 /TNF- -Induced Glucocorticoid-Sensitive
Changes in Multiple Gene Expression and Altered Responsiveness in
Airway Smooth Muscle
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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The pleiotropic cytokines interleukin (IL)-1
and tumor necrosis factor (TNF)-
have been implicated in the pathophysiology of asthma. To elucidate the role of these cytokines in the
pro-asthmatic state, the effects of IL-1 and TNF- on airway
smooth muscle (ASM) responsiveness and ASM expression of
multiple genes, assessed by high-density oligonucleotide array analysis, were examined in the absence and presence of
the glucocorticoid dexamethasone (DEX). Administration of
IL-1/TNF- increased ASM contractility to acetylcholine and
impaired ASM relaxation to isoproterenol. These pro-asthmatic-
like changes in ASM responsiveness were associated with IL-1/
TNF--induced mRNA expression of a host of proinflammatory genes that regulate transcription, cytokines and chemokines,
cellular adhesion molecules, and various signal transduction
molecules that regulate ASM responsiveness. In the presence
of DEX, the changes induced in ASM responsiveness were abrogated, and most of the IL-1/TNF--mediatied changes in
proinflammatory gene expression were repressed, although
mRNA expression of a small number of genes was enhanced
by DEX. Collectively, the observations support the concept
that, together with its role as a regulator of airway tone, in response to IL-1/TNF-, the ASM expresses a host of glucocorticoid-sensitive genes that contribute to the altered structure
and function of the airways in the pro-asthmatic state. We
speculate that glucocorticoid-sensitive, cytokine-induced pathways involved in ASM cell signaling represent important targets for new therapeutic interventions.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Altered airway responsiveness to bronchoactive constrictor and relaxant stimuli is the characteristic pathophysiologic feature of bronchial asthma (1). Whereas infiltration of the airways with inflammatory cells, principally
involving eosinophils, mast cells, and lymphocytes, is implicated in the etiology of the altered airway responsiveness, recent studies have determined that, under specific
conditions, the airway smooth muscle (ASM) itself has the
capacity to autologously induce changes in its constriction and relaxation in response to the induced release and autocrine actions of certain proinflammatory cytokines (4).
Thus, IgE-dependent atopic sensitization and rhinovirus
inoculation of ASM were shown to provoke the release of
Th1- and Th2-type cytokines, IL-1, and other cytokines
from the ASM itself, and these cytokines acting alone or in
combination were found to elicit pro-asthmatic changes in
ASM responsiveness (4, 6, 10). Moreover, in ultimately leading to altered ASM responsiveness, the autologous release of cytokines by ASM was found to display a pattern
of sequential autocrine induction, as exemplified by an initial IL-5-mediated induction of the subsequent release of
IL-1 in atopic sensitized and rhinovirus-inoculated ASM
(5, 9). Given this evidence and the fact that the altered
ASM responsiveness is mechanistically channeled through
the autocrine actions of IL-1 and, to a lesser extent, TNF-
(6, 11), the present study examined whether the latter
pleiotropic cytokines exert an extended downstream effect
in ASM by regulating the expression of other genes that may participate in the overall pro-asthmatic response. Accordingly, using high-density oligonucleotide array analysis, we examined the combined effects of IL-1 and TNF-
administration on ASM cell expression of genes encoding a
variety of cytokines, cellular adhesion molecules (CAMs),
transcription factors, and other cell-signaling molecules
potentially associated with the regulation of ASM responsiveness. Moreover, in an attempt to further elucidate those
genes that may contribute to pro-asthmatic changes in ASM
responsiveness, we examined the effects of glucocorticoid
treatment on ASM responsiveness and its associated pattern of altered gene expression in ASM cells exposed to
IL-1 and TNF-. The rationale for using this strategy was
based on the well established efficacy of glucocorticoids in
the amelioration of asthma symptoms and altered responsiveness in asthmatic airways (14) as well as the ability
of glucocorticoids to generally attenuate the expression of
proinflammatory genes (7, 17). The results provide
new evidence demonstrating that (i) IL-1 and TNF- induce pro-asthmatic-like changes in ASM responsiveness in association with upregulated mRNA expression of a
host of genes encoding cytokines, CAMs, transcription
factors, and other cell-signaling molecules; (ii) the induced
changes in ASM responsiveness are prevented by pretreating the ASM with the glucocorticoid dexamethasone; and (iii) the latter protective effect on ASM responsiveness is associated with glucocorticoid-induced modulation
of expression of a number of upregulated genes. Collectively, these findings provide new insights into those glucocorticoid-sensitive genes expressed by ASM which are
coupled to pro-asthmatic changes in ASM responsiveness.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
Ten adult New Zealand White rabbits were used in this study, which was approved by the Biosafety and Animal Research Committee of the Joseph Stokes Research Institute at Children's Hospital of Philadelphia. The animals had no signs of respiratory disease before the study.
Preparation of ASM Tissues
After anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg),
the animals were killed with systemic air embolism. The tracheas
were removed via open thoracotomy, cleared of loose connective tissue, divided into eight ring segments of 6-8 mm in length, and
incubated for 18 h at room temperature in Dulbecco's modified Eagle's medium containing both IL-1 (10 ng/ml) and TNF-
(100 ng/ml) or in medium alone with no added cytokines, both
conditions in the absence and presence of dexamethazone (DEX;
10
5 M). The medium was aerated with a continuous supplemental O2 mixture (95% O2/5% CO2) during the incubation phase.
Pharmacodynamic Studies
After incubation, the cytokine- and DEX-treated issues were repeatedly rinsed, and each airway segment was suspended longitudinally between stainless steel triangular supports in siliconized
20-ml organ baths (Harvard Apparatus, South Natick, MA). The
lower support was secured to the base of the organ bath, and the
upper support was attached via a gold chain to a force transducer
(FT.03C; Grass Instruments, Quincy, MA) from which isometric
tension was continuously displayed on a multichannel recorder.
Care was taken to place the membranous portion of the trachea
between the supports to maximize the recorded tension generated by the contracting trachealis muscle. The tissues were bathed
in modified Krebs-Ringer solution containing (in mM) 125 NaCl,
14 NaHCO3, 4 KCl, 2.25 CaCl2·2H2O, 1.46 MgSO4·7H2O, 1.2 NaH2PO4·H2O, and 11 glucose. The baths were aerated with 5%
CO2 in oxygen, a pH of 7.35-7.40 was maintained, and the temperature was held at 37°C. Passive resting tension of each tracheal smooth muscle segment was set at 2.0 g after each tissue
had been passively stretched to a tension of 8 g to optimize the
resting length, as previously described (20). The tissues were allowed to equilibrate in the bath for 45 min, at which time each tissue was primed with a 1-min exposure to 104 M acetylcholine
(ACh). Cholinergic contractility was initially assessed in the ASM
by cumulative administration of ACh in final bath concentrations
ranging from 109 to 103 M. Thereafter, following thorough
rinsing, each tissue segment was half-maximally contracted with
ACh, and relaxation dose-response relationships to cumulative
administration of isoproterenol (109 to 104 M) were generated
in paired IL-1/TNF--treated and control tissues in the absence or presence of cotreatment with DEX. The initial constrictor dose-response curves to ACh were analyzed in terms of maximal isometric contractile force (Tmax) and sensitivity to the
agonist, expressed as the negative logarithm of the concentration of ACh producing 50% of Tmax (pD50; i.e., geometric mean
ED50 value). The relaxant responses to isoproterenol were analyzed in terms of percent maximal relaxation (Rmax) from the
active cholinergic contraction, and sensitivity to the relaxing
agent was determined as the corresponding pD50 value associated with 50% of Rmax.
Assessment of Gene Microarray Expression
Simultaneous expression of mRNA from multiple genes was examined in human ASM cells with the Affymetrix (Santa Clara,
CA) expression microarray system using human gene chips
(HUGeneFL array) representing ~ 5,000 genes. The ASM cells were
derived from a 21-yr-old male donor (Clonetics, San Diego, CA)
who had no evidence of pulmonary disease, and the cells were
carefully characterized by the manufacturer with specific markers
to confirm their selective smooth muscle phenotype and to exclude contamination with other cell types. The cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air
and grown in a mixture of 5% Smooth muscle Basal Medium, which
was supplemented with 10% fetal bovine serum, insulin (5 ng/ml),
epithelial growth factor (10 ng/ml; human recombinant), fibroblast
growth factor (FGF) (2 ng/ml; human recombinant), gentamycin
(50 ng/ml), and amphotericin-B (50 ng/ml), as previously described
(4). Once the cells had reached ~ 95% confluence, they were
exposed for 4 h to IL-1 (1 ng/ml) and TNF- (5 ng/ml) combined, or to media alone in the absence and presence of 1 h pretreatment with DEX (105 M). Four experiments were separately
performed from this same primary cell line, at passages 6-8.
Following incubation of the cells, the total RNA used for the Affymertrix microarray expression analysis was extracted and purified using commercially available reagents and in accordance with methods recommended by the manufacturer (21). Briefly, total RNA was extracted using Trizol and purified with Qiagen RNaeasy spin columns. Approximately 5 µg of RNA was used for first- and second-strand cDNA synthesis. After precipitation, the cDNAs were transcribed to cRNAs. Four cRNA reactions were separately performed, one from each experimental culture. The biotinylated cRNA was subsequently hybridized to the Affymetrix gene chips overnight. Nonbound probes were removed by stringency washing. The hybridized chips were developed using a Streptavidin-Phycoerythrin complex and scanned. The scanned images were then analyzed with Affymetrix software, and the data were examined using commercially available software, including Spotfire Net 5.1 (Spotfire, Somerville, MA) (22). The gene expression values were automatically subtracted from background levels, which were calculated by dividing the array into 16 sectors wherein the average of the lowest 2% of intensity in each sector is used as background for genes within that sector. The background was then subtracted from the average difference intensity values. The human 6800K gene array also contains two probe sets (actin and glyceraldehyde phosphate dehydrogenase) in sense direction to control for DNA contamination. For the set of chips used, the average signal for the genes scored as absent is approximately 30 (21). In this study, to avoid errors due to low copy numbers, genes with average difference expression less than 70 were scored as absent. Baseline expression values for all genes included their average difference expression at the 0-h time point for genes with average difference values above 70.
Statistical Analysis
Unless otherwise indicated, results are expressed as means ± SE. Statistical analysis was performed using two-tailed paired Student's t test or ANOVA with multiple comparisons of means, where appropriate. P values < 0.05 were considered significant. The standard errors for the gene array experiments were calculated for each gene from the four separate experiments using a commercially available statistical software program from GraphPad (GraphPad Software, San Diego, CA).
Reagents
The DNA oligonucleotide HuGeneFL chips and analysis system,
including scanner and computer analysis software, were purchased from Affymetrix. Human ASM cells were purchased from
Clonetics (San Diego, CA). Dulbecco's modified Eagle's medium Trizol and cDNA synthesis kits were purchased from Gibco
BRL (Gaithersburg, MD). IL-1 and TNF- were obtained from
R&D Systems (Minneapolis, MN). RNA purification spin columns
were obtained from Qiagen (Qiagen Inc., Valencia, CA). The in
vitro transcription kit was obtained from Affymetrix. Streptavidin
Phycoerythrin was purchased from Molecular Probes (Leiden,
the Netherlands). ACh, isoproterenol hydrochloride, and dexamethasone were purchased from Sigma Chemical (St. Louis,
MO). All drug concentrations are expressed as final bath concentrations. Isoproterenol and ACh were made fresh for each experiment, dissolved in normal saline to prepare 104 M and 103 M
solutions, respectively.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of IL-1 and TNF- on ASM Responsiveness
ASM constrictor dose-response relationships to ACh were
determined in tissues preincubated for 24 h in medium
alone and in the presence of maximally effective concentrations of IL-1 and TNF- (11). As shown in Figure 1,
relative to controls, the IL-1/TNF--treated tissues responded to ACh by constricting significantly more than the
control tissues, with mean ± SE values for Tmax amounting
to 119.4 ± 14.5 g/g ASM weight in the IL-1/TNF--
treated ASM but 93.7 ± 8.9 in the control ASM (P < 0.01).
Additionally, constrictor sensitivity to ACh was relatively
enhanced in the cytokine-treated tissues, with mean ± SE
values for pD50 (i.e., 
log ED50) amounting to 4.95 ± 0.06 log M in the IL-1/TNF--treated ASM but 4.66 ± 0.12 in the control ASM (P < 0.05).
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In separate studies, during comparable levels of initial
sustained ACh-induced contractions, averaging ~ 50% of
Tmax, ASM relaxation responses to cumulative administration of the -adrenergic agonist isoproterenal were generated in control and IL-1/TNF--treated tissues. As shown
in Figure 2, relative to controls, the Rmax responses and
pD50 values for isoproterenol were significantly attenuated in the IL-1/TNF--treated tissues. Accordingly, the Rmax values amounted to 41.3 ± 6.0% in the cytokine-treated ASM and 57.7 ± 7.1% in the control ASM (P < 0.01), and the corresponding pD50 values amounted to
5.87 ± 0.05 and 6.09 ± 0.11 log M, respectively (P < 0.05).
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Effects of IL-1/TNF- on ASM Cell Gene Expression
In light of the above observations, to uncover gene pathways associated with IL-1/TNF--induced changes in ASM
responsiveness, we examined in cultured human ASM
cells the effects of these cytokines on mRNA expression of
multiple genes putatively involved in various cell-signaling
processes in ASM. Using a high-density oligonucleotide DNA microarray analysis, we found in four separate experiments that ~ 40% of genes were expressed in untreated ASM cells and that treatment of cells with IL-1/
TNF- did not significantly alter the total number of genes
expressed. More than 400 genes, however, demonstrated
up- or downregulation of their mRNA signals in response
to IL-1/TNF- administration. Given the established sensitivity of the expression technique applied (21), a 2-fold increase in signal intensities from baseline was considered
significant. Accordingly, ~ 70 genes that play a potential
role in cell signaling in ASM demonstrated a 
2-fold (i.e.,
2 to ~ 150-fold) increase in mRNA expression in response
to IL-1/TNF-. The latter collection of genes is categorically displayed in Figures 3-6 with the genes in each category identified by their symbols and Gene Bank accession numbers and plotted in relation to their respective magnitudes (mean ± SE values of fold-increase) of altered
mRNA expression.
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Within the cytokine/chemokine category of genes, those
depicting upregulated mRNA expression by an average of
10-fold above baseline in response to IL-1/TNF- included the small inducible cytokine subfamily B (SCYB)
members -2, -3, -1, -5, and -6, IL-1, IL-8, colony-stimulating factor (CSF)-2 (i.e., GM-CSF), TNF-IP3, IL-6, and
CSF-3 (Figure 3). Within the CAM/extracellular matrix (ECM)-related category of genes, those upregulated 10-fold included ICAM-1, matrix metalloproteinase (MMP)-12,
and VCAM-1 (Figure 4), and, within the category of transcription factors, the genes comparably upregulated included NR4A3 and BCL-2A1 (Figure 5). Other genes related to various aspects of cellular signaling/metabolism, including those encoding various proteases, kinases, and
other molecules involved in signal transduction were also
upregulated in ASM cells in response to IL-1/TNF- administration, most notably including phosphodieseterase
(PDE)D4, superoxide dismutase-2, and inducible cyclooxygenase (COX)-2 (Figure 6). Contrasting these observations,
treatment of cells with IL-1/TNF- had no effect on mRNA
expression of constitutively expressed "housekeeping" genes such as -actin, ribosomal protein L7, 2-microglobulin, and others (data not shown; available on website).
Glucocorticoid Effect on IL-1/TNF--Induced Changes
in ASM Responsiveness
To assess whether the IL-1/TNF--induced changes in
ASM responsiveness are glucocorticoid sensitive, contractile dose-response relationships to ACh were compared
between IL-1/TNF--treated ASM tissues and their respective paired control ASM segments, both in the absence
and presence of pretreatment of the tissues for 60 min with
dexamethasone (DEX; 105 M). As shown in Figure 7, the
heightened constrictor responses to ACh generated in
IL-1/TNF--exposed ASM were abrogated by pretreating the cytokine-exposed tissues with DEX. Accordingly, in
these DEX-pretreated tissues, the mean ± SE Tmax and
pD50 values were 102.9 ± 13.1 g/g ASM weight and 4.89 ± 0.05 log M, respectively, and the latter determinations
were similar to those obtained in control ASM. In contrast, pretreatment with DEX had no effect on the constrictor responses to ACh in control tissues (Figure 7, open
squares).
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Comparable to the protective effects of DEX on cytokine-induced changes in ASM constrictor responsiveness,
the impaired -adrenoceptor-mediated relaxation responses
to isoproterenol obtained in IL-1/TNF--exposed ASM
were also completely abrogated by pretreating the tissues with DEX (Figure 8). Accordingly, in these DEX-pretreated tissues, the mean Rmax and pD50 values for isoproterenol averaged 55.5 ± 5.7% and 5.99 ± 0.06 log M,
respectively, and the latter determinations were similar to
those obtained in control ASM. In contrast, pretreatment with DEX had no effect on the relaxation responses to isoproterenol in control tissues (Figure 8, open squares).
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Corticosteroid Effect on IL-1/TNF--Induced Gene
Expression in ASM Cells
Given the efficacy of DEX in ablating the effects of IL-1/
TNF- on ASM responsiveness, we next examined whether
DEX also modulates the above-mentioned effects of IL-1/
TNF- on multiple gene expression in ASM cells. In these
experiments, paired cultures of ASM cells were exposed to
vehicle alone (control) or to IL-1/TNF- in the absence
or presence of DEX (105 M) with each condition examined in duplicate. For a given gene, sensitivity to DEX was
then determined as the ratio of the altered (fold-change)
mRNA levels elicited by IL-1/TNF- in the presence and
absence of DEX. Accordingly, a mRNA expression ratio
(MER) of 1.0 implied a lack of effect of DEX, whereas
MER values below and above 1.0 denoted DEX-induced
repression and stimulation of mRNA expression, respectively. The results demonstrate that the upregulated mRNA
levels exhibited by cells exposed to IL-1/TNF- were
largely repressed by pretreating the cells with DEX, as evidenced by MER values below 1.0 for the majority of
genes belonging to each category (Figure 9). Not all genes,
however, displayed DEX sensitivity, and, as shown in Figure 9, a small number of genes in each category exhibited
stimulation of IL-1/TNF--induced mRNA expression
in the presence of DEX (i.e., MERs > 1.0). In evaluating
the variability in DEX sensitivity within each category of
genes, we found no correlation between the MER values
for the different genes and their corresponding magnitudes of IL-1/TNF--induced enhanced mRNA expression in the absence of DEX.
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Of the DEX-sensitive genes depicted in Figure 9, those
that exhibited 10% DEX-induced decrease in mRNA
expression (i.e., MER values 
0.90) are identified in Table 1. It is noted that a strong repressive effect of DEX was
seen for genes known to be involved in the regulation of
cAMP and Ca2+ mobilization, including the phosphodiesterase D4 and plasma membrane Ca2+ ATPase genes,
which provided MER values of 0.40 and 0.34, respectively, corresponding to 60 and 66% inhibition of IL-1/TNF--
induced mRNA expression in the presence of DEX, respectively. Additionally, certain cytokine/chemokine-related
and other cell signaling-related genes were also significantly inhibited by DEX, including the pro-IL-1, IL-8,
IL-13R, small inducible SCY-B2, -B6, -A7, bradykinin receptor-2, and COX-2 genes. Moreover, it is relevant to note that the p50-NF-
B gene, which belongs to the NF-B family of inducible transcription factors that regulate the host
immune and inflammatory responses, was inhibited by 51%
by DEX (i.e., MER = 0.49). Finally, MMP3 and, most notably, MMP10 and MMP12 genes, which are implicated in
tissue remodeling, were also markedly inhibited by DEX.
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Among the collection of DEX-sensitive genes exhibiting 10% augmentation of IL-1/TNF--induced mRNA
expression (i.e., MER values 1.10), as shown in Table 2,
those belonging the cell signaling-related category included
11--hydroxysteroid-dehydroxgenase-1, the MAP kinase
subtype, MAPKKK5, and the ATP-binding cassette gene,
ABC-B2. In the cytokine/chemokine-related category, DEX-induced augmented mRNA expression was most evidenced
by genes encoding epithelial-derived neutrophil-activating
peptide 78 (SCYB5), colony (granulocyte) stimulating factor 3 (CSF3), TNF--induced protein 3 (TNF--IP3), and
TNF--IP6. Finally, other genes upregulated by DEX
included the transcription factor-related gene, CCAAT- enhancer binding protein-delta, and the CAM/ECM molecule-related gene, tenascin C.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The pleiotropic cytokines IL-1 and TNF- have been implicated in the pathophysiology of a host of proinflammatory disorders, including bronchial asthma. Regarding the
latter, elevated levels of these cytokines have been detected in the bronchoalveolar lavage fluid from asthmatic
patients (23), and both IL-1 and TNF- have been
implicated in the development of airway constrictor hyperresponsiveness in animal models of allergic asthma (26, 27). Furthermore, in a series of recent studies, we have
shown that IL-1 and, to a somewhat lesser extent, TNF-,
act directly on ASM to produce pro-asthmatic-like changes
in ASM responsiveness (6, 11), a phenomenon mechanistically coupled to induced upregulated expression and action
of Gi protein, which inhibits cAMP accumulation in ASM
(11, 20). Given this evidence, together with other findings
demonstrating that the induction of pro-asthmatic changes
in ASM responsiveness by atopic sensitization (IgE-mediated) of ASM or rhinovirus inoculation of ASM is mechanistically channeled through the autocrine release and actions
of IL-1 and TNF- in the ASM itself (10, 28, 29), this
study examined whether these cytokines exert an extended downstream effect in ASM by regulating the expression of other putative genes participating in the overall pro-asthmatic response in ASM. Using high-density gene microarray technology for simultaneous analysis of
multiple gene expression in a high-throughput fashion, our
results demonstrated that (i) in association with induced
pro-asthmatic-like changes in ASM responsiveness, IL-1/
TNF--exposed ASM cells exhibited altered mRNA expression of more than 400 genes, of which ~ 70 genes involved with various aspects of cell signaling were upregulated by 2-fold; (ii) of the latter collection of genes
stimulated by IL-1/TNF-, the majority were inhibited
by cotreatment of ASM cells with dexamethasone, in association with ablation of the pro-asthmatic effects of IL-1/
TNF- on ASM responsiveness by the glucocorticoid; and
(iii) a minority of genes modulated by IL-1/TNF- were
upregulated in the presence of dexamethasone. Collectively, these observations show that IL-1/TNF- elicits glucocorticoid-sensitive pro-asthmatic-like changes in ASM
responsiveness, which are associated with glucocorticoid-sensitive modulation of proinflammatory gene expression
patterns in ASM that potentially contribute to the altered
airway structure and function in asthma.
Treatment of ASM tissues with the combination of IL-1
and TNF- elicited pro-asthmatic-like changes in ASM
responsiveness, including heightened constrictor responsiveness to cholinergic stimulation (Figure 1) and impaired
relaxation to -adrenoceptor stimulation (Figure 2) (11).
We previously determined that these actions of IL-1 and
TNF- are associated with an induced upregulated expression and action of Gi protein, principally involving Gi2 and Gi3, which inhibit -adrenoceptor-mediated cAMP
accumulation (6, 11, 20). Interestingly, the present results
demonstrated that, among the collection of cell-signaling/
metabolism genes expressed by ASM cells, mRNA expression of a number of genes known to regulate smooth muscle contractility and relaxation was significantly upregulated in response to IL-1/TNF- (Figure 6). Accordingly, phosphodiesterase (PDE)-4B, which acts in ASM cells to
inhibit cAMP accumulation (30, 31), was most markedly
upregulated in the presence of IL-1/TNF-, suggesting
the induction of an extended mechanism of attenuated
cAMP accumulation (i.e., downstream to Gi protein activation) accompanying the pro-asthmatic-like changes in
ASM responsiveness. The relevance of the latter mechanism is supported by the findings described in a recent report that inhibition of PDE-4 largely ablates the airway
constrictor hyperresponsiveness and airway inflammation
in an in vivo murine model of allergic asthma (32, 33). In
addition to PDE-4, our results also identified IL-1/TNF--
induced upregulated mRNA expression of certain genes regulating intracellular Ca2+ levels, including plasma membrane Ca2+-ATPase (i.e., ATP2B1), stanniocalcin-1 (i.e.,
STC1), and others. Moreover, upregulated mRNA expression was also displayed by a host of proinflammatory genes
that may further contribute to the observed changes in ASM
responsiveness. These include COX-2 (Figure 6), as well as
various cytokine/chemokine genes (Figure 3) that have been
previously implicated in the allergic asthmatic state, such as
IL-8, CSF-2 (GM-CSF), IL-6, RANTES, various numbers
of the small inducible cytokine SCYB, and others. Thus,
downstream to activation with IL-1 and TNF-, ASM cells
upregulated mRNA expression of a pattern of proinflammatory genes in association with the induction of pro-asthmatic changes in ASM responsiveness. Whereas the relative contributions of these individual or combined changes
in cytokine/chemokine gene expression remain to be elucidated, given the diverse proinflammatory nature of these
genes, it is reasonable to speculate that, beyond inducing
autologous changes in its responsiveness through autocrine mechanisms, the ASM may also act as a paracrine
source for the activation of other cell types (e.g., leukocytes) contributing to the overall proinflammatory asthmatic state.
Within the category of genes expressing CAM/ECM
molecules, the pronounced upregulation of ICAM-1 mRNA
following exposure of ASM cells to IL-1/TNF- (Figure
4) is consistent with previous findings demonstrating increased ICAM-1 expression and signaling in ASM exposed to atopic asthmatic serum (34) and to rhinovirus (35), conditions that are both associated with the induction of pro-asthmatic changes in ASM responsiveness (36). Moreover, in accordance with the present observations (Figure
4), enhanced expression of various extracellular matrix
metalloproteinases has also been demonstrated in asthmatic airways and lungs (37, 38), as well as in various animal models of allergic asthma (39). To the extent that IL-1
and TNF- have been shown to induce ASM cell proliferation (40), a phenomenon associated with altered expression and function of specific matrix metalloproteinases (41), the present observations lend further support to the
notion that the ASM itself may autologously regulate its
own state of proliferation under pro-asthmatic conditions
(37). This notion is consistent with our observation that
IL-1/TNF--induced upregulated mRNA expression of
various transcription factors (Figure 5) associated with altered cellular proliferation and differentiation (e.g., BCL-2A1, STAT5A, p50-NF-B, NFATC1, etc.). Taken together,
these results concur with recent evidence demonstrating
an autocrine role for the ASM in the overall process of airway remodeling in the asthmatic state (42).
The observed ability of dexamethasone to abrogate
IL-1/TNF--induced changes in ASM constrictor (Figure 7) and relaxant responsiveness (Figure 8) parallels the
known efficacy of glucocorticoids both in the treatment of
asthma and attenuation of the altered bronchial responsiveness associated with this disease (15). Moreover, in
accordance with the inherent antiinflammatory properties
of glucocorticoids, our results demonstrate that DEX represses the upregulated mRNA expression displayed by a
majority of genes stimulated in the presence of IL-1/
TNF- (Figure 9). Accordingly, with the exception of tenascin C (i.e., HXB), all the other genes belonging to the
CAM/ECM molecules category were suppressed to varying extents by DEX (Table 1). Most genes belonging to
the cytokines/chemokines category were also repressed by
DEX, notably including IL-1, RANTES, IL-8, IL-6,
IL-13 receptor-, members of the small inducible cytokine
(SCY) subfamilies A and B, and others (Table 1). Moreover, within the cell-signaling/metabolism category, it is
relevant to note that DEX markedly repressed the IL-1/
TNF--induced expression of genes associated with altered cAMP metabolism (e.g., PDE-4B and AMPD3) and
Ca2+ mobilization and homeostasis (e.g., ATP2B1 and
STC1). Finally, IL-1/TNF--induced changes in mRNA
expression of most genes in the transcription category
were inhibited by DEX, notably including BCL2A1,
NP4A3, p50-NF-B, and NFATC1. An important consideration raised by the latter observed effect of DEX relates
to the crucial roles played by these transcription factors as
regulators of the host immune and inflammatory responses,
as well as critical aspects of cellular proliferation and apoptosis. For example, with respect to p50-NF-B, the latter
has been implicated in the regulation of cellular expression
of genes encoding nearly 30 different cytokines and chemokines, receptors involved in immune recognition, T- and B-cell differentiation, apoptosis, cellular adhesion molecules, and such proinflammatory enzymes as inducible cyclooxygenase (COX-2) (43). Similarly, the transcription factor
NFAT-C1 participates in the transactivation of a number of
cytokine genes (46, 47), and its induction by growth factors
in vascular smooth muscle cells (VSMC) has been implicated in such processes as extracellular matrix remodeling
and VSMC growth (48). Given this evidence, in light of
our present observations, it may be argued that the overall
pattern of induced proinflammatory gene expression in response to IL-1/TNF- administration was attributed to
the known stimulatory actions of these cytokines on certain inducible transcription pathways (49) and that the
overall repressive effect of DEX on the collection of proinflammatory genes was attributed to the known inhibitory
effect of the glucocorticoid on various inducible transcription factors, most notably including NF-B (50).
Whereas DEX repressed the enhanced mRNA expression of most genes upregulated by IL-1/TNF-, mRNA
levels of a relatively smaller number of genes were unaffected or augmented in the presence of DEX (Table 2). Interestingly, the observed stimulatory effect of DEX was
displayed by several genes belonging to the cytokines/chemokines category, specifically including SCYB-5, CSF-3,
and TNF-IP6. The observed enhanced mRNA expression of these genes was somewhat unexpected, given their
known proinflammatory roles. Although the mechanism
underlying this effect is not readily explained, it is relevant
to note that, in addition to binding to its DNA glucocorticoid-response element, the activated glucocorticoid receptor is also known to interact and cooperate positively with certain transcription factors that regulate the expression of various proinflammatory genes, including STAT3, STAT5,
OCT, and AP-1 (51). Thus, despite the well established
overall antiinflammatory properties of glucocorticoids, the
latter may upregulate the expression of certain proinflammatory genes. The extent to which this action of glucocorticoids serves to modulate airway function in the asthmatic state remains to be elucidated, although it is likely that the therapeutic efficacy of glucocorticoids in the treatment of
asthma is given by the net result of their suppressive action
on proinflammatory gene expression (see Table 1) and upregulation of other genes potentially regulating airway
structure and function (see Table 2). In addition, other factors, such as altered mRNA stability and mRNA transport
critical to its translation, may, at least in part, explain the
failure of DEX to reverse mRNA levels for a number of
genes. These factors are currently being systematically addressed in new and extended experiments.
An important issue related to the above collection of
present findings is that our observations on the effects of
IL-1/TNF- administration and dexamethasone treatment
were based on studies conducted using rabbit ASM tissues
and cultured human ASM cells. Whereas the experiments
using these different preparations provided results that
were largely complimentary in nature, the issue of potential species differences warrants consideration. In this regard, it is relevant to note that earlier studies using atopic asthmatic serum sensitization and rhinovirus exposure to
elicit pro-asthmatic-like changes in ASM responsiveness
have reported comparable effects of these treatment conditions in rabbit and human ASM cells and tissues. Accordingly, atopic asthmatic serum sensitization was found
to elicit qualitatively similar upregulated expression of the
low affinity receptor for IgE, Fc
RII (i.e., CD23), in both
rabbit and human ASM cells (28, 29), as well as increased
release of IL-1 from both cell types (6, 29). Moreover,
rhinovirus inoculation of rabbit and human ASM cells was
also found to provoke the release of IL- from both cell
types (5, 10). Thus, collectively, the findings from these
earlier reports, together with those of the present study,
suggest that there exists a good concordance between rabbit and human ASM cells, at least with respect to the role
of IL-1 in regulating ASM function. Finally, although it
remains to be established whether such an interspecies
concordance also exists in vivo, it is noteworthy that, in
general agreement with the present in vitro observations, in vivo administration of an IL-1 receptor antagonist in a
guinea pig model of allergic asthma was shown to prevent
allergen-induced pro-asthmatic-like changes in airway responsiveness, as well as the accompanying pulmonary infiltration with eosinophils (26).
Another issue pertaining to the present observations
relates to our use of different durations of exposure to IL-1/
TNF- and DEX to determine the effects of these agents
on gene array expression in the cultured ASM cells and
agonist responsiveness in the ASM tissues. In this regard,
changes in ASM agonist responsiveness were examined after exposure of tissues for 18 h to the different treatment
conditions, whereas a shorter treatment duration of 4 h
was used in the gene array experiments. Our use of these
different treatment durations was based on results obtained in previous studies wherein we determined the differences in duration needed for effective induction of
acute changes in mRNA expression and resultant alterations in ASM tissue agonist responsiveness (6, 11). In this
context, the extended duration needed to evoke changes
in ASM tissue responsiveness likely reflects the time required for effective translation of altered mRNA levels into proteins ultimately responsible for the observed changes in
tissue agonist responsiveness. Notwithstanding this consideration, an extended systematic evaluation of the effects of
varying durations of exposure to IL-1/TNF- and DEX
on gene array expression in ASM warrants future investigation.
In conclusion, using the high-density cDNA microarray
technique, in this study we investigated the pattern of altered expression of multiple genes by ASM cells induced
downstream to activation with IL-1/TNF- and examined
how this induced profile of gene expression is modulated
by glucocorticoid treatment. This study demonstrated that,
in association with pro-asthmatic-like changes in ASM responsiveness elicited by IL-1/TNF-, these cytokines induced upregulated mRNA expression of a host of proinflammatory genes that regulate gene transcription,
cytokines and chemokines, cellular adhesion molecules,
and other signal transduction molecules that regulate ASM
responsiveness. Moreover, in addition to suppression of
the IL-1/TNF--induced changes in ASM responsiveness by the glucocorticoid, dexamethasone, the latter repressed most of the IL-1/TNF--induced changes in proinflammatory gene expression, whereas a small number of
genes were further upregulated by dexamethasone. Collectively, these findings support the concept that, together with its role as a regulator of airway tone, the ASM is capable of expressing a host of glucocorticoid-sensitive proinflammatory genes that may contribute to the altered
structure and function of the airways in the pro-asthmatic
state. Extended studies are needed to elucidate the role of
this inducible pattern of multiple gene expression by the
ASM in the overall pathobiology of asthma.
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Footnotes |
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Address correspondence to: Hakon Hakonarson, M.D., DeCode Genetics, Lynghals 1 110 Reykjavik, Iceland. E-mail: hakonh{at}decode.is
(Received in original form May 14, 2001 and in revised form August 23, 2001).
Abbreviations: acetylcholine, ACh; airway smooth muscle, ASM; cellular adhesion molecule, CAM; cyclooxygenase, COX; colony-stimulating factor, CSF; dexamethasone, DEX; extracellular matrix, ECM; interleukin, IL; mRNA expression ratio, MER; matrix metalloproteinase, MMP; phosphodiesterase, PDE; percent maximal relaxation, Rmax; cytokine subfamily, SCY; maximal isometric contractile force, Tmax; tumor necrosis factor, TNF; TNF--induced protein, TNF--IP; vascular smooth muscle
cells, VSMC.
DeCode's website address to access supplemental data derived from the human ASM gene array analysis: (https://inotes.decode.is/ASM array data.nsf)
Acknowledgments: The authors thank E. A. Adalsteins, G. Finnbogason, D. Shkolny, and J. S. Grunstein for expert technical assistance and M. Brown for assisting with typing the manuscript. This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-59906, HL-31467, HL-58245, and HL-61038.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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