53 results found
Conforti F, Davies ER, Calderwood CJ, et al., 2017, The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis, ONCOTARGET, Vol: 8, Pages: 48737-48754, ISSN: 1949-2553
Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.
Thorley A, Ogger P, Legorburo-Schofield M, et al., 2017, Nintedanib Effectively Inhibits Carbon Nanotube-Induced Fibrotic Responses In Human Alveolar Epithelial Cells, Fibroblasts And Pulmonary Microvascular Endothelial Cells, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Chen S, Goode AE, Skepper JN, et al., 2016, Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments, JOURNAL OF MICROSCOPY, Vol: 261, Pages: 157-166, ISSN: 0022-2720
Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.
Ruenraromgsak P, Chen S, Hu S, et al., 2016, Translocation of Functionalized Multi-Walled Carbon Nanotubes across Human Pulmonary Alveolar Epithelium: Dominant Role of Epithelial Type 1 Cells, ACS NANO, Vol: 10, Pages: 5070-5085, ISSN: 1936-0851
Uptake and translocation of short functionalized multi-walled carbon nanotubes (short-fMWCNTs) through the pulmonary respiratory epithelial barrier depend on physicochemical property and cell type. Two monoculture models, immortalized human alveolar epithelial type 1 (TT1) cells and primary human alveolar epithelial type 2 cells (AT2), which constitute the alveolar epithelial barrier, were employed to investigate the uptake and transport of 300 and 700 nm in length, poly(4-vinylpyridine)-functionalized, multi-walled carbon nanotubes (p(4VP)-MWCNTs) using quantitative imaging and spectroscopy techniques. The p(4VP)-MWCNT exhibited no toxicity on TT1 and AT2 cells, but significantly decreased barrier integrity (*p < 0.01). Uptake of p(4VP)-MWCNTs was observed in 70% of TT1 cells, correlating with compromised barrier integrity and basolateral p(4VP)-MWCNT translocation. There was a small but significantly greater uptake of 300 nm p(4VP)-MWCNTs than 700 nm p(4VP)-MWCNTs by TT1 cells. Up to 3% of both the 300 and 700 nm p(4VP)-MWCNTs reach the basal chamber; this relatively low amount arose because the supporting transwell membrane minimized the amount of p(4VP)-MWCNT translocating to the basal chamber, seen trapped between the basolateral cell membrane and the membrane. Only 8% of AT2 cells internalized p(4VP)-MWCNT, accounting for 17% of applied p(4VP)-MWCNT), with transient effects on barrier function, which initially fell then returned to normal; there was no MWCNT basolateral translocation. The transport rate was MWCNT length modulated. The comparatively lower p(4VP)-MWCNT uptake by AT2 cells is proposed to reflect a primary barrier effect of type 2 cell secretions and the functional differences between the type 1 and type 2 alveolar epithelial cells.
Sweeney S, Leo BF, Chen S, et al., 2016, Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells, COLLOIDS AND SURFACES B-BIOINTERFACES, Vol: 145, Pages: 167-175, ISSN: 0927-7765
Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf(®) (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag(+) ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P<0.01), 24h after treatment with 25μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf(®), this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag(+) ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs.
Cryer AM, Ruenraroengsak P, Tetley TD, et al., 2015, SYNTHESIS OF GOLD-BASED NANOMEDICINES TO TREAT NON-SMALL CELL LUNG CANCER, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A58-A59, ISSN: 0040-6376
Sweeney S, Berhanu D, Ruenraroengsak P, et al., 2015, Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: Investigating crystalline phase as a critical determinant, NANOTOXICOLOGY, Vol: 9, Pages: 482-492, ISSN: 1743-5390
There can be significant variability between bioreactivity studies of nanomaterials that are apparently the same, possibly reflecting differences in the models used and differing sources of experimental material. In this study, we have generated two crystal forms of titanium dioxide nanoparticles (nano-TiO2), pure anatase and pure rutile to address the hypothesis that the bioreactivity of these nanoparticles with human alveolar epithelium will depend on their crystal phase. We used a human alveolar type-I-like epithelial cell model (TT1; generated in-house from primary human alveolar epithelial type II cells); these cells cover 95% of the alveolar epithelial surface area and are an important target cell for inhaled nanomaterials. Using literature as a guide, we hypothesised that pure anatase nano-TiO2 would display greater bioreactivity with TT1 cells in comparison to pure rutile nano-TiO2. However, we found the profile and pattern of inflammatory mediator release was similar between these two nano-TiO2 formats, although pure rutile treatment caused a small, but consistently greater, response for IL-6, IL-8 and MCP-1. Interestingly, the temporal induction of oxidative stress (increased reactive oxygen species levels and depleted glutathione) varied markedly between the different nano-TiO2 formats. We have shown that a combination of using nanomaterials synthesised specifically for toxicological study and the use of a highly relevant, reproducible human lung cell model, offers a useful approach to delineating the physicochemical properties of nanomaterials that may be important in their cellular reactivity.
Chen S, Hu S, Smith EF, et al., 2014, Aqueous cationic, anionic and non-ionic multi-walled carbon nanotubes, functionalised with minimal framework damage, for biomedical application, BIOMATERIALS, Vol: 35, Pages: 4729-4738, ISSN: 0142-9612
The use of a thermochemical grafting approach provides a versatile means to functionalise as-synthesised, bulk multi-walled carbon nanotubes (MWNTs) without altering their inherent structure. The associated retention of properties is desirable for a wide range of commercial applications, including for drug delivery and medical purposes; it is also pertinent to studies of intrinsic toxicology. A systematic series of water-compatible MWNTs, with diameter around 12 nm have been prepared, to provide structurally-equivalent samples predominantly stabilised by anionic, cationic, or non-ionic groups. The surface charge of MWNTs was controlled by varying the grafting reagents and subsequent post-functionalisation modifications. The degree of grafting was established by thermal analysis (TGA). High resolution transmission electron microscope (HRTEM) and Raman measurements confirmed that the structural framework of the MWNTs was unaffected by the thermochemical treatment, in contrast to a conventional acid-oxidised control which was severely damaged. The effectiveness of the surface modification was demonstrated by significantly improved solubility and stability in both water and cell culture medium, and further quantified by zeta-potential analysis. The grafted MWNTs exhibited relatively low bioreactivity on transformed human alveolar epithelial type 1-like cells (TT1) following 24 h exposure as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase release (LDH) assays. The exposure of TT1 cells to MWNTs suppressed the release of the inflammatory mediators, interleukin 6 (IL-6) and interleukin 8 (IL-8). TEM cell uptake studies indicated efficient cellular entry of MWNTs into TT1 cells, via a range of mechanisms. Cationic MWNTs showed a more substantial interaction with TT1 cell membranes than anionic MWNTs, demonstrating a surface charge effect on cell uptake.
Mukherjee D, Royce SG, Sarkar S, et al., 2014, Modeling in vitro cellular responses to silver nanoparticles., J Toxicol, Vol: 2014, Pages: 852890-13, ISSN: 1687-8191
Engineered nanoparticles (NPs) have been widely demonstrated to induce toxic effects to various cell types. In vitro cell exposure systems have high potential for reliable, high throughput screening of nanoparticle toxicity, allowing focusing on particular pathways while excluding unwanted effects due to other cells or tissue dosimetry. The work presented here involves a detailed biologically based computational model of cellular interactions with NPs; it utilizes measurements performed in human cell culture systems in vitro, to develop a mechanistic mathematical model that can support analysis and prediction of in vivo effects of NPs. The model considers basic cellular mechanisms including proliferation, apoptosis, and production of cytokines in response to NPs. This new model is implemented for macrophages and parameterized using in vitro measurements of changes in cellular viability and mRNA levels of cytokines: TNF, IL-1b, IL-6, IL-8, and IL-10. The model includes in vitro cellular dosimetry due to nanoparticle transport and transformation. Furthermore, the model developed here optimizes the essential cellular parameters based on in vitro measurements, and provides a "stepping stone" for the development of more advanced in vivo models that will incorporate additional cellular and NP interactions.
Novak P, Shevchuk A, Ruenraroengsak P, et al., 2014, Imaging Single Nanoparticle Interactions with Human Lung Cells Using Fast Ion Conductance Microscopy, NANO LETTERS, Vol: 14, Pages: 1202-1207, ISSN: 1530-6984
Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle-cell interactions.
Sweeney S, Berhanu D, Misra SK, et al., 2014, Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells, CARBON, Vol: 78, Pages: 26-37, ISSN: 0008-6223
Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT.
Thorley AJ, Ruenraroengsak P, Potter TE, et al., 2014, Critical Determinants of Uptake and Trans location of Nanoparticles by the Human Pulmonary Alveolar Epithelium, ACS NANO, Vol: 8, Pages: 11778-11789, ISSN: 1936-0851
The ability to manipulate the size and surface properties of nanomaterials makes them a promising vector for improving drug delivery and efficacy. Inhalation is a desirable route of administration as nanomaterials preferentially deposit in the alveolar region, a large surface area for drug absorption. However, as yet, the mechanisms by which particles translocate across the alveolar epithelial layer are poorly understood. Here we show that human alveolar type I epithelial cells internalize nanoparticles, whereas alveolar type II epithelial cells do not, and that nanoparticles translocate across the epithelial monolayer but are unable to penetrate the tight junctions between cells, ruling out paracellular translocation. Furthermore, using siRNA, we demonstrate that 50 nm nanoparticles enter largely by passive diffusion and are found in the cytoplasm, whereas 100 nm nanoparticles enter primarily via clathrin- and also caveolin-mediated endocytosis and are found in endosomes. Functionalization of nanoparticles increases their uptake and enhances binding of surfactant which further promotes uptake. Thus, we demonstrate that uptake and translocation across the pulmonary epithelium is controlled by alveolar type I epithelial cells, and furthermore, we highlight a number of factors that should be considered when designing new nanomedicines in order to improve drug delivery to the lung.
Chen S, Goode AE, Sweeney S, et al., 2013, Sulfidation of silver nanowires inside human alveolar epithelial cells: a potential detoxification mechanism, NANOSCALE, Vol: 5, Pages: 9839-9847, ISSN: 2040-3364
Silver nanowires (AgNWs) are being developed for use in optoelectronics. However before widespread usage, it is crucial to determine their potential effects on human health. It is accepted that Ag nanoparticles (AgNPs) exert toxic effects by releasing Ag(+) ions, but much less is known about whether Ag(+) reacts with compounds, or any downstream bioactive effects of transformed AgNPs. Analytical high-resolution transmission electron microscopy has been employed to elucidate cellular uptake and reactivity of AgNWs inside human alveolar epithelial type 1-like cells. AgNWs were observed in the cytoplasm and membrane-bound vesicles, and precipitation of Ag2S within the cell occurred after 1 h exposure. Cell viability studies showed no evidence of cytotoxicity and reactive oxygen species were not observed on exposure of cells to AgNWs. We suggest that Ag2S formation acts as a 'trap' for free Ag(+), significantly limiting short-term toxicological effects - with important consequences for the safety of Ag-nanomaterials to human health.
Nestorowa S, Tetley TD, Thorley AJ, et al., 2013, PULMONARY SURFACTANT PROTECTS AGAINST SILVER NANOPARTICLE-INDUCED INFLAMMATION IN THE PERIPHERAL HUMAN LUNG, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A138-A139, ISSN: 0040-6376
Solomon A, Smyth E, Mitha N, et al., 2013, Induction of platelet aggregation after a direct physical interaction with diesel exhaust particles, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 325-334, ISSN: 1538-7933
BACKGROUND: There is a proven link between exposure to traffic-derived particulate air pollution and the incidence of platelet-driven cardiovascular diseases. It is suggested that inhalation of small, nanosized particles increases cardiovascular risk via toxicological and inflammatory processes and translocation of nanoparticles into the bloodstream has been shown in experimental models. We therefore investigated the ability of diesel exhaust particles (DEP) to interact physically and functionally with platelets. METHODS: The interaction of DEP and carbon black (CB) with platelets was examined by transmission electron microscopy (TEM), whereas the functional consequences of exposure were assessed by measuring in vitro and in vivo platelet aggregation via established methods. RESULTS: Both DEP and CB were internalized and seen in proximity with the open canalicular system in platelets. DEP induced platelet aggregation in vitro whereas CB had no effect. DEP induced Ca(2+) release, dense granule secretion and surface P-selectin expression, but not toxicologic membrane disruption. Low concentrations of DEP potentiated agonist-induced platelet aggregation in vitro and in vivo. CONCLUSIONS: DEP associate physically with platelets in parallel with a Ca(2+) -mediated aggregation response displaying the conventional features of agonist-induced aggregation. The ability of DEP to enhance the aggregation response to platelet stimuli would be expected to increase the incidence of platelet-driven cardiovascular events should they be inhaled and translocate into the blood. This study provides a potential mechanism for the increased thrombotic risk associated with exposure to ambient particulate air pollution.
Tetley TD, Thorley AJ, 2013, Health effects, Biochemist, Vol: 35, Pages: 14-20, ISSN: 0954-982X
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.
Zambianchi MZ, Tetley TD, Thorley AJ, et al., 2013, ALVEOLAR EPITHELIAL DNA DAMAGE, INFLAMMATION AND ALTERED AUTOPHAGY FOLLOWING EXPOSURE TO SILVER NANOPARTICLES IS EXACERBATED BY VIRAL LIGANDS IN VITRO, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A52-A52, ISSN: 0040-6376
Beverly A, Tetley TD, Thorley AJ, et al., 2012, PRO-FIBROTIC EFFECTS OF MULTI-WALLED CARBON NANOTUBE EXPOSURE ON PRIMARY HUMAN ALVEOLAR TYPE II EPITHELIAL CELLS AND FIBROBLASTS, Winter Meeting of the British-Thoracic-Society 2012, Publisher: BMJ PUBLISHING GROUP, Pages: A60-A60, ISSN: 0040-6376
Misra SK, Tetley TD, Thorley A, et al., 2012, Engineered Nanomaterials, Pollutants, Human Health and the Environment: A Risk Based Approach, Editors: Plant, Ragnarsdottir, Voulvoulis, Voulvoulis, Publisher: Wiley-Blackwell, Pages: 287-318, ISBN: 9780470742617
Ruenraroengsak P, Novak P, Berhanu D, et al., 2012, Respiratory epithelial cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles, NANOTOXICOLOGY, Vol: 6, Pages: 94-108, ISSN: 1743-5390
The respiratory epithelium is a significant target of inhaled, nano-sized particles, the biological reactivity of which will depend on its physicochemical properties. Surface-modified, 50 and 100 nm, polystyrene latex nanoparticles (NPs) were used as model particles to examine the effect of particle size and surface chemistry on transformed human alveolar epithelial type 1-like cells (TT1). Live images of TT1 exposed to amine-modified NPs taken by hopping probe ion conductance microscopy revealed severe damage and holes on cell membranes that were not observed with other types of NPs. This paralleled induction of cell detachment, cytotoxicity and apoptotic (caspase-3/7 and caspase-9) cell death, and increased release of CXCL8 (IL-8). In contrast, unmodified, carboxyl-modified 50 nm NPs and the 100 nm NPs did not cause membrane damage, and were less reactive. Thus, the susceptibility and membrane damage to respiratory epithelium following inhalation of NPs will depend on both surface chemistry (e.g., cationic) and nano-size.
Bolasco G, Tracey-White DC, Tolmachova T, et al., 2011, Loss of Rab27 function results in abnormal lung epithelium structure in mice, AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, Vol: 300, Pages: C466-C476, ISSN: 0363-6143
Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.
Thorley AJ, Grandolfo D, Lim E, et al., 2011, Innate Immune Responses to Bacterial Ligands in the Peripheral Human Lung - Role of Alveolar Epithelial TLR Expression and Signalling, PLOS ONE, Vol: 6, Pages: e21827-e21827, ISSN: 1932-6203
It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens.
Grandolfo D, Thorley AJ, Salmon GP, et al., 2010, Differential Effect Of Toll-like Receptor Activation On Primary Human Alveolar Epithelium And Alveolar Macrophage Mediator Release In Vitro; Synergistic Effect Of Co-culture, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 181, ISSN: 1073-449X
Maher TM, Evans IC, Bottoms SE, et al., 2010, Diminished Prostaglandin E-2 Contributes to the Apoptosis Paradox in Idiopathic Pulmonary Fibrosis, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 182, Pages: 73-82, ISSN: 1073-449X
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.
Nuseibeh S, Thorley AJ, Tetley TD, 2010, Effect of serum proteins on the cytotoxic and pro-inflammatory response of human alveolar type I epithelial cells to metal oxide nanoparticles, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Ruenraroengsak P, Thorley AJ, Tetley TD, 2010, Induction of oxidative stress and cell death in human alveolar epithelium following nanoparticle exposure depends on surface charge, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Sweeney SS, Ruenraroengsak P, Berhanu D, et al., 2010, Differential reactivity of nano-TiO2 with human lung alveolar epithelium in vitro: importance of physicochemistry, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Thorley AJ, Tetley TD, Thorley AJ, et al., 2010, Mechanisms of uptake of nano-sized latex beads by human alveolar type I epithelial (ATI) cells, 2nd NanoImpactNet Conference, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Mercer PF, Johns RH, Scotton CJ, et al., 2009, Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 179, Pages: 414-425, ISSN: 1073-449X
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.
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