0022-3565/02/3022-814 –821$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:814–821, 2002
Vol. 302, No. 2
33951/996955
Printed in U.S.A.
Goblet Cell Hyperplasia, Airway Function, and Leukocyte
Infiltration after Chronic Lipopolysaccharide Exposure in
Conscious Guinea Pigs: Effects of Rolipram and
Dexamethasone
TOBY J. TOWARD and KENNETH J. BROADLEY
Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom
Received February 6, 2002; accepted April 8, 2002
Chronic mucus hypersecretion is an important symptomatic and pathological feature of a heterogeneous group of
chronic respiratory diseases that includes chronic bronchitis,
chronic obstructive pulmonary disease (COPD), and asthma
(Rogers, 1994; Jackson, 2001). Persistent mucus overproduction contributes to reduced airway caliber and the occlusion
of small airways (reduced FEV1), productive cough, and labored breathing (Jackson, 2001). Individuals with chronic
mucus hypersecretion also suffer from an increased frequency and duration of respiratory infection, causing further
exacerbation of their original respiratory pathology (Jackson,
2001).
The two major sources of mucus secretion in the respira-
This work was financially supported through a GlaxoSmithKline studentship (to T.J.T.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.033951.
sessed a marked goblet cell hyperplasia and evidence of inflammatory edema, features contributory to reduced airway
caliber. Treatment with dexamethasone (20 mg 䡠 kg⫺1) or rolipram (1 mg 䡠 kg⫺1), administered (i.p.) 24 and 0.5 h before
exposure and 24 and 47 h after each subsequent exposure,
attenuated the inflammatory cell infiltration into the airway,
goblet cell hyperplasia, and inflammatory edema. Dexamethasone exacerbated, whereas rolipram reversed, the chronic
LPS-induced bronchoconstrictions. This study demonstrates
that chronic LPS causes persistent bronchoconstriction,
neutrophilic airway inflammation, goblet cell hyperplasia, and
edema. These rolipram-sensitive features suggest the potential
of PDE4 inhibitors in chronic inflammatory lung diseases.
tory tract are the surface epithelial goblet cells and mucous
cells of the submucosal glands. In normal lungs, goblet cells
are present in the large bronchi, becoming increasingly
sparse toward the bronchioles. The submucosal glands are
restricted to the large airways with their density decreasing
with airway caliber, such that they are absent in the bronchioles. In chronic respiratory diseases, such as COPD and
asthma, submucosal glands increase in size (hypertrophy),
and the number of goblet cells is increased (hyperplasia),
becoming more dense in the peripheral airways, via a phenotypic conversion of nongoblet epithelial cells (metaplasia)
(Rogers, 1994; Jackson, 2001). The increased ratio of goblet
cells to ciliated cells and the increased goblet cell density in
terminal bronchioles, under conditions of hypersecretion, impairs clearance of mucus through mucociliary mechanisms or
coughing, respectively. Lung histology from patients affected
by COPD and asthma also shows the presence of edema,
which can further reduce airway caliber and compromise
ABBREVIATIONS:COPD, chronic obstructive pulmonary disease; AHR, airway hyperreactivity; IL, interleukin; TNF-␣, tumor necrosis factor-␣;
COX, cyclooxygenase; PDE4, phosphodiesterase isoenzyme-4; LPS, lipopolysaccharide; sGaw, specific airway conductance; BAL, bronchoalveolar lavage; ABPAS, Alcian Blue-periodic acid Schiff; PAF, platelet-activating factor; PG, prostaglandin; FEV1, forced expiratory volume in 1 s;
TH, T-helper cells; GC, goblet cell.
814
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ABSTRACT
The effects of chronic exposures (nine, 48 h apart) of conscious
guinea pigs to lipopolysaccharide (LPS) (30 ␮g 䡠 ml⫺1, 1 h) on
airway function, airway histology (in particular, goblet cell numbers), and inflammatory cell infiltration of the lungs were examined as a model of chronic inflammatory lung disease, such as
chronic obstructive pulmonary disease. The sensitivity of these
parameters to treatment with the corticosteroid, dexamethasone, or the phosphodiesterase-4 (PDE4) inhibitor, rolipram,
was determined. As the number of LPS exposures increased,
there was a progressively persistent bronchoconstriction after
each exposure. After nine LPS exposures, there was evidence
on histological examination of airway infiltration of, predominantly, neutrophils in perivascular, peribronchial, and alveolar
tissues. After chronic LPS exposure, the airway epithelium pos-
Chronic LPS Inhalation: Lung Function and Goblet Cells
as the functional parameters of acute and chronic LPS-induced inflammation.
Materials and Methods
Animals. Groups of six male Dunkin-Hartley guinea pigs, weighing 300 to 400 g, were used. Animals received food and water ad
libitum, and room temperature (22 ⫾ 2°C) and lighting (maintained
on a 12-h cycle) were regulated. This work complied with the Guidelines for Care and Use of Laboratory Animals, according to the
Animals (Scientific Procedures) Act of 1986 and GlaxoSmithKline
policy.
Measurement of Respiratory Function. Airway function [specific airway conductance (sGaw)] was monitored in conscious guinea
pigs, using whole body plethysmography as previously described by
Griffiths-Johnson et al. (1988). A computerized data acquisition system replaced the original oscilloscope and angle resolver (Danahay
and Broadley, 1997). Guinea pigs with a close-fitting face mask were
placed in a restrainer that was then slid into the plethysmography
chamber. A computer with a Biopac data acquisition system and
AcqKnowledge software (Biopac Systems Inc., Santa Barbara, CA)
acquired and stored data referring to the airflow across a pneumotachograph (Mercury FIL; GM Instruments, Ltd., Scotland, UK) as
the animal breathed. The resulting change in box volume (pressure)
was also simultaneously measured. Changes in airflow and box
pressure were measured by two UP pressure transducers (Pioden
Controls Ltd., Canterbury, UK). The resultant waveforms could then
be rapidly analyzed by comparing the gradients of the flow and the
box pressure waves at a point where flow tended toward zero, i.e., in
the first 30 ms of expiration. A function of these parameters, correcting for ambient pressure and the weight of the animal, determined a
value for sGaw. At least five breaths were analyzed for each animal
at each time point. Before all experiments, the animals were handled
and familiarized with the apparatus to reduce stress.
Inhalation Exposures and Administration of Anti-Inflammatory Compounds. Groups (n ⫽ 6) of guinea pigs were exposed to
LPS or the LPS vehicle (saline) with or without treatment with
dexamethasone or rolipram as shown in Fig. 1 and as previously
described (Toward and Broadley, 2001). In single exposure studies,
guinea pigs were exposed in an exposure chamber (620 ⫻ 300 ⫻ 420
mm) for 1 h to an aerosolized solution of LPS (30 ␮g 䡠 ml⫺1, endotoxin
from Escherichia coli serotype O26:B6) (Sigma Chemical Co., Poole,
Dorset, UK) or saline (NaCl for infusion British Pharmacopoea, 0.9%
w/v) (Baxter Healthcare, Thetford, Norfolk, UK). The aerosol was
generated by a Wright nebulizer driven by compressed air at
20 p.s.i., at a rate of 0.5 ml 䡠 min⫺1. In chronic exposure studies, the
animals received nine exposures, 48 h apart. The lethal dose of LPS
(LD50 within 24 h, 0.7 mg 䡠 kg⫺1, i.p.) in guinea pigs was considered
substantially higher than that administered in this study (Matsuda
Fig. 1. Protocol for single or chronic (nine exposures, 48 h apart) exposure
(60 min) to nebulized LPS (30 ␮g 䡠 ml⫺1) or vehicle (pathogen-free saline)
of conscious guinea pigs, with and without dexamethasone or rolipram
dosing. Animals were terminated 24 h after the first or ninth exposure,
bronchoalveolar lavage fluid was removed, and lung samples were taken
for histology.
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lung function. A marked airway infiltration of macrophages
and granulocytes is also present, principally neutrophils in
COPD and eosinophils in asthma (Postma and Kerstjens,
1998). In clinical studies, these inflammatory parameters
have been shown to correlate with a reduction in lung function (FEV1) and an exaggerated bronchoconstriction [airway
hyperreactivity (AHR)] to nonspecific stimuli (Postma and
Kerstjens, 1998).
Anti-inflammatory steroids are the current mainstay of
severe asthma treatment (British Thoracic Society et al.,
1993), by inhibiting the transcription of proinflammatory
mediators [e.g., eicosanoids, interleukins (IL), and tumor necrosis factor-␣ (TNF-␣)], inducible enzymes [e.g., nitric-oxide
synthase, and cyclooxygenase-2 (COX-2)], and adhesion molecules (Laitinen et al., 1992; Barnes and Adcock, 1993). However, little evidence exists of their clinical benefit on disease
progression in COPD (Burge, 1999). Recently, attention has
focused on the inhibition of phosphodiesterase isoenzyme-4
(PDE4) as a molecular target for COPD (and asthma) (Torphy et al., 1999). Evidence suggests that the subsequent
intracellular elevation in cAMP induces airway smooth muscle relaxation, alleviates inflammatory edema, and suppresses immunocompetent cell activation and migration in
models of acute pulmonary inflammation (Sekut et al., 1995;
Torphy et al., 1999).
The acute symptoms of mucus hypersecretion, as in
chronic bronchitis, can be modeled by exposure of rats to
ozone or sodium metabisulfite (Murlas and Roum, 1985;
Shore et al., 1995). Features of severe asthma (goblet cell
hyperplasia, AHR, and eosinophilic airway infiltration) have
been mimicked by chronic antigen exposure of atopic mice
and guinea pigs (Blyth et al., 1998; Danahay and Broadley,
1998). However, few in vivo models emulate the chronic
inflammation of COPD, afford the examination of lung function over many days (without anesthesia influencing vagal
tone or sensory reflexes), and stimulate the mucus hypersecretion associated with neutrophilia and AHR. A single exposure of rats to lipopolysaccharide (LPS) has been shown to
cause an acute lung neutrophilia and AHR, attenuated by
TNF-␣ inhibition (Kipps et al., 1992). Previously, we have
demonstrated in conscious guinea pigs that chronic (nine,
60-min exposures, 48 h apart) inhalation of aerosolized LPS
causes further features analogous to COPD, namely, a progressive decline in lung function, persistent AHR, and a
neutrophilic inflammatory cell population in the bronchoalveolar fluid, together with nitric oxide overproduction (Toward and Broadley, 2001). Mediators derived from inflammatory cell activation, recruitment, and LPS are thought to
induce epithelial proliferation, permeability, and a mucus
hypersecretory phenotype (Rogers, 1994; Jackson, 2001). In
this study, we therefore extend our previous research to
examine the morphological changes to the lung that underlie
the functional pathology associated with chronic LPS exposure.
The first aim of this study was to characterize the relationship between the previously described lung function and
inflammatory cell influx and the lung morphology after single or chronic exposures to LPS. We regard the latter as more
clinically relevant to chronic pulmonary inflammatory diseases, such as COPD. The second aim was to examine
whether the corticosteroid, dexamethasone, or the PDE4 inhibitor, rolipram, affected the morphological changes as well
815
816
Toward and Broadley
Fig. 2. Airway function of conscious guinea pigs expressed as the change
in specific airway conductance (sGaw). The effects of the first or ninth
(eighth and ninth in rolipram-treated animals) exposures from a chronic
exposure (nine, 60-min exposures, 48 h apart) regimen to nebulized saline
(LPS vehicle) or LPS (30 ␮g 䡠 ml⫺1) are shown. A, first and ninth saline;
B, first and ninth LPS; C, ninth LPS with and without dexamethasone
(20 mg 䡠 kg⫺1); and D, eighth and ninth LPS with and without rolipram
(1 mg 䡠 kg⫺1) treatment. Treatments were administered (i.p.) 24 and 0.5 h
before exposure and 24 and 47 h after each subsequent exposure. The last
dexamethasone and rolipram doses were administered 47 and 24 h after
the eighth exposure, respectively. Each point represents the mean ⫾
S.E.M. (n ⫽ 6) change in sGaw expressed as a percentage of the baseline
sGaw values [sGaw (s⫺1 䡠 cm of H2O)]: saline ⫽ 0.45 ⫾ 0.01 (A), LPS ⫽
0.46 ⫾ 0.3 (B), LPS and dexamethasone treatment ⫽ 0.40 ⫾ 0.01 (C), and
LPS and rolipram treatment ⫽ 0.32 ⫾ 0.01 (D). Negative values represent bronchoconstriction. Significance of differences from baseline values
(ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001), between first and last exposures
(§, p ⬍ 0.05; §§§, p ⬍ 0.001), or between LPS only and dexamethasone or
rolipram treatment (†, p ⬍ 0.05; ††, p ⬍ 0.01; †††, p ⬍ 0.001) was
determined by analysis of variance (single factor), followed by a paired,
paired and unpaired Student’s t test, respectively. Lung function during
chronic exposure to saline with dexamethasone or rolipram treatment
(data not shown) was not significantly different (p ⬎ 0.05) from saline
alone (A).
Data Analysis. To reduce intersubject variability, changes in
sGaw from the baseline sGaw values taken before a procedure are
presented as a percentage of the mean baseline value preceding the
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et al., 1995). The average of two sGaw measurements was obtained
prior to exposure (baseline or 47 h after the previous exposure) and
then at regular intervals (0, 15, 30 min, and hourly) after exposure(s).
Dexamethasone (20 mg 䡠 kg⫺1) or rolipram (1 mg 䡠 kg⫺1) were
administered (i.p.) 24 and 0.5 h before the first of the chronic exposures to LPS or saline and at 24 and 47 h after each subsequent
exposure. Animals treated with rolipram during the chronic LPS
study developed persistent bronchodilation, which would have interfered with an assessment of airway reactivity to histamine 24 h after
the ninth exposure. Consequently, the last dose of rolipram was
given 24 h after the eighth exposure to LPS or saline, which allowed
sGaw to recover to baseline values, at 24 h after the ninth LPS
exposure. Dexamethasone-21-phosphate, disodium salt (Sigma
Chemical), and rolipram (Sigma Chemical) stock solutions were dissolved in 50% dimethyl sulfoxide (Sigma Chemical), 50% saline (Baxter Healthcare) and further diluted with saline for injection (1.0 ml).
The final concentration of dimethyl sulfoxide was less than 5%, and
this vehicle has previously been shown by this laboratory to have no
effect on airway responses or inflammation in a similar chronic
inflammatory model (Danahay and Broadley, 1998). Thus, control
groups treated chronically with LPS and the vehicle for dexamethasone or rolipram were not included; comparisons were made between LPS-exposed animals with or without the drug treatments.
Control groups receiving chronic saline and the drug treatments
were, however, included. Doses of dexamethasone (Whelan et al.,
1995; Toward and Broadley, 1999) and rolipram (Danahay and
Broadley, 1997) were selected based upon the findings from other
studies using similar models of inflammation and those that were
without adverse effects. No animal appeared to be in respiratory
distress or to exhibit other signs of discomfort during the exposure
regimens or during any other part of the protocol described.
All animals underwent a bronchoalveolar lavage (BAL) 24 h after the
last exposure to LPS or saline to determine total cell counts using a
Neubauer hemocytometer and differential cell counts after staining
with Leishman’s stain. The guinea pigs were overdosed with pentobarbitone sodium (400 mg 䡠 kg⫺1, i.p.; Euthatal; Rhone Merieux, Essex,
UK), and the trachea was cannulated. A 1% solution of EDTA disodium
salt (Sigma Chemical) was flushed through the cannula into the lungs
(1 ml 䡠 100 g⫺1 of body weight), recovered 3 min later, and repeated once.
Lung Histology. After lavage, the lungs were fixed by slow in
situ inflation with neutral-buffered formalin (10%, pH 7.0) (1 ml 䡠 100
g⫺1 of body weight) via the tracheal cannula and, following immediate removal from the thoracic cavity, further immersed in neutralbuffered formalin for at least 72 h. After fixation, representative
samples were cut through the large bronchi of the right and left lung
(medial lobe), dehydrated in 70 to 100% ethanol/xylene, and embedded in paraffin wax. Sections were cut (6 ␮m), deparaffinized, and
stained with hematoxylin and eosin or Masson’s trichrome for general morphology. Additional sections were stained with elastic van
Gieson stain to differentiate elastic fibers and collagen, and Alcian
Blue-periodic acid Schiff (ABPAS) was used for identification of
mucin (neutral and acid)-containing cells.
The number of goblet cells in the epithelium of large airways was
determined using light microscopy. Two slide sections (left and right
lobe) from each animal were coded and assessed “blind” to prevent
bias. Only cells that were stained purple/magenta with ABPAS and
were morphologically typical of goblet cells were counted. To reduce
intra- or intergroup variation between the sections derived from
different locations in the bronchial tree, all the airways measured
possessed a similar degree of cartilaginous plating, indicative of the
lower large bronchial region. The internal airway perimeter has been
shown to be unaltered by smooth muscle contraction or lung inflation
(Pare and Hogg, 1989) and was, therefore, measured using Image
Acquisition software (Qwin standard V2.2; Leica, Heidelberg, Germany) to determine the epithelial perimeter. The mucin-containing
cells were then expressed as goblet cells per millimeter of airway
epithelium.
Chronic LPS Inhalation: Lung Function and Goblet Cells
817
first LPS or saline challenge. Absolute values of baseline sGaw are
stated in the figure legends. Significance of differences in the number
of airway epithelial goblet cell was compared using analysis of variance, followed by Scheffe’s post hoc analysis. Changes in airway
function were compared using analysis of variance followed by the
appropriate paired or unpaired Student’s (two-tailed) t test. Differences were considered statistically significant at p ⬍ 0.05 (Motulsky,
1995).
Results
Airway Function
Effects of Chronic Exposure to LPS on Airway Function. The first exposure to LPS in the chronic exposure study
caused an immediate bronchoconstriction (⫺12.4 ⫾ 10.7%
decrease from baseline sGaw values), which recovered 15 to
30 min later but was not significantly different (p ⬎ 0.05)
from the response to saline (⫺20.5 ⫾ 3.6%) (Fig. 2). The
seventh, eighth, and ninth exposures to LPS caused a decline
in sGaw (⫺16.9 ⫾ 5.9, ⫺20.6 ⫾ 2.8, and ⫺23.1 ⫾ 3.6 peak
percentage decrease from baseline sGaw values), with a progressive increase in duration of bronchoconstriction. After
the eighth exposure, there was still significant bronchoconstriction at 30 min (Fig. 2D), whereas after the ninth exposure, the bronchoconstriction remained until 19 h after exposure (Fig. 2B).
Effects of Dexamethasone or Rolipram Treatment on
the Airway Function Responses to Chronic LPS Exposures. Dexamethasone or rolipram treatment did not significantly affect (p ⬎ 0.05) the initial LPS-induced bronchoconstriction after the first exposure. However, dexamethasone
exaggerated the duration of prolonged bronchoconstriction after
the ninth LPS exposure, from 19 h to at least 24 h after exposure (Fig. 2C). In contrast, rolipram-treated animals developed
a significant (p ⬍ 0.001) and persistent bronchodilation at 24
and 47 h after the seventh exposure to LPS (Fig. 2D). When
rolipram was withdrawn 24 h after the eighth exposure to LPS,
bronchodilation returned to baseline sGaw values at 24 h after
the ninth exposure. No bronchodilator activity occurred in rolipram-treated animals exposed to chronic saline (data not
shown). Lung function did not differ in the absence or presence
of dexamethasone or rolipram treatment in saline-exposed animals (data not shown).
Lung Morphology
Effects of Single or Chronic Exposures to LPS on
the Upper Airways. Large bronchial sections, stained
with ABPAS, from the lungs of naive animals or those
removed 24 h after a single or chronic saline exposure (Fig.
3A), appeared to possess a normal composition of epithelial
cells with an occasional darkly stained goblet cell. Compared with naive animals, at 24 h after a single LPS
exposure, the number of goblet cells increased 107% (p ⬎
0.05) (Fig. 4). However, at 24 h after chronic LPS exposure,
the ratio of goblet cells containing both acid (purple) and
neutral (magenta) mucins to normal columnar-ciliated
cells was greatly increased (Fig. 3B). Chronic LPS exposure caused significant (p ⬍ 0.05) 4.6- and 2.5-fold increases, respectively, in the number of goblet cells (hyperplasia) compared with naive and chronic saline-exposed
animals (Fig. 4). In chronically LPS-exposed animals,
there was also an increased number of Clara cells, anatomically defined by their nonciliated, dome-shaped appearance and protrusion into the bronchiolar lumen, although they were not quantified. In all the sections
analyzed, no obvious evidence of airway smooth muscle
hypertrophy, collagen disposition beneath the basement
membrane, or change in elastic fiber composition was
present.
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Fig. 3. Bronchiolar epithelial changes in
guinea pig airways exposed chronically
(nine 60-min exposures, 48 h apart) to nebulized vehicle (saline) (A), LPS (30 ␮g 䡠
ml⫺1) (B), or LPS with dexamethasone (20
mg 䡠 kg⫺1) (C) or rolipram (1 mg 䡠 kg⫺1) (D)
treatment. Treatment was administered
(i.p.) 24 and 0.5 h before exposure and 24
and 47 h after each subsequent exposure.
The last dexamethasone and rolipram
doses were administered 47 and 24 h after
the eighth exposure, respectively. Airway
lumen (L) sections were stained with ABPAS to aid the light microscope counting of
mucus (neutral and acid mucins)-containing goblet cells (G) and depict airway
smooth muscle (ASM), alveoli (Al), and
cartilaginous plating (C). In chronic LPSexposed animals (B), goblet cell hyperplasia was attenuated with both dexamethasone (C) and rolipram (D) treatment. The
airway epithelium from naive animals or
those 24 h after a single saline exposure
did not appear to be different from that of
animals chronically exposed to saline (A).
Bar ⫽ 50 ␮m.
818
Toward and Broadley
exposure, there was an increased migration of macrophages
and neutrophils into the perivascular, peribronchial tissues
and alveoli, which was further increased at 24 h after chronic
LPS exposure (Fig. 5B). In chronic LPS-exposed animals,
there was also some evidence of an increase in the alveolar
wall thickness and edema. Edematous protein-rich fluid (defined by heavy eosin staining) was also present, presumably
exuded from the vasculature into the alveolar lumen.
Effect of Dexamethasone or Rolipram Treatment on
Alveoli after Chronic Exposure to LPS. Treatment with
dexamethasone or rolipram reduced the chronic LPS-induced
infiltration of leukocytes from perivascular sites into the
airways and alveoli. Treating the animals with rolipram
greatly attenuated the amount of proteinaceous fluid in the
alveolar spaces (Fig. 6C), whereas dexamethasone also appeared to reduce the chronic LPS-induced edema, but to a
lesser extent (Fig. 6D).
Fig. 4. Goblet cells per millimeter of airway epithelium from guinea pigs
before (naive) and 24 h after a single or chronic (nine, 24 h apart)
exposure (60 min) to nebulized LPS (30 ␮g 䡠 ml⫺1) or vehicle (pathogenfree saline), in the absence and presence of dexamethasone (20 mg 䡠 kg⫺1)
or rolipram (1 mg 䡠 kg⫺1) treatment. Treatment was administered (i.p.) 24
and 0.5 h before exposure and 24 and 47 h after each subsequent exposure. The last dexamethasone and rolipram doses were administered 47
and 24 h after the eighth exposure, respectively. Two representative
sections of large bronchi, from right and left medial lobes, were stained
purple/magenta with ABPAS to identify and count blind morphologically
typical goblet cells. The internal airway perimeter was measured using
Image Acquisition software (Leica), and the goblet cell number was
expressed per millimeter (GC mm⫺1) of airway epithelium. Each point
represents the mean ⫾ S.E.M. (n ⫽ 6) of two GC mm⫺1 of airway
epithelium determinations per animal. Significance of differences in the
GC mm⫺1 of airway epithelium was compared with those of animals
exposed to chronic saline (ⴱ, p ⬍ 0.05) or chronic LPS (†, p ⬍ 0.05) and
were determined by analysis of variance (single factor), followed by Scheffe’s post hoc analysis.
Effect of Dexamethasone or Rolipram Treatment on
Airway Goblet Cells after Single or Chronic Exposure
to LPS. Treatment with dexamethasone or rolipram greatly
attenuated the chronic LPS-induced goblet cell hyperplasia
(Fig. 3, C and D, respectively). The increased population of
goblet cells 24 h after chronic LPS exposure was significantly
(p ⬍ 0.05) inhibited by 89 and 71% in animals treated with
dexamethasone or rolipram, respectively (Fig. 4). Neither
dexamethasone nor rolipram treatment caused a significant
change in the airway goblet cell population 24 h after chronic
saline (Fig. 4).
Effects of Single or Chronic Exposures to LPS on
Lung Alveoli. Peripheral lung sections, stained with Masson’s trichrome, from the lungs of naive animals or those
removed 24 h after a single or chronic saline exposure (Fig.
5A) had normal alveolar pathology and no evidence to indicate an increased number of resident macrophages or granulocytes in the alveoli. However, at 24 h after a single LPS
Chronic airflow obstruction and AHR are characteristic
features of patients with COPD and severe asthma (Laitinen
et al., 1992; Postma and Kerstjens, 1998). In COPD, there are
increased numbers of inflammatory cells (predominantly
neutrophils) in the airway wall, particularly in the epithelial
layer and around the bronchial submucosal glands (Postma
and Kerstjens, 1998). Persistent airway inflammation in
these patients results in airway wall edema, deposition, and
remodeling of connective tissue components (e.g., submucosal and adventitial collagen disposition), together with hypertrophy and hyperplasia of submucosal glands or the goblet cell phenotype, respectively (Barnes, 1998; Jackson,
2001). These pathological features reduce airway caliber and
are thought to contribute to the heightened constrictor response from spasmogenic stimuli (AHR) and airflow obstruction in COPD (Pare and Hogg, 1989). In this study, we
examined the functional and morphological effects of chronic
pulmonary inflammation derived from repeated exposure of
guinea pigs to LPS as a model for the progressive inflammatory processes of COPD and the ability of dexamethasone and
rolipram to suppress these changes.
The first exposure to LPS caused a small bronchoconstriction that was no different from that observed after saline
inhalation (Toward and Broadley, 2000), which we have previously attributed to the saline condensing in the airways or
to obstructed airway conductance (Toward and Broadley,
2001). By the ninth exposure to LPS, there was a prolonged
period of bronchoconstriction, which did not occur with repeated saline exposure. Previously, we have reported that
the influx of inflammatory cells into the lungs as measured
by BAL, particularly neutrophils, increases with the number
of exposures. Also, AHR to histamine was prolonged from 2 h
after a single exposure to at least 24 h after the eighth
exposure (Toward and Broadley, 2001). In the present study,
we further demonstrate infiltration of neutrophils and macrophages into the alveolar, perivascular, and peribronchial
spaces after chronic LPS exposure.
The increased infiltration of neutrophils into the BAL fluid
and lung tissues was likely to be initially orchestrated by
chemotactic factors, such as TNF-␣ and IL-8, released into
the airways by resident macrophages, epithelial cells, and
lymphocytes (Snella and Rylander, 1985; Brigham and Mey-
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Discussion
Chronic LPS Inhalation: Lung Function and Goblet Cells
819
rick, 1986; Kipps et al., 1992). The early synthesis of TNF-␣
(Brigham and Meyrick, 1986) in response to LPS, activates
other proinflammatory mediators, including arachidonic acid
metabolites, deleterious cytotoxins (proteases and reactive
oxygen species), and cytokines. Bronchial biopsies from patients with COPD show similar inflammatory processes, and
sputum samples have elevated TNF-␣, IL-8, reactive oxygen
species, and proteolytic enzyme levels (Barnes, 1998). The
eicosanoid products of arachidonic acid, namely leukotrienes
(B4, C4, D4, and E4), platelet-activating factor (PAF), and
inducible COX-2-derived prostanoids [thromboxane, prostaglandin (PG)-F2, -D2, and 8-epi-PGF2] have all been implicated in animal models, COPD, and asthma as causes of
AHR, bronchoconstriction, increased airway permeability or
leukocyte influx (Brigham and Meyrick, 1986; Laitinen et al.,
1992; Barnes, 1998; Barnes et al., 1999).
In this study, histological examination of the lungs after a
single LPS exposure showed no morphological features to
support a geometric reduction in airway caliber that would
potentiate a spasmogen-induced airway narrowing and explain the AHR seen previously with this model (Pare and
Hogg, 1989). However, in chronically LPS-exposed animals,
the airway histology showed extensive migration of neutrophils but little evidence of airway collagen or elastic fiber
remodeling, smooth muscle hypertrophy, or epithelial shedding. Both dexamethasone and rolipram were equieffective
at attenuating the chronic LPS-induced airway infiltration of
inflammatory cells, possibly due to their inhibitory effect on
TNF-␣ production (Barnes et al., 1999; Torphy et al., 1999).
Histological examination after chronic LPS exposure also
revealed increased alveolar wall thickness and evidence of
edema and plasma exudation. The inflammatory edema observed after chronic LPS exposure may be a result of an increased capillary blood pressure by vasoconstrictor eicosanoids
or an increased permeability of the capillary wall from the
release of reactive oxygen species (including NO and peroxynitrite), eicosanoids, or proteolytic enzymes (Brigham and Meyrick, 1986; Barnes et al., 1999). Edematous swelling of the
airway wall and increased airway exudate in the lung have
been shown to reduce airway caliber and correlate with AHR in
sheep (Hwang et al., 2001), and may contribute to the AHR
observed in the present chronic LPS model. Edema is also a
probable contributor to the prolonged bronchoconstriction seen
in this study after chronic LPS exposure. The edema and accumulation of proteinaceous fluid in the alveoli was inhibited by
rolipram to a greater extent than by dexamethasone. This may
indicate an increased potency of rolipram on granulocyte infiltration into the airway and a subsequent release of edemainducing mediators. It may also explain why the prolonged
bronchoconstriction following the final LPS challenge was attenuated by rolipram but not by dexamethasone and adds
weight to the conclusion that the prolonged bronchoconstriction
was associated with the edema. The generation of inducible
COX-2-derived prostanoids, PAF, and leukotrienes may also
contribute to the prolonged chronic LPS-induced bronchoconstrictions. In airway epithelial and smooth muscle cell cultures,
dexamethasone inhibits COX-2 expression and the subsequent
release of bronchoconstrictor prostanoids (Barnes et al., 1999).
However, in this study, dexamethasone exacerbated the later
LPS-induced bronchoconstrictions. This may be due to inhibition of expression of the functionally antagonistic COX-2-derived bronchodilator, PGE2 (Barnes et al., 1999). The persistent
bronchodilation in rolipram-treated animals during later LPS
challenges, but not saline exposures, may be due to an induction
of the COX-2-derived PGE2 by rolipram. The bronchodilatory
second messenger of PGE2 is cAMP, the levels of which will be
elevated by PDE4 inhibition with rolipram (Uhlig et al., 1995;
Barnes et al., 1999).
AHR after chronic LPS exposures may have been due to
the formation of the powerful oxidant peroxynitrite from the
interaction of inflammatory-derived superoxide with NO
(Beckman, 1996; Barnes et al., 1999), excessive airway levels
of which occur in chronic LPS-exposed animals (Toward and
Broadley, 2001). Peroxynitrite can induce AHR in guinea
pigs (Sadeghi-Hashijin et al., 1996), possibly through cytotoxic damage of the airway epithelium to expose sensory
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Fig. 5. Representative alveolar sections taken from guinea pig airways exposed chronically (nine, 60-min exposures, 48 h apart) to nebulized vehicle
(saline) (A) or LPS (30 ␮g 䡠 ml⫺1) (B). Peripheral lung sections were stained with Masson’s trichrome to identify general morphological features. In
lungs removed from naive animals and those after single or chronic saline exposure, the alveolar sections appeared normal, possessing a few sentry
macrophages (M), capillary red blood cells (RBC), and the occasional eosinophil. In single and chronic LPS-exposed animals, alveolar infiltration of
activated (enlarged with multiple vesicles or degranulation, respectively) macrophages and neutrophils (N) was extensive. In chronic LPS-exposed
animals, there was also evidence of edematous proteinaceous fluid (PF), but no evidence of an enlarged airspace was observed as in emphysema. Bar ⫽
50 ␮m.
820
Toward and Broadley
nerves (Barnes et al., 1999) or an impairment of ␤-adrenoceptors (Kanazawa et al., 1999). Levels of peroxynitrite or the
peroxynitrite-induced nitration product, nitrotyrosine, were
not, however, determined in the current study.
The major histological change observed after chronic LPS
exposures was an increase in the density of goblet cells in the
epithelial layer. The close proximity of inflammatory cells to
the epithelial goblet cells and submucosal glands in the histology of patients with COPD suggests a causative association between leukocytes and the hypersecretory mucus phenotype (Postma and Kerstjens, 1998). Human airways
possess a large number of submucosal glands and goblet
cells, whereas in guinea pig airways, goblet cells are the
predominant source of mucus secretion (Jackson, 2001). In
guinea pigs, neuronal (cholinergic, adrenergic, and peptidergic) control of mucus secretion appears to be via the goblet
cells, whereas in humans, it is the submucosal glands that
are predominantly innervated. However, in both guinea pigs
and humans, inflammatory mediators act on goblet cells
directly (and also via neuronal mechanisms in guinea pigs) to
influence mucus secretion. Consequently, despite these species differences in the mechanistic regulation and source of
mucus secretion, this model of LPS-induced goblet cell hy-
perplasia has clinical relevance, as the majority of airflow
obstruction in COPD and asthma occurs in the smaller airways, where goblet cells but not submucosal glands are expressed. In this study, a single exposure to LPS caused only
a slight increase in goblet cells 24 h later. However, after
chronic LPS exposure, the epithelial goblet cells were greatly
increased. The peribronchial migration of activated leukocytes into the airway lumen and persistent exposure to proinflammatory stimuli are likely to contribute to goblet cell
up-regulation, although the degree of hyperplastic and metaplastic mechanisms involved in the derivation of this phenotype are unclear (Rogers, 1994). Contrary to the T-helper 2
(TH2)-derived inflammation in asthmatics, LPS causes a predominantly TH1-favored cytokine response (Blyth et al.,
1998; Barnes et al., 1999). This imbalance in TH expression
appears to affect the mechanism of goblet cell induction.
Shimizu et al. (2000) showed in atopic rats exposed to antigen that leukotrienes (C4, D4, and E4) are potent secretagogues and inhibitors of ciliary beat frequency, and play an
important role in goblet cell hyperplasia, whereas in LPSinoculated animals, neutrophil- and COX-derived products
were important (Barnes et al., 1999). Neutrophil-derived reactive oxygen species have been shown to enhance mucin
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Fig. 6. Typical alveolar changes in guinea pigs exposed chronically (nine, 60-min exposures, 48 h apart) to nebulized vehicle (saline) (A), LPS (30 ␮g 䡠
ml⫺1) (B), or LPS with rolipram (1 mg 䡠 kg⫺1) (C) or dexamethasone (20 mg 䡠 kg⫺1) (D) treatment. Treatment was administered (i.p.) 24 and 0.5 h before
exposure and 24 and 47 h after each subsequent exposure. The last rolipram and dexamethasone doses were administered 24 and 47 h after the eighth
exposure, respectively. Peripheral lung sections were stained with hematoxylin and eosin to identify general morphological features. In lungs removed
from naive animals and those after single or chronic saline exposure, the alveolar sections appeared normal. In chronic LPS-exposed animals (B), there
was evidence of edematous proteinaceous fluid (PF), macrophage (M), and neutrophil (N) infiltration, which was greatly attenuated in rolipramtreated animals (C) and to a lesser extent in dexamethasone-treated animals (D). Both dexamethasone and rolipram reduced the macrophage and
neutrophil infiltration into the alveoli. Bar ⫽ 50 ␮m.
Chronic LPS Inhalation: Lung Function and Goblet Cells
Acknowledgments
We gratefully acknowledge Dr. A. T. Nials of the Respiratory
Diseases Unit, GlaxoSmithKline Research and Development, Stevenage, UK, for assistance in conducting this work and critical reading
of the manuscript. We also thank Derek Scarborough of the Histology Department, Biosciences Department, Cardiff University,
Cardiff, UK, for the invaluable contributions of processing, cutting,
and staining of lung tissue, and Michael Pedrick and Tony Savage of
the Histopathology Group, GlaxoSmithKline Research, Stevenage,
UK, for histological interpretation.
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Address correspondence to: Professor Kenneth J Broadley, Division of
Pharmacology, Welsh School of Pharmacy, Cardiff University, King Edward
VII Avenue, Cathays Park, Cardiff CF10 3XF, UK. E-mail: broadleykj@
cardiff.ac.uk
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release in guinea pig tracheal and human bronchial epithelial cells via a NO-dependent pathway, an effect blocked by
COX inhibition (Wright et al., 1996; Barnes et al., 1999). In the
current study, neutrophil-derived superoxide, the excess airway
NO after chronic LPS inhalation (shown in our previous study,
Toward and Broadley, 2001) and their combined product, peroxynitrite, could stimulate mucus secretion and goblet cell hyperplasia (Beckman, 1996; Wright et al., 1996; Barnes et al.,
1999). Inflammation-derived prostanoids, PAF, and proteolytic
enzymes are also capable of stimulating goblet cell mucus secretion and hyperplasia, exacerbating the reduction in airway
caliber after chronic LPS exposure (Barnes et al., 1999). Both
dexamethasone and rolipram attenuated goblet cell hyperplasia after chronic LPS exposure. Suppression of inflammatory
cell activity with rolipram or dexamethasone reduces the production of reactive oxygen species, eicosanoids, and protease,
which stimulate goblet cell secretion and contribute to the etiology that induces a hypersecretory phenotype and edema after
chronic LPS (Barnes and Adcock, 1993; Torphy et al., 1999).
In conclusion, this study demonstrates that morphological
changes to the airways, including neutrophil infiltration,
edema, and goblet cell hyperplasia, following chronic LPS exposure of conscious guinea pigs are associated with functional
changes of persistent bronchoconstriction. These changes, along
with the persistent AHR observed in our previous study, are
characteristic features of COPD. Both dexamethasone and rolipram attenuated goblet cell hyperplasia and edema, probable
contributors of the reduced airway caliber and AHR, via attenuation of LPS and inflammatory cell-derived proinflammatory
mediators. In common with COPD, in which steroids have only
modest beneficial effects, primarily on quality of life, in this
study, dexamethasone failed to improve a deficit in lung function. Rolipram, however, improved lung function. The conscious
guinea pig chronically exposed to LPS may therefore prove a
useful model of COPD, and the results support the further
development of PDE4 inhibitors for the treatment of COPD or
severe asthma.
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