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School of Medicine, Respiratory Cell and Molecular Biology Division, Southampton General Hospital, Southampton, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, common 
-chain, and
IL-13R1, as well as IL-13R2, which
negatively regulates IL-13 signaling; FACS analysis confirmed
IL-13R2 protein expression. Exposure of epithelial
cultures to either Der p extracts, TNF-, 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- was also increased by TNF- 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-, 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to promoting a Th2-type immune response, many inhalant allergens exhibit enzymatic activity, such as the proteolytic activities associated with the major house dust mite Ag Der p 1, a cysteine protease, and Der p 9, a serine protease (2). In an experimental setting, these proteases disrupt epithelial tight junctions, enhance epithelial apoptosis, and promote the release of a variety of proinflammatory cytokines, including IL-8 and GM-CSF (2, 3). Both disruption of the epithelium and enhancement of cytokine release appear to depend on the proteolytic activity of the allergen because both can be blocked by specific protease inhibitors (3). The functional significance of the direct enzymatic effects of Der p 1 in the airways has been shown using the highly selective cysteine protein inhibitor PTL11028, which attenuates airway inflammation and bronchial hyperresponsiveness in allergen-sensitized Brown Norway rats (4).
IL-4 and its structural homologue, IL-13, are prominent cytokines in
asthma not only on account of their proinflammatory role, but also due
to their effects on mucus hypersecretion and airway wall remodelling,
as revealed in transgenic animal models (5, 6, 7, 8, 9). In vitro
studies have confirmed that IL-4 and IL-13 have direct effects on
epithelial and fibroblast function. For example, both cytokines enhance
mucin expression by bronchial epithelial cells and promote release of
GM-CSF and IL-8 from bronchial epithelial or alveolar cell lines
(9, 10, 11). Using fibroblasts grown from bronchial biopsies
of asthmatic subjects, we have recently shown that IL-4 and IL-13
stimulate eotaxin release, but, unlike TGF-
, they are unable to
cause transformation of these cells into myofibroblasts and they do not
stimulate collagen I gene expression (12). However,
because we also demonstrated that IL-4 and IL-13 both enhance release
of TGF- from bronchial epithelial cells, this provides an indirect
mechanism through which these cytokines could drive remodelling
responses in asthmatic airways.
IL-4 and IL-13 exhibit overlapping, but not identical, effector
profiles, which is due to the shared use of the IL-4R -chain
(IL-4R) and IL-13R1 in the multimeric IL-4R
and IL-13R complex (13, 14, 15, 16). IL-4 can also bind to
IL-4R complexed with the common -chain of the IL-2R, while IL-13
can interact with the high affinity IL-13R2,
which has a negative influence on IL-13 signal transduction (17, 18). Both IL-4R and IL-13R1 are
expressed by bronchial epithelium (18, 19), but
expression of IL-13R2 has not been
investigated. IL-4 and IL-13 both signal via the transcription factor
STAT-6 (14, 20), whose expression is prominent in the
bronchial epithelium and is further increased in severe asthma
(21). Genetic variation in several components of the IL-4
and IL-13 signal transduction pathway has been implicated in asthma
susceptibility or severity (13, 22). For examples, the
IL-4R gene on chromosome 16p12.1 has eight polymorphisms in the
coding region leading to amino acid changes (23, 24).
These include an extracellular variant, I50V, that up-regulates
receptor responses to IL-4, leading to increased STAT-6
activation and IgE synthesis (25, 26). Q576R and a single
nucleotide polymorphism in the intracellular domain located in the
STAT-6 binding region enhance signaling and are strongly associated
with asthma severity (27).
In view of the ability of allergen to affect epithelial function directly by protease-dependent mechanisms and indirectly through immune cell activation and secretion of Th2 cytokines, the purpose of this study was to analyze the single and combined effects of IL-4, IL-13, and allergen extracts on the secretory profile of normal and asthmatic bronchial epithelial cells. As cell responses may be influenced by the type of receptors expressed, we also analyzed the IL-4R and IL-13R subunits present on bronchial epithelial cells and related their expression to functional outcome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For bronchial brushing, nonatopic, nonasthmatic control subjects
(n = 19) and asthmatic subjects (n =
19) were characterized according to symptoms, pulmonary function, and
medication. Assessment of asthma severity was in accordance with the
Global Initiative for Asthma guidelines on the diagnosis and
management of asthma (28). The mild asthmatics were
receiving inhaled 2-agonists (salbutamol)
only, while the moderate-severe group was maintained on inhaled
corticosteroids, plus or minus long acting
2-agonists (Table I
). All subjects were nonsmokers and were
free from respiratory tract infections for a minimum of 4 wk before
inclusion to the study. The moderately severe asthmatic subjects
treated with inhaled corticosteroids withheld this medication for a
minimum of 1 wk before bronchoscopy. Written informed consent was
obtained from all volunteers before participation, and ethical approval
was obtained from the Joint Ethics Committee of Southampton University
Hospital Trust.
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Fiberoptic bronchoscopy
Bronchial brushings were obtained by bronchoscopy using a fiberoptic bronchoscope (Olympus FB-20D, Tokyo, Japan) in accordance with standard published guidelines (29). Bronchial epithelial cells were obtained using a standard sterile single-sheathed nylon cytology brush (Olympus BC 9C-26101). This was passed by direct vision via the bronchoscope channel into the lower airways, and five to six consecutive brushings were sampled from the bronchial mucosa of the second and third generation bronchi. Cells were harvested into 5 ml sterile PBS after each brushing. On completion of the procedure, 5 ml RPMI with 10% FBS was added and the sample was centrifuged at 150 x g for 5 min to pellet the cell suspension. Epithelial cell purity was assessed by performing differential cell counts on cytospins of the harvested cell suspension.
Epithelial cultures
Primary cultures were established by seeding freshly brushed bronchial epithelial cells into culture dishes containing 3 ml serum-free hormonally supplemented bronchial epithelium growth medium (Clonetics, San Diego, CA) supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin. When confluent, the cells were passaged (p1) using trypsin and were allowed to further expand until used for experimentation at passage 2 or 3; control experiments confirmed that there was no significant difference between the responses of the cells at p2 or p3. Viability was assessed by exclusion of trypan blue dye, and the epithelial nature of cells was assessed by immunohistochemical staining of cultures grown on culture chamber slides (Labtek II eight-well chamber slides; Fisher Scientific, Loughborough, U.K.) using a pan-cytokeratin Ab as well as Abs specific for cytokeratin 13 and 18.
Cytokine release by bronchial epithelial cells
Primary bronchial epithelial cells were seeded into 24-well
plates (Nunc; Life Technologies) at a density of 5 x
104/well and allowed to grow to 80–90%
confluence. The bronchial epithelium growth medium was then replaced
with basal medium (Clonetics) containing insulin, transferrin, and
sodium selenite supplement (Sigma-Aldrich, Poole, U.K.), and the cells
were rendered quiescent for 24 h before exposure to enzymatically
active extracts of house dust mite (Der p, 5000 U/ml) (ALK,
Copenhagen, Denmark), TNF- (PeproTech, London, U.K.) (20 ng/ml),
IL-4 (20 ng/ml), or IL-13 (20 ng/ml) for 24 h, as detailed in
Results. Release of IL-8, GM-CSF, or TGF- into culture
supernatants was measured using ELISA kits according to the
manufacturer’s instructions (IL-8, Pelikine Research Diagnostics,
Flanders, NJ; GM-CSF, BioSource, Nivelles, Belgium; TGF-,
Oncogene Research Products, San Diego, CA). Cell number was determined
by uptake of methylene blue, and cytokine release was expressed as
pg/106 cells.
IL-4R and IL-13R analysis
Total RNA was extracted from primary bronchial epithelial cells
using TRIzol (Life Technologies, Paisley, U.K.), according to the
manufacturer’s instructions. Total RNA was extracted from whole blood
(1.5 ml) using a RNeasy blood kit (Qiagen, Crawley, U.K.), according to
the manufacturer’s instructions. RNA integrity was assessed by
electrophoresis of the RNA samples on a 1% ethidium bromide-stained
agarose gel and observation of intact 28S and 18S ribosomal bands.
Reverse transcription was performed for 1 h at 37°C using 1 µg
total RNA with 1 µM oligo(dT)15 as a primer and
4 U Omniscript Reverse Transcriptase (Qiagen) in the presence of 0.5 mM
dNTPs, 10 U RNase inhibitor (Ambion, Austin, TX), and 1x
reverse-transcriptase buffer in a total volume of 20 µl. For PCR
analysis, 2 µl cDNA was amplified in a reaction mixture containing 10
mM Tris-HCl (pH 8.3), 50 mM KCL, 0.2 mM dNTPs, 0.2 µM sense and
antisense oligonucleotide primers, and 0.025 U/µl JumpStart
Taq DNA polymerase (Sigma-Aldrich) in a total volume of 25
µl using a TETRAD thermocycler (MJ Research, Boston, MA).
The following MgCl2 concentrations and primers
were used: IL-13R1, 2 mM MgCl2, sense 5'-TCA
TGG TCC CTG GTG TTC-3' and antisense 5'-CGG TGC GCG ACT CAA CAT AAA-3';
IL-13R2, 1.5 mM MgCl2, sense 5'-CAA GGG CAT
TGA AGC GAA GA-3' and antisense 5'-CCA AAT TCC GTC ATC TGA GCA-3';
IL-2R (common -chain (c)), 1 mM
MgCl2, sense 5'-TAC CGG ACT GAC TGG GAC CAC-3'
and antisense 5'-TGG GGG AAT CTC ACT GAC GA-3'; IL-4R, 1.5 mM
MgCl2, sense 5'-CTG ACC TGG AGC AAC CCG TAT-3'
and antisense 5'-CCG CTT CTC CCA CTG TGA CCC-3'. After an
initial denaturation at 95°C for 1 min and 30 s, samples were
amplified using a touchdown protocol for a total of 48 cycles: 94°C,
30 s; X°C, 30 s; and 72°C, 30 s, in which X is 68
for 1 cycle, 67 for 2 cycles, 66 for 3 cycles, 64 for 4 cycles, 62 for
5 cycles, 60 for 6 cycles, and 58 for 28 cycles. Aliquots of PCR
products were run on 2% agarose gels and visualized by ethidium
bromide staining. All amplicons were designed to span introns and were
tested to ensure they would not amplify genomic DNA. The specificity of
amplicons was confirmed by direct sequencing using a BigDye dye
terminator cycle sequencing (Applied Biosystems, Warrington, U.K.) with
the products run on an ABI377 automated sequencer (Applied
Biosystems).
Flow cytometry analysis
Epithelial cell cultures were detached from the culture plates
using trypsin. The single cell suspension was then washed in PBS
containing 2% FCS and resuspended at a concentration of 1 x
107 cells/ml. A total of 100 µl aliquots of
this cell suspension was then incubated for 1 h at 4°C with the
appropriate primary Abs IL-13R2 (Diaclone; IDS, Tyne and Wear,
U.K.) and c, and then washed and
resuspended in 100 µl PBS/1% BSA containing FITC-conjugated
anti-mouse or anti-rabbit secondary Abs. In the case of
IL-4R, detection was with an IL-4 fluorokine (R&D Systems, Abingdon,
U.K.). After 30-min incubation in the dark at 4°C, the cells were
washed in cold PBS for analysis using a FACScan flow cytometer (BD
Biosciences, Oxford, U.K.) with WinMDI 2.8.
Statistical analyses
Data were analyzed initially using the Kolmogorov-Smirnoff test, which indicated that they were not normally distributed. Therefore, the Wilcoxon Signed Rank test was used for within group comparisons, and the Mann Whitney U test was used for between group comparisons. A p value of <0.05 was considered statistically significant. For analysis of cytokine release that involved multiple testing, a Bonferroni correction was applied.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The growth properties of primary cultures of bronchial epithelial
cells established from epithelial brushings of normal and asthmatic
volunteers are described elsewhere (30). RT-PCR analysis
demonstrated that epithelial brushings and cultured epithelial cells
expressed mRNA for IL-4R, IL-13R1,
IL-13R2, and the c of
the IL-2R (Fig. 1a), whereas
IL-13R2 was absent from PBMCs. Cell surface
expression of IL-4R, IL-13R2, and
c protein was confirmed by FACS analysis using
epithelial cultures derived from three normal and three asthmatic
subjects. The presence of IL-13R2 and IL-4R
was readily detectable in every case; however, in the case of
IL-13R2, levels of expression were found to be
variable (Fig. 1b and Table II). In contrast, only low levels of
c were detectable.
IL-13R1 was not evaluated due to the lack of a
suitable Ab for use by FACS analysis.
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on IL-8 and GM-CSF
release by bronchial epithelial cells
Although TNF- and the proteolytic activity of Der p
both have the potential to cause apoptosis (3), neither
Der p (5000 U/ml) nor TNF- (10 ng/ml) caused significant
induction of apoptosis in the primary cultures at the concentrations
used (data not shown). However, exposure of bronchial epithelial cells
to Der p caused a dose-dependent increase in IL-8 and GM-CSF
release by normal and asthmatic bronchial epithelial cell cultures
(Fig. 2). For normal cultures, median
(range) IL-8 release increased from 2.5 (0.1–8.2) to 7.7 (1.0–59.9)
and 11.9 (1.7–59.7) ng/106 cells
(n = 12), and GM-CSF increased from 101 (0–1274) to
128 (0–5358) and 173 (27
5308) pg/106 cells in
response to 2500 and 5000 U/ml Der p, respectively;
corresponding values for the asthmatic cultures were 2.9 (0.1–14.5)
increasing to 11.9 (0.2–46.9) and 13.9 (0.4–37.7)
ng/106 cells (n = 14) for IL-8,
and 200 (0–798) increasing to 375 (42–1708) and 361
(29–1785) pg/106 cells for GM-CSF. TNF- also
significantly stimulated IL-8 and GM-CSF release from the epithelial
cultures (Fig. 2). In each case, cytokine release was significantly
(p < 0.05) reduced by the presence of
dexamethasone, but, in the case of TNF-, this remained significantly
(p < 0.05) above basal levels. There was no
significant difference in the magnitude of cytokine release by the
normal and asthmatic cultures.
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All experiments were performed using primary epithelial cell
cultures, which were >80% confluent and which had been rendered
quiescent by growth factor removal. Under these conditions, neither
IL-4 nor IL-13 had any significant effect on cell number. In the
majority of subjects, exposure of bronchial epithelial cells to IL-4
caused a significant increase in IL-8 and GM-CSF release, and this was
suppressible by corticosteroid treatment, irrespective of disease
status (Fig. 3). However, in the
IL-4-treated asthmatic epithelial cell cultures, the level of IL-8 was
still significantly (p < 0.05) greater than
the untreated control (Fig. 3a), even though there was
significant (p < 0.01) suppression of the
IL-4-induced response by dexamethasone. Although IL-13 significantly
enhanced IL-8 release, the potency of IL-4 usually exceeded that of
IL-13. Similarly, stimulation of GM-CSF release by IL-13 was less than
that by IL-4 and failed to reach statistical significance after
correcting for multiple testing. When either IL-4 or IL-13 was tested
in the presence of Der p, release of GM-CSF and IL-8 was
enhanced and was at least equivalent to the sum of the amounts released
in the presence of either agent alone (Fig. 4).
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release
To determine whether IL-4 and IL-13 might affect remodelling
responses independently of inflammation, we tested their effects on
epithelial release of the potent epithelial and fibroblast mitogen,
TGF-. Using the asthma-derived epithelial cell cultures, significant
stimulation of TGF- occurred in the presence of IL-4, IL-13, or
TNF-, and this was blocked by dexamethasone (Fig. 5a). As found for IL-8 and
GM-CSF, TGF- release was also enhanced by Der p, and this
was further increased in the presence of IL-4 or IL-13 (Fig. 5b); however, in this case, the effect was always less than
additive. In the 9 of 12 normal epithelial cell cultures, a small
increase in TGF- release was observed under the same treatment
conditions; however, this failed to achieve statistical significance
even though significant IL-8 release was observed from these cultures
(Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the effects of IL-4 and IL-13 on epithelial cell lines have been reported previously (9, 10, 11), in this study, we provide the first detailed comparative analysis of the responsiveness of normal and asthmatic bronchial epithelial cells. Although experiments with cell lines tend to show reproducible changes in response to stimulation, considerable variation was observed with the primary cell cultures, both at baseline and after stimulation; this most likely reflects differences arising from the outbred human donor population. However, while individual cultures tended to vary in the amount of cytokine released, the overall responses showed consistency in that they tended to move in the same direction, and a donor who had a low response in one assay usually responded weakly in other assays.
In our study, we set out to test the hypothesis that asthmatic bronchial epithelial cells have a heightened response to an allergic environment by comparing responses to IL-4 and IL-13 that would result from the effect of allergen on immune cell function with any direct effects of allergen on epithelial responses. Significantly, even though all atopic asthmatic subjects were sensitive to house dust mite, we found no differences in cytokine release from normal or asthmatic epithelial cell cultures in response to Der p and Th2 cytokines, either alone or in combination. This suggests that for cytokine release, there is no difference in epithelial cell sensitivity to the direct effects of inhaled house dust mite allergen in asthma when compared with normal subjects and that, if present, IL-4 or IL-13 will promote IL-8 and GM-CSF release irrespective of disease status. Because all forms of asthma are associated with enhanced production of Th2 cytokines, it seems likely that provision of IL-4 and IL-13 is a key difference between asthmatic and normal subjects. This proposal is consistent with the association between atopy and asthma (32, 33) and with the occurrence of promoter and functional polymorphisms in the genes encoding IL-4 and IL-13 (13), which lead to increased cytokine production or enhanced receptor binding. However, as our experiments were performed at maximally stimulating doses of IL-4, we cannot exclude the possibility that certain single nucleotide polymorphisms in components of the IL-4R signaling cascade that are also associated with asthma and atopy (13) may also enhance sensitivity to IL-4 when this cytokine is limiting.
The ability of IL-4 and IL-13 to augment cytokine release from bronchial epithelial cells in the presence of Der p may provide one explanation for the extent of inflammation in asthmatic airways. Because allergen is universally inhaled, predisposition toward Th2 inflammation in asthmatic airways will amplify epithelial release of GM-CSF and IL-8, leading to exaggerated inflammatory responses in asthma. Consistent with this role, IL-8 is a potent chemoattractant for neutrophils and eosinophils (34, 35) and its levels are increased in asthmatic bronchial epithelium and lavage fluid (36, 37). GM-CSF promotes maturation of dendritic cells and eosinophil survival, and, in an animal model of allergic inflammation, expression of GM-CSF as a transgene in the bronchial epithelium causes persistent inflammation by preventing T cell anergy (38, 39).
In contrast with TNF-, which gave a consistent increase in cytokine
release when used as a control proinflammatory stimulus, the responses
of the epithelial cell cultures to IL-4 and IL-13 were much more
variable, with some cultures failing to show any response to these
cytokines. Although several polymorphisms have been identified, which
have positive effects on IL-4 and IL-13 signaling, this system is also
tightly controlled by a variety of negative regulators. These include
SHP phosphatase, silencing of cytokine signaling proteins
(40, 41, 42, 43), a dominant-negative STAT-6 splice variant whose
expression we have recently described in normal and asthmatic bronchial
epithelial cells (21), and
IL-13R2 (18). The latter appears
to negatively regulate IL-13 function by competing for IL-13 binding
and promoting receptor internalization (17). In our study,
we provide the first evidence that this receptor is expressed in human
bronchial epithelium, suggesting that it may be involved in regulating
epithelial responses to IL-13 and IL-4. Although we did not undertake a
systematic analysis of surface IL-13R2
expression in each of the cultures that we studied, it is interesting
that of the six individuals analyzed, the culture that expressed the
highest level of IL-13R2 failed to respond to
IL-13 and IL-4 in the face of a normal response to TNF- (fold
stimulation above baseline using IL-4, IL-13, and TNF- as stimuli
was 0.6, 0.2, and 6.0, respectively, for IL-8, and 1.1, 0.6, and 2.2
for GM-CSF). This suggests that IL-13R2 may be
an important modifier of epithelial responses to Th2 cytokines and that
further work examining the function and regulation of
IL-13R2 expression in normal and
asthmatic bronchial epithelium is warranted.
The lack of any overall significant difference between responses of normal and asthmatic bronchial epithelial cells to Der p and IL-4 or IL-13 differs from a previous study that reported that asthmatic bronchial epithelial cells are more sensitive to diesel exhaust particles when assessed by release of GM-CSF, IL-8, and RANTES (44, 45). This suggests that some epithelial responses are regulated by conditions in the airway microenvironment (e.g., the provision of Th2 cytokines), while other responses are fundamental to the nature of the asthmatic epithelium. Because we have found that asthmatic bronchial epithelial cells are more sensitive to oxidant-induced apoptosis (30), it seems likely that agents such as diesel exhaust particles, which have the capacity to generate reactive oxygen, impact on an underlying difference in the ability of asthmatic epithelial cells to deal with oxidant stress provided by environmental pollutants. However, it is also evident that Th2 inflammation has the potential to impinge on this susceptibility through stimulation of endogenous, inflammatory cell-derived oxidants.
In addition to their proinflammatory role, IL-4 and IL-13 have been
implicated in goblet cell metaplasia by enhancing IL-8 release that
causes oxidant-induced activation of the epidermal growth factor
receptor as a consequence of attraction of neutrophils into the
epithelium (46). However, as IL-4 has been shown to affect
(mucin 5 subtypes A and C) expression in the absence of
neutrophils in vitro (9), we explored the possibility that
IL-4 and IL-13 may activate the epidermal growth factor receptor
directly by increasing release of TGF-, one of its activating
ligands. Recent studies have implicated TGF- in the process of
retinoic acid-induced goblet cell differentiation in air-liquid
interface cultures grown in the presence of IL-13 (47).
TGF- release was readily detected in asthmatic cell culture
supernatants, while for most of the normal cell supernatants, TGF-
levels were close to the limit of detection. Consistent with its
effects in hepatocytes (48), we found that TNF- also
increased TGF- release into asthmatic epithelial cell culture
supernatants. Because neutrophils, mast cells, and macrophages are all
sources of TNF-, this finding suggests a novel mechanism whereby
TNF- can make a significant contribution to remodelling responses
via TGF- mobilization.
In contrast with IL-8 and GM-CSF, which were released with similar
efficacies from normal and asthmatic epithelial cells, no significant
stimulation of TGF- release was detected in the nonasthmatic culture
supernatants under any of the conditions tested; however, there was a
trend for increased release. Failure to achieve statistical
significance probably reflects the lower levels of TGF- detectable
in the normal epithelial cell culture supernatants and may be due to a
difference in the expression, processing, or use of TGF-, rather
than to any selective suppression of responses to the Th2 cytokines.
Consistent with this proposal, IL-4 and IL-13 were found to stimulate
IL-8 release even though the same cultures failed to respond with
TGF- release. TGF- is synthesized as a transmembrane precursor
whose cleavage is catalyzed by metalloproteases including
TNF--converting enzyme (49). Thus, failure to detect
TGF- in nonasthmatic culture supernatants may be due to either low
TGF- gene expression or failure to process and release the growth
factor (or a combination of both factors). Alternatively, as epithelial
cells express epidermal growth factor receptors, TGF- may be
synthesized and cleaved equivalently by normal and asthmatic cell
cultures, but it may be used more rapidly by the nonasthmatic cell
cultures, resulting in an apparent lack of growth factor release.
Systematic analysis of each of these processes will be required to
determine the underlying cause of this potentially important difference
between the normal and asthmatic epithelial cells. On the one hand,
excessive production of TGF- in asthma may contribute to airway
remodelling by affecting both goblet cell metaplasia and fibroblast
proliferation, whereas in contrast, failure of asthmatic epithelial
cells to use TGF- may underlie an abnormal repair response in asthma
and explain the extent of epithelial disruption that is characteristic
of this disease.
Our finding that Der p enhances release of TGF- from
epithelial cells has never been reported previously. Der p
allergens are known to contain cysteine and serine protease activity
(50); however, it is not known whether they directly
cleave the TGF- precursor or activate cell surface metalloproteases.
As many growth factors are produced as cell surface precursors, it will
be important to determine to what extent allergen-derived proteases
modify the cell surface leading to production of biologically active
molecules, as well as causing disruption of adhesion junction
proteins.
In conclusion, we have provided evidence for functional interactions
between allergen and Th2 cytokines in the bronchial epithelium in
asthma linked to proinflammatory and remodelling responses. Our data
identify the IL-13R2 as a potential regulator
of IL-13 and IL-4 signaling in these cells and suggest an important
phenotypic difference between normal and asthmatic epithelial cells
linked to TGF- metabolism. The availability of a reliable method for
the growth of primary cultures from normal and asthmatic bronchial
epithelium should now pave the way toward dissection of the underlying
mechanisms that control bronchial epithelial cell function in an
allergic environment.
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Footnotes |
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2 Current address: Human Anatomy Section, University of Palermo, Palermo, Italy.
3 Address correspondence and reprint requests to Dr. Donna E. Davies, Respiratory Cell and Molecular Biology Division (810), Southampton General Hospital, Southampton, SO16 6YD, U.K. E-mail address: donnad{at}soton.ac.uk
4 Abbreviations used in this paper: Der p, Dermatophagoides pteronyssinus; c, common -chain.
Received for publication October 5, 2001. Accepted for publication April 26, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway. Blood 97:2673. subunit of the interleukin-4 receptor. N. Engl. J. Med. 337:1720. up-regulates IgE synthesis and associates with atopic asthma. Nat. Genet. 19:119.[Medline]
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D. M. Murphy, I. A. Forrest, P. A. Corris, G. E. Johnson, T. Small, D. Jones, A. J. Fisher, J. J. Egan, T. E. Cawston, C. Ward, et al. Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L592 - L599. [Abstract] [Full Text] [PDF]
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 I. H. Heijink, P. M. Kies, H. F. Kauffman, D. S. Postma, A. J. M. van Oosterhout, and E. Vellenga Down-Regulation of E-Cadherin in Human Bronchial Epithelial Cells Leads to Epidermal Growth Factor Receptor-Dependent Th2 Cell-Promoting Activity J. Immunol., June 15, 2007; 178(12): 7678 - 7685. [Abstract] [Full Text] [PDF]
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I. H. Heijink, P. Marcel Kies, A. J. M. van Oosterhout, D. S. Postma, H. F. Kauffman, and E. Vellenga Der p, IL-4, and TGF-beta Cooperatively Induce EGFR-Dependent TARC Expression in Airway Epithelium Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 351 - 359. [Abstract] [Full Text] [PDF]
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L. Cameron, R. B. Webster, J. M. Strempel, P. Kiesler, M. Kabesch, H. Ramachandran, L. Yu, D. A. Stern, P. E. Graves, I. C. Lohman, et al. Th2 Cell-Selective Enhancement of Human IL13 Transcription by IL13-1112C>T, a Polymorphism Associated with Allergic Inflammation J. Immunol., December 15, 2006; 177(12): 8633 - 8642. [Abstract] [Full Text] [PDF]
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 K. F. Chung Intrinsic differences of the airway epithelium in childhood allergic asthma. Am. J. Respir. Crit. Care Med., November 15, 2006; 174(10): 1066 - 1067. [Full Text] [PDF]
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A.-L. Andrews, J. W. Holloway, S. T. Holgate, and D. E. Davies IL-4 Receptor {alpha} Is an Important Modulator of IL-4 and IL-13 Receptor Binding: Implications for the Development of Therapeutic Targets. J. Immunol., June 15, 2006; 176(12): 7456 - 7461. [Abstract] [Full Text] [PDF]
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 C. Bergeron and L.-P. Boulet Structural changes in airway diseases: characteristics, mechanisms, consequences, and pharmacologic modulation. Chest, April 1, 2006; 129(4): 1068 - 1087. [Abstract] [Full Text] [PDF]
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C. Boxall, S. T. Holgate, and D. E. Davies The contribution of transforming growth factor-{beta} and epidermal growth factor signalling to airway remodelling in chronic asthma Eur. Respir. J., January 1, 2006; 27(1): 208 - 229. [Abstract] [Full Text] [PDF]
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I. A. Forrest, D. M. Murphy, C. Ward, D. Jones, G. E. Johnson, L. Archer, F. K. Gould, T. E. Cawston, J. L. Lordan, and P. A. Corris Primary airway epithelial cell culture from lung transplant recipients Eur. Respir. J., December 1, 2005; 26(6): 1080 - 1085. [Abstract] [Full Text] [PDF]
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 C Ward, I A Forrest, D M Murphy, G E Johnson, H Robertson, T E Cawston, A J Fisher, J H Dark, J L Lordan, J A Kirby, et al. Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients Thorax, October 1, 2005; 60(10): 865 - 871. [Abstract] [Full Text] [PDF]
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 M. Lindstedt, A. Schiott, A. Bengtsson, K. Larsson, M. Korsgren, L. Greiff, and C. A. K. Borrebaeck Genomic and functional delineation of dendritic cells and memory T cells derived from grass pollen-allergic patients and healthy individuals Int. Immunol., April 1, 2005; 17(4): 401 - 409. [Abstract] [Full Text] [PDF]
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 P. A.B. Wark, S. L. Johnston, F. Bucchieri, R. Powell, S. Puddicombe, V. Laza-Stanca, S. T. Holgate, and D. E. Davies Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus J. Exp. Med., March 21, 2005; 201(6): 937 - 947. [Abstract] [Full Text] [PDF]
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 E. Bouzigon, M.-H. Dizier, C. Krahenbuhl, A. Lemainque, I. Annesi-Maesano, C. Betard, J. Bousquet, D. Charpin, F. Gormand, M. Guilloud-Bataille, et al. Clustering patterns of LOD scores for asthma-related phenotypes revealed by a genome-wide screen in 295 French EGEA families Hum. Mol. Genet., December 15, 2004; 13(24): 3103 - 3113. [Abstract] [Full Text] [PDF]
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S. Yamamoto, I. Kobayashi, K. Tsuji, N. Nishi, E. Muro, M. Miyazaki, M. Zaitsu, S. Inada, T. Ichimaru, and Y. Hamasaki Upregulation of Interleukin-4 Receptor by Interferon-{gamma}: Enhanced Interleukin-4-Induced Eotaxin-3 Production in Airway Epithelium Am. J. Respir. Cell Mol. Biol., October 1, 2004; 31(4): 456 - 462. [Abstract] [Full Text] [PDF]
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A. Hoshino, T. Tsuji, J. Matsuzaki, T. Jinushi, S. Ashino, T. Teramura, K. Chamoto, Y. Tanaka, Y. Asakura, T. Sakurai, et al. STAT6-mediated signaling in Th2-dependent allergic asthma: critical role for the development of eosinophilia, airway hyper-responsiveness and mucus hypersecretion, distinct from its role in Th2 differentiation Int. Immunol., October 1, 2004; 16(10): 1497 - 1505. [Abstract] [Full Text] [PDF]
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T. D. Le Cras, W. D. Hardie, G. H. Deutsch, K. H. Albertine, M. Ikegami, J. A. Whitsett, and T. R. Korfhagen Transient induction of TGF-{alpha} disrupts lung morphogenesis, causing pulmonary disease in adulthood Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L718 - L729. [Abstract] [Full Text] [PDF]
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T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and D. J. Tschumperlin Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L119 - L126. [Abstract] [Full Text] [PDF]
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 S.T. Holgate Lessons learnt from the epidemic of asthma QJM, May 1, 2004; 97(5): 247 - 257. [Full Text] [PDF]
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 S. T. Holgate, J. Holloway, S. Wilson, F. Bucchieri, S. Puddicombe, and D. E. Davies Epithelial-Mesenchymal Communication in the Pathogenesis of Chronic Asthma Proceedings of the ATS, April 1, 2004; 1(2): 93 - 98. [Abstract] [Full Text] [PDF]
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W. D. Hardie, T. D. Le Cras, K. Jiang, J. W. Tichelaar, M. Azhar, and T. R. Korfhagen Conditional expression of transforming growth factor-{alpha} in adult mouse lung causes pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L741 - L749. [Abstract] [Full Text] [PDF]
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T. D. Le Cras, W. D. Hardie, K. Fagan, J. A. Whitsett, and T. R. Korfhagen Disrupted pulmonary vascular development and pulmonary hypertension in transgenic mice overexpressing transforming growth factor-{alpha} Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L1046 - L1054. [Abstract] [Full Text] [PDF]
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S.T. Holgate, D. E. Davies, S. Puddicombe, A. Richter, P. Lackie, J. Lordan, and P. Howarth Mechanisms of airway epithelial damage: epithelial-mesenchymal interactions in the pathogenesis of asthma Eur. Respir. J., September 20, 2003; 22(44_suppl): 24s - 29s. [Full Text] [PDF]
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I.S. Patel, N.J. Roberts, S.J. Lloyd-Owen, R.J. Sapsford, and J.A. Wedzicha Airway epithelial inflammatory responses and clinical parameters in COPD Eur. Respir. J., July 1, 2003; 22(1): 94 - 99. [Abstract] [Full Text] [PDF]
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S. M. Stick and P. G. Holt The Airway Epithelium as Immune Modulator: The LARC Ascending Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 641 - 644. [Full Text] [PDF]
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N. Asokananthan, P. T. Graham, D. J. Stewart, A. J. Bakker, K. A. Eidne, P. J. Thompson, and G. A. Stewart House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1 J. Immunol., October 15, 2002; 169(8): 4572 - 4578. [Abstract] [Full Text] [PDF]
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