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Departments of
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Biochemistry and Molecular Biology and
Pediatrics, University of Texas Health Science Center, Houston Medical School, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenosine has been implicated to play a role in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (7, 8). Asthmatics have elevated lung adenosine levels (9), and adenosine receptor transcripts are increased in inflamed lungs (10). Many cell types that play important roles in the exacerbation of asthma express adenosine receptors and have demonstrated activities through these receptors. These include mast cells (11, 12), lymphocytes (6), eosinophils (10), neutrophils (13), macrophages (14, 15), and airway epithelial cells (16). Further evidence linking adenosine signaling to asthma comes from reports demonstrating that asthmatics challenged with adenosine elicit a pronounced bronchoconstriction, whereas nonasthmatics show no response to such challenges (17, 18). Whether the bronchoconstrictive actions of adenosine have a direct effect on airway smooth muscle cells or involve the stimulation of mast cell degranulation is still controversial. However, it is clear that this action involves adenosine receptor signaling, because adenosine-induced bronchoconstriction can be inhibited by broad-spectrum adenosine receptor antagonists such as theophylline (19, 20). Collectively, this evidence suggests that adenosine signaling plays a role in the regulation of pulmonary inflammation and physiology.
Despite the extensive data implicating adenosine in asthma, a clear understanding of its actions in the inflamed lung is lacking. This is due in part to the absence of adequate animal models with which to examine the effects of endogenously generated adenosine on lung inflammation and physiology. Toward this end, we have been studying the effects of endogenously elevated adenosine in mouse models that have been engineered to lack the purine catabolic enzyme adenosine deaminase (ADA),3 which is one of the enzymes responsible for controlling adenosine levels in tissues and cells. Completely ADA-deficient mice die from respiratory distress at 3 wk of age (21). The pulmonary phenotype in these mice is characterized by defects in alveolar development early in life, followed by the development of severe lung inflammation and airway occlusion (21). The major inflammatory component found in the lungs is the accumulation of activated alveolar macrophages and the infiltration of eosinophils. This is followed by mucus hypersecretion and airway occlusion that is believed to lead to asphyxiation and death of the animals by 3 wk of age. Adenosine accumulates to very high levels in the lungs of these mice and the lung eosinophilia and mucus hypersecretion seen can be reversed by lowering adenosine levels using ADA enzyme therapy (21). The similarity of this phenotype to that seen in asthmatics, together with the association of inflammation and damage with elevated adenosine, makes this model useful for studying the role of elevated adenosine in lung inflammation and damage. However, the severe alveolar defects and the death of these animals at an early age have made assessment of the impact of elevated adenosine on airway physiology difficult.
In the current study we have characterized the impact of endogenously elevated lung adenosine on airway inflammation and physiology in a mouse model of partial ADA deficiency. Partially ADA-deficient mice were generated by the ectopic expression of an ADA minigene in the gastrointestinal tract of otherwise ADA-deficient mice. These mice do not exhibit the defects in alveogenesis seen in completely ADA-deficient mice. However, partially ADA-deficient mice developed lung inflammation and damage at a much later stage than that seen in completely ADA-deficient mice, and died from respiratory distress at 4–5 mo of age instead of 3 wk of age. Associated with this phenotype was a progressive increase in lung adenosine levels. Examination of airway physiology at 6 wk of age revealed an increase in airway responsiveness. These changes were shown to be dependent on elevated adenosine in that treatment with ADA enzyme therapy reversed these features in conjunction with lowering lung adenosine levels. Furthermore, treatment with the broad-spectrum adenosine receptor antagonist theophylline prevented airway hyperresponsiveness, implicating the involvement of adenosine receptors. All four of the adenosine receptors were shown to be present in control lungs, and transcript levels for the A1, A2B, and A3 adenosine receptors were elevated in the lungs of partially ADA-deficient mice. Elevations in the expression of the C-C chemokine monocyte chemoattractant protein-3 (MCP-3) were detected in the bronchial epithelium, which may be important in the regulation of pulmonary inflammation and airway hyperresponsiveness in this model. Collectively, these findings suggest that elevations in endogenous adenosine can directly impact lung inflammation and physiology in partially ADA-deficient mice. This model will prove useful in the study of specific mechanisms through which adenosine signaling regulates airway inflammation and physiology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male mice homozygous for the null Ada allele and carrying a placental-specific ADA minigene that allows for prenatal rescue were intercrossed with females heterozygous for the null Ada allele (22). Southern blot analysis of genomic DNA obtained from tails at weaning was used to determine the genotypes of the resulting progeny (21). Wild-type mice and mice heterozygous for the null Ada allele were used as controls. Mice were housed in contaminant-controlled environments to minimize pathogen exposure.
Histological analysis
Age-matched control and experimental animals were anesthetized and sacrificed. The lungs were perfused with 5–10 ml of PBS containing heparin and then infused with 0.5 ml of fixative (4% paraformaldehyde) and fixed overnight at 4°C. Fixed lungs were rinsed in PBS, dehydrated through graded ethanol washes, and embedded in paraffin. Sections (5 µm) were collected on slides and stained with H&E (Shandon Lipshaw, Pittsburgh, PA) according to the manufacturer’s instructions, or subjected to immunohistochemistry.
Airway physiology
Airway hyperresponsiveness was assessed by measuring the
responsiveness to 
-methylcholine (methylcholine) in conscious,
unrestrained mice using a whole-body noninvasive plethysmograph (Buxco
Electronics, Troy, NY) as described (23). This system
estimates total pulmonary airflow in mice using a dimensionless
parameter known as enhanced pause (Penh). Pressure differences were
used to extrapolate Penh values, which are a function of the sum of the
airflows in the upper and lower respiratory tracts during a respiratory
cycle. This parameter has been shown to correlate with airway
resistance measured by invasive techniques (23). Baseline
Penh was determined by exposing mice to nebulized saline for 2 min and
then recording and averaging Penh values for 3 min. The mice were then
exposed to increasing concentrations of aerosolized methylcholine
dissolved in saline. Methylcholine was aerosolized using an ultrasonic
nebulizer, and the aerosol was drawn through the chamber at a constant
rate for 2 min, after which Penh values were taken for 3 min and
averaged. Methylcholine response curves for control animals were
consistent with those shown previously for naive 129 mice
(24).
Quantification of lung adenosine levels
Mice were anesthetized, the thoracic cavity was exposed, and the lungs were rapidly removed and frozen in liquid nitrogen. Adenine nucleosides were extracted from frozen lungs using 0.4 N perchloric acid as described (25), and adenosine was separated and quantified using reversed phase HPLC.
ADA enzyme therapy and determination of ADA enzymatic activity
Polyethylene glycol-modified ADA (PEG-ADA, Adagen) was kindly provided by Enzon (Piscataway, NJ). Mice were treated i.p. with 6.25 U of PEG-ADA (26, 27) (1 U defined as the amount necessary to convert 1 µM of adenosine to inosine per min at 25°C) immediately following initial airway physiology evaluation (day 1), and on days 3 and 6 following this. On day 7 airway physiology was measured again and the mice were then sacrificed to obtain blood, tissues, and bronchial alveolar lavage fluid (BALF). Levels of ADA in the tissues and blood were measured by zymogram analysis using procedures previously described (21, 27).
Theophylline treatments
Six-week-old control and partially ADA-deficient mice were given two i.p. injections, 3 h apart, of theophylline (10 mg/kg body weight) or PBS. Based on pharmacokinetic data (not shown), this dosage regimen maintains plasma theophylline levels at between 10 and 40 µM, which is sufficient to antagonize adenosine receptors but not to inhibit phosphodiesterases (28). Airway physiology was examined 1 h after the second injection of theophylline or PBS.
Bronchial alveolar lavage and cellular differentials
Mice were anesthetized and tracheally intubated with a blunted 21-gauge needle. Lungs were lavaged with 1–2 ml of PBS and the recovered BALF was processed as previously described (21) for the determination of cellular differentials. Briefly, total cell counts were performed on initial lavaged aliquots and cellular differentials (200 cells/sample) were conducted on cell pellets resuspended in PBS, cytospun onto slides, and stained with Diff-Quick (Dade Behring, Newark, DE).
Immunohistochemical localization of MCP-3
Paraffin-embedded tissues were sectioned (5 µm), exposed to two changes of histoclear, and rehydrated in a series of graded alcohols to water. Ag unmasking was performed before MCP-3 localization using target retrieval solution following the manufacturer’s guidelines (DAKO, Carpinteria, CA). Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide for 5–10 min. MCP-3 immunohistochemistry and blocking procedures were followed according to the manufacturer’s guidelines using goat IgG VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA). MCP-3 localization was performed by incubating slides for 30 min at room temperature with a 1/4 dilution of goat anti-mouse MCP-3 Ab (R&D Systems, Minneapolis, MN) as primary Ab. After incubation with appropriate biotinylated secondary Abs, the slides were incubated with avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. The slides were developed using 3,3'-diaminobenzidine-tetrachloride (DAKO) for 7–10 min, dehydrated, and mounted.
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR was performed using a 7700
Sequence Detector (Applied Biosystems, Foster City, CA)
(29). Specific quantitative assays for the various
adenosine receptors and MCP-3 were developed using Primer Express
software (Applied Biosystems) following the recommended guidelines
based on sequences from GenBank. The sequences of all oligonucleotides
used are given in Table I
. Total
RNA was isolated from whole lung tissue using the TRIzol reagent from
Life Technologies/BRl followed by DNase treatment to eliminate
potential genomic DNA contamination. This was followed by cDNA
synthesis and real-time PCR using established protocols
(30). The resulting data were analyzed using sequence
detector system software (Applied Biosystems) with TAMRA as the
reference dye. The final data were normalized to -actin and are
presented as molecules of transcript per molecules of -actin x
100 (percentage of -actin). Results are expressed as the mean
± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ADA-deficient fetuses die prenatally due to the absence of ADA in
their placentas (22). ADA-deficient fetuses were rescued
from prenatal lethality by the expression of an ADA minigene in their
placentas (21, 22). With the loss of their placentas,
these rescued pups were completely ADA-deficient and went on to die at
3 wk of age from severe pulmonary inflammation and damage that was
linked to pronounced elevations in lung adenosine levels (21, 26). The severity of this phenotype and the early death of the
mice made examination of the impact of adenosine on lung physiology
difficult. We have recently identified an independent line of rescued
ADA-deficient mice that were not completely ADA-deficient, but
contained ectopic expression of the placental ADA minigene in the
gastrointestinal tract (Fig. 1). The
small and large intestines of these animals were the only tissues found
to express the ADA minigene on this otherwise ADA-deficient background.
Therefore, these animals were referred to as partially ADA-deficient
mice. Unlike completely ADA-deficient mice, which die at 3 wk of age
(21), partially ADA-deficient mice appeared relatively
healthy until the third month of life, when evidence of respiratory
distress became evident. Partially ADA-deficient mice died between 4
and 5 mo of age from apparent respiratory distress. Therefore,
expression of ADA in the gastrointestinal tract of otherwise
ADA-deficient mice could extend the lifespan of these mice, making them
amenable to the analysis of airway physiology.
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Histological analysis of the lungs of partially ADA-deficient mice
was conducted to characterize lung inflammation and damage in this
model. At 6 wk of age the lungs of partially ADA-deficient mice
appeared normal, except for the accumulation of macrophages in the
alveolar spaces (compare Fig. 2, A and B). At 10 wk of age, partially
ADA-deficient lungs exhibited severe lung inflammation, characterized
by the presence of activated alveolar macrophages, perivascular and
peribronchial accumulation of leukocytes, and pronounced alveolitis
(Fig. 2C). By 12 wk of age, the lungs of partially
ADA-deficient mice were severely inflamed and exhibited extensive
bronchial plugging (Fig. 2D). Therefore, partially
ADA-deficient mice develop progressive lung inflammation and damage,
but at a much slower rate than do completely ADA-deficient
mice.
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A major function of ADA is to control the levels of adenosine in
tissues and cells. Hence, adenosine levels are commonly elevated in
tissues of ADA-deficient humans and mice (21, 31). To
determine the status of adenosine in the lungs of partially
ADA-deficient mice, adenosine was quantified in whole lungs collected
from control and partially ADA-deficient mice at 6 and 15 wk of age
(Fig. 3A). At both stages,
there was a marked increase in the levels of adenosine in the lungs of
partially ADA-deficient mice. Furthermore, lung adenosine levels
increased as the lung phenotype progressed. 2'-Deoxyadenosine, another
substrate of ADA, was not detected in the lungs of control or partially
ADA-deficient mice at any stage examined (data not shown). These
findings demonstrated that adenosine levels were elevated in partially
ADA-deficient mice, even at stages when there was not overt lung
inflammation and damage.
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Exogenous adenosine has been shown to induce bronchoconstriction
in asthmatics (17) and various animal models
(32). To determine whether the endogenous accumulation of
adenosine in the lungs was associated with alterations in airway
physiology, airway hyperresponsiveness was measured in 6-wk-old
partially ADA-deficient mice. The 6-wk time point was chosen because at
this stage there was marked adenosine accumulation in the lungs (Fig. 3
A) but relatively little lung inflammation and damage (Fig. 1B). Partially ADA-deficient mice exhibited an 
50%
increase in baseline Penh over control animals (Fig. 4A). Furthermore, partially
ADA-deficient mice demonstrated airway hyperresponsiveness as
determined by a heightened responsiveness to methylcholine challenges
(Fig. 4B). To confirm that these alterations in airway
physiology were due to complications related to ADA deficiency, these
parameters were examined in 6-wk-old partially ADA-deficient and
control mice treated with ADA enzyme therapy (PEG-ADA treatment).
PEG-ADA treatments had no effect on baseline Penh (Fig. 4A)
or methylcholine responsiveness (Fig. 4B) in control mice.
However, this enzyme therapy was able to reverse both the increase in
baseline Penh (Fig. 4A) and the airway hyperresponsiveness
(Fig. 4B) seen in untreated partially ADA-deficient mice.
These findings demonstrated that the metabolic consequences associated
with ADA-deficiency in this model lead to increased baseline Penh and
airway hyperresponsiveness.
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In an attempt to correlate alterations in lung physiology with the
metabolic disturbances seen in the lungs of partially ADA-deficient
mice, adenosine levels were quantified in the lungs of 6-wk-old control
and partially ADA-deficient mice treated with PEG-ADA. Results
demonstrated that the ADA enzyme therapy was able to restore lung
adenosine to control levels (Fig. 3B). These findings
suggested that the increased baseline Penh and airway
hyperresponsiveness seen were directly due to the accumulation of
endogenous adenosine in the lungs of these animals.
Airway hyperresponsiveness in partially ADA-deficient mice is reversed following treatment with a nonselective adenosine receptor antagonist
The extracellular effects of adenosine are mediated through
cell-surface adenosine receptors (1). To begin to
determine whether the effects of elevated adenosine in the lungs of
partially ADA-deficient mice were due to engagement of adenosine
receptors, mice were treated with the broad-spectrum adenosine receptor
antagonist theophylline. Six-week-old control or ADA-deficient mice
were injected i.p. with either PBS or theophylline at a dosing regimen
designed to maintain serum theophylline levels below 40 µM, a
concentration favorable for adenosine receptor antagonism over
phosphodiesterase inhibition (28). Theophylline treatment
had no effect on baseline Penh (Fig. 5A) or methylcholine
responsiveness (Fig. 5B) in control mice, verifying a lack
of effect of this dosage on phosphodiesterase inhibition that could
potentially lead to bronchodilation. Theophylline treatment did not
lower the increased baseline Penh seen in partially ADA-deficient mice
(Fig. 5A); however, the airway hyperresponsiveness seen in
these mice was reversed following theophylline treatment (Fig. 5B). These findings suggested that signaling through
adenosine receptors was responsible for the adenosine-dependent airway
hyperresponsiveness seen in partially ADA-deficient mice.
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Mammals are known to possess four adenosine receptors, the
A1, A2A,
A2B, and A3 adenosine
receptors (1). Although these receptors have a wide tissue
distribution, little attention has been given to the relative
expression levels of these receptors in the lung. To quantify the
expression of adenosine receptors in the lung, specific primers and
probes were developed to conduct quantitative real-time RT-PCR
(Table I). Adenosine receptor transcript levels were quantified in
total cellular RNA extracts isolated from whole lungs of 6-wk-old
control or partially ADA-deficient mice or mice treated with PEG-ADA
(Fig. 6). All four of the adenosine
receptors were readily detected in the control lung, with
A1 receptor transcripts being the most abundant
(Fig. 6A). There was a significant increase in transcript
levels for the A1, A2B, and
A3 adenosine receptors in the lungs of partially
ADA-deficient mice, with the greatest relative increase being seen for
the A3 receptor (Fig. 6D). PEG-ADA
treatment had no effect on adenosine receptor transcript levels in
control lungs, and this treatment lowered transcript levels for only
the A3 receptor in partially ADA-deficient lungs
(Fig. 6D). Transcript levels for the adenosine receptors
were examined at 15 wk of age to determine whether increases in
adenosine receptor transcript levels were progressive in the lungs of
partially ADA-deficient mice. Transcript levels of the
A2A receptor did not change from 6 to 15 wk in
partially ADA-deficient lungs (data not shown). However, 6-wk
transcript levels for the A1 (0.8),
A2B (0.48), and A3 (0.15%
of -actin) receptors in partially ADA-deficient lungs increased to
1.72, 0.7, and 2.9% of -actin, respectively, in 15-wk-old partially
ADA-deficient lungs. These findings demonstrated that all of the
adenosine receptors were expressed in the mouse lung and that receptor
transcripts were elevated in the lungs of partially ADA-deficient mice,
suggesting the potential for increased adenosine receptor signaling in
this adenosine-rich environment.
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Airway hyperresponsiveness has been associated with airway
inflammation in humans and in various animal models (33).
Histological examination demonstrated that there was an increase in
alveolar macrophages in the lungs of partially ADA-deficient lungs at 6
wk of age (Fig. 2B). To more accurately characterize the
inflammatory changes seen in these mice the cellularity of BALF was
examined (Fig. 7). There was a >6-fold
increase in the number of alveolar macrophages in BALF collected from
partially ADA-deficient mice. This increase was reduced by 50%
following PEG-ADA treatment. A significant increase in lymphocytes was
also seen, but this increase was not altered by PEG-ADA treatment.
Interestingly, there was no increase in neutrophils or eosinophils in
the BALF of partially ADA-deficient mice at this stage. These findings
suggested that the elevation in alveolar macrophages may be related to
increases in lung adenosine levels.
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MCP-3 is a C-C chemokine that has been implicated in the
regulation of lung inflammation (34). We have recently
shown that expression of MCP-3 is closely linked to elevated adenosine
levels in completely ADA-deficient mice, suggesting adenosine signaling
may regulate the expression of this chemokine (35). In an
attempt to determine whether increased MCP-3 expression was also
associated with increased lung adenosine levels in partially
ADA-deficient mice, we examined the expression of MCP-3 in the lungs of
these mice using real-time RT-PCR (Fig. 8). Transcripts for MCP-3 were detected
in total cellular RNA isolated from control lungs. There was a 5-fold
increase in MCP-3 transcript levels in the lungs of partially
ADA-deficient mice, and this increase was reversed following PEG-ADA
treatment. MCP-3 immunoreactivity was localized to the bronchial
epithelium (Fig. 9). Furthermore, the
immunoreactivity was more intense in the bronchial epithelium of
partially ADA-deficient mice (Fig. 9B) as compared with the
immunoreactivity seen in the bronchial epithelium of control mice (Fig. 9A), or control mice treated with PEG-ADA (Fig. 9C), or partially ADA-deficient mice treated with PEG-ADA
(Fig. 9D). These findings suggested that the elevated levels
of adenosine in the lungs of partially ADA-deficient mice regulate the
expression of MCP-3 in the bronchial epithelium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenosine mediates its effects by engaging G protein-coupled receptors on the surface of target cells (1). We found that all four of the adenosine receptors were expressed in the normal mouse lung, and that transcript levels for the A1, A2B, and A3 adenosine receptors were elevated in partially ADA-deficient lungs. The greatest relative increase was seen for the A3 receptor. Interestingly, transcript levels for this receptor have also been shown to be elevated in the lungs of humans suffering from lung inflammation (10), where expression was localized to eosinophils. At the stage when receptor transcript levels were examined, there was not an increase in eosinophils in the lung. However, there was an increase in alveolar macrophages, on which A3 receptor activity has also been demonstrated (15, 36). Consistent with this, A3 receptor transcripts and the number of alveolar macrophages in the lungs of partially ADA-deficient mice decreased following ADA enzyme therapy. These data suggest that the increase in A3 transcript levels seen in 6-wk-old partially ADA-deficient mice may be accounted for by increased inflammation. This is supported by the observation that A3 receptor transcripts are even higher in the lungs of 15-wk-old partially ADA-deficient mice that exhibit heightened inflammation. Additional studies are needed to localize the expression of the A3 receptor in these mice to appreciate the functional significance of altered A3 transcript levels in this model.
Elevation in A1 and A2B adenosine receptor transcripts in inflamed lungs was a novel observation and raises the question as to whether or not these increases are directly associated with the changes in lung physiology seen in partially ADA-deficient mice. In support of this possibility, acute treatments with theophylline were able to reverse the adenosine-dependent effects on airway hyperresponsiveness. Theophylline is a broad-spectrum adenosine receptor antagonist that has activity at the murine A1, A2A, and A2B receptor, with little activity at the murine A3 receptor (8). Therefore, engagement of the A1, A2A, or A2B adenosine receptors may be responsible for the airway hyperresponsiveness seen. Whether the effect of receptor engagement in this model is a direct effect on airway smooth muscle or a secondary effect due to activation of other mediator cells (e.g., mast cells, nerves, airway epithelium, macrophages) cannot be determined from these studies. However, there is evidence that the A2B adenosine receptor is expressed in human airway smooth muscle cells (37), and the A1 adenosine receptor has been implicated in direct effects of adenosine on airway smooth muscle in rabbit models of asthma (38, 39). Clearly, determining the cell-specific expression of the adenosine receptors at both the message and protein level in the normal and inflamed lung will be paramount to understanding these issues. Furthermore, because the mechanisms of exogenous adenosine on bronchoconstriction differ depending on the species examined (32), it will be necessary to correlate our findings with adenosine receptor expression profiles in inflamed human lungs before attempting to design adenosine-based therapeutics.
MCP-3 belongs to a family of C-C chemokines that has attracted recent attention by its diverse role in lung inflammation (34, 40). MCP-3 was originally identified from cytokine-stimulated osteosarcoma cells by its ability to induce monocyte migration in vitro (41). Through its interactions with CCR-1 and CCR-3, MCP-3 functions as a potent monocyte and eosinophil chemoattractant (42, 43). We found that MCP-3 expression was elevated in the bronchial epithelium of partially ADA-deficient mice and that this elevation was reversed following ADA enzyme therapy. In addition, MCP-1, a closely related member of the C-C chemokine family, was also elevated in the bronchial epithelium of partially ADA-deficient mice (J. L. Chunn and M. R. Blackburn, unpublished data). These findings suggest that the regulation of chemokines may play an important role in the regulation of leukocyte accumulation in the lungs of these animals. In addition, these findings suggest that adenosine signaling may regulate the expression of MCP-3. Both the transcriptional and posttranscriptional regulation of MCP-3 expression may be mediated by cAMP signaling. The promoter region of the MCP-3 gene contains a cAMP response element (44) that may regulate transcription, and the 3' untranslated region contains an adenosine-uridine-rich element (45), which may mediate mRNA degradation (46). Because adenosine can regulate cAMP levels by engaging its receptors (2), one can speculate that increased MCP-3 expression in the airways of partially ADA-deficient mice may be due to the elevated adenosine levels seen. Localizing adenosine receptor expression to the airways of ADA-deficient lungs in association with MCP-3 will help to strengthen this hypothesis.
There is an outstanding literature base to suggest that adenosine plays a role in the regulation of inflammatory lung diseases such as asthma. In our model of partial ADA deficiency we have characterized specific changes in lung inflammation and physiology that are dependent on elevated lung adenosine levels. This model will prove useful for examining the specific mechanisms involved in these adenosine-dependent changes. As the specific expression patterns for the various adenosine receptors are elucidated, their function can be tested using specific pharmacologic or genetic approaches. For example, selective adenosine receptor antagonists can be used in this model in a similar manner as was the broad-spectrum adenosine receptor antagonist theophylline. In so doing, relevant targets for adenosine antagonism can be identified. In addition, mice deficient in the various adenosine receptors can be crossed onto the partially ADA-deficient background to assess the function of the various receptors in an adenosine-rich environment. The reversal of lung inflammation and airway hyperresponsiveness in association with lowering lung adenosine levels suggests that there may be an eventual therapeutic benefit to lowering the elevated levels of adenosine seen in the lungs of asthmatics by the use of ADA enzyme therapy. Continued work addressing this issue in ADA-deficient mouse models as well as other models of experimental asthma will be necessary before such ideas can be pursued.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael R. Blackburn, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston Medical School, 6431 Fannin Street, Houston, TX 77030. E-mail address: Michael.R.Blackburn{at}uth.tmc.edu
3 Abbreviations used in this paper: ADA, adenosine deaminase; BALF, bronchial alveolar lavage fluid; PEG-ADA, polyethylene glycol-modified ADA; MCP-3, monocyte chemoattractant protein-3; Penh, enhanced pause.
Received for publication June 6, 2001. Accepted for publication August 23, 2001.
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, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol. 157:4634.[Abstract]
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 T. Nakano, H. Inoue, S. Fukuyama, K. Matsumoto, M. Matsumura, M. Tsuda, T. Matsumoto, H. Aizawa, and Y. Nakanishi Niflumic Acid Suppresses Interleukin-13-induced Asthma Phenotypes Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1216 - 1221. [Abstract] [Full Text] [PDF]
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J. L. Chunn, A. Mohsenin, H. W. J. Young, C. G. Lee, J. A. Elias, R. E. Kellems, and M. R. Blackburn Partially adenosine deaminase-deficient mice develop pulmonary fibrosis in association with adenosine elevations Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L579 - L587. [Abstract] [Full Text] [PDF]
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J. Roman, H. N. Rivera, S. Roser-Page, S. V. Sitaraman, and J. D. Ritzenthaler Adenosine induces fibronectin expression in lung epithelial cells: implications for airway remodeling Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L317 - L325. [Abstract] [Full Text] [PDF]
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 A. Mejias, S. Chavez-Bueno, A. M. Rios, M. F. Aten, B. Raynor, E. Peromingo, P. Soni, K. D. Olsen, P. A. Kiener, A. M. Gomez, et al. Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency Antimicrob. Agents Chemother., November 1, 2005; 49(11): 4700 - 4707. [Abstract] [Full Text] [PDF]
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J. L. Chunn, J. G. Molina, T. Mi, Y. Xia, R. E. Kellems, and M. R. Blackburn Adenosine-Dependent Pulmonary Fibrosis in Adenosine Deaminase-Deficient Mice J. Immunol., August 1, 2005; 175(3): 1937 - 1946. [Abstract] [Full Text] [PDF]
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 E. Ghanem, C. Lovdahl, E. Dare, C. Ledent, B. B. Fredholm, J.-M. Boeynaems, W. Van Driessche, and R. Beauwens Luminal adenosine stimulates chloride secretion through A1 receptor in mouse jejunum Am J Physiol Gastrointest Liver Physiol, May 1, 2005; 288(5): G972 - G977. [Abstract] [Full Text] [PDF]
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H. W. J. Young, J. G. Molina, D. Dimina, H. Zhong, M. Jacobson, L.-N. L. Chan, T.-S. Chan, J. J. Lee, and M. R. Blackburn A3 Adenosine Receptor Signaling Contributes to Airway Inflammation and Mucus Production in Adenosine Deaminase-Deficient Mice J. Immunol., July 15, 2004; 173(2): 1380 - 1389. [Abstract] [Full Text] [PDF]
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S. Ryzhov, A. E. Goldstein, A. Matafonov, D. Zeng, I. Biaggioni, and I. Feoktistov Adenosine-Activated Mast Cells Induce IgE Synthesis by B Lymphocytes: An A2B-Mediated Process Involving Th2 Cytokines IL-4 and IL-13 with Implications for Asthma J. Immunol., June 15, 2004; 172(12): 7726 - 7733. [Abstract] [Full Text] [PDF]
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A. J. Archer, J. L. H. Cramton, J. C. Pfau, G. Colasurdo, and A. Holian Airway responsiveness after acute exposure to urban particulate matter 1648 in a DO11.10 murine model Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L337 - L343. [Abstract] [Full Text] [PDF]
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S. K. Banerjee, H. W. J. Young, A. Barczak, D. J. Erle, and M. R. Blackburn Abnormal Alveolar Development Associated with Elevated Adenine Nucleosides Am. J. Respir. Cell Mol. Biol., January 1, 2004; 30(1): 38 - 50. [Abstract] [Full Text] [PDF]
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 Z. H. Neemeth, S. J. Leibovich, E. A. Deitch, E. S. Vizi, C. Szabo, and G. Hasko cDNA Microarray Analysis Reveals a Nuclear Factor-{kappa}B-Independent Regulation of Macrophage Function by Adenosine J. Pharmacol. Exp. Ther., September 1, 2003; 306(3): 1042 - 1049. [Abstract] [Full Text] [PDF]
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H. Zhong, S. G. Shlykov, J. G. Molina, B. M. Sanborn, M. A. Jacobson, S. L. Tilley, and M. R. Blackburn Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor J. Immunol., July 1, 2003; 171(1): 338 - 345. [Abstract] [Full Text] [PDF]
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M. Fan, W. Qin, and S. J. Mustafa Characterization of adenosine receptor(s) involved in adenosine-induced bronchoconstriction in an allergic mouse model Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1012 - L1019. [Abstract] [Full Text] [PDF]
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 J. A. Elias, T. Zheng, C. G. Lee, R. J. Homer, Q. Chen, B. Ma, M. Blackburn, and Z. Zhu Transgenic Modeling of Interleukin-13 in the Lung Chest, March 1, 2003; 123(2007): 339S - 345S. [Abstract] [Full Text] [PDF]
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A. M. Khan, O. Elidemir, C. E. Epstein, K. P. Lally, H. Xue, M. Blackburn, G. L. Larsen, and G. N. Colasurdo Meconium aspiration produces airway hyperresponsiveness and eosinophilic inflammation in a murine model Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L785 - L790. [Abstract] [Full Text] [PDF]
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R. Polosa Adenosine-receptor subtypes: their relevance to adenosine-mediated responses in asthma and chronic obstructive pulmonary disease Eur. Respir. J., August 1, 2002; 20(2): 488 - 496. [Abstract] [Full Text] [PDF]
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0.05 using
Student’s t test.
