Linda D. Martin

A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma

Mucus hypersecretion is a crucial feature of pulmonary diseases such as asthma, chronic bronchitis and cystic fibrosis. Despite much research, there is still no effective therapy for this condition. Recently, we showed that the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is required for mucus secretion by human bronchial epithelial cells in culture. Having synthesized a peptide corresponding to the N-terminal domain of MARCKS, we now show that the intratracheal instillation of this peptide blocks mucus hypersecretion in a mouse model of asthma. A missense peptide with the same amino acid composition has no effect. Based on quantitative histochemical analysis of the mouse airways, the peptide seems to act by blocking mucus release from goblet cells, possibly by inhibiting the attachment of MARCKS to membranes of intracellular mucin granules. These results support a pivotal role for MARCKS protein, specifically its N-terminal region, in modulating this secretory process in mammalian airways. Intratracheal administration of this MARCKS-related peptide could therapeutically reduce mucus secretion in the airways of human patients with asthma, chronic bronchitis and cystic fibrosis.

Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators

Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).

Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase Cδ-mediated mechanism

The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC 5 AC and MUC 5 B, but not MUC 2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the delta isoform.

Effects of TNFα on expression of ICAM-1 in human airway epithelial cells in vitro: Oxidant-mediated pathways and transcription factors

Abstract We demonstrate that two different cell-permeable antioxidants, pyrrolidine dithiocarbamate (PDTC) and dimethylthiourea (DMTU), inhibit TNFα-induced ICAM-1 surface and gene expression in primary cultures of differentiated normal human bronchial epithelial (NHBE) cells. In addition, TNFα stimulates binding of nuclear proteins to the nuclear factor kappa beta (NFκB) and the CAAT/enhancer binding protein (C/EBP) consensus sites in the ICAM-1 promoter in these cells. Because these transcription factors have been suggested to be oxidant-sensitive and important in ICAM-1 expression, the potential involvement of reactive oxygen species (ROS) in the response to TNFα was investigated. Interestingly, neither PDTC nor DMTU altered binding of NFκB complexes. In contrast, either the proteasome inhibitor carbobenzoxy-L-leucy-L-leucy-L-leucinal (MG 132) or the IκBα inhibitor BAY 11-7082 ablated TNFα-induced ICAM-1 gene expression and MG132 inhibited TNFα-induced NFκB complexes. Surprisingly, either PDTC or DMTU inhibited the binding of TNFα-enhanced C/EBP complexes to the consensus site directly adjacent to the NFκB site. These results suggest that although TNFα enhances binding of C/EBP and NFκB complexes in NHBE cells, C/EBP binding seems to involve an oxidant-dependent mechanism, whereas activation of NFκB complexes utilizes the ubiquitin-proteasome pathway, a mechanism that seems to be unaltered by the presence of antioxidants. Because interference with either signaling pathway abrogates TNFα-induced ICAM-1 expression, activation of both complexes seems to be involved in this response to TNFα, but this activation occurs via different intracellular pathways.

IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17

BackgroundThe pleiotrophic cytokine interleukin (IL)-13 features prominently in allergic and inflammatory diseases. In allergic asthma, IL-13 is well established as an inducer of airway inflammation and tissue remodeling. We demonstrated previously that IL-13 induces release of transforming growth factor-α (TGFα) from human bronchial epithelial cells, with proliferation of these cells mediated by the autocrine/paracrine action of this growth factor. TGFα exists as an integral membrane protein and requires proteolytic processing to its mature form, with a disintegrin and metalloproteinase (ADAM)17 responsible for this processing in a variety of tissues.MethodsIn this study, normal human bronchial epithelial (NHBE) cells grown in air/liquid interface (ALI) culture were used to examine the mechanisms whereby IL-13 induces release of TGFα and cellular proliferation. Inhibitors and antisense RNA were used to examine the role of ADAM17 in these processes, while IL-13-induced changes in the intracellular expression of TGFα and ADAM17 were visualized by confocal microscopy.ResultsIL-13 was found to induce proliferation of NHBE cells, and release of TGFα, in an ADAM17-dependent manner; however, this IL-13-induced proliferation did not appear to result solely from ADAM17 activation. Rather, IL-13 induced a change in the location of TGFα expression from intracellular to apical regions of the NHBE cells. The apical region was also found to be a site of significant ADAM17 expression, even prior to IL-13 stimulation.ConclusionResults from this study indicate that ADAM17 mediates IL-13-induced proliferation and TGFα shedding in NHBE cells. Furthermore, they provide the first example wherein a cytokine (IL-13) induces a change in the intracellular expression pattern of a growth factor, apparently inducing redistribution of intracellular stores of TGFα to the apical region of NHBE cells where expression of ADAM17 is prominent. Thus, IL-13-induced, ADAM17-mediated release of TGFα, and subsequent epithelial cell proliferation, could contribute to the epithelial hypertrophy, as well as other features, associated with airway remodeling in allergic asthma.

Susceptibility of inflamed alveolar and airway epithelial cells to injury induced by diesel exhaust particles of varying organic carbon content.

Exposure to traffic-related ambient air pollution, such as diesel exhaust particles (DEP), is associated with adverse health outcomes, especially in individuals with preexisting inflammatory respiratory diseases. Using an analogous novel in vitro system to model both the healthy and inflamed lung, the susceptibility of epithelial cells exposed to DEP of varying organic carbon content was studied. Murine LA-4 alveolar type II-like epithelial cells, as well as primary murine tracheal epithelial cells (MTE), were treated with exogenous cytokines (tumor necrosis factor [TNF] α + interleukin [IL]-1 β + interferon [IFN] γ) to model a mild inflammatory state. Epithelial cells were subsequently exposed to DEP of varying organic carbon content, and the resultant cytotoxic, cytoprotective, or antioxidant cell responses were inferred by changes in lactate dehydrogenase (LDH) release, heme oxygenase-1 (HO-1) expression, or glutathione levels, respectively. Data showed that exposure of healthy LA-4 cells to organic carbon-rich DEP (25 μg/cm2; 24 h) induced adaptive cytoprotective/antioxidant responses with no apparent cell injury. In contrast, exposure of inflamed LA-4 cells resulted in oxidative stress culminating in significant cytotoxicity. Exposure of healthy MTE cells to organic carbon-rich DEP (20 μg/cm2; 24 h) was seemingly without effect, whereas exposure of inflamed MTE cells resulted in increased epithelial solute permeability. Thus, surface lung epithelial cells stressed by a state of inflammation and then exposed to organic carbon-rich DEP appear unable to respond to the additional oxidative stress, resulting in epithelial barrier dysfunction and injury. Adverse health outcomes associated with exposure to traffic-related air pollutants, like DEP, in patients with preexisting inflammatory respiratory diseases may be due, in part, to similar mechanisms.

MODELING THE AIRWAY EPITHELIUM IN ALLERGIC ASTHMA: INTERLEUKIN-13- INDUCED EFFECTS IN DIFFERENTIATED MURINE TRACHEAL EPITHELIAL CELLS

SummaryMucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.

ErbB2 activity is required for airway epithelial repair following neutrophil elastase exposure

In cystic fibrosis and chronic bronchitis, airways are chronically injured by exposure to neutrophil elastase (NE). We sought to identify factors required for epithelial repair following NE exposure. Normal human bronchial epithelial cells were treated with NE (50 nM, 22 h) or control vehicle. Following NE treatment, we found a marked and sustained decrease in epithelial proliferation as detected by Ki67 immunostaining. 3H‐thymidine incorporation was also initially depressed but increased over 72 h in NE‐treated cells, which suggests that DNA synthesis constitutes an early repair process following NE exposure. We hypothesized that ErbB2 receptor tyrosine kinase, a regulator of cancer cell proliferation, was required for epithelial DNA synthesis following NE exposure. Immediately following NE treatment, by flow cytometry analysis, we found a decrease in ErbB2 surface expression. Protein levels of the full‐length 185 kD ErbB2 receptor significantly decreased following NE treatment and smaller ErbB2‐positive bands, ranging in size from 23 to 40 kD, appeared, which suggests that NE caused ErbB2 degradation. By real‐time RT‐PCR analysis, we found no change in ErbB2 mRNA expression following NE treatment, which suggests that changes in ErbB2 protein levels were regulated at the post‐translational level. Following NE treatment, full‐length 185 kD ErbB2 levels increased to pretreatment levels, correlating with the increase in thymidine incorporation during the same time period. Importantly, inhibition of ErbB2 activity with AG825 (5 µM) or Herceptin (3.1 µM), an ErbB2‐neutralizing antibody, blocked thymidine incorporation only in NE‐treated cells. These results suggest ErbB2 is a critical factor for epithelial recovery following NE exposure.

Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro

BackgroundMucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways.MethodsTo study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)].ResultsThe anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins.ConclusionGiven the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates.

Reconstruction of ribosomal subunits and rDNA chromatin imaged by scanning force microscopy

Scanning force microscopy (SFM) reveals surface topography by scanning a sharp tip in close proximity to the sample. Due to tip–sample interaction, artificial broadening of the real surface structure with the tip geometry occurs. One approach for image reconstruction is the use of calibration standards, preferably in the size range of the samples. In the present study, an image reconstruction method based on colloidal gold as a geometric standard was used to reconstruct SFM images of biomolecules. Sample and calibration standard size were in the nanometer range, and the standards were coadsorbed with the specimen. Raw and reconstructed images of the biomolecules were compared, and the reconstruction was characterized by difference images as well as determination of the difference volume. The application of image reconstruction based on colloidal gold as a calibration standard for SFM of biomolecules is discussed.