COPD人肺成纤维细胞IL-6、IL-8、弹性蛋白的生成及其炎症调节的研究
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摘要
[目的]
成纤维细胞是肺的重要结构细胞,并在肺组织破坏后的修复和/或重构中起着重要作用。近年来认为成纤维细胞也参与炎症反应,例如成纤维细胞在风湿性关节炎等疾病中参与局部炎症反应网络的调控。目前关于肺成纤维细胞炎症反应和修复功能关系的相关研究较少。为此,本研究拟评价慢性阻塞性肺疾病(COPD)患者肺成纤维细胞炎症因子水平、增殖能力和弹性蛋白合成的情况；在此基础上深入探讨脂多糖(LPS)、肿瘤坏死因子-α(TNF-α)、凋亡细胞、坏死细胞等常见炎症刺激对人肺成纤维细胞分泌细胞因子和增殖功能的影响,并观察低剂量茶碱的调节作用。
[方法]
(1)从因肺癌等手术切除标本的正常肺组织中分离培养原代人肺成纤维细胞,建立细胞株库,取第2、3代细胞进行进一步实验和检测。所有组织及其应用按照伦理学要求获取知情同意签字后实施。根据术前肺功能将研究对象分为非COPD、COPDⅠ-Ⅲ级。通过对照研究评价COPD患者肺成纤维细胞白介素(IL)-6和IL-8的mRNA和蛋白水平、增殖能力以及弹性蛋白合成。
(2)将人肺成纤维细胞分别和1μg/ml LPS、1ng/ml TNF-α、凋亡细胞或坏死细胞共培养24h后收集培养液上清,并裂解细胞提取RNA保存备测。
(3)在人肺成纤维细胞培养中加入凋亡细胞或坏死细胞,通过多重免疫荧光染色共聚焦显像分析肺成纤维细胞的吞噬功能。
(4)采用逆转录实时定量聚合酶链反应(qRT-PCR)检测IL-6、IL-8、组蛋白去乙酰基酶2(HDAC2)和弹性蛋白的mRNA转录水平。
(5)使用酶联免疫吸附法(ELISA)测定细胞培养液上清中的TNF-α、IL-6、转化生长因子(TGF)-β1、TGF-β2、TGF-β3、IL-8、IL-12p70、IL-1β以及IL-10。
(6)使用Fastin Elastin Assay定量检测弹性蛋白浓度。
(7)用AlamarBlue(?) (AB)法检测LPS、TNF-α、IL-8、IL-6以及经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞增殖的影响。
(8)检测低剂量茶碱(5μg/ml)对LPS引起的肺成纤维细胞IL-6、IL-8分泌、HDAC2 mRNA转录以及增殖能力的影响。
(9)用GraphPad Prism 5.0 for Windows进行统计学分析。符合正态分布的数据以平均值±标准差表示；未通过正态分布检验的数据以中位数(最小值,最大值)表示,其中部分增殖实验结果以中位数(25%四分位数,75%四分位数)表示。根据是否正态分布以及统计分析类型选择相应的统计学方法。P<0.05为有显著性差异。
[结果]
(1)分离得到的原代细胞在显微镜下具有成纤维细胞的典型形态,细胞免疫荧光染色显示99%以上的细胞表达波形蛋白；而细胞角蛋白、von Willebrand因子、结蛋白染色阴性则证明无上皮细胞、间皮细胞、内皮细胞或平滑肌细胞混杂,说明原代分离的人肺成纤维细胞纯度高。
(2)在COPD人肺成纤维细胞研究部分,根据肺功能情况分为COPD s组(包括GOLDⅡ、Ⅲ级)和对照组(包括非COPD和GOLDⅠ级),两组在年龄、性别、吸烟史方面无显著性差异。COPDs组(12例)和对照组(10例)FEV1占预计值%分别为(59.8±9.5)%和(92.3±8.4)%,FEV1/FVC%分别为(54.9±8.3)%和(73.6±10.8)%,P值均<0.001。
(3) qRT-PCR显示COPDs组人肺成纤维细胞IL-6和IL-8的mRNA转录均显著高于对照组,与之相对应的,其蛋白分泌量也明显升高,分别为(701.6±288.0)pg/ml vs (355.7±330.8) pg/ml (P=0.02)和(754.3±297.4) pg/ml vs (373.4±243.1) pg/ml (P=0.02)。IL-6、IL-8的转录及分泌水平和FEV1占预计值%和FEV1/FVC%均呈负相关。
(4) COPDs组人肺成纤维细胞体外增殖能力弱于对照组(1.18±0.32 vs1.63±0.22,P=0.004)。
(5) COPDs组人肺成纤维细胞弹性蛋白mRNA代偿性表达升高[6.194(3.118,33.000) vs 2.241(0.916,22.140),P=0.0276],伴随可溶性弹性蛋白升高[(26.40±6.50)μg/ml vs (15.00±3.90)μg/ml, P=0.001)],但是不可溶性弹性蛋白无差别。
(6)在1μg/ml LPS刺激24h后人肺成纤维细胞分泌IL-6的水平从(1156±455)pg/ml上升至(1535±439) pg/ml (P=0.0046); IL-8从(510.5±170.6) pg/ml上升至(856.5±418.8) pg/ml (P=0.0122); TGF-β1从(1188±623) pg/ml上升至(1773±847) pg/ml (P=0.0038)。IL-1β、TNF-α、IL-12p70、IL-10和TGF-β2基础分泌较低,TGF-β3测不到；其中TGF-β2有较大幅度上升(P=0.0274),IL-1β有小幅度升高(P=0.0027),IL-10从(37.6±17.9) pg/ml下降至(25.1±7.5) pg/ml (P=0.0382),而TNF-α和IL-12p70则基本无改变。
(7)加入1ng/ml TNF-α培养24h后体外人肺成纤维细胞也出现了和LPS刺激相仿的反应,其中以IL-8上升尤为明显,具体表现为IL-6从(1066±305) pg/ml上升至(1398±466) pg/ml (P=0.0358)、IL-8从(532.3±71.7) pg/ml上升至(1660.0±389.2) pg/ml (P=0.0009)、TGF-β1从(218.9±32.6) pg/ml上升至(247.9±29.4) pg/ml (P=0.0495)。
(8) qRT-PCR结果显示HDAC2 mRNA在人肺成纤维细胞的相对表达量在LPS刺激后从3.546(0.511,8.886)下降至1.793(0.174,3.284)(P=0.0355)；TNF-α刺激后HDAC2 mRNA表达略下降,从1.057(0.090,8.886)至0.933(0.073,2.965),但无显著性差异(P=0.0625)。
(9)人肺成纤维细胞具有吞噬周围的凋亡细胞和坏死细胞的能力,并在和凋亡细胞接触24h后IL-6从(991.9±463.8) pg/ml上升至(1616.0±216.5) pg/ml(P<0.01)。和坏死细胞共培养24h后,则出现IL-8从(371.5±57.74) pg/ml上升至(740.0±204.7) pg/ml (P＜0.01); IL-1β和IL-10基础分泌水平较低,前者上升,而后者有下降,P值均小于0.05。
(10)LPS共培养48h对人肺成纤维细胞的增殖有抑制作用,并且这种抑制作用随着浓度和时间的增加而增加。当浓度达到0.1μg/ml时,抑制幅度为4%左右(P=0.0306)；浓度为1μg/ml时抑制幅度约为8%(P=0.0112)；作用时间达到24h时抑制作用开始具有显著性差异。TNF-α对体外人肺成纤维细胞的增殖功能的抑制作用有剂量依赖效应,10ng/ml时抑制程度约为10%(P=0.0241)；IL-8和IL-6具有相似效应。经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞的增殖能力有抑制作用(P<0.05)。
(11)在低剂量茶碱作用研究部分,IL-6从无干预对照组的(1150±426) pg/ml上升至LPS组的(1559±406) pg/ml (P＜0.001);而茶碱+LPS组为(1336±400)pg/ml,较LPS组低(P<0.05)。IL-8的趋势和IL-6相似,空白对照组、LPS组、茶碱+LPS组分别为(560±205)pg/ml、(1087±359) pg/ml和(933±338)pg/ml。但是5μg/ml茶碱不影响细胞的HDAC2 mRNA水平,对1μg/ml LPS引起的HDAC2 mRNA下调也无作用。在0.1-10μg/ml的浓度范围内茶碱对人肺成纤维细胞的增殖能力无影响,而在LPS刺激的同时加入5μg/ml茶碱可以改善LPS导致的细胞增殖抑制(P<0.05)。
[结论]
(1) COPD患者肺成纤维细胞IL-6和IL-8的合成分泌上调,其水平和肺功能呈负相关；伴随着细胞增殖功能受损以及可溶性弹性蛋白向不可溶性弹性蛋白转化的障碍,说明人肺成纤维细胞参与COPD的炎症反应,同时修复功能受损。
(2)在受到LPS和TNF-α刺激后,体外人肺成纤维细胞出现以IL-6、IL-8、TGF-β1升高为特征的改变；人肺成纤维细胞能够吞噬凋亡细胞和坏死细胞,并释放不同的细胞因子,前者以IL-6分泌上升为主,而后者表现为IL-8、IL-1β上升、IL-10下调,这提示人肺成纤维细胞可以随着局部环境的变化释放不同的细胞因子从而参与炎症反应的调节。LPS引起的细胞因子释放和HDAC2转录下调有关。
(3) LPS、TNF-α、IL-6、IL-8以及经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞增殖功能有抑制作用,这说明LPS可以直接或者通过炎性细胞因子以自分泌和旁分泌的形式抑制人肺成纤维细胞的增殖能力。
(4)低剂量茶碱能够部分阻断LPS引起的人肺成纤维细胞IL-6和IL-8分泌,并改善其对增殖能力的抑制。
OBJECTIVE
Fibroblasts are major cells responsible for the production and maintenance of extracellular matrix. Apart from the functions in tissue repair and reconstruction, recent research has demonstrated that fibroblasts participate in the modulation of local inflammation in diseases associated with chronic inflammation such as rheumatic arthritis. Regarding the relationship between the inflammatory response and injury repair capacity of human lung fibroblasts, data is limited. The current study therefore was designed to investigate the cytokine secretion, cellular proliferation and elatin synthesis of lung fibroblasts from subjects with chronic obstructive pulmonary disease (COPD). Furthermore, the response of primary human lung fibroblasts to the common inflammatory stimuli including lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-a), apoptosis cells and necrotic cells were studied. In addition, the effect of low-dose theophylline on the LPS initiated alterations in lung fibroblasts was analyzed.
METHODS
(1) Primary human lung fibroblasts were isolated and cultured from surgical peripheral lesion free lung tissue for further in vitro investigation. Samples were collected after written informed consent for the acquisition of material for research was obtained according to the ethnical principles. Based on preoperative lung function, the subjects were divided into non-COPD, COPD stageⅠ-Ⅲ. The mRNA and protein levels of IL-6 and IL-8 as well as proliferatory fuction and elatin systhesis of COPD lung fibroblasts were analysed using case-control design.
(2) For the evaluation of the response to inflammatory stimuli, the primary human lung fibroblasts were co-cultured with 1μg/ml LPS, 1ng/ml TNF-α, apoptosis cells or necrotic cells for 24h resrespectively, and then the supernatants and RNA were obtained and kept for further analysis.
(3) Multiple fluorescent staining and confocal microscopic technique was used to evaluate the phagocytic function of primary human lung fibroblasts.
(4) The concentration of TNF-α, interleukin (IL)-6, IL-8, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, IL-12p70, IL-1βand IL-10 in supernatants were examined by enzyme linked immunosorbent assay (ELISA).
(5) The mRNA transcription of IL-6, IL-8, histone deacetylase (HDAC) 2 and elastin was analyzed using reverse-transcription real-time quantitative polymerase chain reaction (qRT-PCR).
(6) Insoluble and soluble elastin in cell culture was quantitied by Fastin Elastin Assay.
(7) The AlamarBlue(?) (AB) assay was used for measuring cellular proliferation. The alterations in human lung fibroblast proliferation were examined after exposure to LPS, TNF-α, IL-8, IL-6 as well as conditioned medium from cultured macrophages that had been pretreated with LPS.
(8) The effect of theophylline at the concentration of 5μg/ml on the IL-6 and IL-8 secretion, HDAC2 mRNA regulation and proliferation alteration induced by LPS was analyzed.
(9) Statistics were performed using Prism 5 for windows. For normally distributed data, results are expressed as mean±SD. For abnormally distributed data, results are expressed as medians (range), with the exception of some proliferation data which are expressed as medians (25th percentile,75th percentile). Statistical methods were used for relavant kind of data. Differences were considered significant at the level of P<0.05.
RESULTS
(1) The primary cultured cells appeared to have typical morphology of fibroblast and immunostaining showed that>99% of the cells were vimentin positive. Staining with antibodies to cytokeratin, vonWillebrand factor, and desmin was negative, indicating that the cultures did not contain significant numbers of epithelial or mesothelial cells, endothelial cells, or smooth muscle cells.
(2) Totally 22 subjects were included and divided into two groups, i.e. Controls (10 cases, including non COPD and GOLD stage 1 COPD) and COPDs (12 cases, including GOLD stage 2 and 3). The two groups were similar in age, gender and smoking status. The two groups differed significantly in lung function. As expected, the subjects in COPDs group had lower FEV1% predicted and FEV1/FVC% (p<0.001), with values as (59.8±9.5)% vs (92.3±8.4)% and (54.9±8.3)% vs (73.6±10.8)% respectively.
(3) There was a significant increase in the expression of mRNA for IL-6 and IL-8 for human fibroblasts in COPDs group. In addition, protein levels were increased in the supernatants from the human fibroblasts in COPDs group compared with controls for IL-6 [(701.6±288.0) pg/ml vs (355.7±330.8) pg/ml, p=0.02] and for IL-8 [(754.3±297.4)pg/ml vs (373.4±243.1) pg/ml, p=0.02]. The mRNA expression and protein production of IL-6 and IL-8 was negatively correlated with FEV1 pred% and FEV1/FVC%.
(4) The proliferation was lower in the human lung fibroblasts from COPDs group (1.18±0.32) than that from controls group (1.63±0.22) (p=0.004).
(5) A significantly higher mRNA expression for elastin and protein level for soluble elatin was observed in human lung fibroblasts from COPDs group, however, no change was seen for insoluble elastin.
(6) After exposure to 1μg/ml LPS for 24h, the concentration of IL-6 in human lung fibroblast supernatants increased from (1156±455) pg/ml to (1535±439) pg/ml (p=0.0046). A similar increase was observed for IL-8 [from (510.5±170.6) pg/ml to (856.5±418.8) pg/ml, p=0.0122] and TGF-β1 [(1188±623) pg/ml to (1773±847) pg/ml, p=0.0038]. The constitutional secretion of IL-1β、TNF-α、IL-12p70、IL-10 and TGF-β2 was low and TGF-β3 was undetectable. The level of TGF-β2 also increased by almost 50% with p value being 0.0274, while a small increase for IL-1β(p=0.0027). In contrast, IL-10, an anti-inflammatory cytokine, decreased from (37.6±17.9) pg/ml to (25.1±7.5) pg/ml (p=0.0382). No alteration was observed for TNF-a and IL-12p70.
(7) In response to 24-hour incubation of 1ng/ml TNF-α, the IL-8 release by human lung fibroblasts increased by 2 folds from (532.3±71.7) pg/ml to (1660.0±389.2) pg/ml (p=0.0009). The concentration of IL-6 and TGF-β1 also went up.
(8) A LPS induced down-regulation of HDAC2 mRNA in human lung fibroblast was shown by qRT-PCR, with the relative expression from 3.546 (0.511,8.886) to 1.793 (0.174,3.284) (p=0.0355). Atrend of down-regulation by TNF-αtreatment was also observed without statistical significance (p=0.0625).
(9) A process of phagocytosis with apoptosis cells and necrotic cells was observed under confocal microscope. The apoptosis cells led to an increase of IL-6 in human lung fibroblasts, with the values from (991.9±463.8) pg/ml to (1616.0±216.5) pg/ml (p<0.01). For necrotic cells, a different cytokine profile was presented. Briefly, IL-8 and IL-1βwas increased and IL-10 was decreased.
(10) LPS appeared to have an inhibitory effect on the human lung fibroblast proliferation. The inhibition was dose-dependent and time-dependent. The inhibition became significant (p<0.05) when the concentration of LPS reached 0.1μg/ml or incubation period was longer than 24h. The proliferation was inhibited by about 8%(p=0.0112) with the concentration at 1μg/ml for 24-48h. TNF-α, IL-6 and IL-8 had a significant dose-dependent inhibitory effect on the proliferation of human lung fibroblasts. The proliferation of human lung fibroblasts was consistently inhibited by LPS-pretreated macrophage conditioned medium (p<0.05).
(11)Human lung fibroblasts were treated with 1μg/ml LPS or 1μg/ml LPS+5μg/ml theophylline or without treatment. The concentration of IL-6 for control, LPS treatment and LPS+theophylline treatment was (1150±426) pg/ml, (1559±406) pg/ml and (1336±400) pg/ml respectively (p<0.05). For IL-8, the value for different treatment was (560±205) pg/ml, (1087±359) pg/ml and (933±338) pg/ml (p<0.05). However, the HDAC2 mRNA was not altered by low-dose theophylline. Theophylline did not affect the fibroblast proliferation at the concentration of 0.1-10μg/ml. The theophylline of 5μg/ml alleviated the inhibition by LPS on proliferation (P<0.05).
CONCLUSIONS
(1) Primary human lung fibroblasts from subjects with COPD have an enhanced production of IL-6 and IL-8, associated with a profile of impaired proliferation and transformation from soluable elastin to insoluble elatin. This indicates that the up-regulated inflammation in lung fibroblasts is related to its insufficiency in injury repair function.
(2) The current data demonstrate that human lung fibroblasts have increased IL-6, IL-8 and TGF-β1 release in response to LPS and TNF-α, accompanied with a down-regulation of HDAC2 transcription. In addition, the fibroblasts responses differently in cytokines production to different inflammatory stimulus, indicating that the fibroblasts could participate in the modulation of pulmonary inflammation by producing relevant cytokines.
(3) LPS, TNF-α, IL-6, IL-8 and conditioned medium from LPS-pretreated macrophages can inhibit the proliferation of in vitro human lung fibroblasts, suggesting that the inflammation in milieu exert an effect on the function of lung fibroblasts.
(4) Low-dose theophylline can partly block the increased production of IL-6 and IL-8 as well as inhibited proliferation initiated by LPS.
成纤维细胞是肺的重要结构细胞,并在肺组织破坏后的修复和/或重构中起着重要作用。近年来认为成纤维细胞也参与炎症反应,例如成纤维细胞在风湿性关节炎等疾病中参与局部炎症反应网络的调控。目前关于肺成纤维细胞炎症反应和修复功能关系的相关研究较少。为此,本研究拟评价慢性阻塞性肺疾病(COPD)患者肺成纤维细胞炎症因子水平、增殖能力和弹性蛋白合成的情况；在此基础上深入探讨脂多糖(LPS)、肿瘤坏死因子-α(TNF-α)、凋亡细胞、坏死细胞等常见炎症刺激对人肺成纤维细胞分泌细胞因子和增殖功能的影响,并观察低剂量茶碱的调节作用。
[方法]
(1)从因肺癌等手术切除标本的正常肺组织中分离培养原代人肺成纤维细胞,建立细胞株库,取第2、3代细胞进行进一步实验和检测。所有组织及其应用按照伦理学要求获取知情同意签字后实施。根据术前肺功能将研究对象分为非COPD、COPDⅠ-Ⅲ级。通过对照研究评价COPD患者肺成纤维细胞白介素(IL)-6和IL-8的mRNA和蛋白水平、增殖能力以及弹性蛋白合成。
(2)将人肺成纤维细胞分别和1μg/ml LPS、1ng/ml TNF-α、凋亡细胞或坏死细胞共培养24h后收集培养液上清,并裂解细胞提取RNA保存备测。
(3)在人肺成纤维细胞培养中加入凋亡细胞或坏死细胞,通过多重免疫荧光染色共聚焦显像分析肺成纤维细胞的吞噬功能。
(4)采用逆转录实时定量聚合酶链反应(qRT-PCR)检测IL-6、IL-8、组蛋白去乙酰基酶2(HDAC2)和弹性蛋白的mRNA转录水平。
(5)使用酶联免疫吸附法(ELISA)测定细胞培养液上清中的TNF-α、IL-6、转化生长因子(TGF)-β1、TGF-β2、TGF-β3、IL-8、IL-12p70、IL-1β以及IL-10。
(6)使用Fastin Elastin Assay定量检测弹性蛋白浓度。
(7)用AlamarBlue(?) (AB)法检测LPS、TNF-α、IL-8、IL-6以及经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞增殖的影响。
(8)检测低剂量茶碱(5μg/ml)对LPS引起的肺成纤维细胞IL-6、IL-8分泌、HDAC2 mRNA转录以及增殖能力的影响。
(9)用GraphPad Prism 5.0 for Windows进行统计学分析。符合正态分布的数据以平均值±标准差表示；未通过正态分布检验的数据以中位数(最小值,最大值)表示,其中部分增殖实验结果以中位数(25%四分位数,75%四分位数)表示。根据是否正态分布以及统计分析类型选择相应的统计学方法。P<0.05为有显著性差异。
[结果]
(1)分离得到的原代细胞在显微镜下具有成纤维细胞的典型形态,细胞免疫荧光染色显示99%以上的细胞表达波形蛋白；而细胞角蛋白、von Willebrand因子、结蛋白染色阴性则证明无上皮细胞、间皮细胞、内皮细胞或平滑肌细胞混杂,说明原代分离的人肺成纤维细胞纯度高。
(2)在COPD人肺成纤维细胞研究部分,根据肺功能情况分为COPD s组(包括GOLDⅡ、Ⅲ级)和对照组(包括非COPD和GOLDⅠ级),两组在年龄、性别、吸烟史方面无显著性差异。COPDs组(12例)和对照组(10例)FEV1占预计值%分别为(59.8±9.5)%和(92.3±8.4)%,FEV1/FVC%分别为(54.9±8.3)%和(73.6±10.8)%,P值均<0.001。
(3) qRT-PCR显示COPDs组人肺成纤维细胞IL-6和IL-8的mRNA转录均显著高于对照组,与之相对应的,其蛋白分泌量也明显升高,分别为(701.6±288.0)pg/ml vs (355.7±330.8) pg/ml (P=0.02)和(754.3±297.4) pg/ml vs (373.4±243.1) pg/ml (P=0.02)。IL-6、IL-8的转录及分泌水平和FEV1占预计值%和FEV1/FVC%均呈负相关。
(4) COPDs组人肺成纤维细胞体外增殖能力弱于对照组(1.18±0.32 vs1.63±0.22,P=0.004)。
(5) COPDs组人肺成纤维细胞弹性蛋白mRNA代偿性表达升高[6.194(3.118,33.000) vs 2.241(0.916,22.140),P=0.0276],伴随可溶性弹性蛋白升高[(26.40±6.50)μg/ml vs (15.00±3.90)μg/ml, P=0.001)],但是不可溶性弹性蛋白无差别。
(6)在1μg/ml LPS刺激24h后人肺成纤维细胞分泌IL-6的水平从(1156±455)pg/ml上升至(1535±439) pg/ml (P=0.0046); IL-8从(510.5±170.6) pg/ml上升至(856.5±418.8) pg/ml (P=0.0122); TGF-β1从(1188±623) pg/ml上升至(1773±847) pg/ml (P=0.0038)。IL-1β、TNF-α、IL-12p70、IL-10和TGF-β2基础分泌较低,TGF-β3测不到；其中TGF-β2有较大幅度上升(P=0.0274),IL-1β有小幅度升高(P=0.0027),IL-10从(37.6±17.9) pg/ml下降至(25.1±7.5) pg/ml (P=0.0382),而TNF-α和IL-12p70则基本无改变。
(7)加入1ng/ml TNF-α培养24h后体外人肺成纤维细胞也出现了和LPS刺激相仿的反应,其中以IL-8上升尤为明显,具体表现为IL-6从(1066±305) pg/ml上升至(1398±466) pg/ml (P=0.0358)、IL-8从(532.3±71.7) pg/ml上升至(1660.0±389.2) pg/ml (P=0.0009)、TGF-β1从(218.9±32.6) pg/ml上升至(247.9±29.4) pg/ml (P=0.0495)。
(8) qRT-PCR结果显示HDAC2 mRNA在人肺成纤维细胞的相对表达量在LPS刺激后从3.546(0.511,8.886)下降至1.793(0.174,3.284)(P=0.0355)；TNF-α刺激后HDAC2 mRNA表达略下降,从1.057(0.090,8.886)至0.933(0.073,2.965),但无显著性差异(P=0.0625)。
(9)人肺成纤维细胞具有吞噬周围的凋亡细胞和坏死细胞的能力,并在和凋亡细胞接触24h后IL-6从(991.9±463.8) pg/ml上升至(1616.0±216.5) pg/ml(P<0.01)。和坏死细胞共培养24h后,则出现IL-8从(371.5±57.74) pg/ml上升至(740.0±204.7) pg/ml (P＜0.01); IL-1β和IL-10基础分泌水平较低,前者上升,而后者有下降,P值均小于0.05。
(10)LPS共培养48h对人肺成纤维细胞的增殖有抑制作用,并且这种抑制作用随着浓度和时间的增加而增加。当浓度达到0.1μg/ml时,抑制幅度为4%左右(P=0.0306)；浓度为1μg/ml时抑制幅度约为8%(P=0.0112)；作用时间达到24h时抑制作用开始具有显著性差异。TNF-α对体外人肺成纤维细胞的增殖功能的抑制作用有剂量依赖效应,10ng/ml时抑制程度约为10%(P=0.0241)；IL-8和IL-6具有相似效应。经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞的增殖能力有抑制作用(P<0.05)。
(11)在低剂量茶碱作用研究部分,IL-6从无干预对照组的(1150±426) pg/ml上升至LPS组的(1559±406) pg/ml (P＜0.001);而茶碱+LPS组为(1336±400)pg/ml,较LPS组低(P<0.05)。IL-8的趋势和IL-6相似,空白对照组、LPS组、茶碱+LPS组分别为(560±205)pg/ml、(1087±359) pg/ml和(933±338)pg/ml。但是5μg/ml茶碱不影响细胞的HDAC2 mRNA水平,对1μg/ml LPS引起的HDAC2 mRNA下调也无作用。在0.1-10μg/ml的浓度范围内茶碱对人肺成纤维细胞的增殖能力无影响,而在LPS刺激的同时加入5μg/ml茶碱可以改善LPS导致的细胞增殖抑制(P<0.05)。
[结论]
(1) COPD患者肺成纤维细胞IL-6和IL-8的合成分泌上调,其水平和肺功能呈负相关；伴随着细胞增殖功能受损以及可溶性弹性蛋白向不可溶性弹性蛋白转化的障碍,说明人肺成纤维细胞参与COPD的炎症反应,同时修复功能受损。
(2)在受到LPS和TNF-α刺激后,体外人肺成纤维细胞出现以IL-6、IL-8、TGF-β1升高为特征的改变；人肺成纤维细胞能够吞噬凋亡细胞和坏死细胞,并释放不同的细胞因子,前者以IL-6分泌上升为主,而后者表现为IL-8、IL-1β上升、IL-10下调,这提示人肺成纤维细胞可以随着局部环境的变化释放不同的细胞因子从而参与炎症反应的调节。LPS引起的细胞因子释放和HDAC2转录下调有关。
(3) LPS、TNF-α、IL-6、IL-8以及经LPS预处理的巨噬细胞条件培养液对人肺成纤维细胞增殖功能有抑制作用,这说明LPS可以直接或者通过炎性细胞因子以自分泌和旁分泌的形式抑制人肺成纤维细胞的增殖能力。
(4)低剂量茶碱能够部分阻断LPS引起的人肺成纤维细胞IL-6和IL-8分泌,并改善其对增殖能力的抑制。
OBJECTIVE
Fibroblasts are major cells responsible for the production and maintenance of extracellular matrix. Apart from the functions in tissue repair and reconstruction, recent research has demonstrated that fibroblasts participate in the modulation of local inflammation in diseases associated with chronic inflammation such as rheumatic arthritis. Regarding the relationship between the inflammatory response and injury repair capacity of human lung fibroblasts, data is limited. The current study therefore was designed to investigate the cytokine secretion, cellular proliferation and elatin synthesis of lung fibroblasts from subjects with chronic obstructive pulmonary disease (COPD). Furthermore, the response of primary human lung fibroblasts to the common inflammatory stimuli including lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-a), apoptosis cells and necrotic cells were studied. In addition, the effect of low-dose theophylline on the LPS initiated alterations in lung fibroblasts was analyzed.
METHODS
(1) Primary human lung fibroblasts were isolated and cultured from surgical peripheral lesion free lung tissue for further in vitro investigation. Samples were collected after written informed consent for the acquisition of material for research was obtained according to the ethnical principles. Based on preoperative lung function, the subjects were divided into non-COPD, COPD stageⅠ-Ⅲ. The mRNA and protein levels of IL-6 and IL-8 as well as proliferatory fuction and elatin systhesis of COPD lung fibroblasts were analysed using case-control design.
(2) For the evaluation of the response to inflammatory stimuli, the primary human lung fibroblasts were co-cultured with 1μg/ml LPS, 1ng/ml TNF-α, apoptosis cells or necrotic cells for 24h resrespectively, and then the supernatants and RNA were obtained and kept for further analysis.
(3) Multiple fluorescent staining and confocal microscopic technique was used to evaluate the phagocytic function of primary human lung fibroblasts.
(4) The concentration of TNF-α, interleukin (IL)-6, IL-8, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, IL-12p70, IL-1βand IL-10 in supernatants were examined by enzyme linked immunosorbent assay (ELISA).
(5) The mRNA transcription of IL-6, IL-8, histone deacetylase (HDAC) 2 and elastin was analyzed using reverse-transcription real-time quantitative polymerase chain reaction (qRT-PCR).
(6) Insoluble and soluble elastin in cell culture was quantitied by Fastin Elastin Assay.
(7) The AlamarBlue(?) (AB) assay was used for measuring cellular proliferation. The alterations in human lung fibroblast proliferation were examined after exposure to LPS, TNF-α, IL-8, IL-6 as well as conditioned medium from cultured macrophages that had been pretreated with LPS.
(8) The effect of theophylline at the concentration of 5μg/ml on the IL-6 and IL-8 secretion, HDAC2 mRNA regulation and proliferation alteration induced by LPS was analyzed.
(9) Statistics were performed using Prism 5 for windows. For normally distributed data, results are expressed as mean±SD. For abnormally distributed data, results are expressed as medians (range), with the exception of some proliferation data which are expressed as medians (25th percentile,75th percentile). Statistical methods were used for relavant kind of data. Differences were considered significant at the level of P<0.05.
RESULTS
(1) The primary cultured cells appeared to have typical morphology of fibroblast and immunostaining showed that>99% of the cells were vimentin positive. Staining with antibodies to cytokeratin, vonWillebrand factor, and desmin was negative, indicating that the cultures did not contain significant numbers of epithelial or mesothelial cells, endothelial cells, or smooth muscle cells.
(2) Totally 22 subjects were included and divided into two groups, i.e. Controls (10 cases, including non COPD and GOLD stage 1 COPD) and COPDs (12 cases, including GOLD stage 2 and 3). The two groups were similar in age, gender and smoking status. The two groups differed significantly in lung function. As expected, the subjects in COPDs group had lower FEV1% predicted and FEV1/FVC% (p<0.001), with values as (59.8±9.5)% vs (92.3±8.4)% and (54.9±8.3)% vs (73.6±10.8)% respectively.
(3) There was a significant increase in the expression of mRNA for IL-6 and IL-8 for human fibroblasts in COPDs group. In addition, protein levels were increased in the supernatants from the human fibroblasts in COPDs group compared with controls for IL-6 [(701.6±288.0) pg/ml vs (355.7±330.8) pg/ml, p=0.02] and for IL-8 [(754.3±297.4)pg/ml vs (373.4±243.1) pg/ml, p=0.02]. The mRNA expression and protein production of IL-6 and IL-8 was negatively correlated with FEV1 pred% and FEV1/FVC%.
(4) The proliferation was lower in the human lung fibroblasts from COPDs group (1.18±0.32) than that from controls group (1.63±0.22) (p=0.004).
(5) A significantly higher mRNA expression for elastin and protein level for soluble elatin was observed in human lung fibroblasts from COPDs group, however, no change was seen for insoluble elastin.
(6) After exposure to 1μg/ml LPS for 24h, the concentration of IL-6 in human lung fibroblast supernatants increased from (1156±455) pg/ml to (1535±439) pg/ml (p=0.0046). A similar increase was observed for IL-8 [from (510.5±170.6) pg/ml to (856.5±418.8) pg/ml, p=0.0122] and TGF-β1 [(1188±623) pg/ml to (1773±847) pg/ml, p=0.0038]. The constitutional secretion of IL-1β、TNF-α、IL-12p70、IL-10 and TGF-β2 was low and TGF-β3 was undetectable. The level of TGF-β2 also increased by almost 50% with p value being 0.0274, while a small increase for IL-1β(p=0.0027). In contrast, IL-10, an anti-inflammatory cytokine, decreased from (37.6±17.9) pg/ml to (25.1±7.5) pg/ml (p=0.0382). No alteration was observed for TNF-a and IL-12p70.
(7) In response to 24-hour incubation of 1ng/ml TNF-α, the IL-8 release by human lung fibroblasts increased by 2 folds from (532.3±71.7) pg/ml to (1660.0±389.2) pg/ml (p=0.0009). The concentration of IL-6 and TGF-β1 also went up.
(8) A LPS induced down-regulation of HDAC2 mRNA in human lung fibroblast was shown by qRT-PCR, with the relative expression from 3.546 (0.511,8.886) to 1.793 (0.174,3.284) (p=0.0355). Atrend of down-regulation by TNF-αtreatment was also observed without statistical significance (p=0.0625).
(9) A process of phagocytosis with apoptosis cells and necrotic cells was observed under confocal microscope. The apoptosis cells led to an increase of IL-6 in human lung fibroblasts, with the values from (991.9±463.8) pg/ml to (1616.0±216.5) pg/ml (p<0.01). For necrotic cells, a different cytokine profile was presented. Briefly, IL-8 and IL-1βwas increased and IL-10 was decreased.
(10) LPS appeared to have an inhibitory effect on the human lung fibroblast proliferation. The inhibition was dose-dependent and time-dependent. The inhibition became significant (p<0.05) when the concentration of LPS reached 0.1μg/ml or incubation period was longer than 24h. The proliferation was inhibited by about 8%(p=0.0112) with the concentration at 1μg/ml for 24-48h. TNF-α, IL-6 and IL-8 had a significant dose-dependent inhibitory effect on the proliferation of human lung fibroblasts. The proliferation of human lung fibroblasts was consistently inhibited by LPS-pretreated macrophage conditioned medium (p<0.05).
(11)Human lung fibroblasts were treated with 1μg/ml LPS or 1μg/ml LPS+5μg/ml theophylline or without treatment. The concentration of IL-6 for control, LPS treatment and LPS+theophylline treatment was (1150±426) pg/ml, (1559±406) pg/ml and (1336±400) pg/ml respectively (p<0.05). For IL-8, the value for different treatment was (560±205) pg/ml, (1087±359) pg/ml and (933±338) pg/ml (p<0.05). However, the HDAC2 mRNA was not altered by low-dose theophylline. Theophylline did not affect the fibroblast proliferation at the concentration of 0.1-10μg/ml. The theophylline of 5μg/ml alleviated the inhibition by LPS on proliferation (P<0.05).
CONCLUSIONS
(1) Primary human lung fibroblasts from subjects with COPD have an enhanced production of IL-6 and IL-8, associated with a profile of impaired proliferation and transformation from soluable elastin to insoluble elatin. This indicates that the up-regulated inflammation in lung fibroblasts is related to its insufficiency in injury repair function.
(2) The current data demonstrate that human lung fibroblasts have increased IL-6, IL-8 and TGF-β1 release in response to LPS and TNF-α, accompanied with a down-regulation of HDAC2 transcription. In addition, the fibroblasts responses differently in cytokines production to different inflammatory stimulus, indicating that the fibroblasts could participate in the modulation of pulmonary inflammation by producing relevant cytokines.
(3) LPS, TNF-α, IL-6, IL-8 and conditioned medium from LPS-pretreated macrophages can inhibit the proliferation of in vitro human lung fibroblasts, suggesting that the inflammation in milieu exert an effect on the function of lung fibroblasts.
(4) Low-dose theophylline can partly block the increased production of IL-6 and IL-8 as well as inhibited proliferation initiated by LPS.
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