Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Abstract

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-a activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-a inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-a activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI) 1/2 /arginase II (AII) 2/2 C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI 1/1 /AII 2/2 counterparts, indicating an important role for arginase I in PLYinduced endothelial hyperpermeability. These results identify PKC-a and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.Keywords: PKC; arginase; pneumococcus; pneumolysin; TNF Although severe pneumonia remains the leading cause of mortality worldwide in children aged less than 5 years (1), community-acquired pneumonia represents a major cause of morbidity and mortality mainly in elderly patients (2). Despite the use of potent antibiotics and aggressive intensive care support, the fatality rate associated with Streptococcus pneumoniae, accounting for 45% of all cases of community-acquired pneumonia, is approximately 20% (1, 2). Pulmonary permeability edema, a major complication of severe pneumonia characterized by endothelial hyperpermeability, can occur days after initiation of antibiotics therapy when tissues are sterile and the pneumonia is clearing and correlates with the presence of the bacterial virulence factor pneumolysin (PLY) (3, 4). This cytoplasmic hemolytic protein is released during bacterial lysis, as occurs after treatment with b-lactam antibiotics (5). PLYinduced lung injury was suggested to result from direct pneumotoxic effects on the alveolar-capillary barrier rather than from resident or recruited phagocytic cells (6).Upon binding of PLY to cholesterol in cell membranes, oligo...

A Novel Tumor Necrosis Factor–mediated Mechanism of Direct Epithelial Sodium Channel Activation

“…In previous studies, our research group and other investigators have demonstrated that the lectin-like domain of TNF represents a potent stimulator of ALC in several mammalian species and involves activation of ENaC via an unknown mechanism (21,24,(27)(28)(29)(30)(31)38) . This study was designed to determine the mechanism by which the lectin-like domain of TNF can promote ENaC activity.…”

“…Yet, this dose allowed us to specifically investigate the effect of TIP on PLY-induced impairment of ALC rather than on barrier function. Moreover, we have previously shown that the TIP peptide possesses barrier-protective effects in permeability edema induced by instillation of higher doses of PLY (30).…”

“…The lectin-like domain of TNF is mimicked by the 17-amino acid circular TIP peptide (26), which increases amiloride-sensitive Na 1 uptake in type II AECs (27,28). The TIP peptide has been shown to activate ALC in several animal models of hydrostatic and permeability edema (21,(29)(30)(31). In a recently performed, placebocontrolled, double-blind Phase I clinical trial, TIP peptide (also named AP301) was observed not to induce serious adverse effects upon inhalation (32) Bacterial and viral toxins have been shown to induce significant changes in ENaC P o and/or expression, leading to impaired ALC (33)(34)(35)(36).…”

FMS-Like Tyrosine Kinase 3 Ligand Treatment of Mice Aggravates Acute Lung Injury in Response to Streptococcus pneumoniae: Role of Pneumolysin

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Statin-Conferred Enhanced Cellular Resistance against Bacterial Pore-Forming Toxins in Airway Epithelial Cells

“…Genetic deletion of PLY from S. pneumoniae results in reduction of virulence by several orders of magnitude (23)(24)(25). In addition, PLY has been reported to be a critical virulence factor involved in pneumonia, acute lung injury, and pulmonary permeability edema (26)(27)(28)(29). Hla is another PFT expressed in many strains of S. aureus.…”