interleukin-8 and Hyperoxia

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Topics: Animals; Aquaculture; Erythropoietin; Female; Fish Diseases; Fish Proteins; Fishes; Gene Expression Regulation, Developmental; Hyperoxia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Male; Receptors, Glucocorticoid; Respiratory Physiological Phenomena; Sodium-Hydrogen Exchanger 1

Hypothermia with xenon gas has been used to reduce brain injury and disability rate after perinatal hypoxia-ischemia. We evaluated xenon gas therapy effects in an in vitro model with or without hypothermia on cultured human airway epithelial cells (Calu-3).. Calu-3 monolayers were grown at an air-liquid interface and exposed to one of the following conditions: 1) 21% FiO2 at 37°C (control); 2) 45% FiO2 and 50% xenon at 37°C; 3) 21% FiO2 and 50% xenon at 32°C; 4) 45% FiO2 and 50% xenon at 32°C for 24 hours. Transepithelial resistance (TER) measurements were performed and apical surface fluids were collected and assayed for total protein, IL-6, and IL-8. Three monolayers were used for immunofluorescence localization of zonula occludens-1 (ZO-1). The data were analyzed by one-way ANOVA.. TER decreased at 24 hours in all treatment groups. Xenon with hyperoxia and hypothermia resulted in greatest decrease in TER compared with other groups. Immunofluorescence localization of ZO-1 (XY) showed reduced density of ZO-1 rings and incomplete ring-like staining in the 45% FiO2- 50% xenon group at 32°C compared with other groups. Secretion of total protein was not different among groups. Secretion of IL-6 in 21% FiO2 with xenon group at 32°C was less than that of the control group. The secretion of IL-8 in 45% FiO2 with xenon at 32°C was greater than that of other groups.. Hyperoxia and hypothermia result in detrimental epithelial cell function and inflammation over 24-hour exposure. Xenon gas did not affect cell function or reduce inflammation.

Topics: Anesthetics, Inhalation; Cells, Cultured; Humans; Hyperoxia; Hypothermia; Hypoxia-Ischemia, Brain; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Respiratory Mucosa; Tight Junctions; Treatment Outcome; Xenon

Topics: Autophagy; CCAAT-Enhancer-Binding Protein-beta; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Endoplasmic Reticulum Stress; Etoposide; Extracellular Matrix; Fetus; Fibroblasts; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Humans; Hyperoxia; Interleukin-1; Interleukin-8; Lung; Matrix Metalloproteinase 3; Oxygen; Plasminogen Activator Inhibitor 1; Primary Cell Culture; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

The aim of this study was to investigate the role of pre-B cell colony-enhancing factor (PBEF) in the pathogenesis of bronchopulmonary dysplasia (BPD) using an established cell model of BPD. For this purpose, EA.hy926 cell cultures were divided into 4 groups as follows: the air group as the blank control, the hyperoxia group, the hyperoxia plus PBEF siRNA group and the hyperoxia plus scramble siRNA group. Cell viability and the generation of reactive oxygen species (ROS) were determined using respective kits. Moreover, the protein and mRNA expression levels of PBEF, interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) were also detected by corresponding methods. Compared with the hyperoxia group, the ROS levels in the hyperoxia plus PBEF siRNA group were significantly reduced (P<0.01). The silencing of PBEF increased cell viability compared with the hyperoxia group. The protein and mRNA expression levels of PBEF, IL-8 and TNF-α were all decreased in the hyperoxia plus PBEF siRNA group compared with the hyperoxia group (P<0.01). Our study thus demonstrates that the inhibition of PBEF attenuates oxidative stress and inflammation induced by hyperoxia in EA.hy926 cells, suggesting that PBEF may be a potential diagnostic and therapeutic target, which may be used for the development of novel treatment strategies for BPD.

Topics: Bronchopulmonary Dysplasia; Cell Line; Cytokines; Humans; Hyperoxia; Inflammation; Interleukin-8; Nicotinamide Phosphoribosyltransferase; Oxidative Stress; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Supplemental oxygen (O2) is used as adjunct therapy in anesthesia, emergency, and intensive care medicine. We hypothesized that excessive O2 levels (hyperoxia) can directly injure human adult cardiac myocytes (HACMs). HACMs obtained from the explanted hearts of transplantation patients were exposed to constant hyperoxia (95% O2), intermittent hyperoxia (alternating 10 min exposures to 5% and 95% O2), constant normoxia (21% O2), or constant mild hypoxia (5% O2) using a bioreactor. Changes in cell morphology, viability as assessed by lactate dehydrogenase (LDH) release and trypan blue (TB) staining, and secretion of vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), and various pro-inflammatory cytokines (interleukin, IL; chemokine C-X-C motif ligand, CXC; granulocyte-colony stimulating factor, G-CSF; intercellular adhesion molecule, ICAM; chemokine C-C motif ligand, CCL) were compared among treatment groups at baseline (0 h) and after 8, 24, and 72 h of treatment. Changes in HACM protein expression were determined by quantitative proteomic analysis after 48 h of exposure. Compared with constant normoxia and mild hypoxia, constant hyperoxia resulted in a higher TB-positive cell count, greater release of LDH, and elevated secretion of VEGF, MIF, IL-1β, IL-6, IL-8, CXCL-1, CXCL-10, G-CSF, ICAM-1, CCL-3, and CCL-5. Cellular inflammation and cytotoxicity gradually increased and was highest after 72 h of constant and intermittent hyperoxia. Quantitative proteomic analysis revealed that hypoxic and hyperoxic O2 exposure differently altered the expression levels of proteins involved in cell-cycle regulation, energy metabolism, and cell signaling. In conclusion, constant and intermittent hyperoxia induced inflammation and cytotoxicity in HACMs. Cell injury occurred earliest and was greatest after constant hyperoxia, but even relatively brief repeating hyperoxic episodes induced a substantial inflammatory response.

Topics: Cells, Cultured; Chemokine CCL3; Granulocyte Colony-Stimulating Factor; Humans; Hyperoxia; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; L-Lactate Dehydrogenase; Myocytes, Cardiac; Time Factors; Vascular Endothelial Growth Factor A

Although the mechanisms by which hyperoxia promotes bronchopulmonary dysplasia are not fully defined, the inability to maintain optimal interleukin (IL)-10 levels in response to injury secondary to hyperoxia seems to play an important role. We previously defined that hyperoxia decreased IL-10 production and pre-treatment with recombinant IL-10 (rIL-10) protected these cells from injury. The objectives of these studies were to investigate the responses of IL-10 receptors (IL-10Rs) and IL-10 signalling proteins (IL-10SPs) in hyperoxic foetal alveolar type II cells (FATIICs) with and without rIL-10. FATIICs were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 hrs. Cells in room air were used as controls. IL-10Rs protein and mRNA were analysed by ELISA and qRT-PCR, respectively. IL-10SPs were assessed by Western blot using phospho-specific antibodies. IL-10Rs protein and mRNA increased significantly in FATIICs during hyperoxia, but JAK1 and TYK2 phosphorylation showed the opposite pattern. To evaluate the impact of IL-8 (shown previously to be increased) and the role of IL-10Rs, IL-10SPs were reanalysed in IL-8-added normoxic cells and in the IL-10Rs' siRNA-treated hyperoxic cells. The IL-10Rs' siRNA-treated hyperoxic cells and IL-8-added normoxic cells showed the same pattern in IL10SPs with the hyproxic cells. And pre-treatment with rIL-10 prior to hyperoxia exposure increased phosphorylated IL-10SPs, compared to the rIL-10-untreated hyperoxic cells. These studies suggest that JAK1 and TYK2 were significantly suppressed during hyperoxia, where IL-8 may play a role, and rIL-10 may have an effect on reverting the suppressed JAK1 and TYK2 in FATIICs exposed to hyperoxia.

Topics: Alveolar Epithelial Cells; Animals; Fetus; Gene Expression Regulation; Hyperoxia; Interleukin-10; Interleukin-8; Oxygen; Rats, Sprague-Dawley; Receptors, Interleukin-10; Recombinant Proteins; Signal Transduction

The use of hyperoxia for critically ill patients is associated with adverse impacts resulting in lung injury accompanied by inflammation. The aim of this study was to evaluate aspects of mechanisms that contribute to hyperoxia-induced disruption of the epithelial permeability barrier, and also the protective effects of the antioxidants α-tocopherol and ascorbate. 16HBE14o- cells were cultured as monolayers at an air-liquid interface for 6 days, after which transepithelial electrical resistance reached 251.2 ± 4.1 Ω.cm(2) (mean ± standard error of the mean). They were then exposed for 24 h to normoxia (21% O2, 5% CO2), hyperoxia (95% O2, 5% CO2), hyperoxia with 10(-7) M α-tocopherol, hyperoxia with 10(-7) M ascorbate, hyperoxia with 10(-6) M ascorbate, and hyperoxia with a combination of α-tocopherol and ascorbate (10(-7) M and 10(-6) M, respectively). Significant reductions (P < 0.05) in transepithelial electrical resistance seen after hyperoxia (with or without antioxidants) were associated with reductions in the levels of zona occludens-1 (ZO-1) observed by immunohistochemistry, and downregulation of ZO-1 expression (P < 0.01) as compared with normoxia. In contrast, the expression levels of interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) were increased after hyperoxia (P < 0.01), and marked increases in the levels of these cytokines (ELISA) were seen in the medium (P < 0.001) as compared with normoxia. The antioxidant vitamins E and C had a partial protective effect against the hyperoxia-induced reduction in ZO-1 levels and the increase in levels of the proinflammatory cytokines IL-8, IL-6, and TNF-α. In conclusion, hyperoxia-induced epithelial disruption is associated with tight junction weakening, and induction of a proinflammatory environment.

Topics: Antioxidants; Ascorbic Acid; Bronchi; Cell Membrane; Cell Membrane Permeability; Electric Impedance; Epithelial Cells; Gene Expression; Humans; Hyperoxia; Interleukin-6; Interleukin-8; Oxygen; Respiratory Mucosa; Tight Junctions; Tumor Necrosis Factor-alpha; Vitamin E; Zonula Occludens-1 Protein

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.

Topics: Alveolar Epithelial Cells; Animals; Cell Line; Cell Proliferation; Chemokine CCL2; Chemokine CCL5; Cyclin D1; Hyperoxia; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Potassium Channels, Tandem Pore Domain; Proliferating Cell Nuclear Antigen; Pulmonary Alveoli

Hyperoxia plays an important role in the genesis of lung injury in preterm infants. Although alveolar type II cells are the main target of hyperoxic lung injury, the exact mechanisms whereby hyperoxia on fetal alveolar type II cells contributes to the genesis of lung injury are not fully defined, and there have been no specific measures for protection of fetal alveolar type II cells.. The aim of this study was to investigate (a) cell death response and inflammatory response in fetal alveolar type II cells in the transitional period from canalicular to saccular stages during 65%-hyperoxia and (b) whether the injurious stimulus is promoted by creating an imbalance between pro- and anti-inflammatory cytokines and (c) whether treatment with an anti-inflammatory cytokine may be effective for protection of fetal alveolar type II cells from injury secondary to 65%-hyperoxia.. Fetal alveolar type II cells were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 h and 36 h. Cells in room air were used as controls. Cellular necrosis was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry, and cell proliferation was studied by BrdU incorporation. Release of cytokines including VEGF was analyzed by ELISA, and their gene expressions were investigated by qRT-PCR.. 65%-hyperoxia increased cellular necrosis, whereas it decreased cell proliferation in a time-dependent manner compared to controls. 65%-hyperoxia stimulated IL-8-release in a time-dependent fashion, whereas the anti-inflammatory cytokine, IL-10, showed an opposite response. 65%-hyperoxia induced a significant decrease of VEGF-release compared to controls, and similar findings were observed on IL-8/IL-10/VEGF genes expression. Preincubation of recombinant IL-10 prior to 65%-hyperoxia decreased cellular necrosis and IL-8-release, and increased VEGF-release and cell proliferation significantly compared to hyperoxic cells without IL-10.. The present study provides an experimental evidence that IL-10 may play a potential role in protection of fetal alveolar type II cells from injury induced by 65%-hyperoxia.

Topics: Alveolar Epithelial Cells; Animals; Anti-Inflammatory Agents; Apoptosis; Biomarkers; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gestational Age; Hyperoxia; In Situ Nick-End Labeling; Interleukin-10; Interleukin-8; L-Lactate Dehydrogenase; Necrosis; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Vascular Endothelial Growth Factor A

Exposure of lung epithelial cells to hyperoxia results in the generation of excess reactive oxygen species (ROS), cell damage, and production of proinflammatory cytokines (interleukin-8; IL-8). Although activation of the NF-kappaB and c-Jun N-terminal kinase (JNK)/activator protein (AP)-1 transcription pathways occurs in hyperoxia, it is unclear whether activation of the AP-1 pathway has a direct impact on IL-8 production and whether overexpression of superoxide dismutase (SOD) can mitigate these proinflammatory processes. A549 cells were exposed to 95% O(2), and ROS production, AP-1 activation, and IL-8 levels were determined. Experimental groups included cells transduced with a recombinant adenovirus encoding CuZnSOD or MnSOD (two- to threefold increased activity) or transfected with a JNK1 small interfering RNA (RNAi). Hyperoxia resulted in significant increases in ROS generation, AP-1 activation, and IL-8 production, which were significantly attenuated by overexpression of either MnSOD or CuZnSOD. JNK1 RNAi also moderated IL-8 induction. The data indicate that activation of JNK1/AP-1 and subsequent IL-8 induction in hyperoxia are mediated by intracellular ROS, with SOD having significant protective effects.

Topics: Blotting, Western; Cell Line, Tumor; Enzyme Activation; Epithelial Cells; Humans; Hyperoxia; Interleukin-8; Lung; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Transcription Factor AP-1; Transfection

We tested the hypothesis that hyperoxia or pressure exposure differentially activates expression of cytokines and/or matrix modeling proteins in human airway epithelial cells. Calu-3 epithelial cell monolayers were cultured on transwell plates with the apical surface exposed to gas. Following establishment of baseline, plates were placed in a chamber and exposed to: control (21% O (2); atm), hyperoxia (60% O (2); atm), pressure (21% O (2); 40 cm H (2)O), and combination (60% O (2); 40 cm H (2)O). At 72 hour of exposure, monolayers were assessed for integrity, viability, and expression of interleukin (IL)-6, IL-8 and matrix metalloproteinases (MMPs) -2, -7, and -9. Compared with controls, hyperoxia had lower transepithelial resistance ( P < 0.001) and greater IL-6 secretion ( P < 0.01), and pressure had lower cell viability ( P < 0.001) and greater IL-8 secretion ( P < 0.001). Hyperoxia resulted in more latent MMP-2 ( P < 0.05) and MMP-7 ( P < 0.001). Pressure was associated with a rise in MMPs independent of oxygen exposure ( P < 0.05). Hyperoxia and pressure differentially affected MMP activities in Calu-3 cells and may lead to the different functional and structural abnormalities observed in these in vitro studies.

Topics: Bronchi; Cells, Cultured; Epithelial Cells; Humans; Hyperoxia; Image Processing, Computer-Assisted; Immunohistochemistry; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Pressure; Respiratory Mucosa

To explore the temporal expression and significance of Caspase-3 and interleukin-8 (IL-8) in hyperoxia-induced lung injury in preterm rats.. Two-day-old Sprague-Dawley preterm rats were randomly divided into Air group and Hyperoxia group (each rats in each group). Rats in the Hyperoxia group were exposed to 85% O(2), while rats in the Air group were exposed to air. The rats in each group were sacrificed at 1, 4, 7, 14 and 21 days after exposure (8 rats at each time point), and lung tissues were collected. Pathomorphology of lungs was observed by hematoxylin-eosine staining. The contents of IL-8 in the homogenate of lungs were detected using ELISA. The expression of Caspase-3 was detected by immunohistochemistry and Western blot.. (1) Lung histopathology: after hyperoxia exposure 1 day, no significant effects on alveoli were found; but on days 4 and 7, alveolitis appeared: there were necrotic abscission cells in alveolar space, increased inflammatory cells infiltration, lung interstitial edema; on days 14 and 21, lung structure derangement, decreased number of alveoli, simplified and vesicular lung structure, all of which showed the retardation of alveolar formation. (2) the contents of IL-8 in the homogenate of lungs had no significant change on day 1 but increased significantly on days 4, 7 and 14 compared with air control group (P < 0.01 for all). (3) Immunohistochemistry detected the expression of Caspase-3 in the lung: the intensity of Caspase-3 expression increased significantly on days 4, 7 and 14 compared with air control group (P < 0.01). (4) Western blotting detected the expression of caspase-3 in the lung: the pattern of dynamic expression of Caspase-3 was similar to the results of immunohistochemistry.. Both apoptosis and necrosis contribute to cell death during hyperoxia. Apoptosis and necrosis may both play an important role in hyperoxia-induced lung injury in preterm rats.

Topics: Animals; Animals, Newborn; Apoptosis; Caspase 3; Female; Hyperoxia; Interleukin-8; Lung; Lung Injury; Male; Necrosis; Rats; Rats, Sprague-Dawley; Time Factors

Exogenous surfactant is critical in the treatment of neonates with respiratory distress syndrome. Lucinactant (Surfaxin; Discovery Laboratories, Inc.) is a surfactant replacement therapy containing sinulpeptide, which may reduce lung inflammation. This study tested whether Lucinactant reduces markers of inflammation, damage and remodeling in human airway epithelial cells exposed to hyperoxia. Calu-3 monolayers cultured at an air-liquid interface were treated apically with 140 microL of normal saline, Lucinactant or Beractant (Survanta; Abbott Laboratories, Inc.). Treated monolayers were exposed to 60% O(2)/5% CO(2) for 24 or 72 h. Transepithelial resistance (TER; p < 0.001) and cell viability (p < 0.05) were greater in both surfactant groups compared with saline; by 72 h Lucinactant cells had greater TER than Beractant (p < 0.001). Surfactant treated groups secreted less IL-8 than saline (p < 0.001), whereas Lucinactant cells secreted less IL-6 than saline and Beractant (p < 0.001). Matrix metalloproteinase 7, expressed by saline and Beractant treated cells at 24 h, was attenuated by 72 h by Beractant (p < 0.001), but was never detected in Lucinactant cells. Histology indicated less injury with Lucinactant relative to Beractant and saline. These data suggest that Lucinactant was protective compared with Beractant and control.

Topics: Anti-Inflammatory Agents; Biological Products; Cells, Cultured; Humans; Hyperoxia; Intercellular Signaling Peptides and Proteins; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Peptides; Pneumonia; Pulmonary Alveoli; Respiratory Mucosa

To determine the protective effect of perflubron (PFB), a type of perfluorochemical liquid, in hyperoxia-induced cellular injury in the human airway epithelial cells.. A controlled, in vitro laboratory study.. Tertiary-care children's hospital.. Human airway epithelial cells.. Human airway epithelial cells, Calu-3 cells, grown on polycarbonate porous filters at an air-liquid interface culture were exposed to normoxic (Fico(2) = 5%, balance air) or hyperoxic (Fio(2) = 95%, balance CO(2)) conditions. Hyperoxia-induced cellular changes were monitored by measuring transepithelial resistance (TER) of monolayers, histology of cells, total protein, and interleukin-8 (IL-8) secretion in apical surface fluid (ASF) washings. Under hyperoxic conditions, the protective effect of PFB was assessed by directly adding PFB liquid to the apical surface of monolayers.. During hyperoxic gas-liquid interface culture, Calu-3 monolayers exhibited a loss of cellular integrity morphologically, decreased protein concentration, and IL-8 level in ASF washings. During hyperoxic PFB-liquid interface culture, there was an overall increase in TER value of monolayers, improved histology, decreased total protein secretion in ASF washings, and unaltered IL-8 secretion. Cytomorphologic observations of PFB-treated Calu-3 cells indicated the presence of varying numbers of differently sized intracellular vacuoles during both normoxic and hyperoxic conditions.. We conclude that the air-liquid interface culture of Calu-3 may be helpful in understanding mechanisms of lung injuries caused in clinical practice, and PFB protects against hyperoxia-induced airway epithelial cell injury by promoting cellular integrity as well as cytologic modifications. PFB-liquid interface culture of Calu-3 may be a useful in vitro model for studying the cytoprotective role of liquid ventilation.

Topics: Cell Culture Techniques; Epithelial Cells; Fluorocarbons; Humans; Hydrocarbons, Brominated; Hyperoxia; Interleukin-8; Liquid Ventilation; Lung; Respiratory Insufficiency

The purpose of this study was to determine the retinal expression of angiogenic chemokines/cytokines in a mouse model of oxygen-induced retinopathy.. C57BL/6 (B6) mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and then recovered in room air. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine relative mRNA levels of KC, macrophage inflammatory protein-2 (MIP-2), interleukin-1alpha (IL-1alpha), and interferon gamma (IFN-gamma). Immunohistochemistry was used to localize KC in the retina. IL-1alpha was also injected into the vitreous of mouse eyes, and KC expression was examined by RT-PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry.. KC expression at both the mRNA and protein levels was increased in P14, P17, and P21 of hyperoxia-injured eyes. KC immunoreactivity was localized along the nerve fiber layer and in radial Müller cell processes. IL-1alpha mRNA was modestly increased in hyperoxia-injured eyes on P14 and P17. INF-gamma mRNA was not detected in the retina. Adult mouse eyes injected with IL-1alpha demonstrated increased levels of both KC mRNA and protein, with KC immunoreactivity localized to Müller cell processes.. Oxygen-induced injury to the developing retina results in the induction of the CXC chemokine KC at both the mRNA and protein levels during the peak time points of neovascularization, suggesting a possible role in the pathogenesis of retinopathy of prematurity.

Topics: Animals; Animals, Newborn; Chemokine CXCL1; Chemokine CXCL2; Chemokines; Chemokines, CXC; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression; Hyperoxia; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Interleukin-1; Interleukin-8; Mice; Mice, Inbred C57BL; Oxygen; Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The alveolar macrophage is an important source of interleukin (IL)-8 during pulmonary injury. The IL-8 gene promoter sequence contains nuclear factor (NF)-kappa B, NF-IL6, and activator protein (AP)-1 binding sequences. These sites may have differing regulatory roles in hyperoxia-exposed macrophages than in those stimulated by bacterial lipopolysaccharide (LPS). U-937 and THP-1 macrophage-like cells were exposed to air-5% CO2 or 95% O2-5% CO2, with or without 1.0 microg/ml of LPS, and transfected with an IL-8 promoter-reporter containing NF-kappa B, NF-IL6, or AP-1 mutations. Hyperoxia and LPS caused additive increases in IL-8 production by U-937 cells, whereas THP-1 cells responded only to LPS. An NF-kappa B mutation ablated baseline and O2- and LPS-stimulated reporter activity in both cell lines, whereas NF-IL6 mutations had little effect. An AP-1 mutation had an intermediate effect. LPS, but not hyperoxia, stimulated nuclear translocation of NF-kappa B in both cell lines. Pharmacological blockade of NF-kappa B nuclear translocation ablated LPS-, but not hyperoxia-, stimulated IL-8 production. Although an intact promoter NF-kappa B site is crucial to macrophage IL-8 production, only LPS-stimulated production appears to require additional nuclear translocation of NF-kappa B.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Nucleus; Gene Expression Regulation; Humans; Hyperoxia; Interleukin-8; Lipopolysaccharides; Macrophages; NF-kappa B; Oxidative Stress; Oxygen; Promoter Regions, Genetic; RNA, Messenger; Transcription Factor AP-1; U937 Cells

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-alpha (TNF-alpha)-mediated activation of NF-kappaB and NF-kappaB-dependent gene expression by a mechanism involving increased activation of IkappaB kinase (IKK). Because the signal transduction mechanisms induced by IL-1beta are distinct to that of TNF-alpha, herein we sought to determine whether hyperoxia modulates IL-1beta-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1beta caused increased activation of IKK, prolonged the degradation of IkappaBalpha, and prolonged the nuclear translocation and DNA binding of NF-kappaB compared with cells treated with IL-1beta alone in room air. Hyperoxia did not affect IL-1beta-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-beta alone. In contrast to the effects on the IKK/IkappaBalpha/NF-kappaB pathway, simultaneous treatment with hyperoxia and IL-1beta did not augment NF-kappaB-dependent gene expression compared with treatment with IL-1beta alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1beta caused hyperphosphorlyation of the NF-kappaB p65 subunit compared with treatment with IL-1beta alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1beta augments activation of IKK, prolongs degradation of IkappaBalpha, and prolongs nuclear translocation and DNA binding of NF-kappaB. This activation, however, is not coupled to increased expression of NF-kappaB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.

Topics: Cell Line; Epithelial Cells; Gene Expression; Humans; Hyperoxia; I-kappa B Kinase; I-kappa B Proteins; Interleukin-1; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Phosphorylation; Pneumonia; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Respiratory Mucosa; Signal Transduction; Transcription Factor RelA

To examine whether prematurity significantly changes the lung inflammatory response to oxygen, rabbit lung explant cultures were exposed to 95% or 5% oxygen for 24 hours. Interleukin (IL)-8 protein concentrations from homogenates of the premature lung rose significantly after hyperoxia (6.8 +/- 1.8 in 5% O2 to 45.7 +/- 21.3 pg/microg protein in 95% O2) but not in the term lung (15.9 +/- 6.7 to 20.4 +/- 4.3 pg/microg protein). There was no change in IL-8 mRNA after hyperoxia in either age group. Preterm lungs demonstrated higher IL-8 levels by fluorescence-activated cell sorting (FACs) analysis and immunohistochemistry. This model may help determine why premature lungs are more susceptible to oxygen-induced disease.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation; Hyperoxia; In Vitro Techniques; Interleukin-8; Lung; Oxygen; Pregnancy; Premature Birth; Rabbits; RNA, Messenger; Term Birth

Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.

Topics: Adenocarcinoma; Bacterial Adhesion; Cell Line, Tumor; Humans; Hyperoxia; Interleukin-8; Mitochondria; Oxidoreductases; Pseudomonas aeruginosa; Recombinant Proteins; Respiratory Mucosa; Staphylococcus aureus; Superoxide Dismutase; Transfection

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1beta and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2',7'-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.

Topics: Animals; Animals, Newborn; Blotting, Southern; Bronchoalveolar Lavage Fluid; Cell Separation; Cells, Cultured; Cytokines; Female; Gestational Age; Hyperoxia; Interleukin-1; Interleukin-8; Lung; Macrophages, Alveolar; Rabbits; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Monocyte chemoattractant protein-1 (MCP-1), acting through its C-C chemokine receptor 2 (CCR-2), has important roles in inflammation, angiogenesis, and wound repair. The individual and combined effects of inhaled nitric oxide (NO) and hyperoxia on lung MCP-1 and CCR-2 in relation to lung leukocyte dynamics are unknown. Because MCP-1 gene is up-regulated by oxidants, we hypothesized that inhaled NO with hyperoxia will increase MCP-1 production and CCR-2 expression more than either gas alone. We randomly assigned young piglets to breathe room air (RA), RA+50 ppm NO (RA+NO), O(2), or O(2)+NO for 1 or 5 d before sacrifice. Lungs were lavaged and tissues preserved for hybridization studies, Western blotting, histology, and immunohistochemistry. The results show that lung MCP-1 production and alveolar macrophage count were significantly elevated in the 5-d O(2) and O(2)+NO groups relative to the RA group (p < or = 0.05). In contrast, lung CCR-2 abundance was diminished in the O(2) group (p

Topics: Administration, Inhalation; Animals; Animals, Newborn; Chemokine CCL2; Female; Hyperoxia; Immunohistochemistry; Interleukin-8; Lung; Macrophages, Alveolar; Male; Nitric Oxide; Receptors, CCR2; Receptors, Chemokine; RNA, Messenger; Swine

Interleukin (IL)-8 is an important mediator of acute lung injury. Hyperoxia induces IL-8 production in some cell types, but its effect on IL-8 gene expression in respiratory epithelium is not well described. In addition, IL-8 gene expression resulting from the combined effects of hyperoxia and proinflammatory cytokines has not been well characterized. We treated cultured respiratory epithelial-like cells (A549 cells) with hyperoxia alone, tumor necrosis factor (TNF)-alpha alone, or the combination of TNF-alpha and hyperoxia and evaluated IL-8 gene expression. Hyperoxia alone had a minimal effect on IL-8 gene expression, and TNF-alpha alone increased IL-8 gene expression in a time-dependent manner. In contrast, the combination of TNF-alpha and hyperoxia synergistically increased IL-8 gene expression as measured by ELISA (TNF-alpha alone for 24 h = 769 +/- 89 pg/ml vs. hyperoxia + TNF-alpha for 24 h = 1, 189 +/- 89 pg/ml) and Northern blot analyses. Experiments involving IL-8 promoter-reporter assays, electromobility shift assays, and Western blot analyses demonstrated that hyperoxia augmented TNF-alpha-mediated activation of the IL-8 promoter by a nuclear factor (NF)-kappaB-dependent mechanism and increased the duration of NF-kappaB nuclear translocation after concomitant treatment with TNF-alpha. Additional reporter gene assays demonstrated, however, that increased activation of NF-kappaB does not fully account for the synergistic effect of hyperoxia and that the NF-IL-6 site in the IL-8 promoter is also required for the synergistic effect of hyperoxia. We conclude that hyperoxia alone has a minimal effect on IL-8 gene expression but synergistically increases IL-8 gene expression in the presence of TNF-alpha by a mechanism involving cooperative interaction between the transcription factors NF-kappaB and NF-IL-6.

Topics: Binding Sites; CCAAT-Enhancer-Binding Protein-delta; CCAAT-Enhancer-Binding Proteins; Cell Nucleus; DNA-Binding Proteins; Gene Expression; Genes, Reporter; Humans; Hyperoxia; Interleukin-8; Luciferases; Mutation; NF-kappa B; Nuclear Proteins; Plasmids; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Animals; Capillaries; Cells, Cultured; Erythrocytes; Fluorescent Dyes; Hyperoxia; Interleukin-8; Kinetics; Leukocytes; Luminescent Measurements; Microcirculation; Microscopy, Confocal; Myocardium; Polysaccharides; Pulmonary Alveoli; Rats; Selectins

In the development of lung damage induced by oxidative stress, it has been proposed that changes in alveolar macrophages (AM) function with modifications in cytokine production may contribute to altered repair processes. To characterize the changes in profiles of cytokine production by macrophages exposed to oxidants, the effects of hyperoxia (95% O2) on interleukin (IL)-1 beta, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) expression were studied. Experiments were first performed using AM obtained from control subjects and children with interstitial lung disease. Results showed that a 48 h O2 exposure was associated with two distinct patterns of response: a decrease in TNF-alpha, IL-1 beta and IL-6 expression, and an increase in IL-8. To complete these observations we used U937 cells that were exposed for various durations to hyperoxia. We confirmed that a 48 h O2 exposure led to similar changes with a decrease in TNF-alpha, IL-1 beta and IL-6 production and an increase in IL-8. Interestingly, this cytokine response was preceded during the first hours of O2 treatment by induction of TNF-alpha, IL-1 beta and IL-6. These data indicate that hyperoxia induces changes in the expression of macrophages inflammatory cytokines, and that these modifications appear to be influenced by the duration of O2 exposure.

Topics: Bronchoalveolar Lavage Fluid; Cells, Cultured; Child; Cytokines; Gene Amplification; Gene Expression Regulation; Humans; Hyperoxia; Interleukin-1; Interleukin-6; Interleukin-8; Lung Diseases, Interstitial; Macrophages, Alveolar; Oxidative Stress; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

We previously demonstrated that 48 h of 100 ppm inhaled nitric oxide (NO) and 90% O2 causes surfactant dysfunction and pulmonary inflammation in mechanically ventilated newborn piglets. Because peroxynitrite (the product of NO and superoxide) is thought to play a major role in the injury process, recombinant human superoxide dismutase (rhSOD, a scavenger of superoxide) might minimize this insult. Four groups of newborn piglets (1-3 days of age) were ventilated with 100 ppm NO and 90% O2 for 48 h. Piglets received no drug, 5 mg/kg rhSOD intratracheally at time 0, 5 mg/kg rhSOD intratracheally at 0 and 24 h, or 10 mg/kg rhSOD by nebulization at time 0. At 48 h, bronchoalveolar lavage (BAL) was performed, and lung tissue was analyzed for markers of inflammation, oxidative injury, acute lung injury, and surfactant function. There were significant differences between rhSOD-treated piglets and untreated controls with respect to BAL neutrophil chemotactic activity, cell counts, and protein concentration as well as lung tissue malondialdehyde concentrations. Minimum surface tension of BAL surfactant from all groups studied was increased, with no differences found among groups. These data suggest that rhSOD, at the doses used, mitigated the inflammatory changes, oxidative damage, and acute lung injury from exposure to 100 ppm NO and 90% O2 but did not appear to improve surfactant function. This has important clinical implications for infants treated with hyperoxia and NO for neonatal lung disorders.

Topics: Administration, Inhalation; Animals; Animals, Newborn; Humans; Hyperoxia; Interleukin-8; Lung; Nitric Oxide; Pulmonary Surfactants; Recombinant Proteins; Superoxide Dismutase; Swine

Topics: Cell Division; Cytokines; DNA; DNA Damage; Endothelium, Vascular; Humans; Hyperoxia; Interleukin-1; Interleukin-8; Oxygen; Reactive Oxygen Species; RNA, Messenger; Thymidine; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbilical Veins