Abstract
BackgroundPrevious studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability.MethodsHuman pulmonary alveolar epithelial cells (cell line and primary cells) were transfected with human PBEF cDNA or PBEF siRNA and then cultured in the presence or absence of TNFα. PBEF and IL-8 expression were analyzed by RT-PCR and Western blotting. In addition, changes in pulmonary alveolar epithelial and artery endothelial cell barrier regulation with altered PBEF expression was evaluated by an in vitro cell permeability assay.ResultsOur results demonstrated that, in human pulmonary alveolar epithelial cells, the overexpression of PBEF significantly augmented basal and TNFα-stimulated IL-8 secretion by more than 5 to 10-fold and increased cell permeability by >30%; the knockdown of PBEF expression with siRNA significantly inhibited basal and TNFα-stimulated IL-8 secretion by 70% and IL-8 mRNA levels by 74%. Further, the knockdown of PBEF expression also significantly attenuated TNFα-induced cell permeability by 43%. Similar result was observed in human pulmonary artery endothelial cells.ConclusionThese results suggest that PBEF may play a vital role in basal and TNFα-mediated pulmonary inflammation and pulmonary epithelial barrier dysfunction via its regulation of other inflammatory cytokines such as IL-8, which could in part explain the role of PBEF in the susceptibility and pathogenesis of ALI. These results lend further support to the potential of PBEF to serve as a diagnostic and therapeutic target to ALI.
Highlights
Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI)
Dose-response and time-course of TNFD induced interleukin 8 (IL-8) protein expression in A549 cells In order to examine the role of PBEF in human pulmonary alveolar epithelial cell inflammation, we began by quantifying TNFD induced IL-8 protein expression within A549 cells
Secreted IL-8 levels was significantly increased in a time-dependent manner after TNFD (15 ng/ml) treatment compared with control group (Figure 1, panel B), especially at 6, 18, and 24 h time points
Summary
Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. The mortality and morbidity of ALI/ARDS remain high since the etiology and molecular pathogenesis are still incompletely understood. Our previous study, based on extensive microarray gene expression profiling in canine, murine, and human ALI, revealed pre-B-cell-colony-enhancing factor (PBEF) as a significantly upregulated gene [5]. Our findings were confirmed and extended by Bajwa et al [6], who showed that the PBEF T-1001G variant allele and related haplotype are associated with increased odds of developing ARDS and increased hazard of intensive care unit mortality among at-risk patients. We found that PBEF is critically involved in thrombin-induced lung endothelial cell barrier dysregulation [7]
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