The Chitinase-like Protein YKL-40 Is Secreted by Airway Epithelial Cells at Base Line and in Response to Compressive Mechanical Stress

The chitinase-like protein YKL-40, encoded by the CHI3L1 gene, is a biomarker and functional effector of chronic inflammatory and allergic diseases. In the lung it is associated with asthma severity and reduced lung function. The cellular sources of YKL-40 in human airways and the mechanisms regulating YKL-40 expression are poorly understood. We previously showed that mechanical stress similar to that experienced during bronchoconstriction triggers epithelial cell signaling through epidermal growth factor receptor (EGFR), fibrotic mediator release, and goblet cell hyperplasia consistent with airway remodeling in asthma. We now show that well differentiated normal human bronchial epithelial cells express CHI3L1 and secrete YKL-40 under base-line culture conditions. Mechanical stress (30-cm H₂O transcellular compressive stress) applied for 3 h induces CHI3L1 expression by ∼4-fold compared with time matched controls, resulting in increased secretion of YKL-40 by 3.6-fold 24 h after onset of the 3-h stimulus. Inhibition of EGFR or MEK1/2 (ERK kinase) significantly but incompletely attenuates mechanical stress-induced up-regulation of CHI3L1 expression in normal human bronchial epithelial cells. Direct activation of EGFR utilizing EGF-family ligands induces CHI3L1 expression. Our results reveal that human airway epithelial cells are a source of YKL-40 and demonstrate that mechanical stress potently induces CHI3L1 expression leading to increased secretion of YKL-40 protein in an EGFR and MEK1/2-dependent pathway. In the asthmatic airway mechanical stress may contribute to enhanced YKL-40 levels.     

Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1

Acute lung injury is characterized by injury to the lung epithelium that leads to impaired resolution of pulmonary edema and also facilitates accumulation of protein-rich edema fluid and inflammatory cells in the distal airspaces of the lung. Recent in vivo and in vitro studies suggest that mesenchymal stem cells (MSC) may have therapeutic value for the treatment of acute lung injury. Here we tested the ability of human allogeneic mesenchymal stem cells to restore epithelial permeability to protein across primary cultures of polarized human alveolar epithelial type II cells after an inflammatory insult. Alveolar epithelial type II cells were grown on a Transwell plate with an air-liquid interface and injured by cytomix, a combination of IL-1β, TNFα, and IFNγ. Protein permeability measured by ¹³¹I-labeled albumin flux was increased by 5-fold over 24 h after cytokine-induced injury. Co-culture of human MSC restored type II cell epithelial permeability to protein to control levels. Using siRNA knockdown of potential paracrine soluble factors, we found that angiopoietin-1 secretion was responsible for this beneficial effect in part by preventing actin stress fiber formation and claudin 18 disorganization through suppression of NFκB activity. This study provides novel evidence for a beneficial effect of MSC on alveolar epithelial permeability to protein.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na⁺ channel β-subunit (βENaC-Tg) suggest that raised airway Na⁺ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na⁺ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na⁺ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na⁺ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na⁺ transport and ASL volume, not absolute Na⁺ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.     

Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.     

Loss of βarrestin1 and βarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice

Less is known about the connection between the malfunction of βarrestins and developmental defects as the mice with either of two βarrestin isoforms knockout appear normal. In order to address the biological function of βarrestins during developmental process, we generate βarrestin1/2 double knockout mice. We found that βarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only βarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but βarrestin1 and βarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing βarrestin1 or βarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that βarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that βarrestin1 and βarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.     

Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.     

eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

Oxygen toxicity is the most severe side effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to the overproduction of reactive oxygen species (ROS). In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC) and mouse lungs. Incubation of PAEC under hyperoxia (95% O₂) for 24 h resulted in an increase in peroxynitrite formation. Uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in peroxynitrite formation is accompanied by increases in nitric oxide (NO) release and endothelial NO synthase (eNOS) activity. We have previously reported that association of eNOS with β-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-β-actin association contributes to increased peroxynitrite production, eNOS-β-actin interaction were inhibited by reducing β-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to the actin binding site on eNOS. We found that disruption of eNOS-β-actin interaction prevented hyperoxia-induced increases in eNOS-β-actin association, eNOS activity, NO and peroxynitrite production, and protein tyrosine nitration. Hyperoxia failed to induce the increases in eNOS activity, NO and peroxynitrite formation in COS-7 cells transfected with plasmids containing eNOS mutant cDNA in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Exposure of mice to hyperoxia resulted in significant increases in eNOS-β-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with β-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.     

Pulmonary Collectins Protect Macrophages against Pore-forming Activity of Legionella pneumophila and Suppress Its Intracellular Growth

Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca²⁺-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1–4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.