 1 mRNA Expression and Airways
Fibrosis in Bronchial Asthma
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The histopathology of bronchial asthma is associated with structural changes within the airways, including
subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this
tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming
growth factor-
(TGF-) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-1 mRNA and immunoreactivity
within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the
techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the
asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-1 mRNA
and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater
expression of TGF-1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control
subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the
severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-1
mRNA and immunoreactivity. These results provide evidence that TGF-1 may play a role in the fibrotic
changes occurring within asthmatic airways and that activated eosinophils are a major source of this cyto-kine.
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Introduction
Materials & Methods
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Discussion
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Pathologic features associated with asthma include subepithelial fibrosis, epithelial desquamation, smooth muscle hypertrophy/hyperplasia, and an eosinophilic inflammatory cell infiltration (1). While initially described in postmortem studies, these structural alterations can be observed even in mild and newly diagnosed asthmatics (4, 5). Because any thickening of the airway wall will profoundly increase the maximal degree of airway narrowing when the smooth muscle constricts, it has been proposed that architectural changes similar to those observed in asthmatics contribute to the development of chronic airways hyperresponsiveness and the progressive deterioration in lung function over time (6, 7). While many studies have focused on the mechanisms underlying the acute presentations of bronchial asthma, the factors responsible for the more chronic structural changes within the lungs remain to be elucidated. Several reports have suggested that thickening of the reticular lamina results from interstitial collagen and fibronectin deposition (4, 5). Although myofibroblasts are recognized as being the cell type responsible for subepithelial collagen deposition in these individuals (8), the mechanisms contributing to the onset of airways fibrosis in asthma and the role of inflammatory cells, in particular eosinophils, remain to be established.
Transforming growth factor-beta (TGF-) is a potent
profibrotic cytokine which stimulates fibroblasts to promote the synthesis and secretion of many proteins of the
extracellular matrix (9, 10). In addition, TGF- is an immunomodulatory cytokine and a potent chemoattractant
for several cell types including monocytes (11), fibroblasts
(12), and mast cells (13). Due to its actions in promoting
growth and repair, TGF- is thought to play an important
role at sites of wound healing and tissue remodeling. However, the role of TGF- in contributing to the structural alterations evident in bronchial asthma remains to be established.
Mammalian cells synthesize three isoforms of TGF-,
each encoded by its own gene, which appear to be differentially regulated (14). TGF-1 was the first isoform of
TGF- to be purified and is the most extensively characterized to date. As such, the biologic functions reported
for TGF- are generally those of TGF-1 (14). In lung diseases associated with fibrosis, the cellular source of TGF-1
includes lymphocytes, macrophages, and epithelial cells (15,
16). Recently, several reports have demonstrated that eosinophils infiltrating nasal polyps represent a major source of
TGF-1 (17, 18). Furthermore, expression of TGF-1 is evident in circulating eosinophils from hypereosinophilic individuals (19). Since bronchial asthma is intimately associated with the recruitment and activation of eosinophils
within the airways, the expression of TGF-1 by these cells
may link chronic eosinophilic inflammation to the fibroblast activation and characteristic deposition of collagen
fibers seen in this disease (4, 5).
The aims of the present study were to examine the expression of TGF-1 messenger ribonucleic acid (mRNA)
and immunoreactivity within the airways of mild to severe
asthmatics compared with control individuals, and to determine the potential role of eosinophils in contributing to
the synthesis of this cytokine in bronchial asthma. Furthermore, we investigated the association between TGF-1 mRNA expression, the extent of subepithelial fibrosis, and
baseline lung function, using a combination of histochemical and molecular biologic techniques.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Subjects Studied and Tissue Preparation
The technique of fiberoptic bronchoscopy with bronchial
biopsies in asthmatic volunteers and normal control subjects has been described elsewhere in detail (20). To study
the expression of TGF-1 mRNA and immunoreactivity in
asthmatics compared with normal controls, 28 subjects
were recruited. These included 18 atopic asthmatics and 10 nonatopic normal controls, whose clinical data is shown in
Table 1. The subjects were recruited from the Asthma
Clinic at the Montréal General Hospital and Montréal Chest Institute (Montréal, PQ, Canada), and the General
Clinical Research Center at the National Jewish Center
for Immunology and Respiratory Medicine (Denver, CO).
All patients fulfilled the ATS criteria for asthma (21), had
typical clinical symptoms, documented airways reversibility,
and increased airways responsiveness to methacholine
(PC20 < 8 mg/ml). Informed consent, approved by the
Montréal Chest Institute, the Montréal General Hospital, and the National Jewish Center Institutional Review Board,
was obtained from all patients before entry into this study.
Of the 18 asthmatic individuals, 6 were classified as severe
asthmatics with forced expiratory volume in 1 s (FEV1)
values less than 60% predicted and required the regular
use of inhaled steroids. Six asthmatics had frequent symptoms which were controlled by regular use of 2-agonists
or inhaled steroids with FEV1 values between 60 and 80%
predicted, and were classified as moderately severe. The
remainder (n = 6) were mild asthmatics who required only
occasional 2-agonists, had FEV1 values greater than 80%
predicted, and had been asymptomatic for at least 1 wk before fiberoptic bronchoscopy. None of the asthmatics had
taken oral corticosteroids in the preceding 12 wk and all
patients were atopic as defined by a positive skin-prick test
to one or more aeroallergens. The normal control subjects had negative skin-prick tests to aeroallergens. All of the
subjects were nonsmokers and none had any other serious
lung disease. Two to five endoscopic bronchial biopsies
were obtained from each subject. Biopsies from asthmatics
and normal control subjects were fixed immediately in 4%
paraformaldehyde for 2 h, washed in 15% phosphate-buffered saline-sucrose, blocked, and sectioned on a cryostat at
20°C. Sections (10 µm-thick) were then baked in an oven
at 37°C and stored at 80°C until further use.
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Probe Preparation
A radiolabeled complementary RNA probe coding for
TGF-1 mRNA was prepared from complementary DNA
(cDNA) as described previously (22). The probe was constructed to be specific for the TGF-1 isoform. In brief,
cDNA was inserted into PGEM vectors, linearized, and
transcribed in vitro in the presence of 35S-uridine triphosphate and either SP6 or T7 polymerases. Antisense (complementary to mRNA) and sense probes (identical to
mRNA) were prepared.
In Situ Hybridization
In situ hybridization was performed as previously described (23, 24). Briefly, cryostat sections from biopsies were permeabilized with Triton X-100 and proteinase K solution (1 µg/ml) in 0.1 M Tris containing 50 mM EDTA for 20 min at 37°C. The samples were subsequently incubated with 0.1 M triethanolamine and 0.5% acetic anhydride for 20 min at 37°C in order to prevent the nonspecific binding of the 35S-labeled complementary RNA (cRNA) probes. Prehybridization of the samples was carried out in 50% formamide in 2X standard saline citrate (SSC) for 15 min at 37°C. Hybridization was carried out using a hybridization mixture containing the appropriate sense or antisense probes. Posthybridization involved high-stringency washing of the samples in decreasing concentrations of SSC at 42°C. In order to remove any unbound RNA probes, the samples were washed with RNase solution for 20 min at 42°C. The samples were then dehydrated with increasing concentrations of ethanol, and air-dried. Following this, the samples were dipped in Amersham LM-2 emulsion (Oakville, Ontario, Canada) and exposed for a period of 10 days. The autoradiographs were then developed in Kodak D-19 developer (Eastman Kodak, Toronto, Canada), fixed, and counterstained with hematoxylin. As negative controls, tissue sections were hybridized with the sense probe and/or pretreated with RNase A, then hybridized to antisense probes.
Combined Immunocytochemistry and In Situ Hybridization
To ascertain the expression of TGF-1 mRNA by activated eosinophils, we simultaneously applied radiolabeled
in situ hybridization (ISH) with EG2-immunoreactivity as
previously described in detail elsewhere (25). Briefly, cryostat sections (5 µm) of frozen bronchial biopsies were cut
and the sections were immunostained with an anti-EG2
monoclonal antibody directed against the cleaved form of
eosinophil cationic protein (ECP) (Pharmacia, Uppsala, Sweden). Following visualization of the positive immunostaining using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique, the sections underwent a
modified ISH for TGF-1 mRNA.
Immunocytochemistry for TGF-
TGF-1 immunoreactivity was detected in 5-µm tissue sections by the use of a TGF-1 specific polyclonal antibody
(AB-101-NA; R&D Systems, Minneapolis, MN). Briefly,
endogenous peroxidase activity in cryostat sections of nasal biopsies was blocked using 1% H2O2 (plus 0.02% sodium
azide in Tris-buffered saline [TBS]) for 30 min. Immunocytochemistry using the avidin-biotin-peroxidase complex
method was then performed as previously described (26). For negative control preparations, the primary antibody
was replaced by either nonspecific rabbit immunoglobulin
or TBS.
Double Immunocytochemistry
To confirm the phenotype of TGF-1 immunoreactive positive cells, we undertook double sequential immunocyto-chemistry as previously described (27). Briefly, endogenous peroxidase activity in cryostat sections of bronchial
biopsies was blocked using 1% H2O2 (plus 0.02% sodium
azide in TBS) for 30 min. A mixture of primary antibodies was then applied consisting of the polyclonal anti-TGF-1
antibody used to detect TGF-1 immunoreactivity, and the
appropriate monoclonal antibody to determine the presence of eosinophils (EG2+). After incubating with the appropriate secondary antibodies, a tertiary layer of streptavidin peroxidase and murine APAAP conjugate was then
applied. Sections were developed sequentially in Fast Red (the APAAP substrate) and diaminobenzidine (a peroxide
substrate). TGF-1 immunoreactive cells stained brown,
the eosinophils stained red, and eosinophils expressing eotaxin-immunoreactivity stained a reddish-brown. In all immunochemical studies the appropriate negative controls
were included, which included TBS alone, omission of the
primary antibodies, and the use of an irrelevant mouse or
rabbit IgG antibody.
Histochemistry
For the detection of collagen fibers, sections of bronchial biopsies were stained with van Gieson's stain. Using this technique, the collagen fibers could be identified by their characteristic red staining.
Quantification
Slides were coded and positive cells counted blindly using ×100 magnification with an eyepiece graticule. Hybridization signals between cytokine mRNA and cRNA probes were localized as dense collections of silver grains in photographic emulsion overlying individual cells. Positively staining cells were counted to a depth of 0.45 mm below the basement membrane (BM) and the results were expressed as the mean number of positive cells per mm BM. The severity of airway fibrosis was quantitated on a scale of 1 to 4 according to the extent of fibrosis within the lamina propria, with a score of 4 representing extensive fibrosis within the airways which extended from the BM to the smooth-muscle layer. The within observer coefficient of variation for repeated measures was less than 5%.
Statisical Analysis
The numbers of cells expressing TGF-1 in mild, moderate, and severe asthmatic airways and normal control subjects were compared using a nonparametric Kruskal-Wal-lis analysis of variance. Statistically significant differences
between groups were subsequently analyzed using a Mann-Whitney U test. Correlation coefficients were calculated from Pearson's moment coefficient and were corrected for
multiple comparisons by the use of Bonferroni's correction factor (Systat v5.0, v6.1; SPSS Inc., Chicago, IL). Results were considered statistically significant for P < 0.05.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Hybridization between the labeled cRNA probes and mRNA
encoding TGF-1 was demonstrated by specific deposits of
silver grains in the photographic emulsion overlying the
tissue sections. No positive hybridization signals were observed when the sense probe was used, nor when the tissues were pretreated with RNase. TGF-1 mRNA was detected in bronchial biopsies from control and asthmatic individuals (Figure 1c). The expression of this cytokine
was significantly increased in severe asthmatics (Figure 2;
mean mRNA positive cells/mm BM ± SEM; 18.5 ± 3.1;
n = 6) compared with moderate asthmatics (mean ± SEM;
10.8 ± 1.3; n = 6; P < 0.05), mild asthmatics (mean ± SEM; 7.8 ± 1.5; n = 6; P < 0.01), and normal control subjects (mean ± SEM; 3.5 ± 0.8; n = 10; P < 0.001). As a
group, asthmatics (mean ± SEM; 12.4 ± 1.6; n = 18) had
significantly greater TGF-1 mRNA expression than did
normal individuals (Figure 2; P < 0.05). The expression of
TGF-1 mRNA was also increased in moderate asthmatics compared with normal control subjects (Figure 2; P < 0.05).
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The numbers of cells expressing immunoreactivity for
TGF-1 were also enumerated in bronchial biopsies from
the severe, moderate, and mild asthmatic individuals, as
well as the normal control subjects. Compared with the
normal individuals (mean ± SEM; 5.2 ± 1.0; n = 5), there
was a significant increase in TGF-1 immunoreactive cells
in mild (mean ± SEM; 9.2 ± 1.6; n = 5; P < 0.05), moderate (mean ± SEM; 12.25 ± 1.0; n = 4; P < 0.05), and severe asthmatics (mean ± SEM; 18.8 ± 4.9; n = 5; P < 0.01). There were no significant differences in TGF-1 immunoreactivity between the groups of asthmatics classified according to disease severity (P > 0.05).
Cells exhibiting positive immunoreactivity for ECP were detected by the presence of discrete red staining within the airway submucosa (Figures 1a and 1b). Numbers of EG2-positive cells were increased in severe asthmatics (Figure 2; mean EG2-positive cells/mm BM ± SEM; 15.1 ± 1.5; n = 6) compared with moderate asthmatics (mean ± SEM; 8.8 ± 0.9; n = 6; P < 0.05), mild asthmatics (mean ± SEM; 5.3 ± 1.0; n = 6; P < 0.01), and normal control subjects (mean ± SEM; 0.4 ± 0.2; n = 10; P < 0.001). Moderate and mild asthmatics also had a greater degree of airway eosinophilia than normal controls (Figure 2; P < 0.001).
Histochemically, the extent of airways fibrosis could be visualized by the use of van Gieson's stain, which results in a characteristic red staining of the subepithelial collagen layer (Figures 1e through 1g). Individuals with severe asthma had the greatest degree of airway fibrosis (Figure 3; mean score ± SEM; 3.1 ± 0.2; n = 6) compared with moderate asthmatics (mean score ± SEM; 2.2 ± 0.2; n = 6; P < 0.05), mild asthmatics (mean score ± SEM; 1.4 ± 0.1; n = 6; P < 0.005) and normal control subjects (mean score ± SEM; 0.4 ± 0.2; n = 10; P < 0.001). Significantly increased airway fibrosis was observed even in moderate and mild asthmatics compared with normal control subjects (Figure 3; P < 0.005).
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When examining the numbers of TGF-1 mRNA-positive cells with respect to the numbers of EG2-positive cells
and extent of airways fibrosis in asthmatic individuals, positive correlations could be observed (TGF-1 mRNA and
EG2-positive cells, r2 = 0.86; n = 18; TGF-1 mRNA and
airways fibrosis, r2 = 0.70; n = 18; P < 0.05). These observed increases in subepithelial fibrosis and EG2-positive
cells could also be correlated to baseline FEV1 values in
the asthmatic individuals (Figures 4a and 4b). There were
also significant correlations between the FEV1 value and
the extent of subepithelial fibrosis (r2 = 0.78; P < 0.05)
and EG2-positive cells (r2 = 0.84; P < 0.05).
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In phenotyping the cells expressing mRNA for TGF-1,
combined immunocytochemistry and ISH revealed that
approximately 65% of the TGF-1 mRNA-positive cells
were cells expressing immunoreactivity for the cleaved
form of ECP. The other 35% of the TGF-1 mRNA and
immunoreactive cells exhibited a phenotype consistent with macrophages and fibroblasts. Only approximately 75% of
the EG2+ cells expressed TGF-1 mRNA.
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Discussion |
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Introduction
Materials & Methods
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Discussion
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Since subepithelial fibrosis resulting from the deposition
of collagen beneath the BM has been reported in mild and
newly diagnosed asthmatics (4, 5), we hypothesized that
the mRNA expression of the potent profibrotic cytokine
TGF-1 would be markedly upregulated in bronchial biopsies from patients with more severe asthma, and that the
increased expression of TGF-1 would be associated with
the degree of airway fibrosis. The results of this study
demonstrate that TGF-1 mRNA is upregulated within the airways of asthmatic individuals and that the expression of this cytokine correlates with the degree of subepithelial fibrosis. We also show that the major source of this
cytokine mRNA is activated eosinophils.
Upregulation of TGF-1 mRNA and its association
with airways fibrosis is a novel observation in the pathogenesis of bronchial asthma. To date, there has been only
one previous study examining the expression of TGF-1 in
bronchial asthma (28). Using Northern blot analysis, these
investigators found no difference between asthmatic subjects and individuals with chronic obstructive pulmonary disease or smokers with no fixed airway obstruction. The
discrepancies between these results and our own may lie in
the severity of the asthmatic subjects examined. Indeed,
we found that expression of TGF-1 mRNA was greatest
in the most severe asthmatics, with no significant differences noted between mild asthmatics and the normal control subjects (Figure 2). Other possible explanations include the use of appropriate control individuals, and/or the inability of the Northern blot technique to detect local
increases in TGF-1 mRNA.
Using the ISH techniques, it is possible to determine
the presence of transcribed mRNA only for TGF-1. Thus,
from the results of this study, we are unable to determine
whether this mRNA is being translated into the active protein. Indeed, even if translation were taking place, we still
could not be certain that the TGF-1 was secreted in a biologically active form (29). However, several lines of evidence do suggest that eosinophil-derived TGF-1 mRNA is transcribed into the active protein. Previous immunocytochemical and ISH studies examining TGF-1 in eosinophils have confirmed the presence of active cytokine secretion (17). We have also conducted preliminary studies
which show that supernatants from activated eosinophils
are able to induce the synthesis of collagen types I, II, III,
and IV in a fibroblastic cell line (unpublished observations). Furthermore, our own data show that there is a positive correlation between the extent of subepithelial fibrosis and the number of cells positive for TGF-1 mRNA.
In order to examine asthmatic individuals with a wide
range of disease severity, we used subjects who were on
regular inhaled steroids. It may be argued that the use of
these agents could have influenced the data obtained in
the moderate and severe asthmatics. The ability of corticosteroids to modulate TGF-1 mRNA expression has not
been systematically examined to date. However, there are reports that steroids do not directly influence TGF-1
mRNA production (30, 31). Indeed, steroids may indirectly reduce TGF-1 mRNA production within the lungs
of asthmatic individuals by attenuating eosinophil recruitment (32). The low FEV1 values and increased numbers of
EG2-positive cells in our moderate and severe asthmatics would suggest that these individuals were not being well
controlled by these agents. Alternatively, it would suggest
that inhaled steroids have a limited ability to resolve tissue
eosinophilia compared with oral steroids. In addition, significant amounts of TGF-1 mRNA were present in cells
not staining for EG2 and may be coming from cells already resident within the lung, such as epithelial cells,
macrophages, lymphocytes, and fibroblasts themselves (15,
16). The lack of effect of steroids on fibrosis formation obviously has important clinical considerations and may explain why even aggressive steroid therapy fails to restore normal airways responsiveness (33, 34).
There has been a relative paucity of data examining the
molecular mechanisms underlying the structural abnormalities observed in bronchial asthma. Structural changes
within the airways of asthmatic individuals may contribute
to the development of persistent airways hyperresponsiveness (6) and chronic alterations in airflow obstruction (7),
although the mechanisms by which these alterations occur
is unclear. It has been proposed that collagen types I and
III and fibronectin deposition are involved in the thickening of the reticular layer (4, 5) and that this is accompanied
by a change in the phenotype of fibroblasts to resemble a
contractile cell (8). However, the inflammatory stimulus which induces collagen synthesis from fibroblasts and/or
myofibroblasts remains to be elucidated. Our findings suggest that TGF-1 released locally from beneath the BM
may contribute both to the development of airways fibrosis and the subsequent decline in lung function.
Our colocalization studies show that 65% of the TGF-1
mRNA-positive cells were activated eosinophils. These
cells were located within the reticular lamina (Figure 1)
and therefore were appropriately situated to generate inflammatory stimuli capable of inducing collagen synthesis.
The production by eosinophils of profibrotic agents such
as TGF-1 is not novel and it has been shown previously that these cells are a potential source of platelet-derived
growth factor B-chain in bronchial asthma (35). In addition to its role as a potent profibrotic agent within the airways, the local secretion of TGF-1 by eosinophils is
known to inhibit eosinophil survival and function (36).
The activation of this pathway may be one autoregulatory
mechanism which limits the numbers of tissue-dwelling cells in chronic eosinophilic diseases. Evidence to support
such a role of TGF-1 in bronchial asthma remains to be
established.
In summary, we have demonstrated that TGF-1 mRNA
is upregulated in bronchial asthma and is associated with
the degree of airways fibrosis. The finding that eosinophils
are a potential major source of this cytokine within the reticular lamina links chronic allergic inflammation with structural remodeling and the decline in FEV1 observed in this
disease. Novel therapeutic agents which prevent TGF-1
production may be of use in the restoration of normal lung
function in asthmatic individuals.
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Footnotes |
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Address correspondence to: Dr. Qutayba Hamid, M.D., Ph.D., Meakins-Christie Laboratories, McGill University, 3626 St. Urbain, Montréal, PQ, H3X 2P2 Canada. E-mail: Hamid{at}meakins.lan.mcgill.ca
(Received in original form August 5, 1996 and in revised form December 10, 1996).
Acknowledgments:
The authors thank the Glaxo Institute for Molecular Biology for their generous gift of human cDNA for TGF-1. Dr. Eleanor Minshall is
a recipient of a Canadian Lung Association/Medical Research Council Fellowship. The authors also to thank Ms. Elsa Schotman and Ms. Zivart Yasruel for
their technical assistance. This work was supported by MRC Canada and in
part by NIH grants HL 36577 and RR 00051.
Abbreviations
APAAP, alkaline phosphatase anti-alkaline phosphatase;
BM, basement membrane;
cDNA, complementary deoxyribonucleic acid;
ECP, eosinophil cationic protein;
FEV1, forced expiratory volume in 1 second;
ISH, in situ hybridization;
mRNA, messenger ribonucleic acid;
SSC, standard saline citrate;
TGF-1, transforming growth factor-1.
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References |
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Abstract
Introduction
Materials & Methods
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Discussion
References
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