Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.

Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells.

BACKGROUND: Expression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441. METHODS: Different quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D. RESULTS: SP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone. CONCLUSION: SP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts.

BACKGROUND: Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells. METHODS: The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-ß1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition. RESULTS: Treatment with different glucocorticoids (1 µM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-ß1 mRNA in H441 cells, increased expression of TGF-ß2 and TGF-ß3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression. CONCLUSIONS: In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-ß1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes.

BACKGROUND: Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. AIM: The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. METHODS: Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. RESULTS: Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4+ lymphocytes. CONCLUSION: For the first time, the immunomodulatory capacity of CHF5633 on CD4+ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4+ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.

Airway angiopoietin-2 in ventilated very preterm infants: association with prenatal factors and neonatal outcome.

OBJECTIVE: Pulmonary angiogenesis is a prerequisite for lung development. Angiopoietin-2 (Ang2) destabilizes endothelial cells through its endothelial receptor TIE-2, enabling vascular sprouting. Ang1 stabilizes new blood vessels. Soluble TIE-2 (sTIE-2) modulates these effects. We hypothesized that histological funisitis is associated with alterations of Ang2 in airways and of the systemic angiopoietin-TIE-2 homeostasis in very low birth weight (VLBW) infants, contributing to pulmonary morbidity and mortality. METHODS: We measured Ang2 in tracheobronchial aspirate fluid (TAF) of 42 VLBW <30 weeks of gestation from day 1 through 15 and Ang1, Ang2, and sTIE-2 in umbilical cord serum of 28 infants by enzyme-linked immunosorbent assay. Histological examination distinguished three groups: funisitis, chorioamnionitis, and controls. RESULTS: Funisitis was associated with lower Ang2 values in TAF but not with changes of Ang1, Ang2, and sTIE-2 in umbilical cord serum. Infants who developed bronchopulmonary dysplasia (BPD) or died had a persistently decreased ratio of previously measured Ang1 to Ang2 in TAF on days 1-5 and increased cord serum concentrations of sTIE-2. Moderate BPD/death was associated with an increase of Ang2 in TAF on day 10 and decreased Ang1/Ang2 ratio from day 3-15. Small for gestational age (SGA) infants had increased Ang2 in TAF on day 1-7 and a lower Ang1/Ang2 ratio on days 5-7. CONCLUSIONS: The predominance of Ang2 in airway fluid of infants with BPD/death and SGA infants suggests a link between disrupted placental and fetal pulmonary angiogenesis. Histological funisitis with reduced Ang2 in TAF was of minor relevance for outcome in our cohort.

Clara cell secretory protein in tracheobronchial aspirates and umbilical cord serum of extremely premature infants with systemic inflammation.

BACKGROUND: A systemic fetal inflammatory response, reflected by chorioamnionitis with funisitis, is a risk factor for bronchopulmonary dysplasia. Clara cell secretory protein (CC10), a product of pulmonary Clara cells, has anti-inflammatory properties. Local down-regulation of CC10 has been associated with inflammatory lung disease. Increased serum levels of CC10 can indicate injury to alveolar-capillary integrity. OBJECTIVE: We hypothesized that extremely premature infants with a systemic fetal inflammatory response would have decreased concentrations of CC10 in tracheobronchial aspirates and that CC10 concentrations in umbilical cord serum of these infants would be increased, reflecting alveolar epithelial damage. METHODS: We measured CC10 concentrations in tracheobronchial aspirates of 42 ventilated extremely premature infants during their first week of life and in umbilical cord serum of 24 of them by ELISA. Standardized histological examination of the placenta, membranes and umbilical cord was used to identify infants with funisitis. RESULTS: Seventeen infants with funisitis had lower CC10 concentrations in tracheobronchial aspirates on days 1 (p < 0.01) and 3 (p < 0.05) than the remaining 25. Exogenous surfactant treatment was associated with higher CC10 concentrations on day 1 (p < 0.05). Initial leukocyte count correlated inversely with CC10 in tracheobronchial aspirates on days 1-5. Umbilical cord serum concentrations of CC10 did not differ between the infants with funisitis and the controls. CONCLUSIONS: Reduced anti-inflammatory CC10 concentrations in airways of extremely premature infants with a fetal inflammatory response might make their lungs susceptible for further postnatal injuries. Umbilical cord serum CC10 is not an indicator for a fetal systemic inflammatory reaction.

Airway concentrations of angiopoietin-1 and endostatin in ventilated extremely premature infants are decreased after funisitis and unbalanced with bronchopulmonary dysplasia/death.

A systemic inflammatory response of the fetus, reflected by histologic funisitis, is a risk factor for bronchopulmonary dysplasia (BPD). Impaired pulmonary angiogenesis accompanied by simplification and rarification of alveoli is a histologic hallmark of BPD. Angiopoietin-1 mediates vascular development, maturation, and stabilization. Endostatin mainly acts as an angiostatic factor. We hypothesized that funisitis was associated with changes of endostatin and angiopoietin-1 concentrations in the airways and that an imbalance between the factors might be associated with BPD or death. We measured concentrations of angiopoietin-1 and endostatin by enzyme-linked immunosorbent assay in tracheobronchial aspirate fluid samples of 42 ventilated preterm infants during postnatal days 1 through 15. The secretory component for IgA served as reference protein. A standardized histologic examination was used to distinguish three groups: chorioamnionitis, funisitis, and controls without inflammation. Concentrations of the mediators steadily decreased. Funisitis was associated with lower concentrations of both proteins, which might impair their physiologic activities in pulmonary angiogenesis. An increase of the ratio angiopoietin-1/endostatin until day 7 of life indicated a shift of the mediators potentially favoring angiogenesis. However, infants, who developed BPD or died, had a decreased ratio on days 1, 3, and 15, suggesting an imbalance toward inhibition of pulmonary angiogenesis.

Systemic fetal inflammation and reduced concentrations of macrophage migration inhibitory factor in tracheobronchial aspirate fluid of extremely premature infants.

OBJECTIVE: Macrophage migration inhibitory factor is a proinflammatory mediator of innate immunity, enhances cell growth, and plays a role in preterm delivery. We speculated that funisitis, reflecting fetal systemic inflammation, would be associated with higher concentrations of macrophage migration inhibitory factor in airways of extremely premature infants. STUDY DESIGN: We measured macrophage migration inhibitory factor by enzyme linked immunosorbent assay in tracheobronchial aspirate fluid of 35 ventilated infants less than 30 weeks' gestational age, throughout the first week of life. Three groups were distinguished histologically: chorioamnionitis, funisitis, and control. RESULTS: Unexpectedly, funisitis was associated with significantly decreased macrophage migration inhibitory factor in tracheobronchial aspirate fluid on day 1 (P < .01) and levels remained lower than in the chorioamnionitis group thereafter. For the 35 patients in total, macrophage migration inhibitory factor steadily declined. CONCLUSION: Decreased macrophage migration inhibitory factor concentrations in airways of extremely premature infants with systemic fetal inflammation early in life might predispose them to pulmonary infection and interfere with maturation of the lung, contributing to adverse pulmonary outcome.

Activation of umbilical cord endothelial cells and fetal inflammatory response in preterm infants with chorioamnionitis and funisitis.

Chorioamnionitis and funisitis are associated with preterm labor and postnatal morbidity. Activation of endothelium resulting in up-regulation of adhesion molecules seems to be a key mechanism in development of organ damage. We investigated whether chorioamnionitis with or without funisitis in preterm infants induced expression and shedding of adhesion molecules in the umbilical cord and resulted in increased concentrations of E-selectin, intercellular adhesion molecule (ICAM)-1, IL-1beta, IL-6, and IL-8 in the cord blood. Data were obtained by using immunohistochemistry and ELISA. Thirty-two preterm infants were divided into three groups according to histology: chorioamnionitis with funisitis, chorioamnionitis without funisitis, and controls without signs of inflammation. ICAM-1 expression on arterial endothelium was higher with funisitis compared with chorioamnionitis alone or with the control group. Similar results for ICAM-1 expression were found in venous endothelium, vascular walls, Wharton's jelly, and amnion epithelium. Endothelial E-selectin and vascular cell adhesion molecule (VCAM)-1 expression was only induced significantly with funisitis. Serum-concentrations of soluble ICAM-1 were higher with funisitis compared with chorioamnionitis alone or control group. Similarly, concentrations of soluble E-selectin, IL-1beta, IL-6, and IL-8 were increased exclusively with funisitis. In conclusion, only chorioamnionitis with funisitis was associated with systemic inflammation and endothelial activation with up-regulation and shedding of umbilical cord adhesion molecules. We speculate that this activation of endothelium may not be limited to the umbilical cord but may also involve other organs resulting in neonatal morbidity. This underlines the importance of funisitis as a risk factor for adverse outcome.

Intratracheal albumin reduces interleukin-8 in tracheobronchial aspirates in piglets after meconium aspiration.

Meconium aspiration induces pulmonary inflammation and reduces surfactant function. We hypothesized that albumin mixed with meconium attenuates pulmonary inflammation and improves surfactant function after meconium aspiration. We measured the concentration of free fatty acids (FFA) in the meconium (110 mg dry weight/mL) and added albumin to provide a molar FFA:albumin ratio of 1:1. Newborn piglets, 0-2 day of age, artificially ventilated and exposed to hypoxemia by ventilation with 8% O2, were randomized to group A receiving meconium (n = 12), or group B receiving meconium + albumin (n = 12), 3 ml/kg intratracheally. The animals were reoxygenated for 8 h. Reoxygenation was started when mean arterial blood pressure was < 20 mm Hg or base excess was < -20 mmol/L. During 8 h of reoxygenation the interleukin-8 concentrations in tracheobronchial aspirates increased 5-fold more in the meconium vs. the meconium + albumin groups (93 +/- 56 vs. 18 +/- 4 pg/mL, p < 0.005). There were no differences between the groups for tumor necrosis factor alpha in tracheobronchial aspirates, recruitment of inflammatory cells in the airspaces or surfactant function in bronchoalveolar lavage fluid. In conclusion, albumin significantly decreased interleukin-8 concentrations in tracheobronchial aspirates after meconium aspiration.