Stretch induces a growth factor in alveolar cells via protein kinase

Positive-pressure mechanical ventilation can injure the lung, causing edema and alveolar inflammation in a complication termed ventilator-induced lung injury (VILI). Cytokines such as interleukin-8 (IL-8) reportedly are important in this inflammatory response. On the other hand, hepatocyte growth factor (HGF) promotes regeneration of the lung, and delays pulmonary fibrosis. We postulated that cyclic stretch upregulates production and release of both of mediators. Human alveolar epithelial cells (A549) cultured on a silicoelastic membrane were tested for mRNA expression and release of IL-8 and HGF after cyclic stretch in vitro. Stretch induced mRNA expression and release of these mediators. The signaling pathway from cyclic stretch to release of IL-8 and HGF appeared to involve protein kinase C in the signal transduction pathway.     

Role of Rel A and IkappaB of nuclear factor kappaB in the release of interleukin-8 by cyclic mechanical strain in human alveolar type II epithelial cells A549

BACKGROUND AND OBJECTIVE: Overdistention of the lung tissue during mechanical ventilation may initiate ventilator-induced lung injury (VILI). Release of cytokines, including IL-8, may be responsible for VILI, although the mechanisms remain unclear. This study aimed to determine whether stretch-induced IL-8 production is dependent on degradation of IkappaB (IkappaB) and the resulting Rel A translocation into the nucleus. METHODS: A549 cells were exposed to cyclic stretch of varying amplitude, frequency and duration before the mRNA and protein level of IL-8 were measured. To observe the role of Rel A and IkappaB of nuclear factor kappaB, A549 cells were exposed to cyclic stretch for 5 min to 1 h. Real-time PCR and ELISA respectively were performed to detect mRNA and IL-8 protein. Rel A and IkappaBalpha were assessed by Western blot. Further confirmation was sought using a nuclear factor kappaB inhibitor (PDTC) before mechanical stretch. RESULTS: A549 cells exposed to cyclic stretch produced IL-8 in a time- and strain-dependent manner, but there was no observed effect related to stretch frequency. Activation of Rel A and IkappaBalpha was detected 10 min after the initiation of stretch, peaked at 15 min and returned to baseline within 1 h. IL-8 production was partially inhibited by the presence of PDTC. CONCLUSION: Cyclic mechanical stretch can activate Rel A translocation and IkappaBalpha degradation, thus inducing the secretion of IL-8 in alveolar epithelial type II cells. Pharmacological inhibition of Rel A and IkappaBalpha inhibits IL-8 mRNA and protein levels, suggesting novel approaches to prevent VILI.     

Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cells

OBJECTIVE: Ventilator-induced lung injury (VILI) is characterized by release of inflammatory cytokines, but the mechanisms are not well understood. We hypothesized that stretch-induced cytokine production is dependent on oxidant release and is regulated by intracellular glutathione (GSH) inhibition of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding. METHODOLOGY: Type 2-like alveolar epithelial cells (A549) were exposed to cyclic stretch at 15% strain for 4 h at 20 cycles/min with or without N-acetylcysteine (NAC) or glutathione monoethylester (GSH-e) to increase intracellular GSH, or buthionine sulfoximine (BSO), to deplete intracellular GSH. RESULTS: Cyclic stretch initially caused a decline in intracellular GSH and a rise in the levels of isoprostane, a marker of oxidant injury. This was followed by a significant increase in intracellular GSH and a decrease in isoprostane. Stretch-induced IL-8 and IL-6 production were significantly inhibited when intracellular GSH was further increased by NAC or GSH-e (P < 0.0001). Stretch-induced IL-8 and IL-6 production were augmented when intracellular GSH was depleted by BSO (P < 0.0001). NAC blocked stretch-induced NF-kappa B and AP-1 binding and inhibited IL-8 mRNA expression. CONCLUSIONS: We conclude that oxidant release may play a role in lung cell stretch-induced cytokine release, and antioxidants, which increase intracellular GSH, may protect lung cells against stretch-induced injury.     

Cytokines and biotrauma in ventilator-induced lung injury: a critical review of the literature

BACKGROUND: Mechanical ventilation is known to induce and aggravate lung injury. One of the underlying mechanisms is biotrauma, an inflammatory response in which cytokines play a crucial role. OBJECTIVE: To review the literature on the role of cytokines in ventilator-induced lung injury (VILI) and multiple organ dysfunction syndrome (MODS). MATERIAL AND METHODS: 57 English written, peer-reviewed articles on cytokines in in-vitro settings (n=5), ex-vivo models (n=9) in-vitro models (n=19) and clinical trials (n=24). RESULTS: Mechanical ventilation (MV) can induce cytokine upregulation in both healthy and injured lungs. The underlying mechanisms include alveolar cellular responses to stretch with subsequent decompartimentalisation due to concomitant cellular barrier damage. The cytokines involved are interleukin (IL)-8 and CXC chemokines, and probably IL-6, IL-1beta and tumour necrosis factor (TNF)-alpha. Cytokines are important for signalling between inflammatory cells and recruiting leucocytes to the lung. There is strong circumstantial evidence that the release of cytokines into the systemic circulation contributes to the pathogenesis of MODS. Multiple studies demonstrate the relation between elevated proinflammatory cytokine concentrations and mortality. CONCLUSION: Cytokines are likely to play a role in the various interrelated processes that lead to VILI and other MV-related complications, such as MODS and possibly ventilatorassociated pneumonia. Cytokines are good surrogate endpoints in exploring the pathogenesis and pathophysiology of VILI in both experimental and clinical studies.     

Role of mechanical stretching and lipopolysaccharide in early apoptosis and IL-8 of alveolar epithelial type II cells A549

ObjectiveTo investigate the effects of mechanical stretching and lipopolysaccharide (LPS) on the early apoptosis and IL-8 production of alveolar epithelial type Π cells A549.MethodsThe experimental matrix consisted of three integrated studies. In the first study, A549 cells were subjected to different stretching strain frequency and duration time to see the effects on the early apoptosis. In the second study, A549 cells were subjected to mechanical stretch (15% 4 h, 0.5 Hz) and LPS (1 or 100 ng/mL) to see whether mechanical strain and LPS also have an addictive effect on the early apoptosis. In the third study to investigate whether this addictive effect could be induced by LPS and mechanical stretch on IL-8 production, A549 cells were subjected to LPS (100 ng/mL) and mechanical strain (15%, 0.5 Hz, 4 h). Real time PCR and enzyme linked immunosorbent assay were used to measure mRNA and protein level of IL-8. The early apoptosis was detected by flow cytometry.ResultsMechanical stretch induced the early apoptosis in a force and frequency and time-dependent manner. In the presence of LPS, mechanical stretch enhanced LPS-induced early apoptosis, especially in 100 ng/mL LPS group compared with 1 ng/mL LPS and the control group. Mechanical stretch increased IL-8 production and enhanced LPS-induced IL-8 screation both in mRNA and protein levels.ConclusionsMechanical stretch can induce the early apoptosis and IL-8 secretion. Mechanical stretch and LPS have an addictive effect on the early apoptosis and IL-8 production in alveolar type 2 cells, which is one of the mechanisms of ventilator-induced lung injury.     

普通型间质性肺炎和非特异性间质性肺炎肺组织中不同细胞因子的表达及其意义

目的　研究转化生长因子 (TGF) β1、碱性成纤维细胞生长因子 (b FGF)、白细胞介素 8(IL 8)、白细胞介素 13(IL 13)、γ干扰素 (IFN γ)在普通型间质性肺炎 (UIP)和非特异性间质性肺炎(NSIP)肺组织中的分布、表达及意义。方法　经胸腔镜或开胸肺活检获取 5例UIP和 8例NSIP患者的肺组织。对照组 5例 ,来自手术切除的远离肺癌原发灶的周边肺组织。用免疫组化法半定量分析细胞因子的分布及表达。结果　TGF β1、IL 8、b FGF主要分布在肺泡上皮细胞、肺泡巨噬细胞、细支气管上皮细胞 ,UIP组表达强于NSIP组和对照组。IL 13主要分布在肺泡上皮细胞、肺泡巨噬细胞、间质单个核细胞 ,UIP、NSIP组表达无明显差异 ,但均强于对照组。IFN γ主要分布在间质单个核细胞 ,NSIP组表达强于UIP组和对照组。UIP组的IL 13/IFN γ比值为 (2 18± 0 76 ) ,NSIP组为(0 95± 0 2 8) ,对照组为 (0 91± 0 16 ) ,3组比较差异均有显著性 (P值均 <0 0 5 ) ,而NSIP组与对照组比较差异无显著性。对照组只有肺泡巨噬细胞表达上述各细胞因子。结论　TGF β1、IL 8、b FGF在UIP和NSIP患者肺组织中表达强度的不同和IL 13/IFN γ的是否平衡可能参与了UIP和NSIP不同的发病过程。     

呼吸机所致肺损伤的生物伤机制与易感性

呼吸机所致肺损伤（VILI）的生物伤机制颇受关注。机械牵拉能刺激肺泡上皮细胞和肺内皮细胞、活化多形核白细胞和肺泡巨噬细胞，激活系列信号转导通路，导致细胞因子的释放、黏附分子的表达、细胞外基质及其降解酶的改变、花生四烯酸通路的活化、氧化与抗氧化失衡、核苷酸谱改变、凝血与纤溶异常、凋亡与坏死的变化。VILI存在诸多易感因素，如局部缺血和再灌注、体温、年龄、血流速度、内毒素等。     

Legionella pneumophila-induced NF-kappaB- and MAPK-dependent cytokine release by lung epithelial cells.

Legionella pneumophila causes community-acquired pneumonia with high mortality, but little is known about its interaction with the alveolar epithelium. The aim of this study was to investigate whether L. pneumophila infection of lung epithelial cells (A549) resulted in pro-inflammatory activation. L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-alpha, IL-1beta, interferon-gamma and granulocyte colony-stimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colony-stimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-kappaB subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-kappaB complex dose-dependently lowered IL-8 expression. Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase- and nuclear factor-kappaB/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.     

ST2 gene induced by type 2 helper T cell (Th2) and proinflammatory cytokine stimuli may modulate lung injury and fibrosis

The authors have investigated gene expression of ST2 in the lung tissue of a bleomycin (BLM)-induced lung fibrosis model in vivo and in a human lung fibroblast cell line, WI38, and a human type II alveolar epithelial cell line, A549, reacting to proinflammatory and type 2 helper T cell (Th2)-type cytokine stimuli in vitro. The lung mRNA expression of interleukin (IL)-4, IL-5, IL-1beta, and tumor necrosis factor (TNF)-alpha increased significantly at day 7 after instillation of BLM, whereas interferon (IFN)-gamma mRNA expression did not increase. ST2 and transforming growth factor (TGF)-beta1 mRNA expression of the lung increased significantly between days 7 and 21, and increased to maximal levels at day 14 post-BLM challenge. ST2 mRNA expression statistically correlated with TGF-beta 1 mRNA expression. In addition, the combination of IL-1 beta, TNF-alpha, and IL-4 had an additive effect on ST2 mRNA expression from A549 cells and WI38 cells. These findings suggest that soluble ST2 gene may increase, possibly reflecting the development of the inflammatory process and the Th2-type immune response in the fibrotic lung tissue, and may modulate a process of pulmonary fibrosis.     

Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strain

Background and objective: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. Methods: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. Results: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. Conclusions: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific.     

Transgenic modeling of transforming growth factor-beta(1): role of apoptosis in fibrosis and alveolar remodeling

Inflammation and tissue remodeling with pathologic fibrosis are common consequences of Th2 responses in the lung and other organs. Interleukin (IL)-13 and transforming growth factor-beta1 (TGF-beta(1)) are frequently coexpressed in these responses and are believed to play important roles in the pathogenesis of Th2-induced pathologies. To shed light on the mechanisms of these responses, overexpression transgenic approaches were used to selectively target each of these cytokines to the murine lung. IL-13 proved to be a potent stimulator of eosinophilic inflammation, mucus metaplasia, tissue fibrosis, and alveolar remodeling. CC chemokines, specific chemokine receptors (CCR2, CCR1), adenosine metabolism, vascular endothelial growth factor, and IL-11 contributed to the genesis of these responses. IL-13 also induced tissue fibrosis, at least in part, via its ability to induce and activate TGF-beta(1). In the TGF-beta(1) transgenic mouse, epithelial apoptosis preceded the onset of tissue fibrosis and alveolar remodeling. In addition, chemical (Z-VAD-fmk) and genetic (null mutations of early growth response gene 1) interventions blocked apoptosis and ameliorated TGF-beta(1)-induced fibrosis and alveolar restructuring. These studies define an IL-13-TGF-beta(1) pathway of tissue remodeling that regulates inflammation, mucus metaplasia, apoptosis, vascular responses, and fibrosis in the lung. They also highlight the intimate relationship between apoptosis and fibrosis induced by TGF-beta(1). By defining the complexities of this pathway, these studies highlight sites at which therapies can be directed to control these important responses.     

Effects by 8-bromo-cyclicAMP on basal and organic dust-induced release of interleukin-6 and interleukin-8 in A549 human airway epithelial cells

Inhalation of organic dust from a swine-confinement building leads to an intense inflammatory reaction with an increased number of inflammatory cells and mediators in the upper and lower respiratory tract of previously unexposed subjects. In vitro the dust induces cytokine release from epithelial cells and alveolar macrophages. It is known that intracellular cyclic AMP (cAMP) contributes to the regulation of inflammatory responses. We therefore investigated whether 8-Bromo-cAMP, a cell membrane-permeable cAMP analogue, would influence release of the cytokines interleukin-6 (IL-6) and IL-8 in a human airway epithelial cell line, A549, exposed to a suspension of the organic dust, and to a supernatant prepared by centrifugation (at low g-force) of a suspension of dust. The large particulate matter was thus sedimented, leaving bacteria, whole and cell wall constituents in the supernatant. Cytokine release was measured with enzyme-linked immunosorbent assay (ELISA). The cytokine release induced by a supernatant was 23% (IL-6) and 27% (IL-8) of the release induced by a dust suspension. 8-Bromo-cAMP (1 mM) doubled basal IL-6 release and IL-6 release induced by a dust supernatant (P<0.01), and increased IL-6 release induced by a dust suspension by 19% (P<0.05). 8-Bromo-cAMP did not affect basal IL-8 release, partially inhibited (28%) the release of IL-8 induced by a dust suspension (P<0.01), but increased IL-8 release induced by a dust supernatant by 13% (P<0.05). In summary, expression of the cytokines IL-6 and IL-8 is differentially regulated by 8-Bromo-cAMP, both with regard to basal and dust-induced release. The results indicate that 8-Bromo-cAMP attenuated IL-8 release by affecting signaling transductions induced by the particulate fraction.     

Interleukin-4 and tumour necrosis factor-alpha inhibit transforming growth factor-beta production in a human bronchial epithelial cell line: possible relevance to inflammatory mechanisms in chronic obstructive pulmonary disease

OBJECTIVE: Human bronchial epithelial cells are known to secrete an array of inflammatory cytokines including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-4, which may play a role in immune responses in lung diseases such as chronic obstructive pulmonary disease (COPD). However, the regulatory mechanisms governing cytokine production in bronchial epithelia in COPD are largely unknown. Transforming growth factor-beta (TGF-beta) is an anti-inflammatory cytokine and is involved in airway repair. The purpose of this study was to study the effect of TNF-alpha and IL-4 (pro-inflammatory cytokines known to be up-regulated in COPD), on the production of TGF-beta (a negative regulator of inflammation) by epithelial cells. METHODOLOGY: A bronchial epithelial cell line was used as an in vitro culture model (16HBE). Cell cultures were stimulated with various combinations of TNF-alpha and IL-4 (20 ng/mL) for 24 h. Transforming growth factor-beta production was measured by flow cytometry, enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Exposure to TNF-alpha significantly up-regulated production of IL-4 from cultured epithelial cells. Unstimulated cells spontaneously released TGF-beta. Exposure to TNF-alpha and IL-4 significantly inhibited production of TGF-beta. The inhibitory effects of TNF-alpha and IL-4 on TGF-beta synthesis were summative. CONCLUSIONS: The inhibitory effect of IL-4 and TNF-alpha on production of the regulatory cytokine TGF-beta in a bronchial epithelial cell line suggests that such mechanisms may contribute to the progression of the inflammatory response and compromise repair processes in inflammatory lung diseases such as COPD.     

Lipopolysaccharide enhances substance P-mediated neutrophil adherence to epithelial cells and cytokine release

Lipopolysaccharide (LPS) is implicated in many respiratory tract inflammatory diseases. Tachykinins, especially substance P (SP) through the NK-1 receptor, mediate leukocyte adhesion to the endothelial or airway epithelial cells. Here we assessed the enhancement by LPS of tachykinin-mediated neutrophil adherence to alveolar epithelial cells, and associated interleukin-1 beta (IL-1beta) and tumor necrosis factor (TNF-alpha) release. Neutrophil adherence to A549 epithelial cell was not increased by LPS (100 ng/ml), or SP (10(-)(12)-10(-)(8) M) alone, but was significantly enhanced by their combination (LPS + SP). Neutrophil adherence to epithelial cells induced IL-1beta and TNF-alpha release from A549 cells either spontaneously or stimulated by SP or LPS. LPS + SP significantly enhanced IL-1beta and TNF-alpha release. The NK-1 receptor antagonist L-732,138 inhibited this enhancement response. Prevention of neutrophil adherence by CD11b/CD18 blocking antibody or by placing a filter on the epithelial monolayer diminished spontaneous or LPS + SP-enhanced IL-1beta and TNF-alpha release. Pretreatment with the serine protease inhibitor cocktail also inhibited LPS + SP-enhanced neutrophil adherence-dependent IL-1beta and TNF-alpha release as well as their mRNA expression. In conclusion, we have demonstrated LPS enhanced SP-mediated neutrophil adherence and associated IL-1beta and TNF-alpha release from the A549 epithelial monolayer, partly through NK-1 receptors. Neutrophil adherence to epithelial cells may release serine protease to induce IL-1beta and TNF-alpha release and their synthesis.     

Response of alveolar type II epithelial cells to mechanical stretch and lipopolysaccharide

BACKGROUND: Although many therapeutic strategies have been developed clinically, the mortality associated with acute respiratory distress syndrome remains very high. OBJECTIVES: In this research, we used a cytomechanical method to elucidate the reason for this. METHODS: A549 cells were stimulated with lipopolysaccharide (LPS; 1 or 100 ng/ml) and/or mechanical stretch (5, 15, 30%) in varying frequency (0.2, 0.5, 1 Hz) at indicated time (1, 2, 4 h). Real time PCR and enzyme-linked immunosorbent assay were used to measure mRNA and protein levels of IL-8. RESULTS: In the presence of mechanical stretch, 100 ng/ml LPS significantly increased IL-8 production after 4 h of 5% stretch (p < 0.05). In the presence of LPS, stretch enhanced LPS-induced IL-8 protein production in a force-, time- and frequency-dependent manner. At both the 1- and 4-hour time points, mechanical stretch and LPS increased IL-8 mRNA levels, respectively, and stretch enhanced LPS-induced IL-8 mRNA levels (p < 0.05). CONCLUSIONS: Using cytomechanic methods, we found a synergistic effect of LPS and mechanical stretch on IL-8 production. The response of alveolar type II cells to mechanical stretch depends on their different pathologic states and the applied mechanical stretch, which may reversely influence the outcome of patients with acute respiratory distress syndrome.     

Differential effects of varying concentrations of clostridium difficile toxin A on epithelial barrier function and expression of cytokines

BACKGROUND: Presentation after Clostridium difficile infection may depend on the level of epithelial exposure to toxins. We investigated epithelial barrier function and expression of interleukin (IL)-8 and transforming growth factor (TGF)-beta in response to varying concentrations of C. difficile toxin A. METHODS: T84 cells were either preexposed or continuously exposed to C. difficile toxin A (0.01-1000 ng/mL). Barrier function was assessed by measurements of transepithelial electrical resistance. RESULTS: Preexposure to < or =10 ng/mL toxin A led to an increase in the release of TGF-beta 1, but there was no change in the expression of IL-8. In contrast, after preexposure to >10 ng/mL toxin A, there was enhanced expression of IL-8, but release of TGF-beta 1 was similar to that in control monolayers. After preexposure to >10 ng/mL toxin A, there was complete and irreversible loss of electrical resistance. At lower concentrations, loss of resistance across monolayers was followed by recovery, which was enhanced by all 3 recombinant isoforms of TGF-beta. Pretreatment with recombinant isoforms of TGF-beta or coculture with TGF-beta 3-expressing colonic subepithelial myofibroblasts was also protective. CONCLUSIONS: In C. difficile infection, the development and severity of colonic inflammation may depend on the exposure of intestinal epithelial cells to toxins and the expression of proinflammatory (IL-8) and protective (TGF-beta) factors.     

Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors

Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1β and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses.