Modulation Of IL-1β Mediated Pulmonary Endothelial And Epithelial Cell Inflammation And Permeability By Pre-B-cell Colony Enhancing Factor

Pre-B-cell colony-enhancing factor was first identified as a protein that was secreted by activated lymphocytesin bone marrow stromal cells and that synergized with IL-7 and stem cell factor (SCF) to stimulate early stage B cell formation. PBEF in non-proliferating cells is more abundant in the cells nuclei than in the cytoplasma, whereas in proliferating cells this distribution is inverse, suggesting that PBEF is a cell cycle-associated protein. IL-1βand TNF-α, inflammatory mediators known to inhibit neutrophil apoptosis, lead to an increased PBEF transcription in neutrophils and monocytes. The inhibition of apoptosis by these inflammatory stimuli is abrogated by blocking PBEF translation. PBEF is constitutively expressed by the fetal membranes during pregnancy. It increases the expression of IL-6 and IL-8 and is important in both normal spontaneous labor and infection-induced preterm labor. PBEF acts as a nicotinamide phosphoribosyltransferase involoved in nicotinamide adenine dinucleotide (NAD+) synthesis. This activity is shown to be important for vascular smooth muscle cell (SMC) maturation. PBEF has recently been identified as a new adipokine which exerts insulin-mimetic effects and has a physiological role in lowing plasma glucose levels and improvement of insulin sensitivity. Therefore, PBEF as a multifunctional protein is critically involved in many physiological and pathological processes.Acute lung injury (ALI) is characterized by pulmonary inflammation, non-cardiogenic edema, and severe systemic hypoxemia. Acute respiratory distress syndrome (ARDS) is the severe form of ALI. One of the earliest manifestations of ALI is a diffuse intense inflammatory process and damage to both endothelial and epithelial cell barriers, resulting in marked extravasation of vascular fluid into the alveolar airspace. A number of inflammatory cells and cytokines including neutrophils, macrophages, IL-1βand IL-8 can induce or aggravate the inflammation of endothelial and epithelial cells, leading to this barrier dysfunctions.The mortality and morbidity of ALI/ARDS remain high since the etiology and molecular pathogenesis are still incompletely understood.In our previous study on animal models of ALI, we identified pre-B-cell colony enhancing factor as a significantly upregulated gene in ALI. We discovered single nucleotide polymorphisms in the human PBEF gene promoter. Moreover we found that carriers of the haplotype GC from SNPs T-1001G and C-1543T had a 7.7-fold higher risk of ALI. The T variant from the SNP C-1535T resulted in a significant decrease in the transcription rate (1.8-fold) by the reporter gene assay. We further found that a reduction in PBEF protein expression by siRNA significantly attenuated pulmonary artery endothelial cell barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance. Taken together, these results strongly indicate PBEF as a potential novel candidate gene and biomarker in ALI.The objective of this study was to further elucidate the role of PBEF in pulmonary inflammation and permeability using pulmonary cells as cell models since increased vascular and epithelial cell inflammation and permeability processes are important features of ALI. We investigated the molecular mechanism by which IL-1βinduces the expression of PBEF in pulmonary vascular endothelial cells. We assessed the effect of PBEF knockdown with PBEF siRNA and PBEF overexpression on basal and IL-1β-mediated pulmonary epithelial cell (A549) and human pulmonary artery endothelial cell (HPAEC) IL-8 production and permeability by in vitro cell permeability assay. We also examined the role of PBEF expression on other inflammatory cytokines such as IL-16 and CCR3. The main work is as followings:1. Dose-response and time-course of IL-1βinduced PBEF protein expression in A549 cells were analyzed by Western blotting. The results demonstrated that IL-1βsignificantly increased PBEF protein expression in a time-dependent manner and cell lysate PBEF expression was significantly increased with different dose treatments of IL-1β(5 to 25 ng/ml).2. The mRNA levels of HPAEC cells treated for 4 h with IL1-βin the presence or absence of actinomycin D, a transcription inhibitor were assessed by RT-PCR. The results showed that simultaneous treatment of HPAEC with IL1-βplus act D prevented the IL1-βinduction of PBEF mRNA, supporting that IL1-βinduces the transcription of PBEF gene. The data indicates that a transcriptional regulation mechanism is at least in part responsible for the IL-1βinduced up-regulation of the PBEF gene expression.3. EMSA analyzed whether the T-variant altered the binding affinity to any transcription factor, which may underlie its effect on the decrease in the transcription of PBEF gene expression. The results revealed that -1535 T-variant in the human PBEF gene promoter has less binding to an unknown transcription factor than the common -1535 C-allele, this difference became more pronounced after the IL1-βor TNFαtreatment. The altered transcriptional factor binding may underlie the reduced expression of PBEF and thus less susceptibility to acute lung injury in those -1535T carriers.4. A549 cells and HPAEC were transiently transfected with pCAGGS-hPBEF or PBEF stealth siRNA in order to increase or knock down PBEF expression. Then we assessed the effect of PBEF knockdown and PBEF overexpression on IL-8 production in the basal and IL-1β-mediated A549 cells and HPAEC. The results demonstrated that knockdown of PBEF expression by PBEF siRNA significantly blunted IL-1β-stimulated IL-8 secretion and its production in the A549 cells and HPAEC, and PBEF-overexpression augmented IL-8 secretion and its production from A549 cells, suggesting that PBEF may be a target of IL-1βinvolved in the inflammatory process during the pathogenesis of ALI.5. The role of PBEF expression on other inflammatory cytokines such as IL-16 and CCR3 was examined by RT-PCR. The results showed that the knockdown of PBEF in A549 cells attenuated IL-1β-stimulated increase of IL-16 and CCR3 gene expression, while the overexpression of PBEF in A549 cells promoted IL-1β-stimulated increase of IL-16 and CCR3 gene expression at mRNA level. These data implicate PBEF might be an important"master" signal transducer or initiator in the inflammation pathway to regulate the synthesis of some inflammatory cytokines and PBEF could play a critical role as an inflammatory cytokine during the pathogenesis of ALI.6. The effect of PBEF knockdown and PBEF overexpression permeability on cell permeability in the basal and IL-1β-mediated A549 cells and HPAEC was investigated by in vitro cell permeability assay. The results demonstrated that the overexpression of PPEF significantly augmented IL-1βmediated cell permeability by 44% in A549 cells and 65% in endothelial cells; the knockdown of PBEF expression significantly attenuated IL-1β-induced cell permeability by 29% in epithelial cells and 24% in endothelial cells. These results indicate that overexpression of PBEF in A549 cells and HPAEC augmented IL-1βinduced lung epithelial cell barrier dysfunction and thus increased cell permeability in vitro.These results suggest that PBEF may play a critical role as an inflammatory cytokine in the development of pulmonary inflammation and dysregulation of pulmonary vascular endothelial and alveolar epithelial cell barriers, which may be an important mechanism underlying PBEF in the pathogenesis of ALI. These results lend further support that PBEF may represent a new diagnostic and therapeutic target in ALI.