫活性物质脂多糖（LPS）、巨噬细胞激活脂肽-2（MALP-2）、干扰素γ（IFNγ）和地塞米松孵育后细胞因子产生的变化以及细胞表面标志物的表达进行了表征。通过测量乳酸脱氢酶（LDH）酶活性以及使用共聚焦显微镜进行活/死荧光染色，来控制野生型和CD11c增强型黄色荧光蛋白（CD11-EYFP）转基因小鼠的PCLS的活力。使用Luminex技术和酶联免疫吸附测定（ELISA）检测细胞因子和趋化因子。使用共聚焦显微镜在活体小鼠PCLS中原位研究抗原呈递细胞（APC）标志物。LPS在PCLS中引发了深刻的促炎作用。地塞米松可预防LPS诱导的PCLS中细胞因子/趋化因子如白细胞介素（IL）-5、IL-1α、肿瘤坏死因子α（TNFα）、IL-12（p40）和调节激活正常T细胞表达和分泌的趋化因子（RANTES）的产生。幼稚PCLS的APC中存在主要组织相容性复合体（MHC）II类、CD40和CD11c的表面表达，但不存在CD86的表面表达。与LPS孵育可特异性增强不同细胞上MHC II类的表达。MALP-2仅未能改变细胞因子或趋化因子水平，但与IFNγ联合使用时非常有效，导致TNFα、IL-12（p40）、RANTES和IL-1α水平升高。PCLS对典型的促炎刺激表现出特征性反应，因此可能提供一种合适的体外技术来预测吸入物质的免疫调节效力。
