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Division of Clinical Immunology and Allergy, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224; and
Department of Pharmacology, SmithKline Beecham, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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alone or
in combination with IFN-
was near-maximal after 1 h, peaked at
4 and 8 h, respectively, remained unchanged up to 24 h, and
was protein synthesis independent. In contrast, RANTES mRNA was
detectable only after 2 h and slowly increased to a peak at
24 h, and was protein synthesis dependent. Induction of eotaxin
and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-
compared with RANTES mRNA. IL-4 and IFN- had selective effects on
chemokine expression; IL-4 selectively up-regulated the expression of
eotaxin and MCP-4 and potentiated TNF--induced eotaxin, while
IFN- markedly potentiated only the TNF--induced expression of
RANTES. Although budesonide inhibited the expression of chemokine mRNA
to a variable extent, it effectively inhibited production of eotaxin
and RANTES protein. Budesonide inhibited both RANTES- and eotaxin
promoter-driven reporter gene activity. Budesonide also selectively
accelerated the decay of eotaxin and MCP-4 mRNA. These results point to
IL-4 as a possible mediator by which Th2 cells may induce selective
production of C-C chemokines from epithelium and indicate that
glucocorticoid inhibit chemokine expression through multiple mechanisms
of action. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, IL-13) and eosinophil-selective chemoattractant
molecules, especially C-C chemokines (4). Several members
of the C-C branch of the chemokine family display potent and/or
selective chemoattractant and activating properties toward eosinophils,
basophils, monocytes, and T lymphocytes, cell types associated with
allergic reactions, while being very poor neutrophil chemoattractants.
The potent, eosinophil-selective C-C chemokines include RANTES,
eotaxin, eotaxin-2, monocyte chemoattractant protein
(MCP)3-3, and MCP-4
(5, 6, 7, 8, 9, 10). Cooperation between IL-5
and chemokines in promoting eosinophil migration has been described
both in vitro (11) and in animal models (12, 13). Cutaneous injection of RANTES or eotaxin has been shown to
induce eosinophil accumulation (14, 15, 16, 17). Interestingly,
injection of RANTES induced eosinophil accumulation with a profoundly
faster time course in allergic individuals than in nonallergic
individuals (15).
Within the last few years, numerous studies have documented increased
expression of mRNA and/or protein for several C-C chemokines, including
RANTES, eotaxin, MCP-3, and MCP-4, in diseases characterized by tissue
eosinophilia, such as nasal polyposis, rhinitis, and asthma (16, 18, 19, 20, 21, 22, 23, 24), as well as after experimental Ag
challenge in human skin (25) and lung (26).
Studies in murine models of allergic inflammation provided crucial
information on the nonredundant role of chemokines at different stages
of the development of an inflammatory infiltrate (17, 27, 28). Work by Gonzalo et al. showed that
challenge-induced recruitment of specific leukocyte types in the lung
correlates with a distinct pattern of chemokine expression
(17). Individual blockade of eotaxin, MCP-5, macrophage
inflammatory protein-1, RANTES, and MCP-1 in OVA-induced airway
allergic inflammation in mice has revealed distinct roles for each
chemokine in inducing inflammatory cell recruitment and bronchial
hyper-reactivity (27). The nonredundant role of chemokines
has also been suggested by studies conducted with a gene disruption
strategy. The macrophage inflammatory protein-1 knockout mice were
unable to mount an inflammatory response to influenza virus and showed
resistance to Coxsackievirus-induced myocarditis (29).
Disruption of the eotaxin gene has been shown by Rothenberg et al. to
suppress the early eosinophil recruitment after Ag challenge, but not
the late eosinophil influx (30), although in a different
eotaxin knockout system, the eosinophil influx in the bronchoalveolar
lavage induced by OVA challenge was not modified compared with that in
wild-type mice (31).
Several lines of evidence indicate that epithelial cells play a crucial role in regulating chemokine production and function in the airways. Localization of infiltrating cells to the epithelial region (18, 32) strongly implicates epithelial cells as a relevant source of these chemoattractants. In fact, immunohistochemistry studies of RANTES, MCP-1, MCP-4, and eotaxin show that epithelium is among the most heavily staining cell types, if not the most heavily staining cell type in biopsies of both upper and lower airways of humans and mice (17, 18, 19, 22, 26, 33, 34). Also, numerous in vitro studies have demonstrated that airway epithelial cells produce substantial quantities of RANTES and other C-C chemokines (9, 35, 36, 37). Epithelial cells are also considered to be among the most relevant cellular targets of topically delivered glucocorticoids (GC).
The aim of the present study was to test the hypothesis that the epithelial-derived expression of the C-C chemokines RANTES, eotaxin, and MCP-4 can be differentially regulated by inflammatory and immunomodulatory cytokines as well as by GC, leading to distinct patterns of expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dATP (DuPont-New England Nuclear,
Boston, MA), FuGene (Boehringer Mannheim, Indianapolis, IN),
luciferase detection kit (Analytical Luminescence Laboratory, Ann
Arbor, MI), Bio-ray reagent (Bio-Rad, Hercules, CA). Human
recombinant TNF- and IL-4 (R & D, Minneapolis, MN), IFN-
(Genzyme, Cambridge, MA), and ExpressHyb solution (Clontech, Palo Alto,
CA). Budesonide was a gift from Drs. Per Andersson and Ralph Brattsand
(Astra Draco, Lund, Sweden) and was stored as a 0.1-M stock in DMSO at
-20°C. Isolation of primary bronchial epithelial cells (PBEC)
Isolation of bronchial epithelial cells (n = 6) was performed as previously described (38). The purity of PBEC cultures (>95%) has been confirmed by immunohistochemical staining for cytokeratin (n = 3, not shown), performed as previously described (39).
Culture of BEAS-2B cells and PBEC
The BEAS-2B cell line, derived from human bronchial epithelium
transformed by an adenovirus 12-SV40 hybrid virus
(40), was supplied by Dr. Curtis
Harris. BEAS-2B cells and PBEC were cultured in
75-cm2 tissue culture flasks uncoated (BEAS-2B)
or coated with collagen (PBEC) and maintained in F-12/DMEM medium
containing 5% heat-inactivated FCS, 1% L-glutamine,
penicillin (100 U/ml), and streptomycin (100 mg/ml). This medium is
referred to as complete medium. BEAS-2B cells were used from passages
36–45. Each batch of PBEC was seeded in multiple T-75 flasks and used
only at their first passage. Cells were cultured at 37°C with 5%
CO2 in humidified air. When the cells reached
80–90% confluence, they were stimulated according to the experimental
protocols, then harvested with 0.02% trypsin/EDTA in HBSS and washed
twice in HBSS, with a recovery of 
5–8 x
106 cells/flask. The viability of both PBEC and
BEAS-2B cells, assessed by staining with erythrosin B, was consistently
>95% of the cells harvested.
Northern blot analysis
Total RNA was extracted using the RNAzol B reagent according to a previously described method (41). Northern blot analysis was performed as previously described (9, 36). To compare the expression of eotaxin, MCP-4, and RANTES mRNA from the same cells, three aliquots, 20 µg each, of the total RNA samples were run in parallel on the same 1% agarose/6% formaldehyde gel. Particular effort was made to maintain equal amounts of RNA among the aliquots by measuring the RNA concentration spectrophotometrically and comparing the results with the intensity of ethidium bromide-stained 28S and 18S RNA bands observed with 1 µg (based on the optical density reading) of total RNA for each condition loaded on a 1% agarose gel. After separation by electrophoresis, RNA samples were blotted onto a single piece of GeneScreen Plus nylon membrane by a 1-h transfer with a positive pressure blotter (Stratagene, La Jolla, CA). After the transfer, the membrane was carefully cut into three pieces, each containing one of the three mRNA aliquots. The three membranes were prehybridized for 45 min and then hybridized for 90 min with the three 32P-labeled cDNA probes (for eotaxin, MCP-4, and RANTES) using Expresshyb hybridization solution. The same amounts of eotaxin, MCP-4, and RANTES cDNA probes (100 ng) were labeled using the random priming method. This procedure yields 32P-labeled probes with comparable specific activities, and the same amount of radioactive probe (1 x 106 cpm/ml of hybridization buffer) for each chemokine was used. After hybridization, membranes were washed with 2x SSC/0.2% SDS at room temperature (twice, 15 min each time), then with 2x SSC/0.2% SDS at 65°C (four times, 15 min each time), and finally with 0.2x SSC/0.2% SDS at 65°C (twice, 10 min each time). Membranes were then exposed to the same x-ray film for equal exposure time. Autoradiographs were quantified by video densitometry using a gel documentation system configured by UVP (San Gabriel, CA) interfaced with a Macintosh Centris 610 containing Image 1.60 software (National Institutes of Health Public Software, Bethesda, MD). Results are shown as the ratio of chemokine/GAPDH (housekeeping gene) densitometric units. BEAS-2B cells and PBEC showed low, but detectable, constitutive levels of eotaxin and MCP-4 mRNA in 6 of 20 and 3 of 20 experiments, respectively, whereas constitutive RANTES mRNA was never detected. The probes used were a PCR-amplified fragment spanning 290 bp of the coding region of RANTES, an EcoRI-XhoI fragment spanning 312 bp of the coding region of MCP-4, a BamHI fragment spanning 260 bp of the coding region of eotaxin, and a cDNA probe (1100 bp) for GAPDH (Clontech).
ELISA
Eotaxin and RANTES protein levels in cell supernatants were assayed by commercially available ELISA kits (R & D Systems). The limits of detection in the assays were 5 and 2 pg/ml for eotaxin and RANTES, respectively.
RANTES and eotaxin promoter constructs
The pRANTES/Luc-884 luciferase reporter construct has been prepared by subcloning bp -884 to +64 of human RANTES from the plasmid PCR 1000 (a gift from Dr. Tom Schall) into the HindIII-XhoI sites of pGL2-Basic (Promega, Madison, WI). The promoter region of eotaxin was amplified by a modification of 5' rapid amplification of cDNA ends using the Genome Walker Kit (Clontech, Palo Alto, CA). PCR product was generated from adaptor-ligated genomic DNA fragments using a 5' adaptor primer and a primer complementary to a sequence from the first exon of eotaxin (5'-TAGCAGCTGCCTTCAGCCCCCAGGGG-3') (16), then used as template for a nested PCR reaction using the adaptor primer and a primer derived from the eotaxin 5'-upstream sequence (5'-ACTTCTGTGGCTGCTGCTCATAG-3'). A 1.4-kb product was cloned into a TA vector (Invitrogen, San Diego, CA), and its sequence was verified by the dideoxy method (Johns Hopkins DNA Analysis Facility, Baltimore, MD). The resulting plasmid (referred to as TA-Eotax. 1400 Vector) was used as template to amplify a region of the eotaxin promoter spanning bp -1363 to -1 using a 5' primer containing a restriction site for MluI (5'-ACTATAGGGCACGCGTGGT-3') and a 3' primer containing a restriction site for BglII (5'-GAAGATCTCAGCCTCTCTGCTCCTC-3'). The PCR product was cloned into the MluI and BglII sites of pGL3-Basic (Promega, Madison, WI), and the resulting plasmid is referred to as pEotax/Luc-1363.
Cell transfections and analysis of reporter gene expression
BEAS-2B cells were subcultured in six-well plates in complete
medium at a density of 200,000–250,000/well, then transfected
24 h later, at about 50–60% confluence, using the nonliposomal
cationic vehicle FuGene. Briefly, 3 µl of FuGene was resuspended (5
min at room temperature) in serum-free Opti-MEM, then allowed to
complex with 1 µg of plasmid DNA for 15 min at room temperature. The
plasmid/FuGene mixture (100 µl/well) was overlayed on the cells in a
final volume of 2 ml complete medium. After incubation for 4–24 h at
37°C, cells were treated according to the experimental protocols
indicated in the text, then washed twice with 1x PBS and lysed
using a 1x lysis buffer. The total protein content was assayed in 10
µl of each lysate using a modified Bradford reagent. Luciferase
activity in each sample was measured by light emission and expressed as
relative luciferase units normalized to total protein.
Statistical analysis
Analysis of data was performed using StatView II software (Abacus Concepts, Berkeley, CA). Data are expressed as the mean ± SEM. Statistical analysis between pairs was performed using the nonparametric Mann-Whitney U test. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
To characterize and compare the kinetics of expression of
chemokines by epithelial cells, we stimulated epithelial cells with
TNF- and IFN- (100 ng/ml each) for increasing periods of time and
analyzed mRNA levels by Northern blot. Upon stimulation with TNF-
and IFN-, eotaxin and MCP-4 mRNA were very rapidly up-regulated,
being detectable as early as 30 min after stimulation (not shown) and
reaching maximum expression levels within the first 2–8 h of
stimulation, respectively (Fig. 1
A). In contrast, induction of
RANTES mRNA displayed a slow kinetic, being detectable after 2–4 h and
reaching maximum expression only at 18–24 h. Similar results were
obtained for eotaxin and RANTES with freshly isolated PBEC (Fig. 1B) and using TNF- alone as the stimulus
(n = 3; data not shown). To further explore the
response to TNF- and IFN-, we performed concentration-response
experiments, incubating BEAS-2B cells with increasing concentrations of
TNF- (1, 10, and 100 ng/ml) alone or in combination with a fixed
concentration (100 ng/ml) of IFN- for 8 h, a time point at
which all three TNF--induced chemokine mRNAs were detectable. The
results shown in Fig. 2 reveal that
TNF- was 10- to 100-fold more potent in inducing eotaxin and
MCP-4 than RANTES. In fact, 1 ng/ml of TNF- was near
maximal for both eotaxin and MCP-4, but 100 ng/ml was required for
maximal induction of RANTES. The same concentration-response curve to
TNF- observed for RANTES mRNA expression after 8 h was
maintained after 24 h of TNF- treatment (36).
IFN- alone induced small amounts of mRNA for eotaxin and MCP-4, but
not RANTES. We have previously reported (36) that
stimulation of BEAS-2B cells with IFN- induces small amounts of
RANTES mRNA and protein, but it requires longer (18–24 h) incubation
times. As expected (9, 36), IFN- produced a strong
potentiation of TNF--induced RANTES and MCP-4 mRNA (Fig. 2), whereas
potentiation of eotaxin expression by IFN- was less pronounced.
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and IFN-
The characteristics of RANTES and eotaxin protein secretion,
assessed by ELISA in the supernatants of BEAS-2B cells, were similar.
The data in Fig. 3 demonstrate that
secretion of eotaxin induced by TNF- preceded RANTES secretion,
becoming significant at 4 h after stimulation, whereas RANTES
secretion was significantly elevated only after 18 h of
incubation. The addition of IFN- to TNF- induced a faster
production of RANTES and resulted in a 3-fold increase in RANTES
secretion. In contrast, IFN- did not affect the kinetics or the
amount of eotaxin produced by BEAS-2B cells in response to TNF-
stimulation.
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The time- and stimulus-dependent differences in chemokine
expression reported above indicate potential differences in mechanisms
regulating their synthesis. To evaluate regulatory mechanisms of
chemokine production, we stimulated cells with TNF- for 8 h
(100 ng/ml) in the absence or the presence of a 2-h preincubation with
the protein synthesis inhibitor cycloheximide (5 µg/ml). Induction of
mRNA for eotaxin and MCP-4 was only weakly inhibited after
cycloheximide treatment, while RANTES mRNA production was significantly
reduced (Fig. 4). These results suggest
that induction of an intermediate protein (e.g., a transcription
factor) may be necessary for expression of RANTES mRNA, but not for
that of eotaxin or MCP-4.
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Incubation of airway epithelial cells with IL-4 induced the
expression of eotaxin and, to a lesser extent, of MCP-4 mRNA (Fig. 5A). Interestingly, in the
same experiments we observed no induction of RANTES mRNA by IL-4. This
result has been reproduced in freshly isolated PBEC (Fig. 5B). Concentration-response experiments in BEAS-2B cells
(10–100 ng/ml; not shown) revealed that chemokine mRNA expression was
detectable after challenge with 10 ng/ml of IL-4, as documented by
others in human fibroblasts (42), and was maximal when 50
ng/ml of IL-4 was used. We then incubated epithelial cells in the
presence of TNF- (100 ng/ml), alone or in combination with IL-4 (50
ng/ml), for 18 h. Densitometric analysis revealed that IL-4
consistently induced an increase in TNF--induced eotaxin mRNA levels
(36 ± 15%) and MCP-4 mRNA (50 ± 38%), while reducing by
27 ± 7% RANTES mRNA, although these changes were not
statistically significant. A more striking differential effect of IL-4
on eotaxin and RANTES production was found at the protein level (Fig. 6). When used alone as a stimulus for
18 h, IL-4 induced a significant release of eotaxin from BEAS-2B
cells (274 ± 95 pg/ml), but not RANTES, while TNF- stimulated
the secretion of both eotaxin and RANTES (582 ± 313 and
8689 ± 3155 pg/ml, respectively). Furthermore, IL-4 caused a
striking (7.6-fold) increase in TNF--induced eotaxin release
(4402 ± 1903 pg/ml), while it did not affect RANTES secretion.
IFN- alone did not induce eotaxin or RANTES protein
release (n = 2; not shown), but it selectively
potentiated TNF--induced RANTES release (Fig. 6; see also Fig. 3
).
Taken together, these results suggest that the Th2-derived cytokine
IL-4 up-regulates eotaxin, but not RANTES, while the Th1-derived
cytokine IFN- enhances the up-regulation of RANTES, but not that of
eotaxin.
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Studies from our laboratory and others have demonstrated that
epithelial production of chemokines is inhibited by GC (9, 36). We first compared the effect of the potent topical GC
budesonide on the epithelial expression of eotaxin, RANTES, and MCP-4
mRNA. Epithelial cells were treated for 24 h with a concentration
of budesonide (10-7 M) previously shown to
maximally suppress epithelial-derived RANTES and MCP-4 (9, 36) and then were stimulated with TNF- (100 ng/ml), alone or
in combination with IFN- or IL-4 for 18 h. Treatment of cells
with the budesonide diluent, DMSO, tested at an amount equivalent to
that contained in the budesonide preparation, did not alter basal or
TNF--induced chemokine expression (n = 2; data not
shown). Kinetic experiments, in which BEAS-2B cells were pretreated
with 10-7 M budesonide at various times before,
simultaneously with, and after stimulation with TNF- revealed that
inhibition of chemokine mRNA expression was similar even if budesonide
was added up to 6 h after cytokine stimulation (data not shown).
Budesonide inhibition of the expression of eotaxin, MCP-4, and RANTES
mRNA varied substantially. TNF--induced RANTES mRNA appearance was
inhibited to the greatest extent (80.9 ± 3.3% inhibition);
MCP-4 mRNA was also inhibited significantly, although to a lesser
extent (43.5 ± 4.9% inhibition), while eotaxin mRNA was reduced
only by 25.5 ± 8.2% (Fig. 7A). In cells activated with
TNF- and IFN-, the effect of budesonide was less marked, although
the same rank of sensitivity was observed among the three chemokines
(data not shown). In contrast to TNF-, when IL-4 was used as a
stimulus, eotaxin mRNA expression was substantially diminished by GC
treatment (Fig. 7B). Similar results to those in Fig. 7, A and B, were obtained in PBEC (n
= 2; data not shown). Despite the relative resistance of the expression
of eotaxin mRNA to budesonide, eotaxin protein production was well
inhibited by the GC (Fig. 8).
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To further dissect the underlying molecular mechanisms responsible
for the differential regulation of epithelial chemokines by cytokines
and budesonide, we compared the effects of cytokine stimulation and
treatment with budesonide in BEAS-2B cells transfected with either
RANTES or eotaxin promoter-driven luciferase reporter constructs
(pRANTES/Luc-884 and pEOTX/Luc-1363). Transfected cells were incubated
in the presence of medium containing the DMSO diluent or budesonide
(10-7 M) and challenged with increasing
concentrations of TNF- (0.1–100 ng/ml), IL-4 (50 ng/ml), or IFN-
(10 ng/ml), alone or combined with TNF- (10 ng/ml). In cells
transfected with pEOTX/Luc-1363 (Fig. 9,
left panel), TNF- elicited a 2-fold increase in
luciferase levels at 0.1 ng/ml, with only a minimal further increase
observed at 1 and 10 ng/ml (2.2- and 2.3-fold increases, respectively).
TNF- produced a concentration-dependent increase in RANTES
promoter-driven luciferase production as well (Fig. 9, right
panel). As observed with Northern blot analysis, higher
concentrations of TNF- (10- to100-fold) were required to
maximally activate the RANTES promoter than the eotaxin promoter
construct. Also in agreement with Northern blot and ELISA results, IL-4
stimulation produced an increase (2.1-fold) in luciferase in cells
transfected with pEOTX/Luc-1363, while it did not affect luciferase
levels in cells transfected with pRANTES/Luc-884. Furthermore, IL-4
increased TNF--induced luciferase production in cells transfected
with pEOTX/Luc-1363 (>5-fold), but not in RANTES/Luc-884-transfected
cells. In fact, IL-4 significantly inhibited TNF--induced luciferase
production in cells transfected with pRANTES/Luc-884 (Fig. 9). Finally,
as observed with Northern blot and ELISA, IFN- significantly
potentiated TNF--induced luciferase production in cells transfected
with pRANTES/Luc-884, but not in cells transfected with pEOTX/Luc-1363.
The sensitivities to inhibition by GC of the cytokine-induced response
driven by the eotaxin and RANTES promoter also differed. Budesonide
treatment inhibited the TNF--mediated RANTES promoter activity more
markedly than the eotaxin promoter activity (Fig. 9).
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and IFN- (100 ng/ml each) for 8 h.
Cells were then either harvested as controls or treated with the
transcription inhibitor actinomycin D (3 µg/ml) for increasing times
to determine the decay of mRNA for eotaxin, MCP-4, and RANTES. For all
three chemokines, the cytokine-induced mRNA were very stable, with
>70% of mRNA still detectable after 8-h incubation with actinomycin D
in cells activated by cytokines but not treated with budesonide (Fig. 10). Budesonide treatment significantly
accelerated the decay of eotaxin and MCP-4 mRNA, but not that of RANTES
mRNA. The same results were obtained when TNF- alone was used as the
stimulus (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although it is now well established that multiple C-C chemokines are
produced by airway epithelial cells, little is known about the
differences in their specific profiles of activation. In this study, we
obtained evidence for distinct regulatory pathways for three
epithelial-derived, eosinophil-active C-C chemokines: eotaxin, MCP-4,
and RANTES. We found remarkable differences in the rate of expression
of mRNA for these three chemokines. Eotaxin and MCP-4 were rapidly
induced by TNF- and IFN- and reached near-maximal mRNA expression
within 2 h, while RANTES mRNA induction was much slower,
reaching peak expression only after 18–24 h. The rapid induction of
eotaxin mRNA levels is in agreement with findings using the eotaxin
knockout mouse model, in which the earliest recruitment of eosinophils
after allergen challenge in sensitized animals is selectively ablated
(30). We also found significant
differences in the sensitivity to TNF- among the three chemokines,
with eotaxin and MCP-4 mRNA being almost maximally induced by 1 ng/ml
of TNF-, whereas RANTES required 10- to 100-fold higher
concentrations to reach maximal induction. It is conceivable,
therefore, that in vivo, low concentrations of TNF- could
selectively induce eotaxin and MCP-4. In contrast, the degree of
synergism of TNF- and IFN- in inducing RANTES was much greater
than that observed for eotaxin and MCP-4, especially at protein level.
Another factor distinguishing the expression of RANTES mRNA from that
of eotaxin and MCP-4 in epithelial cells is the protein synthesis
requirement. The relative lack of de novo protein synthesis requirement
together with the rapid kinetics of activation indicate that eotaxin
and MCP-4 are immediate-early genes, whereas RANTES, being expressed
late after cell activation and requiring de novo protein synthesis,
is not.
A remarkable feature distinguishing RANTES from eotaxin and MCP-4
production is the response to IL-4. We found that both eotaxin and
MCP-4 were up-regulated by incubation of epithelial cells with IL-4,
whereas RANTES was not. Furthermore, IL-4 induced a striking
potentiation of TNF--induced eotaxin, but not RANTES release. In
further support of our findings, IL-4-induced eosinophil accumulation
in various mouse models appears to be at least partially mediated by
the endogenous production of eotaxin. Eosinophil accumulation induced
by intradermal injection of IL-4 was significantly inhibited by
anti-eotaxin Ab treatment (46), anti-IL-4
treatment of mice with Schistosoma (type 2) granulomas in
the lungs impaired the local expression of eotaxin mRNA
(47), and administration of IL-4, IL-13 and Th2
supernatants intranasally to naive mice induced lung eosinophilia and
eotaxin expression in airway epithelium (34). In vitro,
IL-4 induces the selective production of eotaxin in human
fibroblasts (42). The divergent regulation of eotaxin and
RANTES by IL-4 may have a relevant role in the establishment of chronic
allergic inflammation. It can be hypothesized that in vivo, epithelial
chemokine expression might be determined by the profile of cytokine
released in the microenvironment. The coordinated release of TNF-
from macrophages and mast cells and of IL-4 from adjacent cells,
particularly Th2 lymphocytes, may act as a strong regulatory signal for
the selective expression of eotaxin from epithelium. In support of this
hypothesis, MacLean et al. found that the increased eotaxin production
induced in the lungs by OVA challenge is blocked by anti-CD3 Ab
treatment of the animals, suggesting that eotaxin production might be
driven by Ag-specific T cells (28). The Th1 cytokine,
IFN-, had a profile of activity in stark contrast with that of IL-4.
Although IFN- profoundly potentiated the production of RANTES
protein and mRNA induced by TNF-, it failed to increase the activity
of the eotaxin promoter luciferase construct, failed to increase
eotaxin protein production induced by TNF-, and only modestly
increased mRNA detected by Northern blot. Taken together, these data
indicate that the Th2 cell-derived cytokine IL-4 may lead to induction
of eotaxin expression (and perhaps even suppression of RANTES; see Fig. 9 and Ref. 48), while the Th1 cytokine IFN- has the
opposite effect, namely, potentiation of RANTES expression with little
or no effect on eotaxin expression.
Several potential cis-regulatory elements in the promoter
regions of the RANTES and eotaxin genes have been identified
(49, 50, 51, 52). The human RANTES promoter
contains four NF-
B binding sites (53), and disruption
of any of these elements dramatically reduces RANTES promoter activity
in T cells (50). The critical involvement of NF-B in
RANTES expression in epithelial cells is consistent with the exquisite
GC sensitivity of this gene. The progressive accumulation of RANTES
mRNA over at least 24 h after stimulation and the protein
synthesis requirements of the response are suggestive of the later
involvement of additional factors mediating sustained transcription of
the gene. One possible candidate responsible for the delayed maximal
expression of RANTES mRNA is STAT-1, which has been shown to cooperate
with NF-B in the synergistic activation of the RANTES gene by
TNF- and IFN- in murine fibroblasts (54). The higher
sensitivity to TNF- stimulation observed with eotaxin and MCP-4 may
be due to different transcriptional activation patterns, such as
differences in the affinity of transcription factors for
cis-regulatory elements within the chemokine promoter
regions. Regarding the ability of IL-4 to stimulate eotaxin expression,
it is noteworthy that at least four potential STAT-6 binding elements
can be recognized within the proximal 1 kb of the human eotaxin
promoter (51, 52).
The inhibitory effect of topical GC on epithelial-derived inflammatory
mediators is increasingly viewed as an important feature of the
clinical efficacy of inhaled GC. Our data suggest that the GC
inhibition of epithelial chemokine expression is a complex process,
possibly involving multiple pathways with different sensitivities to GC
as well as different mechanisms of gene repression. It is now known
that GC can down-regulate gene expression by at least three different
molecular mechanisms: binding of ligand-activated GC receptor to
negative GC-responsive elements present in the promoter regions of
target genes; interference with the DNA binding activity and/or
trans-activation potential of transcription factors, such as
NF-B and AP-1, by protein-protein interaction with ligand-activated
GC receptor; and posttranscriptional regulatory mechanism(s), via
destabilization of mRNA transcripts and degradation of protein products
via mechanisms yet to be fully elucidated (55). Each of
these mechanisms has been implicated or can be speculated for the GC
inhibition of chemokines produced by epithelium. A GRE is present in
the promoter region of eotaxin (51, 52), but its influence
is unknown. The transcription factors AP-1 and NF-B are known to be
involved in the up-regulation of several chemokines induced in
epithelial cells, including RANTES (50) and eotaxin
(56). Recently, GC-mediated inhibition of AP-1 and NF-B
reporter gene constructs has been reported in BEAS-2B cells
(57). Inhibition of IL-8 expression by GC has been shown
to occur through GR-mediated repression of NF-B function
(58). Expression of C-C chemokines induced by TNF-,
such as RANTES, MCP-4, and eotaxin, could also be inhibited by GC
through this process. Based on our results, it can be hypothesized that
the GC effect on epithelial-derived chemokines is mediated by multiple
inhibitory mechanisms, acting at both transcriptional and
posttranscriptional levels, that contribute to a different degree to
the suppression of each chemokine. We have previously demonstrated that
the expression of RANTES mRNA and protein in epithelial cells was
significantly inhibited by GC without an effect on RANTES mRNA
half-life, leading us to hypothesize that suppression occurs mainly at
the transcriptional level (36). This hypothesis is
supported by the finding in the present study that budesonide
suppressed luciferase expression from a construct driven by the RANTES
promoter. We did find, however, that budesonide selectively
destabilized eotaxin and MCP-4 mRNA in epithelial cells, in contrast to
RANTES. In the case of eotaxin, one possible explanation of these
results may be the influence of destabilizing sequences, known as
AU-rich elements (ARE), in the 3' untranslated region (UTR) of the
transcripts (59), acting as binding sites for
trans-acting factors
(60, 61, 62, 63). It has been shown that GC
can influence mRNA decay via AREs (64). Based upon
published sequences, the eotaxin mRNA 3' UTR contains two AUUUA motifs
(37). Interestingly, no ARE are present in the 3' UTR of
MCP-4 and RANTES (10, 65). Other regulatory
cis-elements present in the MCP-4 3' UTR or in other regions
of the unspliced, nuclear form of the mRNA may be targets of GC
regulation. Non-ARE mRNA destabilizing elements have recently been
identified (66, 67). Although the effect of budesonide on
eotaxin mRNA level measured by Northern blot was modest, it suppressed
transcription of luciferase in the eotaxin promoter construct and
destabilized eotaxin mRNA. Together these effects led to a profound
suppression of eotaxin protein release in epithelial cells.
Taken together, our results indicate the existence of independent mechanisms of regulation of epithelial chemokine production that can lead to different profiles of chemokines according to the nature and the timing of the stimulation. Furthermore, we propose that GC suppress the expression of these important genes by both transcriptional and posttranscriptional mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cristiana Stellato, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address:
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; ARE, AU-rich elements; GC, glucocorticoids; PBEC, primary bronchial epithelial cells; UTR, untranslated region; pRANTES, RANTES promoter plasmid; pEOTX, eotaxin promoter plasmid; BUD, budesonide.
Received for publication April 27, 1999. Accepted for publication August 31, 1999.
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;
n = 3 for eotaxin; n = 5 for
RANTES) or combined with IFN-
; n
= 3). Eotaxin release in supernatants from unstimulated cells after
24-h incubation was 33 ± 17 pg/ml, while RANTES release was not
detected. *, p < 0.05 compared with unstimulated
cells.
