Comparison of nasal and bronchial epithelial cells obtained from patients with COPD

David M Comer, J Stuart Elborn, Madeleine Ennis, David M Comer, J Stuart Elborn, Madeleine Ennis

Abstract

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Immunocytochemistry for cytokeratin 5 in…
Figure 1. Immunocytochemistry for cytokeratin 5 in nasal and bronchial epithelial cell cultures.
Primary nasal (A) and bronchial (B) epithelial cell cultures were stained with a rabbit anti-human antibody against cytokeratin 5 (1∶100). The primary antibody was detected using a secondary antibody coupled to Alexafluor 568 (1∶500). Nuclei were stained blue with DAPI (×40).
Figure 2. (A) IL-8 dose response and…
Figure 2. (A) IL-8 dose response and (B) linear regression analysis from paired nasal and bronchial epithelial cells from COPD subjects after 24 h treatment with LPS.
Paired nasal and bronchial epithelial cells were with various concentrations of LPS [5–25 µg/ml] for 24 h (n = 8). Supernatants were collected and assessed for IL-8 by ELISA in all cases. Data are displayed as median (interquartile range). * indicates a significant difference (p<0.05).
Figure 3. IL-6 dose response from paired…
Figure 3. IL-6 dose response from paired PNECs and PBECs from COPD subjects after 24 h treatment with LPS.
Paired nasal and bronchial epithelial cells were with various concentrations of LPS [0–25 µg/ml] for 24 h (n = 8). Supernatants were collected and assessed for IL-6 by ELISA in all cases. Data are displayed as median (interquartile range). * indicates a significant difference (p<0.05).
Figure 4. Effects of (A) 5% CSE…
Figure 4. Effects of (A) 5% CSE and (B) acrolein on IL-8 release from nasal and bronchial epithelial cultures.
Cells were treated 5% CSE prepared with a single cigarette for 4 h or 24 h (n = 5). In separate experiments, cells were treated with increasing concentrations of acrolein for 4 h. Supernatants were collected and assessed for IL-8 by ELISA in all cases. Data are displayed as median (interquartile range). * indicates a significant difference (p<0.05).
Figure 5. Representative FACS histograms demonstrating intracellular-location…
Figure 5. Representative FACS histograms demonstrating intracellular-location of TLR-4, and a reduced total TLR-4 after CSE treatment.
Localisation of Toll-like receptor 4 (TLR-4). (A): Representative histogram for surface and cytoplasmic staining for IgG and TLR-4 in bronchial epithelial cells. (B): rMFI for TLR-4 in permeabilised and non-permeabilised nasal and bronchial epithelial cells. (C): Staining for TLR-4 in permeabilised bronchial epithelial cells after stimulated with 25 µg/ml P aeruginosa LPS, with or without 24 h pre-treatment with CSE. (D): rMFI for TLR-4 was lower in bronchial epithelial cells than for nasal epithelial cells (for control, 4 h and 24 h CSE treatment). A 24 h exposure to CSE significantly decreased rMFI for bronchial, but not for nasal, epithelial cells. Data are displayed as median (interquartile range). * indicates a significant difference (p<0.05).
Figure 6. Effect of LPS ±5% CSE…
Figure 6. Effect of LPS ±5% CSE pre-treatment on TLR-4 mRNA expression.
Nasal epithelial cell cultures were pretreated with 5% CSE prepared for (A) 4 h,(B) 24 h or vehicle, and subsequently stimulated with P aeruginosa LPS [10–30 µg/ml]for 24 h. Experiments were repeated in bronchial epithelial cell cultures, again for (C) 4 h and (D) 24 h 5% CSE exposure.

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