A2B Adenosine Receptors Induce IL-19 from Bronchial
Epithelial Cells, Resulting in TNF-␣ Increase
Hongyan Zhong, Yuzhi Wu, Luiz Belardinelli, and Dewan Zeng
Department of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc., Palo Alto, California
Adenosine is a signaling nucleoside that has been proposed to
contribute to the pathogenesis of asthma and chronic obstructive
pulmonary disease. Previous studies suggest that adenosine might
play an important role in modulating levels of inflammatory media-
tors in the lung. Because airway epithelium is an important cellular
source of inflammatory mediators, the objective of the present
study was to determine whether adenosine affects the expression
and release of inflammatory cytokines from human bronchial
epithelial cells (HBECs). Among the four subtypes of adenosine
receptors, the A2B receptor was expressed at the highest level.
5ⴕ-(N-ethylcarboxamido)-adenosine (NECA), a stable analog of
adenosine, increased the release of IL-19 by 4.6- ⴞ 1.1-fold. A selec-
tive antagonist of the A2B receptor, CVT-6694, attenuated this effect
of NECA. The amount of IL-19 released from HBEC was sufficient
to activate a human monocytic cell line (THP-1) and increase the
release of TNF-␣. Furthermore, TNF-␣ was found to upregulate A2B
receptor expression in HBECs by 3.1- ⴞ 0.3-fold. Hence, these data
indicate that NECA increases the release of IL-19 from HBECs via
activation of A2B receptors, and IL-19 in turn activates human mono-
cytes to release TNF-␣, which upregulates A2B receptor expression
in HBECs. The results of this study suggest that there is a novel
pathway whereby adenosine can initiate and amplify an inflamma-
tory response which might be important in pathogenesis of inflam-
matory lung diseases.
Keywords: adenosine; TNF-␣; bronchial epithelial cells; IL-19
Airway epithelium is known to play an important role in airway
defense mechanism via a mucociliary system and as a mechanical
barrier. Recent studies further indicate that the epithelium is
not only a barrier but can also actively generate a range of
molecules, including lipid mediators, growth factors, and a vari-
ety of cytokines/chemokines, that are important in the inflam-
matory and remodeling responses that occur in the lungs (1).
In asthma, the epithelium seems more sensitive and responds
abnormally to various stimuli (2). In addition, airway epithelial
cells are able to interact with immune and inflammatory cells
via direct adhesion as well as by releasing mediators including
cytokines (1). Thus, the epithelium is actively involved as regula-
tor of airway inflammatory responses important in the pathogen-
esis of airway disorders.
Adenosine is a nucleoside that can elicit many physiologic
and pathophysiologic responses by activating one or more of its
four subtypes of G protein–coupled receptors (A1, A2A, A2B, and
A3) on target cells. Adenosine has been proposed to contribute
(Received in original form December 21, 2005 and in final form June 1, 2006 )
An abstract (less than 250 words) related to this study has been published
for the American Thoracic Society International Conference in 2005: Zhong H,
Wu Y, Belardinelli L, Zeng D. Adenosine indirectly activates monocytes by releasing
IL-19 from human bronchial epithelial cells. Proc Am Thorac Soc 2005;2:A110.
Correspondence and requests for reprints should be addressed to Hongyan Zhong,
Ph.D., CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto, CA 94304. E-mail:
hongyan.zhong@cvt.com
Am J Respir Cell Mol Biol Vol 35. pp 587–592, 2006
Originally Published in Press as DOI: 10.1165/rcmb.2005-0476OC on June 15, 2006
Internet address: www.atsjournals.org
to the pathogenesis of asthma and chronic obstructive pulmonary
disease (COPD) (3). This hypothesis is based on the findings
that the interstitial concentration of adenosine is elevated in the
lungs of individuals with asthma (4) and inhaled adenosine
causes bronchoconstriction in patients with asthma (5). The
bronchoconstrictive effect of adenosine has been attributed to
the activation of lung mast cells (6, 7). In addition to the mast
cells (8, 9), adenosine has been found to modulate the functions
of other inflammatory cells such as lymphocytes (10), eosinophils
(11, 12), neutrophils (13), and macrophages (14), and lung cells
such as bronchial smooth muscle cells (15, 16) and fibroblasts (17).
The physiologic effects of adenosine on lung epithelial cells have
also been investigated. Adenosine is able to modulate the activity
of ion channels (18–20), and induce fibronectin (21) and mucin
gene expression (22). However, it is unknown whether and how
adenosine affects the release of inflammatory cytokines from epi-
thelial cells and epithelial cell–inflammatory cell communication.
In this study, we used the primary cultured human bronchial
epithelial cells (HBECs) as the model system. The objectives of
this study were to determine (1 ) whether adenosine affects the
release of inflammatory cytokines from bronchial epithelial cells,
(2 ) what the potential effects of these cytokines on lung inflam-
mation might be, and (3 ) which adenosine receptor subtype is
responsible for the effect of adenosine.
MATERIALS AND METHODS
Materials
A selective antagonist to the A2B receptor (CVT-6694) was synthesized
by the Department of Bio-Organic Chemistry at CV Therapeutics Inc.
(Palo Alto, CA), and was described in our previous publication (15). All
other reagents, such as rolipram, forskolin, 5⬘-(N-ethylcarboxamido)-
adenosine (NECA), and adenosine deaminase (ADA), were purchased
from Sigma (St. Louis, MO) unless otherwise stated.
Cell Culture
Primary cultured normal HBECs were obtained from Clonetics (San
Diego, CA) and cultured using bronchial epithelial cell growth medium
(Clonetics). HBECs were routinely grown in a humidified incubator with
5% CO2 at 37⬚C. Cells from three different donors and from passages 2–3
were used in the following studies. THP-1 cells were purchased from
ATCC (Manassas, VA) and cultured according to ATCC’s instructions.
Stimulation of HBECs
HBECs were seeded into 12-well tissue culture plates at a density of
1 ⫻ 105 cells/well and allowed to adhere overnight and reach ⵑ 90%
confluence. Cells were washed twice in HEPES-buffered saline, and
cultured in bronchial epithelial cell basal medium (Clonetics) containing
various agonists or antagonists of AdoRs for 1 or 24 h.
RNA Extraction and Real-Time RT-PCR
Total RNA was extracted from HBECs using the Stratagene Absolutely
RNA RT-PCR Miniprep Kit (Stratagene Corp., La Jolla, CA) followed
by DNase treatment to eliminate potential genomic DNA contamina-
tion. Real-time RT-RCR for adenosine receptors was performed as
previously described (15). Specific primers for IL-19 (forward: 5⬘-AAA
CAATCTCCCCAAGGTGGAT-3⬘; reverse: 5⬘-AGGAAATGCTGT
CAAGGTTTGC-3⬘), GRO-␤ (forward: 5⬘-TTTCTTCGTGATGACA
588 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 35 2006

TATCACATGT-3⬘; reverse: 5⬘-TCCTCAGCCTCTATCACAGTGG-

3⬘), and GRO-␥ (forward: 5⬘-TGCTTGTAGGGCATAATGCCT-3⬘;
reverse: 5⬘-GGGAAAGAGAAACGCTGCAG-3⬘) were designed using
Primer Express 2.0 (Applied Biosystems, Foster City, CA) following
the recommended guidelines based on sequences from GenBank. At
the end of the PCR cycle, a dissociation curve was generated to ensure
the amplification of a single product, and the threshold cycle times (Ct
values) for each gene were determined. Relative mRNA levels were
calculated based on the Ct values, normalized to ␤-actin in the same
sample, and presented as percentages of ␤-actin mRNA.

Measurement of cAMP Accumulation
Cells were harvested using 0.0025% trypsin and 2 mM EDTA in PBS,
washed and resuspended in phenol-free DMEM to a concentration of
5 ⫻ 105 cells/ml, and then incubated with 1 U/ml of ADA for 30 min
at room temperature. Cells were then treated with AdoR agonists,
antagonists, and forskolin in the presence of 50 ␮M of the phosphodies-
terase IV inhibitor, rolipram. After incubating for 15 min at 37⬚C, cells
were lysed and cAMP concentrations were determined using cAMP-
Screen Direct System (Applied Biosystems) according to the manufac-
turer’s instructions.
cDNA Array Analysis
Expression of inflammatory cytokines was determined using a cDNA
array kit (Cat. No. HS-033; SuperArray, Frederick, MD). The assay
was performed according to manufacturer’s instructions. Biotinylated
cDNA probes were generated from 1–2 ␮g of total RNA using GEArray
RT-labeling enzyme kit (SuperArray). The labeled cDNA probes were
then hybridized with gene-specific cDNA fragments spotted on nylon
membrane. The signals were detected with the chemiluminescent detec-
tion reagents. The relative expression level of each gene was analyzed
using GEArray analysis software (SuperArray).
Figure 1. The mRNA levels of AdoR subtypes in HBECs. Total RNA iso-
lated from HBECs was subjected to real-time RT-PCR analysis. The rela-
tive levels of the AdoR transcripts are presented as percentages of the
␤-actin transcript. Data shown are averages ⫾ SEM (n ⫽ 6). nd denotes
not detected.
mined. NECA is a stable analog of adenosine, and it activates
all four AdoR subtypes including A2B receptors. As shown in
Figure 2A, NECA increased cellular cAMP accumulation in a
concentration-dependent manner, with potency (EC50 value) of
8.8 ⫾ 1.3 ␮M. In contrast, the A2A-selective agonist CGS-21680
(⭐ 10 ␮M) did not cause a significant increase in cellular cAMP
concentration. In addition, the A1-selective agonist, CPA (1 ␮M),
and the A3-selective agonist, IB-MECA (1 ␮M), failed to inhibit
the cellular cAMP accumulation caused by forskolin (10 ␮M,
Figure 2B). Because there is no selective agonist for A2B re-
ceptors, the effect of a selective antagonist to A2B receptors,
CVT-6694, on NECA-induced cellular cAMP accumulation was

Measurement of IL-19 and TNF-␣

ELISA for IL-19 was developed according to manufacturer’s protocol
(R&D Systems, Minneapolis, MN). Plates (96-well) were pre-coated
with 50 ng/well anti-human IL-19 Ab (R&D Systems). IL-19 was de-
tected using 100 ng/ml of biotinylated polyclonal IL-19 Ab (R&D sys-
tems), streptavidin-HRP, and TMB (tetramethylbenzidine) chromogen
(Biosource, Camarillo, CA). Serial dilutions of recombinant human IL-
19 (rhIL-19; PeproTech, Rocky Hill, NJ) was used as standard. The
detection limit of rhIL-19 was 0.2 ng/ml. The concentrations of TNF-␣
in the cell medium were determined using ELISA kits obtained from
Biosource according to the manufacturer’s instructions. The minimal
detection level of TNF-␣ with these kits was 1.7 pg/ml.

Statistical Analysis
Data are presented as mean ⫾ SEM of at least three separate experi-
ments. The statistical analysis was performed using ANOVA followed
by Bonferroni test. A P value of ⬍ 0.05 was considered significant.

RESULTS
Expression of AdoR Subtypes in HBECs
Real-time RT-PCR was performed to quantify the levels of
transcripts for AdoRs. Among the four subtypes, the A2B recep-
tor had the highest transcript level (0.49 ⫾ 0.04% of ␤-actin
expression) (Figure 1). Lower levels of A1 and A2A receptor
transcripts were also detected (0.0012 ⫾ 0.0002% and 0.0058 ⫾
0.0003% of ␤-actin, respectively), whereas the transcript for A3
receptors was below the detection level. Hence, the rank order
of AdoR mRNA levels was A2B ⬎ ⬎ A2A ⬎ A1 ⬎ ⬎ A3.
Figure 2. Effects of AdoR agonists and antagonist on cellular cAMP
In many cell types, activation of A2A or A2B receptors leads accumulation in HBECs. (A ) Concentration–response curves of CGS-
to increases in cellular cAMP accumulation, whereas activation 21680 (CGS, circles) and NECA in the absence (squares) or presence
of A1 or A3 receptors decreases cellular cAMP accumulation (triangles) of the A2B receptor antagonist CVT-6694 (1 ␮M). (B ) Lack of
caused by the adenylate cyclase activator, forskolin. To identify effect of CPA (1 ␮M) and IB-MECA (IM, 1 ␮M) on forskolin (Fsk,
the AdoR subtype(s) that are functionally expressed in HBECs, 10 ␮M)-induced cellular cAMP accumulation. Data shown are averages ⫾
the effects of a nonselective agonist NECA and several other SEM (n ⫽ 3). *P ⬍ 0.05, compared with control; #P ⬍ 0.05, compared
selective agonists on cellular cAMP accumulation were deter- with NECA-treated cells in A.
Zhong, Wu, Belardinelli, et al.: Adenosine Induces IL-19 and Activates Monocytes
determined. CVT-6694 has a high affinity for the A2B receptor
(Ki ⫽ 7 nM) and very low affinity for three other AdoR subtypes
(Ki values are ⬎ 5 ␮M for A1, A2A, and A3 receptors) (15, 17). As
shown in Figure 2A, CVT-6694 (1 ␮M) significantly attenuated
NECA-induced cellular cAMP accumulation. Thus, using cellu-
lar cAMP concentration as readout for the functional expression
of AdoRs, the results indicate that A2B receptors are functionally
expressed in HBECs, whereas A1, A2A, or A3 receptors are not.
Effects of NECA on Expression of Inflammatory Cytokines
The effect of NECA on the gene expression of the inflammatory
cytokines was determined using a cDNA array containing 85
inflammatory genes involved in asthma. Among the 85 genes on
the microarray, GRO-␤, GRO-␥, and IL-19 genes were increased
above 2-fold by NECA (data not shown).
To confirm and quantify NECA-induced expression of
GRO-␤, GRO-␥, and IL-19, gene-specific real-time RT-PCR
was performed on HBECs treated with NECA (10 ␮M) for
1 h. As shown in Figure 3, NECA increased mRNA expression
of IL-19, GRO-␤, and GRO-␥ up to 4.3- ⫾ 0.9-, 5.6- ⫾ 0.6-, and
4.3- ⫾ 0.6-fold above control levels, respectively.
Activation of the A2B Receptor Increased the Release of
IL-19 from HBECs
Recent publications suggesting a potential role of IL-19 in lung
inflammation prompted us to determine the effect of NECA on
IL-19 release. IL-19 concentrations in the culture media from
cells treated with NECA were measured using ELISA. The
levels of IL-19 in the media from vehicle- and NECA-treated
cells for 24 h were 19.2 ⫾ 6.3 ng/ml and 89.2 ⫾ 20.2 ng/ml,
respectively (Figure 4). Hence, NECA (10 ␮M) caused 4.6- ⫾
1.1-fold increase of IL-19 release compared with vehicle-treated
cells. To determine the role of A2B receptors in NECA-induced
IL-19 production, cells were incubated with CVT-6694 (1 ␮M)
together with NECA. The A2B receptor antagonist CVT-6694
(1 ␮M) reduced the NECA-increased IL-19 release by 88.9 ⫾
0.5%. These results confirmed that NECA-induced IL-19 release
is mediated by the A2B receptor subtype.
Effects of IL-19 Released from HBEC on Activation of a
Human Monocytic Cell Line (THP-1)
To determine whether the amount of IL-19 released from HBEC
can activate inflammatory cells, the effect of IL-19 alone or the
conditional medium from NECA-treated HBEC on the release
of TNF-␣ from a monocytic cell line (THP-1) was determined.
The basal level of TNF-␣ in the media from vehicle-treated
(24 h) THP-1 cells was 7.5 ⫾ 0.5 pg/ml. IL-19 (100 ng/ml) increased
the concentrations of TNF-␣ in the media by 4.3- ⫾ 0.4-fold
(Figure 5A). In contrast, GRO-␤ (100 ng/ml), GRO-␥ (100 ng/ml),
Figure 3. Effects of NECA on
the mRNA levels of GRO-␤,
GRO-␥, and IL-19 determined
using real-time RT-PCR.
HBECs were incubated with
NECA (10 ␮M) for 1 h. Cells
incubated with vehicle were
used as control. The expres-
sion levels of the cytokines
were normalized to that of ␤-actin and are presented as the ratios of
the expression level in NECA-treated cells versus that in vehicle-treated
cells. The expression levels of GRO-␤, GRO-␥, and IL-19 in control cells
are 0.020 ⫾ 0.003%, 0.008 ⫾ 0.002%, and 0.168 ⫾ 0.025% of
␤-actin, respectively. Data shown are averages ⫾ SEM (n ⫽ 3).
589
Figure 4. Effect of NECA
on the release of IL-19 by
HBECs. Cells were treated
with vehicle, NECA in the
absence or presence of
CVT-6694 for 24 h. Media
from treated cells were
collected, and the con-
centrations of IL-19 were
determined using ELISA.
CVT-6994 alone did not
change the release of IL-19 by HBECs (data not shown). Data shown
are the averages ⫾ SEM (n ⫽ 3). *P ⬍ 0.05, compared with control;
#
P ⬍ 0.05, compared with NECA-treated cells.
or NECA (10 ␮M) alone had no significant effect on TNF-␣
release from THP-1 cells (Figure 5A). These data demonstrate
that IL-19, but not NECA or GRO-␤/␥, was able to activate
THP-1 cells. Furthermore, the conditional media collected from
HBECs treated with vehicle (control-M) or NECA (NECA-M)
for 24 h were used to incubate THP-1 cells for an additional
24 h. NECA-M caused a 4.6- ⫾ 1.0-fold increase in TNF-␣ release
from THP-1 cells compared with control-M. This effect was
completely abolished by an IL-19–neutralizing Ab (Figure 5B).
These results suggest that IL-19 released from NECA-stimulated
HBECs is able to and sufficient to activate THP-1 cells.
Effects of TNF-␣ on Expression of AdoRs in HBECs
To further explore the interaction between epithelial cells and
monocytes, the effect of TNF-␣ on AdoR expression in HBECs
was determined using real-time RT-PCR. TNF-␣ caused a sig-
nificant time-dependent increase in mRNA expression of A2B
Figure 5. Effects of cytokines and conditional medium from HBECs on
TNF-␣ release from THP-1 cells. (A ) THP-1 cells were incubated with
vehicle (control), GRO-␤ (100 ng/ml), GRO-␥ (100 ng/ml), IL-19
(100 ng/ml), or NECA (10 ␮M) for 24 h. (B ) Conditional medium col-
lected from HBECs that were treated with vehicle (control-M) or NECA
(NECA-M) for 24 h was used to incubate THP-1 cells in the absence or
presence of polyclonal IL-19 Ab (1 ␮g/ml) for an additional 24 h. The
concentration of TNF-␣ in media was measured using ELISA. Data shown
are the averages ⫾ SEM (n ⫽ 4). *P ⬍ 0.05, compared with control
(A ) or control-M (B ); #P ⬍ 0.05, compared with NECA-M in B.
590 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 35
receptors (Figure 6). After 24 h incubation with TNF-␣, mRNA
level of A2B receptors was increased to 3.1 ⫾ 0.3 fold above
basal levels (Figure 6). Incubation of TNF-␣ for 5 and 24 h
also significantly upregulated the mRNA levels of A1 and A2A
receptors up to 2.0- ⫾ 0.3- and 1.6- ⫾ 0.1-fold above basal levels,
although the expression of both subtypes of receptors was still
very low (below 0.01% of ␤-actin mRNA level). The mRNA
expression of the A3 receptors remained below the detection
level after TNF-␣ treatment (Figure 6).
DISCUSSION
The main objective of this study was to determine the effect of
adenosine on the inflammatory responses mediated by epithelial
cells. The main findings of our study are: (1 ) A2B receptors
are the predominant subtype of AdoRs expressed in primary
HBECs; (2 ) activation of A2B receptors on HBECs increases
the release of IL-19, which is able to and sufficient to induce
TNF-␣ production from human monocytes; and (3 ) TNF-␣ can
upregulate the expression of A2B receptors in HBECs. These
findings support the hypothesis that adenosine can initiate and
amplify inflammatory responses which might be important in
pathogenesis of inflammatory lung diseases.
Among the four subtypes of AdoRs, the A2B receptor has
the lowest affinity for the natural ligand, adenosine, requiring
at least micromolar concentrations of adenosine for its activation
(23). Hence, under normal physiologic conditions, adenosine
levels might be too low to activate the A2B receptor. However,
higher levels of adenosine have been detected during hypoxia,
ischemia, and inflammation (24–26). In addition, results of recent
studies (16, 17, 26, 27) suggest the A2B receptor expression can
markedly increase during inflammation and hypoxia. The molec-
ular mechanism of how A2B receptor expression is modulated
during inflammation is largely unknown. The results of the cur-
rent study demonstrate that TNF-␣, a well-known inflammatory
marker and mediator, upregulates the expression of A1, A2A,
and A2B receptors in HBECs. This is consistent with an earlier
report suggesting that TNF-␣ regulates the expression of A2A
and A2B receptors in microvascular endothelial cells (28).
It should be noted that the expression of A2B receptors are
upregulated in the lung of ragweed-challenged mice (27) and
IL-13–transgenic mice (26). More strikingly, increased expres-
sion of A2B receptors in the lung of IL-13–transgenic mice has
been localized in bronchial epithelium. On the other hand, re-
sults of a recent study suggested that the density of A2B receptor,
determined using saturation binding assays with an antagonist
radioligand, was significantly decreased in the lungs of patients
Figure 6. Effect of TNF-␣
on the mRNA levels of
AdoR subtypes in HBECs.
HBECs were incubated
with TNF-␣ (5 ng/ml) for
0, 1, 5, and 24 h. The
relative levels of the
AdoR transcripts are pre-
sented as percentages of
the ␤-actin transcript.
Data shown are aver-
ages ⫾ SEM (n ⫽ 3). nd
denotes not detected.
*P ⬍ 0.05, compared
with 0 h.
2006
with COPD compared with the control group, whereas the ra-
dioligand affinity for A2B receptors was not altered (29). How-
ever, it is not entirely clear which cells contribute to the decrease
of A2B receptors in COPD, how the expression of A2B receptor
is regulated in allergic diseases versus COPD, and the precise
role of A2B receptor in these diseases. Interestingly, in the same
study, the expression level of the A2B receptor was found to be
highest in mast cells and macrophages. Regardless, the authors
concluded that the A2B receptor might play an important role
in the pathogenesis of COPD. Therefore, future studies to deter-
mine the effect of A2B antagonist in COPD would be of interest.
Functional expression of the A2B receptor in airway epithelial
cells has been reported. This receptor has been shown to mediate
adenosine-modulated ion transport (18, 19); however, the func-
tion of A2B receptor in mediating cytokine release is not known.
The results of our study showed that activation of A2B receptors
upregulates the expression of proinflammatory cytokines such
as IL-19, GRO-␤, and GRO-␥. GRO-␤ (CXCL2) and GRO-␥
(CXCL3) are CXC chemokines. The functions of these two
chemokines are similar to IL-8 (CXCL8), which belongs to the
same chemokine family. GRO-␤ and GRO-␥ chemoattract and
activate neutrophils (30), and promote angiogenesis by activating
the CXCR2 receptor on endothelial cells (31). Because neutro-
phils have been implicated in the pathogenesis of inflammatory
lung diseases including asthma and COPD, and because CXC
chemokine–mediated angiogenesis is associated with idiopathic
pulmonary fibrosis (32), CXCR2 is a potential therapeutic target
for these diseases. A more important finding of the present study
is that NECA increases the release of IL-19 from HBECs.
IL-19 belongs to the IL-10 family, which includes IL-10, IL-19,
IL-20, IL-22, IL-24, and IL-26 (33). In contrast to the pleiotropic
role of IL-10, IL-19 has been shown to have mainly proinflam-
matory roles. IL-19 stimulates T cells to produce IL-4 and
IL-13 (34) and alters the balance of Th1/Th2 in favor of Th2.
IL-19 also induces production of IL-6, TNF-␣, and reactive oxy-
gen species from mouse monocytes (35). IL-19 level is elevated
in the serum of patients with asthma compared with healthy
control subjects, and its transcript is also increased in the lung
of an allergen-challenged mouse model (34). Because adenosine
is elevated in the asthmatic lung to hundred-micromolar range
(4), and at this level it can increase the release of IL-19 from
HBECs (data not shown), it is plausible to postulate that elevated
adenosine in the asthmatic lung may contribute to the elevation
of IL-19. Interestingly, recent studies have shown that elevated
adenosine play a key role in the upregulation of IL-13 and several
other cytokines in mouse models of pulmonary diseases (26).
Consistent with earlier reports on the proinflammatory roles
of IL-19, the results of our study demonstrate that IL-19 released
by bronchial epithelial cells can activate human monocytes. In
the present study, conditional medium of NECA-stimulated
HBECs was able to activate THP-1 cells. This effect is not due
to the activation of AdoRs on THP-1 cells, because this effect was
completely abolished by the IL-19–neutralizing Ab and NECA
alone did not stimulate TNF-␣ production from THP-1 cells.
Furthermore, the results in this study suggest that elevated
TNF-␣ could have a positive feedback on the expression of the
adenosine receptors on the epithelial cells. Hence, adenosine
may play an important role in the interaction between epithelial
cells and inflammatory cells in the airway.
Activation of the A2B receptor increases cAMP, which is
generally believed to elicit anti-inflammatory responses. How-
ever, proinflammatory roles of this receptor have been reported
(36). For example, in human mast cells (HMC-1), activation of
A2B receptors increase cAMP (37) and also increase the release
of IL-4, IL-8, and IL-13 (8). A possible explanation is that A2B
receptor can couple to other cell signaling pathways, such as
Zhong, Wu, Belardinelli, et al.: Adenosine Induces IL-19 and Activates Monocytes
Figure 7. Schematic representation of an inflammatory response initi-
ated and amplified by adenosine involving HBECs and monocytes. Aden-
osine, via activation of the A2B receptors, increases the release of IL-19
from HBECs. IL-19, in turn, stimulates human monocytes to release
TNF-␣. TNF-␣ upregulates A2B receptor expression in HBECs.
Ca/PLC, and MAP kinases (38), and these pathways are likely
to mediate the proinflammatory effect of adenosine or adenosine
analog via activation of A2B receptors. However, it remains to
be established which signaling pathway is responsible for the
A2B-mediated upregulation of IL-19. It should be noted that anti-
inflammatory roles of the A2B receptor have also been reported
in other in vitro studies (39–41). Therefore, further studies using
animal models or patients will be helpful to determine the role
of A2B receptors in the lung.
In summary, adenosine, IL-19, and TNF-␣ are important pro-
inflammatory mediators in lung inflammation. These mediators
may interact with one another to cause the alleviation or exacer-
bation of the disease. The results of the present study demon-
strated that the A2B receptor subtype is the predominant AdoR
expressed in HBECs. Activation of this AdoR subtype increased
the release of IL-19. IL-19 released from these cells, in turn,
stimulated the human monocytes to release TNF-␣. TNF-␣ is
able to upregulate the expression of A2B receptors in HBECs
(Figure 7). Thus, our findings provide a novel mechanism that
could explain the interactions among adenosine, IL-19, and
TNF-␣ in the initiation, maintenance, and amplification of in-
flammatory responses. In addition, our findings suggest that the
A2B receptor might be a novel therapeutic target for inflamma-
tory lung diseases.
Conflict of Interest Statement : H.Z., Y.W., L.B., and D.Z. are employees of CV
Therapeutics, Inc. (CVT) and own stock and stock options in this company. CVT
has patented numerous A2B antagonists, and one such antagonist is currently
being developed for the treatment of asthma.
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