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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have demonstrated that, in addition to their contractile function, human airway smooth-muscle cells
(HASMC) are able to express and to secrete chemokines of the monocyte chemotactic protein (MCP)/
eotaxin subfamily. This group of chemokines is believed to play a fundamental role in the development of
allergic airway diseases such as asthma. The expression levels of MCP (MCP-1, -2, and -3) messenger
RNA (mRNA) were compared with those of regulated on activation, normal T cells expressed and secreted
(RANTES) mRNA in HASMC in culture. HASMC express MCP and RANTES mRNA after stimulation
with interleukin (IL)-1
, tumor necrosis factor-
, and interferon-
. MCP mRNA was maximal at 8 h,
whereas RANTES mRNA expression was delayed to 24 h after stimulation. Further, significant differences were observed in the induction patterns of MCP and RANTES mRNA expression after stimulation
with the individual cytokines. Dexamethasone (DEX) significantly inhibited cytokine-induced accumulation of MCP and RANTES mRNA, in contrast to IL-4, IL-10, and IL-13, which had no inhibitory effect on
cytokine-induced chemokine expression. The cytokine-induced MCP mRNA expression in HASMC was
associated with MCP release, which was inhibited by DEX and post-translationally by IL-4. HASMC can
actively participate in the pathogenesis of asthma by the expression and release of chemokines, which are
likely to play a critical role in the generation and regulation of the inflammatory response characteristic of allergic airway diseases.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Several airway diseases, such as asthma, rhinitis, and chronic bronchitis, are inflammatory disorders that are characterized by an accumulation in the airways of an abnormally large number of leukocytes, such as eosinophils, lymphocytes, or macrophages (1). In asthma, correlations have been described between the numbers of infiltrating lymphocytes and eosinophils and markers of the severity of the disease (4), and even in mild asthma the infiltration of the bronchial mucosa by eosinophils and lymphocytes is a consistent finding (5). Furthermore, the role of the eosinophil in the pathogenesis of asthma is now well known (6). It is believed that toxic eosinophil-derived proteins can cause bronchial mucosal damage in asthmatic airways that may contribute to the symptoms of asthma, such as variable airway obstruction and airway hyperresponsiveness (4). The mechanisms, therefore, that control the recruitment and retention of inflammatory cells in the lung are likely to play a critical role in the regulation of the inflammatory response in asthmatic airways. It is now apparent that chemokines, which are leukocyte-chemotactic and activating proteins, may play a pivotal role in inflammatory diseases (7). It is suggested that in allergic inflammation (e.g., in asthma) the CC chemokines, particularly the monocyte chemotactic protein (MCP)/eotaxin subfamily, are likely to play a critical role in the regulation of the inflammation (8), although the collaboration of other cytokines, especially interleukin (IL)-5, seems to be essential for airway eosinophilia (9).
The MCPs and eotaxin constitute a subfamily of structurally related CC chemokines (8). However, the spectrum of target cells, the specific activities, the production levels, and the inducers of these chemokines differ (10). MCP-3 is one of the most pluripotent chemokines, acting on multiple cell types including monocytes, lymphocytes, eosinophils, basophils, dendritic cells, and natural killer cells (10); in contrast to MCP-1, which is chemotactic mainly for monocytes and lymphocytes (11), and eotaxin, which is a strong chemotactic agent for eosinophils (12, 13). Increased levels of these CC chemokines have been detected in lavage fluid and in bronchial biopsies of patients with asthma, both at the protein and the messenger RNA (mRNA) levels (14), and in epithelial tissue of patients with atopic dermatitis and allergic rhinitis (19, 20). It has therefore been suggested that the MCP/eotaxin chemokines constitute a molecular link between antigen-specific immune activation and the migration of eosinophils into tissues (7). Until now, however, the observations of elevated chemokine mRNA in lavage fluid did not provide information as to the cellular source of the chemokine mRNA. There is now substantial evidence that the epithelium can actively participate in the inflammatory process in the airways by the release of a number of proinflammatory mediators, including chemokines (21). The airway smooth-muscle cell is also capable of synthesizing proinflammatory mediators, such as granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8, and regulated on activation, normal T cells expressed and secreted (RANTES) (22). In the present study we examined whether airway smooth cells in culture can express and release MCPs, which are upregulated in asthmatic airways and may therefore be relevant to the pathogenesis of asthma. Further, we have investigated whether the expression and release of MCP-1, -2, and -3 can be modulated and we have compared these effects with the expression of RANTES mRNA.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Culture of Human Airway Smooth-Muscle Cells
Human airway smooth-muscle cells (HASMC) were grown from explants of human bronchial smooth muscle, as previously described by Hall and colleagues (25). Briefly, airway tissue was obtained from resections of patients undergoing surgery for lung carcinoma. None of the patients had characteristics of asthma. Bronchial smooth-muscle tissue was carefully dissected clear of surrounding tissue. Small explants (2 × 2 mm) of the bronchial muscle were prepared and placed in a petri dish. After allowing the explants to adhere, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (1.25 µg/ml) was added, just to cover the explants. The medium was changed every day until the cells started to grow, whereafter the cultures were supplemented with fresh DMEM containing FCS, glutamine, antibiotics, and amphotericin B every 3 d. When the cells were reaching confluency in some parts of the petri dish the explants were removed, and 24 h later the cells were harvested with trypsin/ethylenediaminetetraacetic acid (EDTA) and plated into a 75-cm2 flask, where they were grown to confluency. After subculturing the cells twice, the cultures were characterized immunohistochemically, using an antihuman smooth-muscle actin antibody. Primary cell cultures used for the experiments showed > 95% of cells staining for smooth-muscle actin. All experiments were carried out between passages 3 and 12.
After reaching near confluency the cells were washed
and incubated with DMEM containing only L-glutamine
(2 mM) for 24 h before stimulation. The cells were stimulated with a mixture of cytokines (IL-1, tumor necrosis
factor [TNF]-, and interferon [IFN]-, all at a concentration of 10 ng/ml) for 1, 2, 4, 8, 16, 24, and 48 h. In separate
experiments, the cells were incubated with IL-1 (10 ng/
ml), TNF- (10 ng/ml), IFN- (10 ng/ml), or lipopolysaccharide (LPS) (10 µg/ml) for 8 or 24 h. For inhibition studies, the cells were incubated for 8 or 24 h with the mixture
of cytokines together with dexamethasone (DEX) (1 µM),
IL-4, IL-10, or IL-13 (all at 25 ng/ml and added 1 h before
stimulation). Protein synthesis was inhibited with cycloheximide (10 µg/ml, added 30 min before stimulation).
Northern Blot Analysis
Total RNA was isolated by guanidinium isothiocyanate
phenol/chloroform extraction and isopropanol precipitation (26). The RNA samples were subjected to a 1% agarose/formaldehyde gel containing 20 mM morpholinosulfonic acid, 5 mM sodium acetate, and 1 mM EDTA (pH 7)
and blotted onto a nylon membrane (Genetic Research Instrumentation, Dunmow, UK) by capillary blotting. A 170-base pair (bp) (KspI/SpeI) fragment specific to the human
MCP-1 complementary DNA (cDNA), a 900-bp (NotI/
XhoI) fragment specific to the human MCP-2 cDNA, a
700-kb (EcoRI/XhoI) fragment specific to the human
MCP-3 cDNA, a 410-bp (EcoRI/ApaI) fragment specific
to human RANTES cDNA, and a 1,200-bp (PstI/PstI) fragment specific to rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were labeled by a random primer
labeling kit (Amersham, Roosendaal, The Netherlands)
using [-32P]deoxycytidine triphosphate (3,000 Ci/mmol,
Amersham). After prehybridization for 4 h at 42°C in a
buffer containing 50% formamide, 4× standard sodium citrate (SSC), 50 mM Tris-HCl (pH 7.5), 5× Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, and 250 µg/ml denatured salmon sperm DNA, the blots
were hybridized overnight at 42°C with the labeled probes
(1 to 2 × 106 cpm/ml). After hybridization, the blots were
washed to a high stringency of 0.1× SSC and 0.1% SDS at
55°C before exposure to X-OMAT-S film. After suitable
exposure time, the autoradiographs were analyzed by a laser densitometer (PDI, New York, NY) and the RNA levels were expressed as the ratio of chemokine to GAPDH mRNA to overcome differences in loading or transfer of
the RNA.
Measurement of Secreted MCP-1 and MCP-2 Protein by Use of Enzyme-Linked Immunosorbent Assay
Recombinant MCP-1 was a gift from Dr. J. J. Oppenheim (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD). MCP-2 was synthesized and purified as described previously (27). Specific polyclonal antibodies (Ab) against recombinant MCP-1 and synthetic MCP-2 were raised in rabbits and purified by protein G-sepharose chromatography. MCP-1 was detected with a sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit antihuman MCP-1 Ab for coating and mouse antihuman MCP-1 monoclonal Ab (mAb) (R&D Systems, Abingdon, UK) for capturing. MCP-2, on the contrary was detected with a sandwich ELISA using mouse antihuman MCP-2 mAb (R&D Systems) for coating and polyclonal rabbit antihuman MCP-2 Ab for capturing. The secondary Ab used were peroxidase-labeled goat antimouse Ab and peroxidase-labeled donkey antirabbit Ab for the MCP-1 and MCP-2 ELISAs, respectively. The detection was performed with 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate (Aldrich Chemical, Milwaukee, WI). The detection limits for MCP-1 and MCP-2 were 0.1 and 0.05 ng/ml, respectively.
Statistical Analysis
Data are expressed as means ± standard error of the mean (SEM). The Mann-Whitney U test was performed between groups to determine significance, and P < 0.05 was considered significant.
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Materials and Methods
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Discussion
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Cytokines Induce the Expression of MCP-1, -2, and -3 and RANTES mRNA in Airway Smooth-Muscle Cells
No detectable levels of mRNA expression of MCP-1, -2, or -3 or RANTES were found in unstimulated HASMC.
However, stimulation with the mixture of cytokines (IL-1, TNF-, and IFN-, all at 10 ng/ml; the "cytomix") for
different time periods induced a significant accumulation
of mRNA, reaching maximal expressions between 4 and
8 h for MCP-1 and MCP-3 and between 8 and 16 h for MCP-2, whereas RANTES mRNA expression still increased after 24 h of stimulation (Figure 1).
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When comparing the effects of the individual cytokines
(Figure 2), we found that IL-1 induced more pronounced
expression of MCP-1 and MCP-3 (67 ± 4% and 28 ± 2%,
respectively, of the cytomix-induced expression, which
was maximal at 8 h) than did TNF- (21 ± 3% and 16 ± 3%, respectively, of the cytomix-induced expression,
which was maximal at 8 h), whereas IFN- had a much less
pronounced effect. In contrast, MCP-2 mRNA expression
was induced by IL-1 and IFN- (17 ± 2% and 15 ± 2%,
respectively, of the cytomix-induced expression, which
was maximal at 8 h), whereas TNF- was ineffective.
RANTES mRNA, on the other hand, was better induced
by TNF- than by IL-1 (70 ± 11% and 40 ± 1%, respectively of the cytomix-induced expression, which was maximal at 24 h), whereas IFN- was ineffective. Costimulation
of the HASMC with IL-1, TNF-, and IFN- resulted in
a distinct synergistic induction of MCP-2 and MCP-3
mRNA, but not of MCP-1 or RANTES mRNA. LPS (10 µg/ml) did not induce an accumulation of any of the
chemokine mRNAs investigated after either 8 or 24 h of
incubation (Figure 2).
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Effects of DEX, IL-4, -10, and -13, and Cycloheximide on the Cytokine-Induced Expression of MCP-1, -2, and -3 and RANTES mRNA
Pretreatment of the cells with DEX (1 µM) significantly
inhibited the cytomix-induced expression of MCP-1, -2, and -3 and RANTES mRNA (at 8 or 24 h of stimulation),
with a decrease of, respectively, 51 ± 6, 49 ± 9, 48 ± 7, and
61 ± 10% compared with cytomix alone. The T-helper
(Th)2 cytokines IL-4, IL-10, and IL-13 (all at 25 ng/ml), on
the other hand, failed to attenuate significantly the cytomix-induced accumulation of MCP-1, -2, and -3 and
RANTES mRNA (Figure 3). No detectable levels of
MCP-1, -2, or -3 or RANTES mRNA were induced by either IL-4, -10, or -13 on their own (results not shown). Cycloheximide (10 µg/ml), a protein synthesis inhibitor, on
its own induced a modest increase of chemokine mRNA
expression in HASMC. Pretreatment with cycloheximide
(30 min) followed by an 8-h stimulation period with the
mixture of cytokines (IL-1, TNF-, and IFN-) caused a
marked increase of MCP mRNA (results not shown).
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Effect of Cytokine Stimulation on the MCP-1 and MCP-2 Protein Levels
In the supernatant of unstimulated HASMC cells, no detectable level of MCP-1 or MCP-2 protein was measured.
After stimulation with the cytomix, however, there was a
significant, time-dependent increase of the cell-supernatant MCP-1 and MCP-2 proteins, attaining a maximum
level at 24 h (362 and 13 ng/ml, respectively), which was
sustained until at least 48 h (300 and 41 ng/ml, respectively) after cytomix stimulation (Figures 4A and 5A). In
accordance with the mRNA levels after stimulation with
the individual cytokines (for 8 h), we found that IL-1 was
the superior inducer of MCP-1 protein (58 ng/ml), whereas
TNF- induced only a slight elevation of MCP-1 protein
(16 ng/ml). No detectable level of MCP-1 protein was observed after IFN- or LPS stimulation (Figure 4B). No detectable level of MCP-2 protein was observed after stimulation with the individual cytokine or LPS.
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Effects of DEX and IL-4, -10, and -13 on the Cytokine-Induced Production of MCP-1 and MCP-2 Protein
Stimulation of the HASMC with a mixture of cytokines (8 h) in the presence of DEX (1 µM), resulted in a reduction of MCP-1 and MCP-2 protein release (115 and 3.3 ng/ml versus 65 and 6.6 ng/ml, respectively, in the presence of DEX). Neither IL-10 nor IL-13 was able to inhibit the cytomix-induced MCP-1 (Figure 4C) and MCP-2 (Figure 5B) protein release from HASMC. In contrast, stimulation of the HASMC with the cytomix (8 h) in the presence of IL-4 resulted in a reduction of MCP-1 protein release (115 and 42 ng/ml in the absence and presence of IL-4, respectively) and MCP-2 protein release (6.6 and 2.6 ng/ml in absence and the presence of IL-4, respectively). No detectable levels of cell supernatant MCP-1 and MCP-2 protein were induced by either IL-4, -10, or -13 on their own (results not shown).
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Abstract
Introduction
Materials and Methods
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Discussion
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In this study we have demonstrated that HASMC in culture are able to express MCP (MCP-1, -2, and -3) and
RANTES mRNA when stimulated with the cytokines IL-1, TNF-, and IFN-. Moreover, the increased accumulation of MCP-1 mRNA was associated with an increase in
the release of MCP-1 and MCP-2 protein in the cell supernatant. Because there is evidence that the chemokines
from the MCP/eotaxin subfamily are likely to play a critical role in the regulation of the allergic inflammatory reponse in the airways (8), our findings demonstrate that airway smooth-muscle cells may contribute directly to the
development of airway inflammation by promoting the recruitment of eosinophils, lymphocytes, and monocytes to
the bronchial mucosa, which is one of the hallmarks of allergic asthma (3).
IL-1, TNF-, and IFN- have already been demonstrated to induce a synergistic upregulation of RANTES
and inducible nitric oxide synthase (iNOS) mRNA expression in airway epithelial cells (28, 29). Moreover, there is
substantial evidence that the proinflammatory cytokines
IL-1 and TNF- are important mediators in initiating
asthmatic airway inflammation. Significantly increased levels of IL-1 and TNF- have been detected in bronchoalveolar lavage fluid from symptomatic asthmatic patients
(30). Intratracheal instillation of TNF- caused a pronounced airway eosinophilia in guinea pigs (33), and inhalation of TNF- or administration of IL-1 was shown to
cause an increase in airway responsiveness (34, 35). Further, an IL-1 receptor antagonist suppressed bronchial
hyperreactivity and inflammatory cell infiltration after allergen challenge in sensitized guinea pigs (36), confirming an important role for IL-1 and TNF- in allergic inflammation and in the development of late asthmatic responses. Increased levels of IFN- have also been found in
bronchial biopsies from subjects with asthma (37). Several
reports have also indicated that IFN- has synergistic effects on cytokine-induced expression of eotaxin and IL-8
mRNA in A549 cells and HASMC, respectively (23, 38),
implicating a role for IFN- in the promotion of tissue
eosinophilia, which may be relevant to allergic airway inflammation.
In our experiments we found that a mixture of IL-1,
TNF-, and IFN- induced an accumulation of MCP-1, -2, and -3 mRNA, which was maximal at 8 h of stimulation, in
contrast to RANTES mRNA, which reached a maximum
at 24 h. The increase in MCP-1 and MCP-2 mRNA steady-state levels was associated with an increase of MCP-1 and
MCP-2 protein in the cell supernatant. At present no radioimmunoassay or ELISA has yet been developed for
MCP-3 protein determinations. The differences observed
between the time courses of MCP and RANTES mRNA
expression might imply that they have different roles in the development of allergic inflammation. Similar differences in the kinetics of the expression of mRNA encoding
MCP-3 and RANTES have also been demonstrated in
skin biopsies of human atopic subjects, obtained after allergen challenge, and these differences in kinetics may be
related to the time course of accumulation of eosinophils and T cells. MCP-3 may therefore be involved in the early
eosinophil response to allergen challenge, whereas RANTES
has more relevance to the later accumulation of T cells (20).
We also found differences in induction patterns between chemokine mRNA expression after stimulation
with the individual cytokines. MCP-1 and MCP-3 mRNA
were better induced by IL-1, whereas MCP-2 mRNA was
induced by both IL-1 and IFN-; and RANTES mRNA,
on the other hand, was best induced by TNF-. IFN- had
a negligible effect on the release of MCP-1, MCP-3, and
RANTES, in contrast to MCP-2 release, which was relatively well-induced by IFN-. This finding is in agreement
with previous studies, demonstrating that in human fibroblasts cytokines differentially regulate chemokine induction, IL-1 and IFN- being potent stimuli of MCP-1 and
MCP-2, respectively (39, 40). Therefore, it was speculated
that MCP-2 is more a Th1-like cytokine because it is relatively better induced by IFN- than is MCP-1, whereas experimental evidence showed that MCP-1 contributes more
to Th2- than to Th1-mediated inflammation (40). The differential regulation of MCPs and RANTES clearly demonstrates differences in signaling pathways and effector elements for transcriptional activation of each chemokine
mRNA gene by cytokines. And for several (but not all)
CC chemokines, promoter studies have been published to
confirm the gene regulation at the molecular level. In particular, the MCP-1 gene promoter contains the following
specific elements: a TATAA box, a GC box, two activator protein-1 elements, a nuclear factor (NF)-
B element, and
an octamer element (41, 42). Similarly, Murakami and associates (43) studied the cis-elements in the promoter region of the human MCP-3 gene. Finally, we recently studied the human MCP-2 gene promoter by transfection in
diploid fibroblasts. IFN- transactivated promoter deletion constructs 300 base pairs 5' upstream of the transcription initiation start. In addition, IL-1 had a stimulatory
effect on the IFN--induced transcriptional activity (Van
Coillie and coworkers, submitted manuscript).
We have also shown that the glucocorticoid DEX significantly inhibited the cytokine-induced expression of MCP and RANTES mRNA in HASMC and inhibited MCP-1 and MCP-2 protein secretion. These findings indicate that HASMC may be a target for (inhaled) glucocorticoids, although epithelial cells but not HASMC are the first cell type to come in contact with inhaled steroids. Inhaled steroids now represent a first-line therapy for most patients with asthma because steroids are extremely effective in suppressing airway inflammation and in controlling the symptoms of asthma (44, 45). Despite the wide use of inhaled steroids, however, the exact mechanisms underlying the anti-inflammatory effects are not yet fully elucidated and their cellular targets remain unknown (45). The inhibition by DEX of the cytokine-induced expression of MCP-1, -2, and -3 and RANTES mRNA in HASMC may, therefore, be one of the mechanisms by which (inhaled) glucocorticoids can inhibit the influx of leukocytes into the bronchial mucosa and thus suppress the airway inflammation in asthma.
In addition to the modulatory effects of DEX on the
expression of MCPs and RANTES mRNA in HASMC,
we have also examined the effects of Th2 cell-derived cytokines, IL-4, IL-10, and IL-13, all of which have been
demonstrated to have some anti-inflammatory effects (46-
48). Several studies have reported differential effects of
these inhibitory cytokines on different cell types, including inhibition of iNOS expression in epithelial cells and inhibition of macrophage inflammatory protein-1, IL-1, TNF-,
IFN-, RANTES, and IL-8 protein production in monocytes, macrophages, epithelial cells, and smooth-muscle
cells (23, 28, 49). Recently, a potential mechanism for
the in vivo anti-inflammatory effects of IL-10 and IL-13
has been identified (52). It has been suggested that IL-10
and IL-13 may operate by preventing degradation of IB
and thus inhibiting the activation of nuclear factor (NF)- B, a transcription factor that regulates many of the inflammatory proteins expressed in asthmatic airways (53).
However, neither IL-4, IL-10, nor IL-13, at concentrations
that have been shown to inhibit iNOS expression in epithelial cells (29), had any inhibitory effect on the cytokine-induced expression of MCP-1, -2, and -3 mRNA in HASMC.
The cytokine-induced release of MCP-1 and MCP-2 protein, on the other hand, was inhibited at the post-transcriptional level by IL-4 but not by IL-10 and IL-13, suggesting
that IL-4 may be a regulator of cytokine-induced MCP-1
and MCP-2 production by HASMC.
The differences in inhibitory effect between IL-4 and
IL-13, however, might be surprising, because IL-13 shares
a common receptor subunit and various biologic activities
with IL-4. However, our data are consistent with the observations of Berkman and colleagues (28), who demonstrated an inhibitory effect of IL-4 on cytokine-induced RANTES release from A549 cells, whereas IL-13 was ineffective, demonstrating that although postreceptor signaling events induced by IL-13 and IL-4 may be similar, end-point function is cytokine-specific (54). Nevertheless, our
data suggest that IL-4 has anti-inflammatory properties;
this was recently confirmed by Cunha and associates (55),
who provided evidence that IL-4 limits inflammatory hyperalgesia by the inhibition of TNF-, IL-1, and IL-8 production.
Differences, however, do seem to exist between cell types and stimuli: increased expression of MCP-1 mRNA and MCP-1 protein was observed after IL-4 stimulation in human endothelial cells (56). Cytokine-induced RANTES release in HASMC has previously been demonstrated to be inhibited by IL-4, -10 and -13; however, the effects at the mRNA level were not investigated (24). In the present study we have shown that none of these anti-inflammatory cytokines had any inhibitory effect on the accumulation of RANTES mRNA in HASMC, suggesting that the modulation of RANTES production by IL-4, -10, and -13 occurred at the post-transcriptional level. The differences in the time course, in the pattern of induction, and in the modulation of MCP and RANTES mRNA expression and protein production in HASMC, therefore, indicate that they might have different roles in airway inflammation.
Together, our results support the hypothesis that HASMC, by synthesizing proinflammatory mediators, may play a role in the generation and regulation of airway inflammation, which is a characteristic feature of asthma. In the pathophysiology of asthma, airway smooth-muscle cells have traditionally been considered a cell type responsible for airway narrowing due to their contractile capacities to stimulatory agents. Further, it has been suggested that, in addition to increases in airway smooth-muscle mass due to both hyperplasia and hypertropy of the smooth muscle (57), structural changes of the airway wall may be responsible for the excessive airway narrowing in asthma (58). Recently, evidence has emerged that suggests that the airway smooth-muscle cell can play an active role in airway inflammation (59) by the synthesis of proinflammatory mediators such as RANTES, IL-8, and GM-CSF (22- 24). Moreover, Hirst (60) has recently postulated the hypothesis of phenotype plasticity in the airway smooth-muscle cell. Repeated inflammatory insults could change the airway smooth-muscle contractile state toward the synthetic phenotype. This would result in a greater proportion of the synthetic phenotype of smooth-muscle cell within the airway wall of patients with asthma, resulting in an increased release of inflammatory mediators and leading to the perpetuation of airway inflammation.
Our findings support the hypothesis that HASMC may play an active role in airway inflammation by the production of MCP-1, -2, -3, and RANTES, which promote the chemotaxis of eosinophils, lymphocytes, and monocytes. However, the actual contribution of the airway smooth-muscle cell to the inflammatory process in vivo, with regard to the expression of mRNA for CC chemokines, has yet to be determined. In situ hybridization of airway tissue of subjects with asthma could possibly reveal whether HASMC can indeed actively participate in the airway inflammation.
This is the first study demonstrating the expression and release of MCPs (MCP-1, -2, and -3) from human airway smooth-muscle cells. Because the MCPs may play a fundamental role in the regulation of airway inflammation in asthma, this study clearly demonstrates that airway smooth-muscle cells may actively participate in the generation and regulation of airway inflammation.
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Footnotes |
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Address correspondence to: Geert M. Verleden, M.D., Ph.D., UZ Gasthuisberg, Dept. of Pneumology, 49 Herestraat, Leuven B-3000, Belgium. E-mail: geert.verleden{at}uz.kuleuven.ac.be
(Received in original form January 11, 1999 and in revised form May 10, 1999).
Abbreviations: antibodies, Ab; base pair, bp; complementary DNA, cDNA; dexamethasone, DEX; Dulbecco's modified Eagle's medium, DMEM; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immunosorbent assay, ELISA; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; human airway smooth-muscle cells, HASMC; interferon, IFN; interleukin, IL; inducible nitric oxide synthase, iNOS; lipopolysaccharide, LPS; monocyte chemotactic protein, MCP; messenger RNA, mRNA; regulated on activation, normal T cells expressed and secreted, RANTES; standard error of the mean, SEM; T helper, Th; tumor necrosis factor, TNF.Acknowledgments: This work was supported by Glaxo-Wellcome Belgium, by the Fund for Scientific Research of Flanders (FWO-Vlaanderen), and by the Cancer Foundation of the General Savings and Retirement Fund (A.S.L.K.). One author (G.M.V.) is holder of the "Glaxo-Wellcome leerstoel voor respiratoire farmacologie" and one author (P.M.) is a research assistant of the FWO-Vlaanderen. The authors thank W. Put for the determination of MCP-2 protein.
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References |
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Materials and Methods
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Discussion
References
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