Audrey Richter

Involvement of the epidermal growth factor receptor in epithelial repair in asthma

Epithelial damage and airway remodeling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. To examine the mechanisms that control bronchial epithelial repair, we investigated expression of the epidermal growth factor receptor (c‐erbB1, EGFR) in asthmatic bronchial mucosa and studied repair responses in vitro. In biopsies from asthmatic subjects, areas of epithelial damage were frequently observed and exhibited strong EGFR immunostaining. EGFR expression was also high in morphologically intact asthmatic epithelium. Using image analysis, EGFR immunoreactivity (% of total epithelial area, median (range) was found to increase from 9.4 (4.1‐20.4) in normal subjects (n=10) to 18.4 (9.3‐28.9) in mild asthmatics (P<0.01, n=13) and 25.4 (15.4‐31.8) in severe asthmatics (P<0.00, n=5). Epithelial EGFR immunoreactivity remained elevated in patients treated with corticosteroids and was positively correlated with subepithelial reticular membrane thickening. Using 16HBE 14o‐ bronchial epithelial cells, we found that EGF accelerated repair of scrape‐wounded monolayers and that the EGFR‐selective inhibitor, tyrphostin AG1478, inhibited both EGF‐stimulated and basal wound closure whereas dexamethasone was without effect. Intrinsic activation of the EGFR was confirmed by analysis of tyrosine phosphorylated proteins, which revealed a rapid, damage‐induced phosphorylation of the EGFR, irrespective of the presence of exogenous EGF. To assess the relationship between EGFR‐mediated repair and tissue remodeling, release of the profibrogenic mediator TGF‐β2 was also measured. Scrape wounding increased release of TGF‐β2 from epithelial monolayers and EGF had no additional stimulatory effect. However, when repair was retarded with AG1478, the amount of TGF‐β2 increased significantly. These data indicate that the EGFR may play an important role in bronchial epithelial repair in asthma and that impairment of this function may augment airway remodeling.—Puddicombe, S. M., Polosa, R., Richter, A., Krishna, M. T., Howarth, P. H., Holgate, S. T., Davies, D. E. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J. 14, 1362–1374 (2000)

Cooperative Effects of Th2 Cytokines and Allergen on Normal and Asthmatic Bronchial Epithelial Cells

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4Rα, common γ-chain, and IL-13Rα1, as well as IL-13Rα2, which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13Rα2 protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-α, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-α was also increased by TNF-α and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-α, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma.

Expression of ErbB receptors and mucins in the airways of long term current smokers

Background: Airway epithelial goblet cell hyperplasia is known to occur in chronic smokers. Although the epidermal growth factor receptor has been implicated in this process, neither ErbB receptor expression nor the mucosecretory phenotype of the epithelium have been characterised in current smokers. Methods: Bronchial biopsies obtained from non-smokers (n = 10) and current smokers, with or without chronic obstructive pulmonary disease (n = 51), were examined immunohistochemically to measure the expression of epidermal growth factor receptor, ErbB2, ErbB3, ErbB4 and mucin subtypes (MUC2, MUC5AC and MUC5B) in the bronchial epithelium. The results were correlated with neutrophil counts measured in the airway wall and induced sputum. Results: Epidermal growth factor receptor, ErbB3 and MUC5AC expression, in addition to PAS staining, were significantly increased in all smokers compared with non-smokers, irrespective of the presence of chronic obstructive pulmonary disease. MUC5AC expression was significantly associated with both PAS staining and ErbB3 expression; no correlation was observed between either mucin or ErbB receptor expression and neutrophil counts. Conclusions: The results suggest that long term current smoking induces enhanced epidermal growth factor receptor, ErbB3, and MUC5AC expression in vivo; these increases are not associated with the presence of neutrophilic inflammation. ErbB receptors may contribute to epithelial responses to cigarette smoke.

Mechanisms of airway epithelial damage: epithelial-mesenchymal interactions in the pathogenesis of asthma

The aims of this article are to understand the shortfalls in thinking of asthma purely as an atopic disorder, to gain insight into the epithelial abnormalities of asthma and how these interface with the environment, and to view asthma as a disease of airway wall restructuring that engages activation of the epithelial mesenchymal trophic unit (EMTU). Furthermore, based on these developments, possible novel therapeutic targets for chronic asthma are identified. It has long been recognised that T‐helper cell (Th) type‐2 airway inflammation underpins airway dysfunction in asthma. Atopy is also a key feature of this disease but accounts for <40% of the population attributable risk. Based on a careful pathological and functional assessment of chronic asthma, the authors propose that altered epithelial-mesenchymal communication is also fundamental to disease pathogenesis. A number of factors contribute to a thickened hyperresponsive airway that provides an ideal microenvironment for the persistence of Th2‐mediated inflammation, including a more susceptible epithelium to injury, delayed epithelial repair, an altered trajectory of epithelial repair to a mucus-secreting phenotype, generation of growth factors that drive mesenchymal cell proliferation and differentiation towards increased matrix deposition and smooth muscle, and the production of neural and vascular growth factors. Cytokines and mediators derived from infiltrating inflammatory cells interact with this EMTU to augment and prolong responses. The “remodelling” changes of asthma have been observed in childhood asthma and may, indeed, precede the development of the disease. The recent recognition that atopy per se is not a key factor in the initiation of asthma (although it is important in aggravating the disease) suggests that gene-environmental interactions involving similar processes to those occurring in branching morphogenesis are critical for the full asthma phenotype to develop. A recent National Institutes for Health Workshop and a European Respiratory Society Task Force both concluded that more work …

Invited Lecture: Activation of the Epithelial Mesenchymal Trophic Unit in the Pathogenesis of Asthma

Background: A recent NIH Workshop and an ERS Task Force concluded that more work was needed to understand mechanisms of severe and chronic asthma. This report describes a series of studies that identify aberrant epithelial mesenchymal signalling in the airways as an important event in maintaining inflammation and driving remodelling in response to environmental injury. Methods: Immunohistochemistry, genotyping and functional studies conducted on cultured asthmatic cells and mucosal biopsies were used to identify biochemical pathways involved in epithelial injury and repair in asthma and their relationship to disease severity. Results: Our findings suggest that the asthmatic state results from an interaction between a susceptible epithelium and Th-2-mediated inflammation to alter the communication between the epithelium and the underlying mesenchyme – the epithelial mesenchymal trophic unit – leading to disease persistence, airway remodelling and refractoriness to corticosteroid treatment. Conclusions: Asthma is more than an inflammatory disorder, but requires engagement of important signalling pathways involved in epithelial repair and tissue remodelling. These pathways involving EGFRs and TGF-βRs provide targets against which to develop novel therapies for chronic asthma.

Growth inhibitory effects of FK506 and cyclosporin A independent of inhibition of calcineurin

The ability of the immunosuppressive agent FK506 to affect growth of the epidermal growth factor-receptor (EGF-R) overexpressing cell line, A431, was compared with that of the structurally unrelated immunosuppressive compound, cyclosporin A (CyA). Both were shown to inhibit growth, although neither of them caused down-regulation of the EGF-R or affected epidermal growth factor (EGF)-induced tyrosine phosphorylation of the EGF-R. Inhibition of growth was not specific to EGF-R pathways, as both FK506 and CyA also inhibited EGF- and platelet-derived growth factor (PDGF)-induced DNA synthesis in fibroblasts. In all assays FK506 was less potent than CyA even though it is 10-100 times more potent as an immunosuppressive agent. The role of calcineurin in CyA- or FK506-induced growth inhibition was investigated using the synthetic pyrethroid insecticides: cypermethrin, deltamethrin and fenvalerate, which are known calcineurin inhibitors. Failure of these agents to block cell growth or influence growth factor-induced mitogenesis indicated that the biochemical pathway(s) by which CyA or FK506 inhibited cell growth did not depend solely on inhibition of calcineurin.

Effects of anthralin and hypericin on growth factor signalling and cell proliferation in vitro

The effect of the anthranoids, anthralin and hypericin, on epidermal growth factor receptor (EGF-R) activation and their degree of specificity was examined. Hypericin, but not anthralin, was found to inhibit binding of [125I]-labelled epidermal growth factor (EGF) to HN5 squamous carcinoma cells that overexpress EGF-R. This effect was a result of dose- and time-dependent reduction of EGF-R number and affinity. Neither compound directly inhibited EGF-induced tyrosine phosphorylation of the EGF-R in HN5 cells. Although anthralin and hypericin both inhibited the mitogenic effect of EGF in NR6/HER cells (IC50S = 100 nM and 10 microM, respectively), they also had comparable effects on DNA synthesis in response to acidic fibroblast growth factor (aFGF) and platelet-derived growth factor (PDGF). When tested in proliferation assays using cells expressing differing numbers of EGF-R, the growth inhibitory effects of both compounds were independent of EGF-R number. We conclude that, although anthralin and hypericin both inhibit EGF signalling, they do not act specifically on the EGF-R pathway. Moreover, their mechanisms of action do not appear to be comparable.

Constrained Peptide Analogues of Transforming Growth Factor-α Residues Cysteine 21-32 Are Mitogenically Active USE OF PROLINE MIMETICS TO ENHANCE BIOLOGICAL POTENCY

Two proline mimetics, the enantiomers of 2-azabicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop β-sheet sequence of human transforming growth factor-α (TGF-α) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 μM; in the presence of low doses of TGF-α, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-α alone. These effects are consistent with the peptides acting via the same mechanism as TGF-α. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the β-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.

A synthesis of 18-O-methyl mycalamide B

Metallated dihydropyran 9 and the dihydropyranone 10 previously used in a synthesis of the insect toxin pederin were adapted to the synthesis of 18-O-methyl mycalamide B, the most potent derivative of the anti-tumour agents isolated from a sponge. Key steps in the synthesis include the oxidation of enol silane 11 from the more hindered face using dimethyldioxirane to introduce the hydroxy group at C-12 and the acylation of 6-lithio-3,4-dihydro-2H-pyran 9 with oxalamide 8 to forge the N-(1-alkoxy-1-alkyl)amide bridge. Biological tests in human tumour cell lines confirm the potent anti-proliferative effect of 18-O-methyl mycalamide B in pM concentrations.