Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.     

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.     

Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.     

克雷伯杆菌分泌因子及菌体成分在细胞内、外刺激肺上皮细胞分泌IL-8的研究

目的:通过比较肺炎克雷伯杆菌(Klebsiellapneumoniae,Kp)分泌因子及菌体成分对经过digitonin处理和未处理的肺上皮细胞株IL8分泌的影响,进一步了解克雷伯杆菌诱导肺上皮细胞炎症反应的信号转导机制。方法:实验分为两组,一组使用能使细胞膜通透性增加的温和去污剂digitonin处理,而另一组不处理。分别用Kp03183的细菌培养上清和超声处理的菌体成分刺激肺上皮细胞株A549,酶联免疫吸附实验(ELISA)检测细胞IL8表达水平。并用RT-PCR的方法检测肺上皮细胞胞内模式识别受体NOD1的表达。结果:Kp培养上清对未经digitonin处理的细胞IL8分泌无明显增强作用,与对照相比差异无显著意义(p>0.05),菌体成分能刺激IL8分泌增加(P<0.01),但增高不超过一倍。经digitonin处理使细胞膜通透性增加后,Kp培养上清及菌体成分刺激细胞IL8的作用都增强,菌体成分刺激IL8效果更为显著,为对照的3倍,而培养上清作用相对较弱。RT-PCR检测结果表明,肺上皮细胞表达胞内模式识别受体NOD1。结论:菌体成分是诱导炎症反应更有效的刺激物,肺炎克雷柏杆菌侵入肺上皮细胞可能是引发细胞炎症反应的始动环节。肺上皮细胞表达胞内模式识别受体NOD1,它是否参与肺上皮细胞识别肺炎克雷伯杆菌菌体成分,值得进一步的研究。     

A role for the Mycoplasma pneumoniae adhesin P1 in interleukin (IL)-4 synthesis and release from rodent mast cells

Mycoplasma pneumoniae is a respiratory tract pathogen associated with exacerbations in patients with chronic asthma, yet relatively little is known about the potential role of this organism in asthma pathogenesis. Our previous studies demonstrated that RBL-2H3 mast cells co-cultured with M. pneumoniae released preformed inflammatory mediators, synthesized multiple cytokine mRNA species, and released IL-4 protein. In this study, we sought to determine the mechanism by which M. pneumoniae activates mast cell cytokine production. Cytokine mRNA upregulation and IL-4 protein production in RBL cells were induced almost exclusively by plastic-adherent M. pneumoniae cultures (MpA). Organisms grown under non-adherent conditions (MpN) were unable to induce cytokine responses efficiently. Western blots demonstrated that MpA was enriched for P1, the major M. pneumoniae adhesin, compared to MpN. M. pneumoniae-induced IL-4 release from RBL cells was inhibited >85% by anti-P1 monoclonal antibodies. Additionally, a P1-deficient strain of the bacteria was unable to efficiently induce IL-4 release. Desialation of RBL cell surface glycoproteins by neuraminidase treatment eliminated IL-4 release. We conclude that P1 plays an important role in M. pneumoniae-induced cytokine responses in RBL mast cells and that direct contact between the organism and sialated residues on the RBL surface mediates this activation.     

Cytokine gene expression profile in monocytic cells after a co-culture with epithelial cells

Epithelial cells represent an important source of cytokines that may modulate the influx and functions of mononuclear phagocytes. The aim of our study was to characterize changes in the gene expression of selected cytokines in human macrophages co-cultured with respiratory epithelial cells. The A549 alveolar type II-like cell line was co-cultured with THP-1 cells (monocyte/macrophage cell line) in filter-separated mode to avoid their cell-cell contact. At different time-points (0, 4, 8, 12 and 24?h), the cells were harvested separately to evaluate their gene and protein expression (IL-1 beta, IL-6, IL-8, IL-10 and GM-CSF). Quantitative RT-PCR analysis showed prominent changes in the THP-1 cytokine gene expression induced by a co-culture with A549 cells. Fourfold upregulation of mRNA expression has been found in 12 genes and 4-fold downregulation in 5 genes as compared to the unstimulated control sample with a p value smaller than 0.05. The induction of inhibin beta A and IL-1 beta mRNA after 12?h and the expression of IL-1 alpha and GM-CSF mRNA after 24?h were the most prominent. When looking at the cytokine levels in culture supernatants, IL-1 beta and IL-8 were induced early (at 8?h) as compared to the release of IL-6 and GM-CSF (at 24?h). We conclude that respiratory epithelial cells constitutively regulate the cytokine gene expression of macrophages located in their environment and might further modulate the release of cytokines by posttranslational pathways.     

Modulation of endothelin-1 expression in pulmonary epithelial cell line (A549) after exposure to RSV

Respiratory syncytial virus (RSV) is one of the most important respiratory tract pathogens in infants and young children. The airway epithelial cells are the primary target cells for RSV infection. The airway epithelial layer is not only a physical barrier, but also plays a role in a synthesis of a variety of major inflammatory cytokines (IL-6, IL-8, GM-CSF etc.) as previously reported. Endothelin-1 (ET-1) is a potent bronchoconstrictor and vasoconstrictor factor, and involved in pathogenesis of various diseases of the respiratory tract. We hypothesized that RSV may induce the release of ET-1 from the bronchial epithelial cell line. No previous data is available regarding association between RSV infection and ET-1 release. We evaluated the effect of RSV with different concentrations of RSV (MOI 0.1, 1 and 3 pfu/cell) on bronchial epithelial cell line (A549) and measured the production of ET-1 at both protein and mRNA level. A549 cells were treated with different conditions by using LPS, heat-inactivated RSV, RSV or medium alone as control. We observed time-dependent ET-1 release by RSV-infected A549 cells at 4 h, 24 h and maximum at 72 h. ET-1 was expressed in unstimulated A549 cells and was further increased by RSV. RSV with concentration MOI 0.1 (pfu/cell) and LPS appeared to have strongest stimulation on production of ET-1. In addition, ET-1 mRNA was increased significantly by 16 h and decreased to relatively low-level at 24 h. These experiments suggested that airway epithelial cells might play a role in the local airway smooth muscle tone through the production of endothelin-1 during RSV infection.     

Type-specific induction of interleukin-8 by adenovirus.

Infection with adenovirus (Ad) causes acute pneumonia in a type-specific fashion because type 7 but not type 5 Ad has been isolated as a causative agent. We postulated that the type specificity of induction of pneumonia may be related to type-specific cytokine induction in lung cells. To test this hypothesis, we infected human fetal lung fibroblasts and the lung epithelial cell line A549 with live type 5 and type 7 Ad. Virus inactivated by irradiation was used as a control. Type 7 but not type 5 Ad induced interleukin (IL)-8 protein production in both cell types in a dose- and time-dependent manner. Inactivated virus had no effect on the production of IL-8 protein. Type 7 but not type 5 virus also stimulated IL-8-specific messenger RNA (mRNA) production in these cells. Because half-life of IL-8 mRNA was prolonged in both type 5- and type 7-infected A549 cells, induction likely involves enhancement of message stability as well as other effects. Virus early gene expression did not consistently correlate with IL-8 message induction and followed induction in fibroblasts. These results suggest that there is type-specific induction of IL-8 production during infection of lung cells with Ad. Induction involves message stabilization and may not require viral gene expression. Because IL-8 is one of the important mediators of lung inflammation, type-specific induction of this and other cytokines may account for the different consequences of lung infection with different types of Ad.     

Caspase-3基因沉默对肺炎链球菌感染的肺泡上皮细胞凋亡的影响

目的:通过siRNA抑制caspase-3基因的表达探讨肺炎链球菌对肺泡上皮细胞凋亡的影响及凋亡基因caspase-3对凋亡的调节作用,寻找治疗肺炎链球菌肺炎的新方法。方法:体外培养肺泡上皮细胞A549,用肺炎链球菌R6作用于A549细胞,使用siRNA技术抑制caspase-3表达,RT-PCT法检测caspase-3转录强度,化学荧光测定法检测caspase-3蛋白含量,ELISA法检测细胞上清中IL-6和IL-10浓度,TUNEL法检测细胞凋亡。结果:肺炎链球菌能诱导A549细胞凋亡、导致caspase-3的表达和IL-6的浓度升高、IL-10的浓度降低;使用siRNA抑制caspase-3表达后,caspase-3的表达降低,A549细胞的凋亡率降低,而IL-6和IL-10的浓度并无明显变化。结论:Caspase-3在肺炎链球菌引起的肺泡上皮细胞的凋亡中占据了重要的地位;运用RNA干扰技术抑制caspase-3的表达能降低肺泡上皮细胞的凋亡率,这可能对肺炎链球菌肺炎的治疗有积极意义。     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Mycoplasma pneumoniae-induced activation and cytokine production in rodent mast cells

BACKGROUND: Mycoplasma pneumoniae is a respiratory tract pathogen that has been associated with severe exacerbations in patients with chronic asthma. Murine models of infection have recently been established, with disease manifestations similar to those observed in human subjects. Previous studies have suggested that this organism is capable of producing activation of a wide range of immunologic cell types. OBJECTIVE: We sought to determine whether M pneumoniae can induce mast cell activation in the rodent mast cell line RBL-2H3. RESULTS: After 4 hours of coculture, morphologic changes indicative of activation were observed by means of electron microscopy, and M pneumoniae was identified, by means of immunoelectron microscopy, adhering to mast cell membranes. Coculture of rat basophilic leukemia cells with viable M pneumoniae for 4 hours resulted in net release of beta-hexosaminidase and serotonin into the supernatant. Live, but not heat-killed, organisms induced the release of IL-4 protein into the culture supernatant, with a peak at 4 hours. During coculture with M pneumoniae, production of mRNA for IL-4, IL-6, and TNF-alpha was upregulated after 2 hours and had returned to near baseline by 24 hours after infection. CONCLUSIONS: We conclude that viable M pneumoniae induces activation of mast cells with release of granule contents, as well as cytokine production.     

MyD88缺去突变基因转染降低病原菌感染的人呼吸道上皮细胞IL-8的分泌

白细胞介素-8（1L-8）是呼吸道炎症反应的重要介质。本实验通过构建突变MyD88真核表达质粒（MyD88 DN），转染人呼吸道上皮细胞株A549及SPC-A-1，探讨其对病原菌感染上皮细胞IL-8表达的影响。结果显示：MyD88 DN转染可降低结核杆菌、绿脓杆菌培养上清诱导的IL-8释放；对肺炎克雷伯杆菌和绿脓杆菌活菌侵袭细胞所刺激的IL-8分泌也有明显的阻断作用。提示突变MyD88能够阻断细菌感染引起的呼吸道上皮细胞IL-8表达，可能成为呼吸道严重炎症反应基因治疗的新靶基因。     

Differential role of CbpA and PspA in modulation of in vitro CXC chemokine responses of respiratory epithelial cells to infection with Streptococcus pneumoniae

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.     

Peroxynitrite enhances interleukin-10 reduction in the release of neutrophil chemotactic activity

Peroxynitrite, formed by nitric oxide and superoxide, has been shown to nitrate and reduce the function of proinflammatory proteins such as interleukin (IL)-8, monocyte chemoattractant protein-1, and eotaxin, but in contrast, to enhance the function of the anti-inflammatory cytokine IL-10 in reducing IL-1 release from blood monocytes. However, the effect of nitrated IL-10 on release of proinflammatory cytokines from lung epithelial cells is unknown. We hypothesized that peroxynitrite would enhance the capacity of human IL-10 to reduce inflammatory mediators released by epithelial cells. To test this hypothesis, recombinant human IL-10 was evaluated for its capacity to attenuate the release of neutrophil chemotactic activity and IL-8 from a human epithelial cell line in response to IL-1 beta and tumor necrosis factor-alpha. Neutrophil chemotactic activity and IL-8 in lung epithelial culture supernatant fluids were significantly lower after culture with nitrated human IL-10 compared with non-nitrated human IL-10 controls (P < 0.05). Consistent with these results, nitrated human IL-10 attenuated IL-8 mRNA expression more than non-nitrated human IL-10 controls (P < 0.05). These data demonstrate that peroxynitrite exposed human IL-10 has enhanced anti-inflammatory activity and suggest that nitration may play a critical role in the regulation of inflammation within the lower respiratory tract.