Teresa D. Tetley

Differential Regulation of Cytokine Release and Leukocyte Migration by Lipopolysaccharide-Stimulated Primary Human Lung Alveolar Type II Epithelial Cells and Macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-α and IL-1β (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene α (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-α and IL-1β during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1β and TNF-α (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-α (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-α and IL-1β were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-α and IL-1β by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.

Diminished Prostaglandin E2 Contributes to the Apoptosis Paradox in Idiopathic Pulmonary Fibrosis

RATIONALE Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.

Fibroblast Growth Factor 2-Mediated Translational Control of IAPs Blocks Mitochondrial Release of Smac/DIABLO and Apoptosis in Small Cell Lung Cancer Cells

ABSTRACT The mitochondrial release of cytochrome c and Smac/DIABLO has been implicated in the activation of apoptosis in response to cell stress. Smac promotes cytochrome c-induced activation of caspases by sequestering the inhibitor of apoptosis protein (IAP) family of potent caspase suppressors. Differential release from mitochondria of cytochrome c and Smac can occur, but the underlying mechanism and physiological significance of this are unclear. Here we show that the mechanism by which fibroblast growth factor 2 (FGF-2) protects small cell lung cancer (SCLC) cells from etoposide-induced cell death involves inhibition of Smac release but not of cytochrome c release. This process is MEK dependent and correlates with an increased expression of XIAP and cellular IAP-1, mediated principally through translational regulation. Exogenous expression of XIAP is sufficient to inhibit caspase 9 activation, Smac release, and cell death induced by etoposide. Prevention of the FGF-2-promoted increase in levels of functional IAPs by RNA interference or the cell-permeant Smac amino-terminal peptide blocked FGF-2-induced protection. FGF-2 can thus protect SCLC cells from chemotherapeutic drugs by modulating IAP levels via posttranscriptional regulation, providing a mechanism for postmitochondrial survival signaling by the MEK/mitogen-activated protein kinase pathway.

Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 microm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 microm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-microm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1–inducible CCL2 in Pulmonary Fibrosis

RATIONALE Studies in patients and experimental animals provide compelling evidence of the involvement of the major thrombin receptor, proteinase-activated receptor-1 (PAR(1)), and the potent chemokine, chemokine (CC motif) ligand-2 (CCL2)/monocyte chemotactic protein-1, in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PAR(1) knockout mice are protected from bleomycin-induced lung inflammation and fibrosis and this protection is associated with marked attenuation in CCL2 induction. OBJECTIVES The aim of this study was to determine which cell types represent the major source of PAR(1)-inducible CCL2 in the fibrotic lung. METHODS Using immunohistochemistry and dual immunofluorescence, we examined PAR(1) and CCL2 expression in the bleomycin model and human IPF lung. PAR(1) and CCL2 gene expression was also assessed in laser-captured alveolar septae from patients with IPF. The ability of PAR(1) to induce CCL2 production by lung epithelial cells was also examined in vitro. MEASUREMENTS AND MAIN RESULTS We report for the first time that PAR(1) and CCL2 are coexpressed and co-up-regulated on the activated epithelium in fibrotic areas in IPF. Similar observations were found in bleomycin-induced lung injury. Furthermore, we show that thrombin is a potent inducer of CCL2 gene expression and protein release by cultured lung epithelial cells via a PAR(1)-dependent mechanism. CONCLUSIONS These data support the notion that PAR(1) activation on lung epithelial cells may represent an important mechanism leading to increased local CCL2 release in pulmonary fibrosis. Targeting PAR(1) on the pulmonary epithelium may offer a unique opportunity for therapeutic intervention in pulmonary fibrosis and other inflammatory and fibroproliferative conditions associated with excessive local generation of thrombin and CCL2 release.

Health effects of nanomaterials

With the rapid growth of nanotechnology and future bulk manufacture of nanomaterials comes the need to determine, understand and counteract any adverse health effects of these materials that may occur during manufacture, during use, or accidentally. Nanotechnology is expanding rapidly and will affect many aspects of everyday life; there are already hundreds of products that utilize nanoparticles. Paradoxically, the unique properties that are being exploited (e.g. high surface reactivity and ability to cross cell membranes) might have negative health impacts. The rapid progress in development and use of nanomaterials is not yet matched by toxicological investigations. Epidemiological studies implicate the ultrafine (nano-sized) fraction of particulate air pollution in the exacerbation of cardiorespiratory disease and increased morbidity. Experimental animal studies suggest that the increased concentration of nanoparticles and higher reactive surface area per unit mass, alongside unique chemistry and functionality, is important in the acute inflammatory and chronic response. Some animal models have shown that nanoparticles which are deposited in one organ (e.g. lung and gut) may access the vasculature and target other organs (e.g. brain and liver). The exact relationship between the physicochemistry of a nanoparticle, its cellular reactivity, and its biological and systemic consequences cannot be predicted. It is important to understand such relationships to enjoy the benefits of nanotechnology without being exposed to the hazards.

Two distinct phosphoinositide 3-kinases mediate polypeptide growth factor-stimulated PKB activation

Eight human isoforms of phosphoinositide 3‐kinases (PI3Ks) exist, but their individual functions remain poorly understood. Here, we show that different human small cell lung carcinoma (SCLC) cell lines overexpress distinct subsets of class IA and II PI3Ks, which results in striking differences in the signalling cascades activated by stem cell factor (SCF). Over expression of class IA p85/p110α in SCLC cells increased SCF‐stimulated protein kinase B (PKB) activation and cell growth, but did not affect extracellular signal‐regulated kinase (Erk) or glycogen synthase kinase‐3 (GSK‐3). This effect was selective, since it was not observed in SCLC cell lines overexpressing p85/p110β or p85/p110δ. The SCF receptor associated with both class IA p85 and class II PI3KC2β, and both enzymes contributed to SCF‐stimulated PKB activity. A dominant‐negative PI3KC2β blocked both PKB activation and SCLC cell growth in response to SCF. Together our data provide novel insights into the specificity and functional significance of PI3K signalling in human cancer.

New perspectives in nanomedicine.

Recent advances in nanotechnology have revolutionised all aspects of life, from engineering to cosmetics. One of the most exciting areas of development is that of nanomedicine. Due to their size (less than 100nm in one aspect), nanoparticles exhibit properties that are unlike that of the same material in bulk size. These unique properties are being exploited to create new diagnostics and therapeutics for application in a broad spectrum of organ systems. Indeed, nanoparticles are already being developed as effective carriers of drugs to target regions of the body that were previously hard to access using traditional drug formulation methods. However, in addition to their role as a vehicle for drug delivery, nanoparticles themselves have the potential to have therapeutic benefit. Through manipulation of their elemental composition, size, shape, charge and surface modification or functionalisation it may be possible to target particles to specific organs where they may elicit their therapeutic effect. In this review we will focus on the recent advances in nanotechnology for therapeutic applications with a particular focus on the respiratory system, cancer and vaccinations. In addition we will also address developments in the field of nanotoxicology and the need for concomitant studies in to the toxicity of emerging nanotechnologies. It is possible that the very properties that make nanoparticles a desirable technology for therapeutic intervention may also lead to adverse health effects. It is thus important to determine, and appreciate, the fine balance between the efficacy and toxicity of nanomedicine.

The Stability of Silver Nanoparticles in a Model of Pulmonary Surfactant

The growing use of silver nanoparticles (AgNPs) in consumer products has raised concerns about their potential impact on the environment and human health. Whether AgNPs dissolve and release Ag(+) ions, or coarsen to form large aggregates, is critical in determining their potential toxicity. In this work, the stability of AgNPs in dipalmitoylphosphatidylcholine (DPPC), the major component of pulmonary surfactant, was investigated as a function of pH. Spherical, citrate-capped AgNPs with average diameters of 14 ± 1.6 nm (n = 200) were prepared by a chemical bath reduction. The kinetics of Ag(+) ion release was strongly pH-dependent. After 14 days of incubation in sodium perchlorate (NaClO4) or perchloric acid (HClO4) solutions, the total fraction of AgNPs dissolved varied from ∼10% at pH 3, to ∼2% at pH 5, with negligible dissolution at pH 7. A decrease in pH from 7 to 3 also promoted particle aggregation and coarsening. DPPC (100 mg·L(-1)) delayed the release of Ag(+) ions, but did not significantly alter the total amount of Ag(+) released after two weeks. In addition, DPPC improved the dispersion of the AgNPs and inhibited aggregation and coarsening. TEM images revealed that the AgNPs were coated with a DPPC layer serving as a semipermeable layer. Hence, lung lining fluid, particularly DPPC, can modify the aggregation state and kinetics of Ag(+) ion release of inhaled AgNPs in the lung. These observations have important implications for predicting the potential reactivity of AgNPs in the lung and the environment.

Novel cross talk between MEK and S6K2 in FGF-2 induced proliferation of SCLC cells

Here, we show that fibroblast growth factor-2 (FGF-2) induces proliferation of H-510 and H-69 small cell lung cancer (SCLC) cells. However, the optimal response to FGF-2 was obtained at 10-fold lower concentrations in H-510 cells. This correlated with the selective activation of the mitogen-activated protein kinase kinase (MEK) pathway in H-510, but not H-69 cells. Moreover, inhibition of MEK with PD098059 blocked FGF-2-induced proliferation in H-510 cells only. Similarly, ribosomal protein S6 kinase 2 (S6K2), a recently identified homologue of S6K1 was activated by FGF-2 in H-510, but not H-69 cells. This activation was independent of phosphatidylinositol-3 kinase, but was sensitive to inhibition of the MEK pathway. These data suggest that S6K2 is a novel downstream target of MEK. The potency of FGF-2 in H-510 cells might reflect this additional MEK/S6K2 signalling. In contrast to S6K2, S6K1 was activated in both SCLC cell lines. Inhibition of the mammalian target of rapamycin with 10 ng/ml rapamycin blocked S6K1 activation and proliferation of both lines. However, even at 100 ng/ml, rapamycin only partially inhibited S6K2. Strikingly, this correlated with inhibition of MEK signalling. Our data indicate that S6K1, and possibly S6K2, are involved in FGF-2-induced SCLC cell growth, a notion supported by the overexpression and higher baseline activity of both isoforms in SCLC lines, as compared to normal human type-II pneumocytes.