Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro

BACKGROUND: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP). AIM: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells. METHODS: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy. RESULTS: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes CONCLUSIONS: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage.     

Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.     

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.     

Leukotriene synthesis by epithelial cells

Leukotrienes (LTs) are intercellular signaling molecules that evoke a variety of responses. They are best known as potent promoters of inflammation. Normally, LTs are produced primarily by leukocytes. As a result, current models regarding the production of LTs in the context of disease focus on the leukocytes as the site of production. Structural cells, including epithelial cells, are typically relegated to supportive roles. It is recognized that epithelial cells normally contain all the components necessary for LT synthesis except the enzyme 5-lipoxygenase (5-LO). There is accumulating evidence that some populations of epithelial cells normally express low levels of 5-LO and can synthesize LTs autonomously. Moreover, certain factors, including bacterial and viral infection, can promote the expression of 5-LO in airway, gastrointestinal and skin epithelial cells. The appearance of active 5-LO enzyme in epithelial cells at these sites may contribute to diseases like cancer, colitis and psoriasis. This paper reviews the state of our knowledge regarding the expression of 5-LO in epithelial cells, the factors that modify that expression, and the implications regarding pathogenesis.     

Formation of endothelin by cultured airway epithelial cells

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co-eluted with endothelin 1 and endothelin 3 respectively.     

Effects of amphotericin B on ion transport proteins in airway epithelial cells

Topical intranasal application of the antifungal Amphotericin B (AmphoB) has been shown as an effective medical treatment of chronic rhinosinusitis. Because this antibiotic forms channels in lipid membranes, we considered the possibility that it affects the properties and/or cell surface expression of ion channels/pumps, and consequently transepithelial ion transport. Human nasal epithelial cells were exposed apically to AmphoB (50 microM) for 4 h, 5 days (4 h daily), and 4 weeks (4 h daily, 5 days weekly) and allowed to recover for 18-48 h. AmphoB significantly reduced transepithelial potential difference, short-circuit current, and the amiloride-sensitive current. This was not due to generalized cellular toxicity as judged from normal transepithelial resistance and mitochondrial activity, but was related to inhibitory effects of AmphoB on ion transport proteins. Thus, cells exposed to AmphoB for 4 h showed decreased apical epithelial sodium channels (ENaC) activity with no change in basolateral Na(+)K(+)-ATPase activity and K(+) conductance, and reduced amount of alphaENaC, alpha1-Na(+)K(+)-ATPase, and NKCC1 proteins at the cell membrane, but no change in mRNA levels. After a 5-day treatment, there was a significant decrease in Na(+)K(+)-ATPase activity. After a 4-week treatment, a decrease in basolateral K(+) conductance and in alphaENaC and alpha1-Na(+)K(+)-ATPase mRNA levels was also observed. These findings may reflect a feedback mechanism aimed to limit cellular Na(+) overload and K(+) depletion subsequently to formation of AmphoB pores in the cell membrane. Thus, the decreased Na(+) absorption induced by AmphoB resulted from reduced cell surface expression of the ENaC, Na(+)K(+)-ATPase pump and NKCC1 and not from direct inhibition of their activities.     

Regulation of endothelin-1 synthesis in cultured guinea pig airway epithelial cells by various cytokines.

To study regulatory mechanisms influencing the synthesis and release of ET-1, a potent bronchoconstrictor, epithelial cells from guinea pig tracheas were cultured to test various cytokines for the synthesis of ET-1 and its precursor, big ET-1. Cytokines tested were divided into 4 groups, based on their potential modes of action. IL-8, TNF alpha and TGF beta transiently increased the synthesis of ET-1, while EGF, PDGF and GM/CSF promoted proliferation of ET-1 synthesizing cells. IL-1 enhanced the synthesis of ET-1 precursor without mitogenesis, whereas IL-2, IL-6 and IGF-1 induced both the synthesis of big ET-1 and mitogenesis. These observations suggest that cytokines involved in damage, inflammation and repair of the airway epithelial layer regulate the synthesis and release of ET-1 by multiple mechanisms, thereby influencing airway muscle tone.     

Cystic fibrosis pathogens activate Ca2+-dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells

Much of the pulmonary disease in cystic fibrosis is associated with polymorphonuclear leukocyte-dominated airway inflammation caused by bacterial infection. Respiratory epithelial cells express the polymorphonuclear chemokine interleukin-8 (IL-8) in response to ligation of asialylated glycolipid receptors, which are increased on damaged or regenerating cells and those with cystic fibrosis transmembrane conductance regulator mutations. Because both Pseudomonas aeruginosa and Staphylococcus aureus, the most common pathogens in cystic fibrosis, bind asialylated glycolipid receptors such as asialoGM1, we postulated that diverse bacteria can activate a common epithelial signaling pathway to elicit IL-8 expression. P. aeruginosa PAO1 but not pil mutants and S. aureus RN6390 but not the agr mutant RN6911 stimulated increases in [Ca(2+)](i) in 1HAEo- airway epithelial cells. This response stimulated p38 and ERK1/2 mitogen-activated protein kinase (MAPK) signaling cascades resulting in NF-kappaB activation and IL-8 expression. Ligation of the asialoGM1 receptor or thapsigargin-elicited Ca(2+) release activated this pathway, whereas P. aeruginosa lipopolysaccharide did not. The rapid kinetics of epithelial activation precluded bacterial invasion of the epithelium. Recognition of asialylated glycolipid receptors on airway epithelial cells provides a common pathway for Gram-positive and Gram-negative organisms to initiate an epithelial inflammatory response.     

TLR4在气道上皮细胞诱导的哮喘气道平滑肌细胞迁移中的作用

目的:探讨Toll样受体4(TLR4)的激活在气道上皮细胞诱导的哮喘气道平滑肌细胞(ASMCs)迁移中的作用。方法:细胞消化法培养原代哮喘ASMCs,TNF-α刺激上皮细胞系RTE细胞收集细胞培养上清液,检测上清液中IL-8和RANTES的含量,改良Boyden趋化小室检测哮喘ASMCs的跨膜迁移,以TLR4抗体作为工具药,观察其在上皮细胞诱导的哮喘ASMCs跨膜迁移中的作用。结果:各TNF-α组培养上清液中IL-8和RANTES水平均显著增高,20 ng/ml组较其他组显著增高(P<0.01)。各组哮喘ASMCs跨膜迁移较正常组均增加(P<0.01);哮喘组和TNF-α+TLR4抗体组哮喘ASMCs跨膜迁移数较TNF-α组显著减少(P<0.01)。TLR4抗体组哮喘ASMCs跨膜迁移数较哮喘组增加(P<0.05)。结论:气道上皮细胞可能通过分泌细胞因子激活哮喘ASMCs表面的TLR4,诱导增强ASMCs的跨膜迁移,在哮喘的气道重构中发挥一定的作用。     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Sulphated proteins secreted by rat mammary epithelial cells

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.