 Expression in Human Asthmatic Airways
Relationship with Proliferation, Apoptosis, and Airway Remodeling |
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Airway inflammation and alterations in cellular turnover are histopathologic features of asthma. We show that the expression of
peroxisome proliferator-activated receptor 
(PPAR), a nuclear hormone receptor involved in cell activation, differentiation, proliferation, and/or apoptosis, is augmented in the bronchial submucosa,
the airway epithelium, and the smooth muscle of steroid-untreated asthmatics, as compared with control subjects. This is associated with enhanced proliferation and apoptosis of airway epithelial and
submucosal cells, as assessed by the immunodetection of the nuclear antigen Ki67, and of the cleaved form of caspase-3, respectively, and with signs of airway remodeling, including thickness of
the subepithelial membrane (SBM) and collagen deposition. PPAR
expression in the epithelium correlates positively with SBM thickening and collagen deposition, whereas PPAR expressing cells in
the submucosa relate both to SBM thickening and to the number
of proliferating cells. The intensity of PPAR expression in the
bronchial submucosa, the airway epithelium, and the smooth muscle is negatively related to FEV1 values. Inhaled steroids alone, or
associated with oral steroids, downregulate PPAR expression in
all the compartments, cell proliferation, SBM thickness, and collagen deposition, whereas they increase apoptotic cell numbers in
the epithelium and the submucosa. Our findings have demonstrated that PPAR (1) is a new indicator of airway inflammation
and remodeling in asthma; (2) may be involved in extracellular
matrix remodeling and submucosal cell proliferation; (3) is a target for steroid therapy.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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Keywords: nuclear antigens; airway smooth muscle; bronchial epithelium; corticosteroids
Asthma is classically defined as a chronic inflammatory disease of the airways characterized by reversible airflow obstruction. Nevertheless, alterations in the structure of the airway wall, defined as airway remodeling, are now also recognized as regular features of asthma (1). These alterations include extensive epithelial damage, collagen deposition, thickness of the subepithelial basement membrane (SBM), subepithelial fibrosis and airway smooth muscle hypertrophy, and/or hyperplasia (1).
Bronchial inflammation and airway structural abnormalities involve an altered expression and/or function of a large variety of proinflammatory cytokines, growth factors and their receptors, as well as a dysregulation in the differentiation, proliferation, and apoptosis of multiple cell types. These include infiltrating leukocytes, particularly eosinophils, and structural cells responsible for the maintenance of airway tissue integrity such as mesenchymal cells and bronchial epithelial and smooth muscle cells (3, 4).
Peroxisome-proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily of ligand-activated transcriptional factors, which include receptors for
steroids, thyroid hormone, vitamin D, and retinoic acid (5).
Among them, PPAR was originally characterized as a regulator of adipocyte differentiation and lipid metabolism (6)
and, more recently, of cellular turnover (7). Indeed, several
lines of evidence indicate that PPAR profoundly affects cell
cycle, differentiation and apoptosis (7). Thus, PPAR activation by natural or synthetic ligands such as the cyclooxygenase metabolite 15-deoxy-
12,14 PGJ2, polyunsaturated fatty acids, different nonsteroidal anti-inflammatory drugs, and the
oral antidiabetic agents thiazolidinediones favor macrophage
differentiation and prevent colorectal, prostate, and breast
cancer by inhibiting cell growth and accelerating apoptosis (7).
Fibroblast, synoviocyte, macrophage, endothelial and T-cell apoptotic death in response to thiazolidinediones has also been documented (8).
In addition, PPAR activation downregulates the synthesis
and release of immunomodulatory cytokines from various cell
types (12).
Although these findings support a major role for PPAR in
immune disorders characterized by tissue inflammation and
remodeling, there is no evidence, as yet, of PPAR expression
in human asthmatic airways.
The present study was aimed at investigating the localization of PPAR in bronchial biopsies from control subjects and
asthmatic patients and at determining whether changes in its
expression correlate with cell proliferation and apoptosis, with
the expression of markers of airway remodeling and with steroid therapy.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patients
Thirty-four nonsmoker asthmatic patients, mainly atopic, and fulfilling the criteria of the Guidelines for the Diagnosis and Management of Asthma of the National Heart, Lung, and Blood Institute (NHLBI) (15) were recruited (Table 1). All patients did not experience exacerbations within the 2 mo preceding the bronchoscopy. A flow-volume curve was performed in all subjects and FEV1, FVC, and the ratio FEV1/FVC were assessed before and after the inhalation of 200 µg of salbutamol.
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The 34 asthmatics were distributed into three groups as follows: 13 asthmatics who were receiving intermittent inhaled short-acting 
2 agonists, taken as needed; 12 asthmatics who were treated with short-
and long-acting 2 agonists and inhaled steroids (
800 µg/d of budesonide or 1,000 µg/d of beclomethasone dipropionate, or 500 µg/d of
fluticasone propionate); 9 asthmatics who received long-acting 2 agonists, inhaled steroids and 10 to 60 mg/d of regular oral prednisone
(Table 1).
A group of eight healthy volunteers receiving no medication was also studied (Table 1).
Fiberoptic Bronchoscopy, Biopsy Processing and Immunohistochemistry
The bronchoscopy was performed according to the guidelines outlined by the American Thoracic Society (16), as described (17). The Bichat Hospital Ethics Committee approved the experimental protocol for fiberoptic bronchoscopy, and all subjects gave their written consent to participate.
Immunohistochemistry was performed on 5-µm sections from endobronchial biopsies, as described (17), using the following antibodies
or their corresponding isotypes: antihuman PPAR (SC7196, 10 µg/ml;
Santa Cruz Biotechnologies, Santa Cruz, CA), anti-CD3 (clone Leu-4,
10 µg/ml; Sigma Chemical, St. Louis, MO), anti-CD68 (clone EBM11,
4 µg/ml; Dako, Trappes, France), anti-ECP (clone EG2, 10 µg/ml; Pharmacia, Uppsala, Sweden), anti-Ki67 (clone B56, 1 µg/ml; PharMingen,
Becton Dickinson, San Diego, CA), an anticleaved subunit of caspase-3 of 20 kD Ab (clone C92-605, 2.5 µg/ml; PharMingen). Mouse
or rabbit alkaline phosphatase antialkaline phosphatase (APAAP)
antibodies were used next.
Immunofluorescence double staining was performed using rhodamine-labeled goat F(ab')2 to rabbit Ig and fluorescein-conjugated goat F(ab')2 to mouse IgG1 (Immunotech, Marseille, France).
A minimum of two serial sections from the same biopsy, or sections from three to five different biopsies, were analyzed. PPAR-, Ki67-, and caspase-3 expression in the bronchial mucosa, the airway epithelium, and the smooth muscle were evaluated as described (17).
Assessment of Airway Remodeling
SBM thickening was quantified by computer-assisted image analysis (Microvision Instruments, Histolab, Evry, France), as described (18). Final results were the means of 20 to 50 measurements performed from each biopsy specimen, and two biopsies from each patient were analyzed.
Collagen deposition was evaluated through staining of bronchial sections using the picrosirius red technique (19) and it was measured at regular intervals with a calibrated image analyzer (Microvision Instruments). Results were expressed as a percentage of picrosirius-stained area over the total biopsy area.
Statistical Analysis
Results are expressed as means ± SEM. A one-way analysis of variance followed by the nonparametric Mann-Whitney U test for unpaired
values were used to determine significance among groups. Correlation coefficients (r') were calculated using Spearman's nonparametric
rank-order method; p values of 0.05 were considered significant.
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RESULTS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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PPAR Expression in Bronchial Biopsies from Control Subjects
and Asthmatics and Modulation by Steroid Therapy
Control subjects showed only rare PPAR positive cells in
their bronchial submucosa and a weak or no expression of
PPAR in the bronchial epithelium and the airway smooth
muscle (Figures 1 and 2). PPAR expression was significantly
augmented in all compartments in steroid-untreated asthmatics (Figures 1 and 2).
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Treatment with inhaled steroids was associated with a decrease in PPAR expression in the bronchial submucosa
(70%, p < 0.001) (Figure 1), in the airway smooth muscle
(92%, p < 0.001) (Figure 1), and, to a lesser extent, in the
bronchial epithelium (42%, difference not statistically significant) (Figure 1). In the inhaled and oral steroid-treated group,
the increase in PPAR expression was significantly reduced in
the three compartments analyzed (Figure 1 and Figure 2E).
By immunofluorescence, we established that PPAR-
expressing cells in the bronchial submucosa were eosinophils
and macrophages, whereas T lymphocytes were consistently
negative (Figure 3). Furthermore, in several biopsy specimens,
particularly from steroid-untreated asthmatics, we observed
increased numbers of subepithelial cells expressing PPAR
and showing a fibroblastlike morphology (Figure 2B and C).
However, the intense spontaneous trapping of fluorescence by
connective tissue, which was frequently observed in these areas, prevented double staining to be assessed.
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PPAR Expression in Relation to Cell Proliferation
and Apoptosis
To determine cell proliferation and apoptosis, the immunohistochemical detection of the Ki67 nuclear antigen (20) and of the cleaved form of caspase-3 (21), respectively, were assessed.
Although these markers were distributed to different degrees along the airway epithelium and the bronchial submucosa in both control subjects and asthmatics, the airway smooth muscle was consistently negative, irrespective of the patient group (data not shown).
Control subjects showed low numbers of Ki67-positive cells in their bronchial epithelium and submucosa (Figure 4A and B and Figure 5). A significant increase in the number of proliferating epithelial and mucosal cells was observed in steroid-untreated asthmatics (Figure 4A and B and Figure 5). Inhaled steroids decreased cell proliferation rate in the epithelium without modifying significantly that of mucosal cells (Figure 4). In the inhaled and oral steroid-treated group, the number of Ki67-positive cells in both the epithelium and the bronchial submucosa decreased down to the levels of control patients (Figures 4 and 5).
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Very few caspase-3-positive cells were enumerated in the bronchial epithelium and the submucosa of control subjects (Figure 4B and D and Figure 5). A significant increment in these numbers was noted in both compartments in steroid-untreated, in inhaled steroid- and in inhaled and oral-treated asthmatics, as compared with control subjects (Figure 4B and D and Figure 5).
To avoid confounding effects of steroid treatment when interpreting the association between the extent of PPAR expression and the number of proliferating and apoptotic cells in
the different compartments, only control subjects and steroid-untreated asthmatics were considered. The individual intensities of PPAR expression in the airway epithelium failed to
correlate with the number of Ki67- or caspase-3-positive cells
(r' = 0.03 and r' = 0.116, respectively, n = 21, not significant).
In contrast, the number of PPAR-expressing cells in the
bronchial submucosa positively correlated with that of proliferating cells (r' = 0.62, n = 21, p < 0.001), but not with that of
apoptotic cells (r'= 0.13, n = 21, not significant).
PPAR Expression in Relation to Airway Remodeling and to the
Degree of Bronchial Obstruction
Morphometric and histochemical analysis of bronchial biopsies from control subjects showed an absence or a moderate thickness of the SBM and collagen deposition (Figure 6). SBM thickening was markedly augmented in steroid-untreated asthmatics (Figure 6A). Treatment with inhaled or inhaled and oral steroids was associated with a significant inhibition in SBM thickness (40.6 and 71.4%, p < 0.01 and p < 0.001, respectively) (Figure 6A).
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A trend toward an increase in collagen deposition was also noted in steroid-untreated asthmatics, as compared with control subjects, but the high degree of variability precluded the results from achieving statistical significance (Figure 6B). In the inhaled and inhaled and oral steroid-treated group, collagen deposition was reduced by 50.5 and 71.4%, respectively, although the differences were not statistically significant (p > 0.05).
The association between PPAR expression, SBM thickness, collagen deposition and FEV1 values in control subjects
and steroid-untreated asthmatics was then examined.
The intensities of PPAR expression in the bronchial submucosa and the airway epithelium and smooth muscle correlated positively with SBM thickness (r' = 0.49, p < 0.001; r' = 0.29, p < 0.01 and r' = 0.67, p < 0.001, respectively, n = 21) and
was negatively related to the individual FEV1 values (Figure 7).
Epithelium-associated PPAR expression also correlated positively with collagen deposition (r' = 0.30, n = 21, p < 0.05).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
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In the present study, we have demonstrated for the first time
the localization of the nuclear antigen PPAR in human airways. This expression was augmented in asthmatics as compared with control subjects, particularly in airway epithelium
and smooth muscle. Although we have been able to identify
the PPAR protein in association with eosinophils and macrophages, a rise in the numbers of subepithelial cells expressing
PPAR and showing a fibroblastlike morphology was also noted.
In keeping with these observations, we detected high levels of
PPAR by immunoblot in protein extracts from unstimulated primary cultured human lung fibroblasts (unpublished results). These findings parallel those recently reported showing the
presence of PPAR protein in human synovial fibroblasts and
in hepatic myofibroblasts (8, 22).
T lymphocytes in the bronchial mucosa failed to express
PPAR, an observation in contrast with previous reports demonstrating the constitutive expression of the PPAR gene and
protein by murine helper T-cell clones and by freshly isolated
murine T lymphocytes (11, 23). Because no additional data
are available in the literature concerning PPAR expression
in human T cells, either in peripheral blood or in lymphoid
and inflamed tissues, it is difficult at this stage to find an explanation for this discrepancy. Nevertheless, we confirmed the
absence of PPAR immunostaining associated with T cells using frozen sections from human tonsils and lung lymph nodes
(data not shown).
Upregulation of PPAR expression in airway epithelium
and smooth muscle of asthmatics reported in this study is reminiscent of previous observations showing an augmented
PPAR immunostaining associated with the colonic epithelium in mice with an inflammatory bowel disease (24), in rat
neointima after balloon injury and in early human atheroma
(25). This enhanced PPAR expression may reflect an inflammatory response of different cell types and structures to natural PPAR ligands generated within the airways during the allergic reaction. Although the nature of these stimuli in the
lung is unknown, it is well established that a range of naturally
occurring substances, including polyunsaturated fatty acids, the
15-lipoxygenase metabolite, 15-hydroxyeicosatetranoic acid
(15-HETE), or cytokines such as IL-4, are potent PPAR expression-promoting agents (12, 26, 27). In particular, 15-HETE and IL-4 are produced in the airways of asthmatics by
different inflammatory and immune cells (28) and contribute to eosinophilic inflammation, mucus secretion, and narrowing (33). The possibility that 15-HETE and IL-4 also
operate in airway inflammation by inducing and/or activating
PPAR in different pulmonary cells remains to be established.
Structural abnormalities characteristic of the remodeling
response in asthma involve, at least in part, a dysregulation in the proliferation and apoptosis of different cell types responsible for the maintenance of airway integrity (36). Of note, synthetic and naturally occurring PPAR ligands greatly ameliorate different features of tissue remodeling, including the
thickening of the bowel wall in mice with inflammatory bowel
diseases (24) and arterial restenosis after endothelial injury in
rats (37). These beneficial effects may be related to the ability
of PPAR ligands to inhibit cell migration, proliferation, and
proinflammatory and toxic mediator production (24, 37)
and to promote apoptotic cell death (8).
These observations led us to determine whether changes in
PPAR expression related to the proliferating and apoptotic
status of the different cell types analyzed and to SBM thickening and submucosal collagen deposition, two histopathologic
features of airway remodeling (3).
When compared with control subjects, asthmatic patients showed an enhanced proliferation and apoptosis in the epithelium and the bronchial submucosa, as assessed by the increment in the number of cells expressing the nuclear antigen Ki67 and the cleaved form of caspase-3, respectively.
As previously reported (3, 40, 41), a remodeling response characterized by SBM thickening and collagen deposition was also noted in steroid-untreated asthmatics as compared with control subjects. Together, these observations argue for an increased cellular turnover, probably in response to different mitogens, growth factors, and toxic products chronically released within the inflammatory airways.
The intensity of PPAR expression in the airway epithelium is related to SBM thickening and to collagen deposition,
but not to proliferation or apoptosis. In contrast, the number
of PPAR-positive cells in the bronchial submucosa significantly correlated with SBM thickening and with the number
of Ki67-, but not of caspase-3-expressing cells. These results
suggest that PPAR may be involved in the regulation of extracellular matrix deposit and of submucosal cell proliferation,
rather than in epithelial cell turnover.
Finally, the intensities of PPAR expression in the bronchial submucosa, the airway epithelium, and the smooth muscle of steroid-treated asthmatics and the individual values of
FEV1 significantly correlated, suggesting that high levels of
PPAR in the airways may represent a marker of the severity
of airflow obstruction.
Corticosteroids offer clinical improvement in airway function most likely by reducing airway inflammation, as a result
of an inhibition of many transcription genes involved in the
synthesis of proinflammatory lipid mediators and cytokines
(42). Here we demonstrated that PPAR expression in the bronchial mucosa, the airway epithelium, and the smooth muscle
was also downregulated in inhaled- and, to a higher extent, in
oral steroid-treated asthmatics. These results indicate that the
anti-inflammatory and immunomodulatory properties of these
drugs may extend to the regulation of PPAR expression in
target cells. However, the possibility that long-lasting 2-agonists, which are regularly administered to most of the asthmatic patients in combination with inhaled or oral steroids,
would also interfere with PPAR expression cannot be ruled
out. This particularly in view of substantial clinical data showing a synergistic benefit of these two therapies in reducing airway inflammation and in improving symptoms, probably as a
result of their complementary modes of action (43).
Our observations showing lower levels of PPAR in steroid-treated asthmatics are in apparent contradiction with some
in vitro findings showing upregulation by dexamethasone of
PPAR gene expression in human adipocytes (44). Because
no additional information is available in the literature concerning the modulation by corticosteroids, alone or in association with long-lasting 2-agonists, of PPAR gene and protein
expression in human tissues, it is difficult to find an explanation for this discrepancy. Further studies with primarily cultured bronchopulmonary cells will certainly help to clarify this matter.
The mechanisms by which steroid therapy may decrease
the levels of PPAR are presently unclear. It may be hypothesized that corticosteroids suppress the synthesis and release of
PPAR expression-promoting agents from different cell types
present in asthmatic airways. This would be the case for IL-4,
whose gene expression is markedly reduced in bronchial biopsies of steroid-treated asthmatics (45). The production of the
lipoxygenase metabolite, 15-HETE is also downregulated by
corticosteroids, although at very high doses, in cultured human bronchial epithelial cells and dermal fibroblasts (46, 47).
Similarly to what we have observed for PPAR expression
in the bronchial epithelium and in the submucosa, inhaled,
and, to a greater extent, oral steroid therapy, inhibited cell
proliferation, SBM thickness, and collagen deposition, whereas
they enhanced apoptotic death. Several in vitro and in vivo
findings have shown the ability of corticosteroids to prevent
epithelial cell proliferation (48) and to induce apoptosis of different mucosal cells, including eosinophils (19, 49, 50) and
T lymphocytes (53). In contrast, the efficacy of these drugs in
modulating extracellular matrix alterations is a matter of controversy. Thus, in some studies inhaled steroids decreased
SBM thickening and collagen deposition (53), whereas
other studies have not been able to detect this benefit (56).
In conclusion, our results identify PPAR as a new factor
expressed in high levels by submucosal and structural cells
during the inflammatory and remodeling response in asthma.
It is difficult at this stage to anticipate a role for PPAR in human asthmatic airways. In view of the results from the literature showing the inhibitory properties of this nuclear antigen
against cell differentiation, proliferation, and activation (12),
it may be hypothesized that its upregulation in asthma would
represent a self-regulatory mechanism aimed at preventing
further cell activation and expansion, thus contributing to the
cessation of airway inflammatory and remodeling, as proposed for other pathologic conditions (8, 12, 24, 25, 60). However, this classic view may be contradicted by our findings demonstrating a clear association between an augmented PPAR
expression, the presence of features of airway remodeling, and
the increase in bronchial obstruction. These observations, together with the efficacy of steroid therapy in downregulating PPAR expression, may lead to consider this nuclear antigen
as a new mediator with proinflammatory and fibrogenic activities. This hypothesis is consistent with the expression of high
levels PPAR within the atherosclerotic plaques of mice and
humans (25, 61) and with the recent debate concerning the
potential atherogenic properties of endogenously produced
PPAR (60).
Future studies with tissue-specific PPAR-deficient mice,
or the use of PPAR agonists in experimental animal models
and in isolated cells are thus required to gain better insight of
the functional properties of PPAR and of the possibility of
using its ligands as alternative or additional therapeutic strategies to resolve airway inflammation and remodeling in asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Marina Pretolani, PhD, INSERM U408, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail: mpretol{at}bichat.inserm.fr
(Received in original form January 17, 2001 and accepted in revised form July 12, 2001).
Dr. Benayoun is a fellow of the Fondation de France.Dr. Letuve is a fellow of the Fondation pour la Recherche Médicale.
Acknowledgments: The writers wish to thank Dr. Pierre Desreumaux for interesting discussion, Mireille Autré for kind assistance in collecting clinical data concerning the patients, and Isabelle Poirier and Gabrielle Beuve for expert technical help during fiberoptic bronchoscopy.
Supported by the Fonds de Recherche Hoechst Marion Roussel and by Grant No. 5367 from the Association pour la Recherche sur le Cancer.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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