Primary bronchial epithelial cell culture from explanted cystic fibrosis lungs

Brodlie, Malcolm; McKean, Michael C; Johnson, Gail; Perry, John D.; Nicholson, A.; Verdon, Bernard; Gray, Michael A.; Dark, John H.; Pearson, Jeffrey P.; Fisher, Andrew J.; Corris, Paul A.; Lordan, James; Ward, Chris

doi:10.3109/01902140903165265

Cited by 16 publications

(14 citation statements)

References 42 publications

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“…Primary human bronchial epithelial (HBE) cells (non-CF and CF) were obtained from endobronchial brushings or extracted from explanted lungs and cultured as previously described1415. HBE cells were obtained with informed written consent from all study patients and in accordance with approval from the Newcastle and North Tyneside Local Regional Ethics Committee (reference number 2001/179 and 07/Q0906/47) The CF Center Tissue Core, University of North Carolina at Chapel Hill Biomedical Institutional Review Board (protocol #03-1396).…”

Section: Methodsmentioning

confidence: 99%

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion

Garnett

Kalsi

Sobotta

et al. 2016

Sci Rep

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The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3− transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3− removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H+ co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H+ secretion by secreting HCO3−, a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD.

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Section: Methodsmentioning

confidence: 99%

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion

Garnett

Kalsi

Sobotta

et al. 2016

Sci Rep

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“…Primary human bronchial epithelial cells (HBECs) were obtained from endobronchial brushings or extracted from explanted lungs and cultured as previously described [17–19]. HBECs were obtained in accordance with approval from the relevant ethics committees: The University of North Carolina at Chapel Hill Biomedical Institutional Review Board (protocol #03-1396).…”

Section: Methodsmentioning

confidence: 99%

A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration

Helassa

Garnett

Farrant

et al. 2014

Biochemical Journal

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Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm2) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R–BADAN (Kd 0.86 ± 0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R–BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung.

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“…PBECs were isolated and cultured from explanted CF lungs as previously described [22]. Brief details of the patients from whom PBECs were cultured are provided in table 1.…”

Section: Stimulation Of Pbecs With Il-17mentioning

confidence: 99%

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease

Brodlie¹,

McKean²,

Johnson³

et al. 2010

European Respiratory Journal

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Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells.Immunohistochemistry was performed on explanted CF lungs and compared with the nonsuppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs.Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p50.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells?mm -1 compared with 9 cells?mm -1 basement membrane, p50.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor.In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

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Primary bronchial epithelial cell culture from explanted cystic fibrosis lungs

Cited by 16 publications

References 42 publications

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion

A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease

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