Results presented in this study demonstrate that the metabolic disturbances associated with ADA deficiency in mice result in abnormal lung development and the promotion of lung inflammation and damage. ADA-deficient mice exhibited alveolar defects that were overcome by genetically restoring ADA enzymatic activity to these animals. In addition, lowering ADA substrates in the lung using enzyme therapy reversed lung eosinophilia and mucus production. The ADA substrates adenosine and 2′-deoxyadenosine both have potent cellular signaling properties, some of which have been implicated to play a role in lung inflammation. These mice will therefore serve as a useful in vivo model system in which to study the role of purinergic signaling in aspects of lung development and disease.
Defects in alveogenesis have been noted in mice deficient in various growth factor signaling pathways, including fibroblast growth factor 42
, platelet-derived growth factor 43
, and transforming growth factor β 44
signaling pathways. In addition, overexpression of cytokines such as IL-11 45
and IL-13 46
in the lungs of mice results in defects in alveogenesis. These findings suggest that this stage of lung development is influenced by complex signaling pathways. Developmental analysis of lung structure in ADA-deficient mice revealed a defect in alveolarization. This defect preceded the onset of lung inflammation. The pronounced elevations of adenosine in ADA-deficient lungs suggested that perturbations in adenosine signaling may play a role in the alveolar defect seen. Nothing is known with regard to the expression of adenosine receptors during lung development, and examining the expression of adenosine receptors in normal and ADA-deficient lungs will help clarify the role of adenosine signaling during normal and abnormal alveogenesis.
ADA-deficient mice develop and succumb to severe pulmonary inflammation and lung damage by 3 wk of age 15
. Characterization of the inflammation revealed a large accumulation of activated macrophages throughout the lungs and an infiltration of eosinophils around pulmonary blood vessels and bronchial airways. The inflammation was progressive, with no inflammation evident until postpartum day 10, after which lung inflammation increased in severity. This inflammation was associated with a pronounced increase in mucus production and a marked increase in lung adenosine levels. The observation that lowering adenosine levels improved pulmonary inflammation and mucus production suggested that adenosine may mediate these processes. Consistent with this suggestion is the extensive literature base showing that adenosine plays a role in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (for a review, see reference 20). The exact functions that this signaling nucleoside plays in lung disease are not known, but they likely depend on the type of inflammatory cells present and the distribution of adenosine receptors on these cells. The ability to control adenosine levels in ADA-deficient mice using enzyme replacement therapy will provide a useful model for examining the influence of adenosine on different inflammatory cells in vivo.
Eosinophils have emerged as a major inflammatory cell type in asthma, and an increase in eosinophils is often observed in the lungs of asthmatics 47
. These cells can release mediators that contribute to the airway damage often associated with asthma such as bronchial epithelial cell damage and the stimulation of mucus production 4849
. The accumulation of eosinophils in the lungs of ADA-deficient mice may be responsible for the increased mucus production seen. This is supported by the observation that decreasing the number of eosinophils in the lungs of ADA-deficient mice using ADA enzyme therapy also resulted in decreased mucus production. Alternatively, the decreased mucus production may be a direct effect of lowering lung adenosine levels since adenosine signaling has been demonstrated to increase mucus secretion in a canine mucus model 50
. Increased mucus production in ADA-deficient mice was not associated with an increase in IL-4 in the BALF, suggesting the production of mucus in this model was not IL-4 dependent. Whether the mucus production was mediated by other Th2 cytokines such as IL-13 or IL-9, or by eosinophil-derived mediators or adenosine itself, remains to be determined.
The involvement of adenosine signaling in eosinophil biology has been demonstrated. The A3 adenosine receptor is expressed on human eosinophils that accumulate in the lung 23
, and engagement of this receptor on eosinophils is thought to mediate the release of Ca2+
from intracellular stores 29
, inhibit superoxide release 28
, and inhibit eosinophil chemotaxis, which may serve a pro- or antiinflammatory role 2327
. Whether or not the A3 receptor is expressed in murine eosinophils and in the lungs of ADA-deficient mice is currently under investigation. However, the large increase in lung eosinophils in ADA-deficient mice and the ability to rapidly reverse this eosinophilia by lowering adenosine concentrations suggest that adenosine signaling may be mediating the lung eosinophilia occurring in these mice.
In addition to an increase in eosinophils, the number and activation of alveolar macrophages were greatly increased in the lungs of ADA-deficient mice. Engagement of adenosine receptors on macrophages elicits both pro- and antiinflammatory events, including the inhibition of TNF-α expression 5152
and nitric oxide production 51
, increased production of IL-10 51
, increased differentiation of monocytes into macrophages 313253
, increased rates of phagocytosis 32
, and stimulation of giant cell formation 30
. Therefore, the increased number and activity of alveolar macrophages and giant cells in ADA-deficient mice may result from aberrant adenosine signaling brought about by persistent elevations in lung adenosine levels. Activated macrophages can contribute to alveolar airway damage 54
. The enlargement of the alveolar airways in ADA-deficient mice is associated with a defect in alveogenesis. However, the enlargement of these airways is progressive from postpartum day 15 to 18, suggesting that damage to these airways is also occurring. The large number of activated macrophages found in the alveolar airways of these mice may contribute to the increased damage seen. The determination of proteolytic enzyme production by these macrophages and the influence of adenosine signaling on this process will help clarify the role of activated macrophages in this model. The number of macrophages found in the lungs of ADA-deficient mice was not altered 72 h after PEG-ADA treatments, nor was there any improvement in the alveolar damage seen. The persistence of macrophages may indicate that these cells are actively involved in the clearance of cellular debris resultant of the severe eosinophilia and tissue damage seen. The ability to control adenosine levels using varying doses of PEG-ADA will provide a useful tool to explore the involvement of adenosine signaling in both eosinophil and macrophage function.
ADA deficiency in humans is most commonly associated with a combined immunodeficiency 7
. However, additional phenotypes have been described, including bone and renal abnormalities 12
, hepatocellular damage 13
, neurological disorders, and pulmonary insufficiencies 7
. Although the treatment of ADA-deficient patients with PEG-ADA has rapid beneficial effects on some of these phenotypes 1455
, it is still not clear whether they are a primary consequence of the ADA deficiency. Here, we demonstrate that ADA enzyme therapy can rapidly reverse respiratory distress in ADA-deficient mice in conjunction with lowering lung adenosine and 2′-deoxyadenosine levels, suggesting that the respiratory distress seen in this model is a direct consequence of ADA deficiency. This suggestion is supported by observations that the ADA enzyme therapy protocol used did not improve the immune status in these animals. Pulmonary insufficiency is common in ADA-deficient patients, and these insufficiencies are most often attributed to bacterial or viral pneumonia that arises from a compromised immune system. However, in many cases of interstitial pneumonia an organism cannot be isolated 7
. Our observations in ADA-deficient mice suggest that it is possible that the adenine metabolic disturbances in ADA-deficient patients may directly contribute to the pulmonary insufficiency occurring in this population.
Some ADA-deficient patients have been shown to have elevated levels of IgE, eosinophilia, and an increased incidence of asthma 83839
. These individuals are typically patients with delayed or late onset ADA deficiency and thus have milder forms of immunodeficiency 78
. ADA-deficient mice exhibited an increase in serum IgE, eosinophilia, and developed lung inflammatory changes, suggesting that they resemble patients with a less severe form of ADA deficiency. Consistent with this is the observation that the immunodeficiency seen in ADA-deficient mice is not as severe as that seen in completely ADA-deficient humans 15
. However, the immunodeficiency seen in these animals must be considered when trying to understand the nature of the lung inflammation seen. Lung eosinophilia is often associated with a Th2 cytokine profile 56
. However, there was not a robust Th2 cytokine profile in BALF collected from ADA-deficient mice. Since Th2 cytokines such as IL-4 are produced largely by CD4 T cells, the immunodeficiency seen in ADA-deficient mice may impact the relative capability to generate Th2 cytokines. Alternatively, the absence of a robust Th2 response suggests that other signaling pathways are involved in mediating the lung eosinophilia seen.
In conclusion, by deleting the enzyme responsible for controlling the levels of adenosine and 2′-deoxyadenosine, we have generated animals that exhibit adenine metabolic disturbances in association with alveolar defects and the development of severe pulmonary inflammation. Lung eosinophilia was reduced and the animals were rescued from respiratory distress by lowering adenosine and 2′-deoxyadenosine levels using ADA enzyme therapy. Although 2′-deoxyadenosine–mediated effects on this phenotype cannot be ruled out, there is substantial evidence to suggest adenosine signaling may be playing an important role in the type of inflammation and tissue damage seen 20
. Defining the adenosine receptors expressed on eosinophils, macrophages, and in the lungs of ADA-deficient mice, and using pharmacological and genetic technologies to assess their function, will help us to understand how adenosine influences lung inflammation in this model. This may in turn help guide new therapies for the treatment of lung conditions in which eosinophils and macrophages are thought to mediate damage, including asthma, idiopathic eosinophilic lung inflammation, chronic obstructive pulmonary disease, and emphysema. The correlation of increased lung adenosine and asthma 21
and the ability to relieve lung eosinophilia in mice by lowering adenosine levels raise the possibility that ADA enzyme therapy may be beneficial in the treatment of eosinophilic lung inflammation. Using ADA-deficient mice as a testing ground to understand the basis for adenosine-dependent lung eosinophilia will aid in evaluating the efficacy of such therapies.