An in vitro model for cigarette smoke induced
goblet cell hyperplasia using flow cytometry
Haswell L. E., Newland N., Breheny D. and Richter A.
British American Tobacco, Group R&D Centre, Southampton SO15 8TL
Cigarette smoking is associated with chronic obstructive
pulmonary disease (COPD) – a term that encompasses Figure 2. Identification of goblet cells.
chronic lung inflammation, excess mucus production and Goblet cells containing the mucin
chronic cough (chronic bronchitis), and destruction of the MUC5AC were visualised by
alveoli (emphysema). Goblet cell hyperplasia is a immunocytochemistry (arrow). Cell
characteristic feature of the lung epithelium in hypersecretory nuclei were counterstained with
diseases such as chronic bronchitis and is thought to Mayer’s haemalum.
contribute to the overproduction of airway mucus observed in
some patients. It is possible to culture primary human
bronchial epithelial cells (HBECs) at an air-liquid interface for
Both TPM-treated and untreated control cultures underwent
28 days and induce epithelial mucociliary differentiation (figure
mucocilliary differentiation as observed by the appearance of
1). In this study we have used this in vitro model to investigate
ciliated cells and goblet cells in the cell populations. TPM
the effect of cigarette smoke total particulate matter (TPM) on
affected the goblet cell density in a dose-dependant manner.
goblet cell population, and assessed the possibility of using
The percentage of goblet cells was positively correlated with
flow cytometry to identify goblet cells.
the TPM dose from 0.31-5 µg/ml. A response was not
observed at the highest dose of 20 µg/ml (Figures 3A & 3B).
The same dose-response was observed, irrespective of the
Figure 1. Ultrastructural detection method employed.
morphology of the differentiated
cell cultures. Scanning electron
micrographs of the cultures A. Immunochemical staining
show the apical surface and the 30
fractured edge of a culture.
% MUC5AC +ve cells
Primary human bronchial epithelial cells were obtained from 5
Cambrex. At passage 3 7.5x104 per transwell were seeded
into 6.5 mm diameter Transwell-Clear cell culture inserts 0 0.31 1.25 5 20
(Corning) and cultured for 28 days at an air-liquid interface at Concentration of TPM (µg/ml)
37°C, 5% CO2 as described by Gray et.al. (1). Serial doses of
cigarette smoke TPM (0-20 µg/ml) were added to the basal B. Flow cytometry
medium of treated cultures. The basal medium ± TPM was 30
%MUC5AC +ve cells
Cells were trypsinised and cytospins prepared. The cytospin 15
slides were immuno-stained using an antibody against the 10
airway mucin MUC5AC (Zymed). To identiify goblet cells 5
bound antibody was detected with an indirect
immunoperoxidase technique, using diaminobenzidine as the
0 0.31 1.25 5 20
chromogen, (figure 2). 450 epithelial cells were analysed and
Concentration of TPM (µg/ml)
the number of goblet cells were expressed as a percentage.
Figure 3. The effect of TPM concentration on goblet cell density.
Flow cytometric analysis Goblet cells were identified using either immunohistochemical
Cells were washed 3 times, trypsinised, resupended in PBS staining (A) or flow cytometry (B). Error bars indicate SD.
and fixed. Resulting samples were permeabilised, and stained
using antibodies recognising MUC5AC and isotype control. CONCLUSIONS
Bound antibody was recognised by a secondary Alexa Fluor This study demonstrates that TPM can promote goblet cell
488 IgG (Invitrogen) to identify goblet cells (figure 3). Cells hyperplasia in vitro. This model may have potential for
were then analysed by flow cytometry (Partec GmbH). 10, 000 assessing the toxicity of cigarette smoke and for mechanistic
events were analysed per sample, and positives were studies of its morphological effects on the lung epithelium. The
expressed as a percentage of the total. flow cytometry method eliminates the subjectivity of manual
scoring, allows the analysis of a larger number of cells per
sample, and facilitates a higher throughput of samples.
1. Gray, T.E., et al., 1996. Am.J.Respir.Cell Mol.Biol. 14:104-112.