Perfluorocarbon blocks tumor necrosis factor-alpha-induced interleukin-8 release from alveolar epithelial cells in vitro

OBJECTIVE: To determine whether tumor necrosis factor (TNF)-alpha-induced interleukin (IL)-8 production by pulmonary alveolar epithelial cells is blocked by perfluorocarbon (PFC). DESIGN: Controlled, laboratory investigation of IL-8 production by pulmonary alveolar epithelial cells after exposure to PFC in vitro. SETTING: University research laboratory. SUBJECT: The human alveolar epithelial cell line with pulmonary type II (A549) cell properties. INTERVENTIONS: The A549 cells on a polycarbonate porous filter were stimulated either on the apical or the basolateral side with TNF-alpha. To determine TNF-alpha-induced IL-8 production, IL-8 was measured by using a human IL-8 kit in both control and experimental groups. MEASUREMENTS AND MAIN RESULTS: TNF-alpha stimulation induced a large increase in IL-8. When PFC was added to the medium immediately after TNF-alpha stimulation, PFC separated the medium from the cells and IL-8 production was markedly reduced (TNF-alpha alone, 8342+/-470 pg vs. TNF-alpha followed by PFC, 417+/-88 pg, p < .05). Preincubation of A549 cells with PFC for 24 hrs before stimulation with TNF-alpha followed by removal of PFC did not affect IL-8 production (8834+/-204 vs. 8342+/-470 pg; p = NS). When added to the lower chamber, TNF-alpha also induced IL-8 production unaffected by the addition of PFC to the upper chamber. The decrease in TNF-alpha-induced IL-8 production depended on the time of PFC administration after the initiation of TNF-alpha stimulation. The earlier PFC was added, the more pronounced the diminution was in IL-8. CONCLUSIONS: PFC appears to function as a physical barrier, thus reducing cytokines produced by alveolar epithelial cells in vitro. This mechanism may partially explain the decreased inflammatory response observed during liquid ventilation in models of acute lung injury.     

Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung.

Cellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8). This interaction appears to occur via the ability of human alveolar macrophage (AM)-derived monokines, tumor necrosis factor (TNF), and interleukin-1 (IL-1) to induce gene expression of IL-8 from pulmonary type II-like epithelial cells (A549). Northern blot analysis demonstrated that steady-state IL-8 mRNA expression, by either TNF- or IL-1 beta-treated A549 cells, occurred in both a dose- and time-dependent fashion. Similarly, extracellular antigenic IL-8, as assessed by specific ELISA, was expressed from TNF- or IL-1 beta-stimulated epithelial cells in a time-dependent fashion with maximal IL-8 antigen detected at 24 h poststimulation. Immunohistochemical staining utilizing rabbit anti-human IL-8 antibody identified immunoreactive, cell-associated IL-8 antigen as early as 8 h post-TNF or IL-1 beta stimulation. A549-generated neutrophil chemotactic bioactivity paralleled IL-8 steady-state mRNA levels. Signal specificity was demonstrated in this system as IL-8 mRNA or protein expression by lipopolysaccharide (LPS)-treated A549 cells was not different from unstimulated cells. Although LPS did not serve as a direct stimulus for the production of IL-8 by type II-like epithelial cells, the condition media from LPS-challenged AM induced a significant expression of IL-8 mRNA by the A549 cells. 24-h conditioned media from LPS-treated cells was as potent as either IL-1 beta or TNF in generating steady-state IL-8 mRNA by A549 cells. Preincubation of LPS-treated AM-conditioned media with anti-human TNF or IL-1 beta neutralizing antibodies resulted in significant abrogation of IL-8 gene expression by A549 pulmonary epithelial cells. These findings demonstrate potential cell-to-cell communication circuits that may be important between AMs and pulmonary epithelial cells during the recruitment phase of acute lung inflammation.     

Role of interleukin-8 and growth-regulated oncogene-alpha in the chemotactic migration of all-trans retinoic acid-treated promyelocytic leukemic cells toward alveolar epithelial cells

OBJECTIVE: Although all-trans retinoic acid (ATRA) can treat acute promyelocytic leukemia (APL), it also causes retinoic acid syndrome with presentations similar to acute respiratory distress syndrome. We investigated the role of interleukin (IL)-8 and growth-regulated oncogene (GRO)-alpha in the chemotactic transmigration of ATRA-treated NB4 (ATRA-NB4) APL cells toward A549 alveolar epithelial cells. DESIGN: An in vitro human cell culture study. SETTING: University hospital research laboratories. SUBJECTS: NB4 and A549 cells. INTERVENTIONS: NB4 and A549 cells were separately cultured with ATRA and/or dexamethasone for 1-3 days. NB4 or ATRA-NB4 cells were then placed in an upper insert and co-incubated with A549 cells or their conditioned medium located in a lower plate. MEASUREMENTS AND MAIN RESULTS: ATRA stimulated NB4 cells to transmigrate toward the A549 cells in a time- and dose-dependent manner. Replacement of A459 condition medium by its original medium abrogated this transmigration. Only A549 cells constitutively secreted GRO-alpha, and both A549 and NB4 cells constitutively secreted IL-8, which was enhanced by ATRA. Exogenous administration of IL-8 or GRO-alpha also promoted the ATRA-NB4 transmigration. The binding assay demonstrated that ATRA-NB4 cells bound IL-8, but not GRO-alpha, more avidly. Pretreatment with antibodies directed against IL-8 and GRO-alpha receptors reduced ATRA-NB4 transmigration by about 60%. Dexamethasone did not suppress their IL-8 secretion and transmigration in ATRA-NB4 cells, but when applied to A549 cells, IL-8 secretion was suppressed but not GRO-alpha secretion, and there was attenuation of ATRA-NB4 transmigration. CONCLUSIONS: IL-8 and GRO-alpha secreted from alveolar epithelial cells play an important role in the cell-cell interaction involved in the chemotactic transmigration of ATRA-treated APL cells toward alveolar epithelial cells.     

Pneumocystis carinii attachment to cultured lung cells by pneumocystis gp 120, a fibronectin binding protein.

Pneumocystis carinii is an extracellular organism which is thought to require attachment to alveolar epithelial cells for its growth and replication in humans. Fibronectin (Fn) binding to P. carinii is essential for optimal P. carinii attachment. This study demonstrates that gp120, a 110-120-kD membrane glycoprotein on P. carinii, mediates attachment of the organism to cultured lung cells and is the site of Fn binding to P. carinii. A 51Cr-labeled P. carinii binding assay was used to quantify attachment of the organism to the alveolar epithelial cell line A549. Addition of free gp120, purified from whole P. carinii organisms, caused a significant decrease in attachment of P. carinii to A549 cells from 44.2 +/- 5.5% to 22.4 +/- 4.2% (P less than 0.01). Preincubation of the P. carinii organisms with a polyclonal antibody to gp120 also resulted in a marked decrease in P. carinii attachment to A549 cells from 46.8% +/- 5.2% to 21.3 +/- 4.8% (P less than 0.01). Furthermore, addition of free gp120 to P. carinii organisms caused a significant reduction in specific binding of 125I-Fn to P. carinii (from 83.3 +/- 8.5 ng to 47.1 +/- 5.9 ng, P less than 0.01). Similarly, anti-gp 120 antibody decreased specific Fn binding to P. carinii from 74.3 +/- 8.4 ng to 25.5 +/- 5.3 ng (P less than 0.001). Solubilized P. carinii organisms separated by gel electrophoresis and blotted with 125I-Fn demonstrated specific binding of the 125I-Fn to gp120. In addition, a specific anti-beta 1-integrin antiserum reacted with gp120 by Western blot, suggesting structural homology between gp120 and the beta-subunit of integrins. Thus, the data suggest that the P. carinii membrane glycoprotein gp120 functions as a Fn binding protein and is required for optimal P. carinii attachment to alveolar epithelial cells.     

菊花总黄酮对转染RSV的A549细胞RANTES及MCP-1表达的影响

【目的】探讨菊花总黄酮对呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)转染 A549细胞诱导激活正常 T 细胞表达和分泌因子(regulated upon activation ,normal Tcell expressed and secreted ,RANTES)及单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)释放作用影响。【方法】实验分为正常对照组,病毒对照组,菊花总黄酮组和巴韦林组。在人喉癌上皮细胞(Hep-2细胞)和气道上皮细胞(A549)分别加入菊花总黄酮和巴韦林的含药维持液,测定上述两种药物的最大无毒浓度；RSV 转染 Hep-2细胞,观察药物对 RSV 的病毒抑制作用；RSV 转染 A549细胞,ELISA 法测细胞趋化因子 RANTES 及 MCP-1含量。【结果】菊花总黄酮组50%有效率优于巴韦林组,差异有统计学意义(P <0.05)；菊花总黄酮组 RANTES、MCP-1明显降低,但高于正常细胞组,优于巴韦林组,差异有统计学意义(P <0.05)。【结论】菊花总黄酮能够抑制 RSV 活性,明显降低 A549细胞释放 RANTES、MCP-1,缓解患儿的呼吸道症状。     

结核杆菌H37Ra对肺的Ⅱ型上皮细胞B7-H2表达的调控

目的探讨结核杆菌H37Ra对诱导共刺激分子(ICOS)的配体B7-H2表达的影响。方法体外培养肺泡Ⅱ型细胞A549并且从人外周血中分离B淋巴细胞,用逆转录聚合酶链反应(RT-PCR)方法分析hGL50和B7-H2在两种细胞中不同的表达。A549细胞分别用结核杆菌H37Ra,脂多糖(LPS)和白细胞介素4(IL-4)诱导4、8、12小时,用RT-PCR方法分析B7-H2的表达。用免疫荧光方法分析B7-H2分子在A549细胞表面的表达。用流式细胞方法分析H37Ra、干扰素γ(IFN-γ)和IL-4诱导4小时和24小时后A549细胞B7-H2和CD40分子表达情况。结果B7-H2mRNA在A549细胞表达,hGL50mRNA在B细胞高表达。与LPS、IL-4相比,结核杆菌H37Ra可以诱导A549细胞高表达B7-H2。免疫荧光染色可以看到A549细胞高表达B7-H2分子。流式细胞结果显示,与IFN-γ和IL-4相比,结核杆菌H37Ra可以显著诱导A549细胞表面表达B7-H2分子。结论 ICOS配体B7-H2表达于肺泡Ⅱ型上皮细胞A549,结核杆菌H37Ra诱导可从mRNA和蛋白水平显著提高B7-H2的表达。     

高氧所致氧化应激状态下肺泡上皮细胞Toll样受体的表达及其功能研究

目的 观察高氧暴露后肺泡上皮细胞内活性氧(ROS)水平与Toll样受体(TLRs)基因、蛋白表达变化及其与信号通路功能之间的关系,探讨高氧所致肺损伤炎症反应的可能机制.方法 将体外培养的人肺腺癌A549细胞株分为空气对照组、高氧组、N-乙酰半胱氨酸(NAC)预处理组;高氧组细胞在＞90％O2的高氧环境中暴露2、6、12、24及48 h,NAC预处理组细胞予以ROS清除剂NAC预处理后高氧暴露6h.于相应时间点用流式细胞术检测细胞内ROS含量以及TLR2/4蛋白在细胞中的分布和表达;用逆转录-聚合酶链反应(RT-PCR)法检测TLR2/4 mRNA表达;用酶联免疫吸附法(ELISA)检测细胞上清液中白细胞介素(IL-6、IL-8)的含量.结果 A549细胞有TLR2/4表达,并且以细胞质内表达为主.与空气对照组比较,高氧组暴露2h细胞内ROS含量(荧光强度)即明显增高(11.820±3.123比7.223±1.170,P＜0.01),随高氧暴露时间延长呈进行性增高,于48 h达高峰(113.837±5.247,P＜0.01);高氧暴露2 h TLR2/4 mRNA表达至高峰(TLR2 mRNA:1.820±0.056比1.263±0.023;TLR4 mRNA:2.080±0.220比1.317±0.107,均P＜0.01),随高氧暴露时间延长,TLR2/4 mRNA表达虽仍有增多,但无显著变化;高氧暴露后TLR2/4蛋白表达显著增高,6h表达至高峰(TLR2蛋白:8.370±1.548比3.930±0.277;TLR4蛋白:25.803±5.783比8.867±2.230,均P＜0.01),以细胞质内表达为主;随高氧暴露时间延长,细胞上清液中IL-6(ng/L)、IL-8(ng/L)水平呈进行性增高,于48 h达高峰(IL-6:2 213.41±69.99比9.76±1.47;IL-8:11 520.38±429.93比159.56±20.80,均P＜0.01).在NAC预处理情况下,高氧刺激后,细胞内ROS水平明显降低(14.050±1.257比31.180±2.336,P＜0.01),细胞TLR2/4 mRNA和蛋白表达显著降低(TLR2 mRNA:1.270±0.061比1.683±0.025; TLR4 mRNA:1.507±0.058比1.650±0.139;TLR2蛋白:3.458±0.258比8.370±1.548;TLR4蛋白:11.611±3.403比25.803±5.783,均P＜0.05),细胞上清液中IL-6、IL-8水平显著下降(IL-6:8.42±0.70比73.51±16.70;IL-8:134.94±5.19比772.82±96.05,均P＜0.05),与空气对照组比较差异均无统计学意义.结论 A549细胞在高氧暴露后,细胞内ROS能够激活人肺泡上皮细胞TLR2/4的表达,导致前炎症细胞因子IL-6和IL-8的大量释放.     

Induction of monocyte chemoattractant protein-1 by Mycoplasma hominis in respiratory epithelial cells.

BACKGROUND: Very small preterm infants who have genital mycoplasmas isolated from the trachea are at increased risk to develop bronchopulmonary dysplasia (BPD). The early stages of BPD are characterized by inflammation. Recruitment and activation of mononuclear cells in response to mycoplasmas may be important in the early stage of the disease. Lung epithelial cell production of monocyte chemoattractant protein-1 (MCP-1), a protein that attracts and activates mononuclear cells, could be critical in the regulation of mononuclear cell migration to the lung. METHODS: We examined the potential of Mycoplasma hominis (Mh) to induce MCP-1 gene expression and protein production in A549 cells, a pulmonary epithelial cell line with characteristics of type II cells. RESULTS: Live or heat-inactivated Mh induces MCP-1 mRNA and protein in a dose- and time-dependent manner. Stimulation of MCP-1 by Mh was not inhibited by 50 micrograms/mL of polymyxin B, interleukin (IL)-1ra, or neutralizing antibodies to IL-1 alpha, IL-1 beta, or tumor necrosis factor alpha (TNF-alpha). IL-1 alpha, IL-1 beta, and TNF-alpha were not detected in conditioned media of Mh-stimulated A549 cells. CONCLUSIONS: These data suggest that Mh may participate in the inflammatory component of BPD by directly inducing epithelial cell production of cytokines that recruit and activate mononuclear cells.     

Clarithromycin has an immunomodulatory effect on ERK-mediated inflammation induced by Pseudomonas aeruginosa flagellin

OBJECTIVES: Pseudomonas aeruginosa exoproducts are potent triggers of immune responses in eukaryotic cells. Clarithromycin initially decreases, then increases and finally produces a sustained suppression of interleukin (IL)-8 secretion from normal human bronchial epithelial (NHBE) cells through inhibition and activation of extracellular signal-regulated kinase (ERK). This polyphasic immune response is referred to as immunomodulation. METHODS: We studied the effects of P. aeruginosa flagellin and alginate on IL-8 secretion from NHBE cells and how this was affected by clarithromycin or dexamethasone. We also assessed the upstream kinase cell signalling intermediates that appear to be responsible for flagellin-induced IL-8 secretion. ELISA was used to measure IL-8 in culture supernatants, and western blots were used to measure kinase and phosphokinase levels. RESULTS: Flagellin dose-dependently increased IL-8 secretion in NHBE cells at 24 h, whereas alginate had no effect on IL-8. Clarithromycin significantly decreased flagellin-induced IL-8 over the first 9 h but not at 24 h. A 60 min exposure to clarithromycin decreased flagellin-induced ERK phosphorylation, but at 24 h, clarithromycin increased phospho-ERK1/2 beyond the effect of flagellin alone. Pre-treatment with PD98059 (MEK inhibitor) decreased flagellin-induced IL-8 secretion by 47.7% (P < 0.0001) compared with control flagellin exposure and decreased basal IL-8 in the absence of flagellin by 27.9% compared with untreated control cells (P < 0.0001). Dexamethasone and PD98059 together had an additive suppressive effect on flagellin-induced IL-8 secretion. CONCLUSIONS: P. aeruginosa flagellin, but not alginate, stimulates IL-8 secretion in NHBE cells in part through ERK1/2. This effect is modulated by clarithromycin, whereas suppression of IL-8 secretion by dexamethasone probably occurs through different pathways.     

p38丝裂原活化蛋白激酶对大鼠肾脏分泌单核细胞趋化因子-1的影响

目的 动态观察单核细胞趋化因子-1(MCP-1)在单侧输尿管结扎(UUO)大鼠肾小管的表达,探讨它与p38丝裂原活化蛋白激酶(p38MAPK)、核转录因子-кB(NF-кB)及肾损害之间的关系. 方法 雌性SD大鼠共36只,体质量150～170 g.随机分为2组:假手术组作为对照组;手术组根据梗阻时间分3组,每组6只.皮下注射水合氯醛(4 mg/kg)麻醉,开腹后结扎右侧输尿管,缝合腹腔,即制备UUO模型.假手术组开腹后不结扎输尿管,缝合腹腔.采用免疫组织化学检测NF-кB、MCP-1,Western印迹分析法检测p38MAPK的磷酸化水平和lVICP-1蛋白水平,逆转录-聚合酶链反应(RT-PCR)方法检测MCP-1 mRNA的表达.光镜检查肾组织的形态改变.生化方法测定血尿素氮、血肌酐. 结果 与对照组比,随着梗阻时间的延长,MCP-1蛋白表达明显上调,[对照组:0.401±0.039,UUO组8h:0.894±0.137;24 h:1.416±0.135;72 h:1.894±0.143,与对照组相比,P<0.05],MCP-1 mRNA表达明显上调,[对照组:50.08±3.210,UUO组8 h:108.25±4.325;24 h:179.34±3.237;72 h:230.12±3.026,与对照组相比,P<0.05],同时NF-кB、p38MAPK蛋白活性增高,[p38MAPK蛋白对照组:110.65±9.734,UUO组8 h:200.15±8.326;24 h:272.74±7.244;72 h:549.11±9.544,与对照组相比,P<0.05],肾小管MCP-1的表达与p38MAPK、NF-кB呈显著正相关(r=0.74、r=0.81,P<0.01). 结论 UUO大鼠.肾小管MCP-1表达增加参与了UUO大鼠.肾小管间质损害的发病机制;p38MAPK可能介导了NF-кB的表达,进而调节MCP-1的表达增多.     

Normal human alveolar macrophages obtained by bronchoalveolar lavage have a limited capacity to release interleukin-1.

Interleukin-1 (IL-1) is a mediator released by stimulated mononuclear phagocytes that is thought to play an important role in modulating T and B lymphocyte activation as well as in contributing to the febrile response and other inflammatory processes. Circulating mononuclear phagocytes, blood monocytes, readily release IL-1 when stimulated. However, the ability of lung mononuclear phagocytes, alveolar macrophages, to dispose of the large daily burden of inhaled antigens without stimulating an inflammatory response suggests that the release of IL-1 by alveolar macrophages may differ significantly from that of blood monocytes. To evaluate this hypothesis, normal autologous alveolar macrophages, obtained by bronchoalveolar lavage, were compared with blood monocytes for their ability to release IL-1 in response to a standard stimulus, lipopolysaccharide (LPS). Alveolar macrophages were found to be at least 1,000 times less sensitive to LPS than blood monocytes. Furthermore, alveolar macrophages released significantly less IL-1 than blood monocytes (26 +/- 11 vs. 128 +/- 21 U/10(6) cells X 24 h, respectively, after stimulation with 10 micrograms/ml of LPS, P less than 0.001). This difference was not due to the release of substances by macrophages, which inhibited lymphocyte proliferation in response to IL-1, or to degradation of IL-1 by macrophages. Culturing macrophages in the presence of indomethacin and dialysis of macrophage supernatants did not affect the difference, and culturing macrophages with monocytes did not decrease detectable IL-1 activity from the monocytes. The IL-1 produced by the two cell types was indistinguishable by anion-exchange chromatography, gel filtration, and isoelectric focusing. In addition, consistent with the findings for alveolar macrophages, macrophages generated by the in vitro maturation of blood monocytes were also deficient in their ability to release IL-1. These findings suggest that if the population of alveolar macrophages obtained by bronchoalveolar lavage represents the total in vivo population of alveolar macrophages, although normal human macrophages are capable of IL-1 release, they are relatively limited in this ability, and this limitation seems to be linked to the maturational state of the mononuclear phagocyte. These observations may explain, in part, the ability of alveolar macrophages to clear the airspaces of foreign antigens without extensive activation of other pulmonary inflammatory and immune effector cells.     

Effects of hypoxia and nitric oxide on ferritin content of alveolar cells

Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with a variety of respiratory disorders. Ferritin synthesis by cells is modulated by iron content but is also influenced by stimuli other than iron. In this study we sought to determine whether in vitro exposure to hypoxia- or nitric oxide (NO)-induced ferritin accumulation or release by human alveolar macrophages (AMs) or a lung cancer-derived epithelial cell line (A549). Changes in cell content of iron and ferritin (L- and H-types), as well as ferritin content of cell supernatants, were determined after in vitro exposure to hypoxia (1% or 10% O(2), 18 hours) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01-1.0 mmol/L, 18 hours). Exposure to 1% O(2) increased ferritin content in both cell types (>fourfold increase; P <.005) without changing iron content. Treatment with SNAP increased ferritin content of A549 cells in a dose-dependent manner, whereas treatment of AMs decreased cellular iron and ferritin content and increased supernate ferritin content. Pretreatment of cells with N-acetylcysteine (500 micromol/L) reduced hypoxia-induced ferritin accumulation in alveolar cells and completely inhibited NO-induced ferritin accumulation in A549 cells. These findings indicate that exposure to 1% O(2)can increase ferritin content in alveolar cells, whereas NO can increase ferritin content (A549 cells) or decrease ferritin content (AMs).     

白细胞介素-1β对小鼠气道成纤维细胞前列腺素E2表达的影响

目的研究白细胞介素-1β（IL-β）对小鼠气道成纤维细胞前列腺素E2（PGE2）表达的影响,以探讨其在吸入性损伤修复中的作用。方法分离雄性昆明种小鼠的气道成纤维细胞并进行体外培养,分别收集不同浓度的IL-1β作用后不同时间点IL-1β的培养上清液及细胞;采用酶联免疫吸附试验法和westem blot技术测定PGE2水平,环氧化酶-2（COX-2）、膜相关前列腺素E2合成酶1（mPGES1）蛋白表达。结果①经IL-1β1．0μg/L处理后不同时间点气道成纤维细胞培养上清液PGE2水平在8h[（148．2±28．3）ng／L]、16h[（267．6±45．4）μg／L]、24h[（210．5±38．6）ng／L]、48h[（198．7±36．5）ng／L]均高于各对照组[分别为（57．8±15．3）、（58．2±15．7）、（57．9±15．8）、（58．1±16．1）ng／L],差异均有统计学意义（P均〈0．01）,其中16h时间点水平最高;气道成纤维细胞COX-2表达在8h[（0．478±0．029）COX-2／β-actin、16h（0．672±0．047）cox-2／β-aetin、24h（0．617±0．036）cox-2／β-actin、48h（0．593±0．034）COX-2／β-actin]均高于对照组[（0．309±0．019）COX-2／β-actin、（0．311±0．019）COX-2／13-actin、（0．309±0．019）COX-2／β-actin、（0．310±0．018）cox-2／β-actin],差异有统计学意义（P均〈0．01）,其中16h时间点表达水平最高;气道成纤维细胞mPGESl的表达在8h（0．300±0．018）mPGES1／β-actin、16h（0．549±0．034）mPGES1／β-actin、24h（0．497±0．030）mPGES1/β-actin、48h（0．443±0．026）mPGES1／β-aetin均高于各对照组[（0．1994±0．012）、（0．201±0．013）、（0．200±0．013）和（0．200±0．012）mPGES1／β-actin],差异有统计学意义（P均〈0．01）,其中16h时间点表达水平最高。②不同浓度IL-1β处理气道成纤维细胞后,气道成纤维细胞培养上清液PGE：水平在IL-1β0．1μg／L组[（142．9±22．3）ng／L]、1．0μg／L组[（267．6±45．4）μg／L和10．0μg／L组[（587．3±106．9）μg／L]均高于对照组[（58．5±16．0）μg／L],差异有统计学意义（P均〈0．01）,并且这3组之间比较差异也有统计学意义（P均〈0．01）;气道成纤维细胞COX-2表达在IL-1β0．1μg／L组（0．525±0．031）ng／L、1．0μg/L组（0．672±0．047）ng／L和10.0μg/L组（1.012±0．064）ng／L均高于对照组（0．309±0．019）ng／L,差异有统计学意义（P均〈0．01）,并且这3组之间比较差异也有统计学意义（P均〈0．01）;气道成纤维细胞mPGES1表达在IL-1β0．1μg／L组（0．380±0．021）ng／L、1．0μg／L组（0．549±0．034）ng／L和10．0μg／L组（0．879±0．045）ng／L均高于对照组（0．199±0．012）ng／L,差异有统计学意义（P均〈0．01）,并且这3组之间比较差异也有统计学意义（P均〈0．01）。结论炎症递质IL-1β可上调气道成纤维细胞PGE2水平、COX-2和mPGES1表达,这表明IL-1β可能是通过COX-2和mPGES1的表达来影响PGE2合成,从而参与气道吸入性损伤修复过程。气道成纤维细胞可能是损伤修复过程中炎症递质的主要来源细胞之一。     

Pneumocystis carinii: inhibition of lung cell growth mediated by parasite attachment.

Pneumocystis carinii pneumonia is a significant cause of mortality in immunocompromised patients. Current concepts suggest that attachment of P. carinii to alveolar epithelium is required for development of pneumonia. We examined the mechanism of P. carinii adherence to cultured A549 cells, a permanent cell line derived from human alveolar epithelium. P. carinii adherence was quantified by measuring attachment of 51Cr-labeled P. carinii to cultured A549 cells. After 8 h of incubation, 37.4 +/- 4.2% of P. carinii were adherent to A549 cells. In the presence of agents known to impair cytoskeletal function, including 10(-5) M cytochalasin B, 10(-5) M colchicine, and 10(-5) M trimethylcolchicinic acid (TMCA), adherence was decreased from 57.4 +/- 4.2% to 9.3 +/- 3.4%, 12.5 +/- 3.6%, and 21.5 +/- 3.6%, respectively (P less than 0.01, all comparisons). Secondly, we examined the effect of P. carinii on the function of A549 cells. P. carinii resulted in significant impairment of A549 cell growth, indicating P. carinii adversely affected the function of target lung cells. A P. carinii:A549 cell ratio of 50:1 resulted in 43.5 +/- 2.9% inhibition of A549 cell growth (P less than 0.001). Additionally, TMCA, which significantly prevented attachment of P. carinii, reversed the impairment of A549 cell growth. These data demonstrate that P. carinii attachment to cultured lung cells can be quantified, is dependent on intact cytoskeletal function and is necessary for impairment of lung cell replication.     

Gamma-interferon inhibits Pneumocystis carinii attachment to lung cells by decreasing expression of lung cell-surface integrins

Pneumocystis carinii (PC) is a leading cause of pneumonia in immunocompromised patients. Previous work has shown that fibronectin (Fn) and Fn-binding integrins mediate PC attachment to lung cells in vitro . Gamma-interferon (γ-IFN) is a major factor in host defence against PC infection. To determine the effect of γ-IFN on PC attachment to lung cells, the alveolar epithelial cell line A549 was incubated with γ-IFN (0–10⁴ U mL⁻¹) and attachment of ⁵¹Cr-labelled PC to the A549 cells was quantified. PC attachment was significantly decreased ( P  < 0.01) by addition of γ-IFN with no evidence of injury to either the PC or A549 cells. Effects of γ-IFN on PC attachment were observed after 24 h and reached a maximum after 48 h of incubation. To investigate the mechanism of this decrease, we examined integrin expression on γ-IFN-treated A549 cells. A549 cell expression of the α₅ and β₁ integrin subunits was decreased, whereas expression of the α_v subunit was unchanged. Northern blot analysis showed a similar decrease in mRNA for the α₅ and β₁ integrins. Therefore, γ-IFN-mediated inhibition of PC infection may, in part, result from inhibition of PC attachment to alveolar epithelial cells caused by γ-IFN-induced decreases in alveolar integrin expression.     

Spontaneous release of interleukin 2 by lung T lymphocytes in active pulmonary sarcoidosis is primarily from the Leu3+DR+ T cell subset.

The inflammation within the lower respiratory tract of individuals with pulmonary sarcoidosis is dominated by large numbers of helper T lymphocytes that proliferate and spontaneously release interleukin 2 (IL-2). To identify the lymphocyte subpopulation that releases IL-2 in this disorder, lung lymphocytes recovered by bronchoalveolar lavage were characterized using the monoclonal antibodies Leu4 (T lymphocyte), Leu3 (helper/inducer), Leu2 (suppressor/cytotoxic), and anti-HLA-DR, and separated by panning and flow cytometry. The majority of the IL-2 spontaneously released by T cells in the sarcoid lung was contributed by the Leu3+ cell population (Leu3+65 +/- 23 IL-2 units released/10(6) cells per 24 h; Leu2+ 9 +/- 8, P less than 0.04). Further characterization of the lung Leu3+ T cells in sarcoid demonstrated that 30 +/- 3% were expressing HLA-DR molecules on their surface compared with 6 +/- 1% in normals (P less than 0.01). Importantly, the subpopulation of Leu3+ lung T lymphocytes expressing a high intensity of HLA-DR molecules on their surface was responsible for the majority of the release of IL-2 in the sarcoid lung (Leu3+ high-intensity DR 42 +/- 17 U/10(6) cells per 24 h, Leu3+ low-intensity DR 8 +/- 1 U/10(6) cells per 24 h; P less than 0.01). Thus, the spontaneous release of IL-2 in the lung of sarcoid patients appears to be localized to a subset of Leu3+ high-intensity DR ("activated" lung helper/inducer) T lymphocytes. Because the sarcoid lung is characterized by markedly increased numbers of these cells, it is likely that this compartmentalized T cell population plays a major role in sustaining the exaggerated localized immune processes of this disorder.     

Effect of ulinastatin on paraquat-induced-oxidative stress in human type II alveolar epithelial cells

BACKGROUND:

Ulinastatin (UTI) is a urinary trypsin inhibitor extracted and purified from urine of males. This study aimed to explore the effects of UTI on paraquat-induced-oxidative stress in human type II alveolar epithelial cells.

METHODS:

The human type II alveolar epithelial cells, A549 cells, were cultured in vitro. The A549 cells were treated with different concentrations of paraquat (200, 400, 600, 800, 1 000, 1 200 µmol/L) and ulinastatin(0, 2 000, 4 000, 6 000, 8 000 U/mL) for 24 hours, the cell viability was measured by cell counting kit-8 and the median lethal concentration was selected. In order to establish an in vitro model of paraquat intoxication and to determine the safe dose of ulinastatin, we calculated LD50 using cell counting kit-8 to determine the survival rate of the cells. A549 cells were divided into normal control group, paraquat group and paraquat+ulinastatin group. The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by biochemistry colorimetry, while the level of reactive oxygen spies (ROS) was detected by DCFH-DA assay.

RESULTS:

The survival rate of A549 cells treated with different concentrations of paraquat decreased in a concentration-dependent manner. Whereas there was no decrease in the survival rate of cells treated with 0–4 000 U/mL ulinastatin. The levels of MDA, MPO, and ROS were significantly higher in the paraquat group than in the normal control group after 24-hour-exposure. And the survival rate of the paraquat+ulinastatin group was higher than that of the paraquat group, but lower than that of the normal control group. The levels of MDA, MPO, and ROS were lower than those of the paraquat group.

CONCLUSION:

Ulinastatin can alleviate the paraquat-induced A549 cell damage by reducing oxidative stress.KEY WORDS: Ulinastatin, Paraquat, Oxidative stress, A549 cell     

Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products.

The aims of this study were (a) to determine if rat alveolar type II (ATII) cells and human pulmonary epithelial-derived cells (A549 cell line) could generate IL-6 in vitro, (b) to characterize the cytokine regulation of IL-6 gene and protein expression in these cells, and (c) to detect the in vivo expression of immunoreactive IL-6 by human ATII cells. Rat ATII cells in primary culture secreted bioactive IL-6 and immunostained with an anti-IL-6 antiserum. Spontaneous IL-6 secretion by rat ATII cells amounted to 5,690 +/- 770 pg/ml/10(6) cells (n = 12) and was fivefold higher than spontaneous rat alveolar macrophages IL-6 secretion (1,052 +/- 286 pg/ml/10(6) cells, n = 8, P = 0.001). Rat alveolar macrophage conditioned media (CM) increased IL-6 secretion by rat ATII cells through the effect of IL-1 and TNF. IL-6 gene expression and IL-6 secretion by A549 cells was induced by IL-1 beta, TNF alpha, and by human alveolar macrophages and THP1 cells CM. Induction was abolished when CM were preincubated with anti-IL-1 beta and anti-TNF alpha antibody. The combination of IFN gamma and LPS induced the expression of IL-6 mRNA by A549 cells whereas LPS alone had no effect. Immunohistochemical staining evidenced the expression of immunoreactive IL-6 by hyperplastic ATII cells in fibrotic human lung, a condition in which alveolar macrophages are known to be activated. ATII cells in normal human lung did not express immunoreactive IL-6. Our findings demonstrate that ATII cells may be an important source of IL-6 in the alveolar space thereby participating to the regulation of the intra-alveolar immune response.     

肺表面活性蛋白-D在高原肺水肿发生发展中的表达及作用

目的探讨肺表面活性蛋白-D（SP-D）在高原肺水肿发生发展中的表达及作用。 方法将64只Wistar雄性大鼠随机分为4组,正常对照组（16只,586 m室温饲养）,低压低氧24 h、48 h及72 h组（各16只,于模拟海拔6000 m低压低氧舱内分别饲养24 h、48 h及72 h）,取右侧肺组织行HE染色,观察肺组织损伤情况,收集左侧肺组织肺泡灌洗液,采用酶联免疫吸附法（ELISA）测定支气管肺泡灌洗液（BALF）中SP-D、白介素（IL）-1β、IL-6、C反应蛋白（CRP）及肿瘤坏死因子α（TNF-α）的含量。 结果成功建造高原肺水肿动物模型;与正常对照组相比,低压低氧24、48、72 h组肺组织病理评分升高,差异有统计学意义（P＜0.05）;SP-D含量均有所下降,IL-1β、IL-6、CRP、TNF-α含量均上升,且各组间两两比较差异均有统计学意义（P＜0.05）;BALF中SP-D含量与肺组织病理评分呈负相关（r=-0.687,P＜0.05）,炎症因子IL-1β、IL-6、CRP及TNF-α与肺组织病理评分均呈正相关（r=0.705,P＜0.05;r=0.728,P＜0.05;r=0.733,P＜0.05;r=0.737,P＜0.05）。 结论SP-D在高原肺水肿的发生发展中起着一定的作用,其可能是防治高原肺水肿的新靶点。     

Risk stratification for heart failure and death in an acute coronary syndrome population using inflammatory cytokines and N-terminal pro-brain natriuretic peptide

BACKGROUND: Inflammation in acute coronary syndrome (ACS) can identify those at greater long-term risks for heart failure (HF) and death. The present study assessed the performance of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) (cytokines involved in the activation and recruitment of leukocytes) in addition to known biomarkers [e.g., N-terminal pro-brain natriuretic peptide (NT-proBNP)] for predicting HF and death in an ACS population. METHODS: In a cohort of 216 ACS patients, NT-proBNP (Elecsys; Roche) and IL-6, IL-8, and MCP-1 (evidence investigator; Randox) were measured in serial specimens collected early after symptom onset (n = 723). We collected at least 2 specimens from each participant: an early specimen (median 2 h; interquartile range 2-4 h) and a later specimen (9 h; 9-9 h), and used the later specimens' biomarker concentrations for risk stratification. RESULTS: An increase in both IL-6 and NT-proBNP was observed but not for IL-8 or MCP-1 early after pain onset. Kaplan-Meier analysis demonstrated that individuals with increased NT-proBNP (>183 ng/L) or cytokines (IL-6 > 6.4 ng/L; above upper limit of normal for IL-8 or MCP-1) had a greater probability of death or HF in the following 8 years (P <0.05). In a Cox proportional hazard model adjusted for both CRP and troponin I, increased IL-6, MCP-1, and NT-proBNP remained significant risk factors. Combining all 3 biomarkers resulted in a higher likelihood ratio for death or HF than models restricted to any 2 of these biomarkers. CONCLUSION: IL-6, MCP-1, and NT-proBNP are independent predictors of long-term risk of death or HF, highlighting the importance of identifying leukocyte activation and recruitment in ACS patients.