Randall S. Frey

TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase

Interactions of polymorphonuclear neutrophils (PMNs) with endothelial cells may contribute to the activation of endothelial cell responses involved in innate immunity. We explored a novel function of PMN NADPH oxidase in the mechanism of Toll-like receptor-2 (TLR2) upregulation induced by LPS-TLR4 signaling in endothelial cells. We showed that LPS induced TLR2 up-regulation through TLR4- and MyD88-dependent signaling. In neutropenic mice, the LPS-induced NF-kB activation and TLR2 expression were significantly reduced, and both responses were restored upon repletion by PMN obtained from WT mice but not by PMNs from NADPH oxidase gp91pho(-/-) mice. These findings were recapitulated in mouse lung vascular endothelial cells cocultured with PMNs, indicating that the augmented NF-kB activation and the resultant TLR2 upregulation in endothelial cells were secondary to oxidant signaling generated by PMN NADPH oxidase. The functional relevance of NADPH oxidase in mediating TLR4-induced TLR2 expression in endothelial cells was evident by markedly elevated and stable ICAM-1 expression as well as augmented PMN migration in response to sequential challenge with LPS and peptidoglycan. Thus, PMN NADPH oxidase-derived oxidant signaling is an important determinant of the cross talk between TLR4 and TLR2 and the control of endothelial cell activation.

PKCζ Regulates TNF-α--Induced Activation of NADPH Oxidase in Endothelial Cells

Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKCzeta, has been implicated in the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKCzeta in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47(phox) and membrane-bound gp91(phox) in human pulmonary artery endothelial (HPAE) cells. TNF-alpha failed to activate oxidant generation in lung vascular endothelial cells derived from p47(phox-/-) and gp91(phox-/-) mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-alpha resulted in the phosphorylation of p47(phox) and its association with gp91(phox). Inhibition of PKCzeta by multiple pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation of p47(phox), and its translocation to the membrane. PKCzeta was shown to colocalize with p47(phox), and inhibition of PKCzeta activation prevented the interaction of p47(phox) with gp91(phox) induced by TNF-alpha. Furthermore, inhibition of association of p47(phox) with gp91(phox) prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKCzeta in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.

Phosphatidylinositol 3-kinase γ signaling through protein kinase Cζ induces NADPH oxidase-mediated oxidant generation and NF-κB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kγ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kγ-mediated oxidant signaling in the mechanism of NF-κB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110γ catalytic subunit of PI3Kγ. Tumor necrosis factor (TNF) α challenge of wild type ECs caused p110γ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110γ–/– ECs. ROS production was the result of TNFα activation of Ser phosphorylation of NADPH oxidase subunit p47phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110γ–/– ECs. Additionally, the TNFα-activated NF-κB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kγ. TNFα challenge of p110γ–/– ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCζ. Thus, PI3Kγ lies upstream of PKCζ in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-κB activation and ICAM-1 expression.

Tumor Necrosis Factor-α Induces Early-Onset Endothelial Adhesivity by Protein Kinase Cζ–Dependent Activation of Intercellular Adhesion Molecule-1

Abstract— We tested the hypothesis that TNF-&agr; induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the &bgr;2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-&agr; resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-&agr; challenge. We observed that TNF-&agr; induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1–dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-&agr;–induced endothelial adhesivity was protein synthesis–independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKC&zgr;, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-&agr;–induced ICAM-1 gene transcription, we determined the possibility that PKC&zgr; was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-&agr; stimulation of endothelial cells induced PKC&zgr; activation and its association with ICAM-1. Inhibition of PKC&zgr; by pharmacological and genetic approaches prevented the TNF-&agr;–induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-&agr; induces early-onset, protein synthesis–independent expression of endothelial adhesivity by PKC&zgr;-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.

Endothelial cell–restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

E3 ubiquitin ligase Cblb regulates the acute inflammatory response underlying lung injury.

The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. Here we show that Cblb also has an unexpected function in acute lung inflammation. Cblb attenuates the sequestration of inflammatory cells in the lungs after administration of lipopolysaccharide (LPS). In a model of polymicrobial sepsis in which acute lung inflammation depends on the LPS receptor (Toll-like receptor 4, TLR-4), the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Loss of Cblb significantly increases sepsis-induced release of inflammatory cytokines and chemokines. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of wild-type Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for the transcription factor nuclear factor-κB (NF-κB) in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of neutrophils is impaired in the absence of Cblb. Our data reveal that Cblb regulates the TLR4-mediated acute inflammatory response that is induced by sepsis.

SHIP2 Is Recruited to the Cell Membrane upon Macrophage Colony-Stimulating Factor (M-CSF) Stimulation and Regulates M-CSF-Induced Signaling

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-κB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.

Blockade of class IA phosphoinositide 3-kinase in neutrophils prevents NADPH oxidase activation- and adhesion-dependent inflammation

We examined the role of class IA phosphoinositide 3-kinase (PI3K) in the regulation of activation of NADPH oxidase in PMNs and the mechanism of PMN-dependent lung inflammation and microvessel injury induced by the pro-inflammatory cytokine TNF-α. TNF-α stimulation of PMNs resulted in superoxide production that was dependent on CD11b/CD18-mediated PMN adhesion. Additionally, TNF-α induced the association of CD11b/CD18 with the NADPH oxidase subunit Nox2 (gp91phox) and phosphorylation of p47phox, indicating the CD11b/CD18 dependence of NADPH oxidase activation. Transduction of wild-type PMNs with Δp85 protein, a dominant-negative form of the class IA PI3K regulatory subunit, p85α, fused to HIV-TAT (TAT-Δp85) prevented (i) CD11b/CD18-dependent PMN adhesion, (ii) interaction of CD11b/CD18 with Nox2 and phosphorylation of p47phox, and (iii) PMN oxidant production. Furthermore, studies in mice showed that i.v. infusion of TAT-Δp85 significantly reduced the recruitment of PMNs in lungs and increase in lung microvascular permeability induced by TNF-α. We conclude that class IA PI3K serves as a nodal point regulating CD11b/CD18-integrin-dependent PMN adhesion and activation of NADPH oxidase, and leads to oxidant production at sites of PMN adhesion, and the resultant lung microvascular injury in mice.

Functional PU.1 in macrophages has a pivotal role in NF-κB activation and neutrophilic lung inflammation during endotoxemia

Although the role of ETS family transcriptional factor PU.1 is well established in macrophage maturation, its role in mature macrophages with reference to sepsis- related animal model has not been elucidated. Here, we report the in vivo function of PU.1 in mediating mature macrophage inflammatory phenotype by using bone marrow chimera mice with conditional PU.1 knockout. We observed that the expression of monocyte/macrophage-specific markers CD 11b, F4/80 in fetal liver cells, and bone marrow-derived macrophages were dependent on functional PU.1. Systemic inflammation as measured in terms of NF-κB reporter activity in lung, liver, and spleen tissues was significantly decreased in PU.1-deficient chimera mice compared with wild-type chimeras on lipopolysaccharide (LPS) challenge. Unlike wild-type chimera mice, LPS challenge in PU.1-deficient chimera mice resulted in decreased lung neu-trophilic inflammation and myeloperoxidase activity. Similarly, we found attenuated inflammatory gene expression (cyclooxygenase-2, inducible nitric-oxide synthase, and TLR4) and inflammatory cytokine secretion (IL-6, MCP-1, IL-1β, TNF-α, and neutrophilic chemokine keratinocyte-derived chemokine) in PU.1-deficient mice. Most importantly, this attenuated lung and systemic inflammatory phenotype was associated with survival benefit in LPS-challenged heterozygotic PU.1-deficient mice, establishing a novel protective mechanistic role for the lineage-specific transcription factor PU.1.

Chapter 8 Reactive Oxygen Species and Endothelial Permeability

Alterations in endothelial permeability are a defining feature of diverse processes including arteriosclerosis, inflammation, ischemia/reperfusion injury, angiogenesis, pulmonary edema in acute lung injury and adult respiratory distress syndrome. Endothelial monolayer permeability increases as a result of both disruption of endothelial cell–cell contacts and EC contraction. Disruption of endothelial cell–cell junctions occurs concomitantly with the redistribution and tyrosine phosphorylation of the VE-cadherin-containing adherens junction (AJ) protein complexes. Little is known about mechanisms of how endothelial permeability is regulated. Reactive oxygen species (ROS) including superoxide (O2−) and hydrogen peroxide (H2O2) generated by activated polymorphonuclear leukocyte (PMNs) and endothelial cells (ECs) impair endothelial barrier integrity by promoting loss of cell–cell adhesions and reorganization of actin cytoskeleton. These responses are involved in promoting transendothelial migration of PMNs and endothelial permeability. Major source of ROS in PMNs and ECs is NADPH oxidase. Phagocyte NADPH oxidase consists of membrane-bound gp91phox and p22hox as well as cytosolic components such as p47phox, p67phox and small GTPase Rac. Recently, several novel homologues of gp91phox (Nox2) of NADPH oxidase (Nox) have been cloned in non-phagocytic cells. In ECs Nox1, Nox2, Nox4 and Nox5 are functionally expressed. NADPH oxidase in ECs is activated by inflammatory cytokines, thrombogenic agents, growth factors, G-protein coupled receptor agonists and shear stress. ROS derived from NADPH oxidase function as signaling molecules to activate various redox signaling pathways through modulating activity of kinases and phosphatases, which may contribute to increase in endothelial permeability. Understanding mechanisms by which ROS regulate endothelial permeability is important for the development of novel therapeutic approaches against various diseases such as inflammation, atherogenesis and acute lung injury.