The importance of COX-1 and COX-2 in the resolution of airway inflammatory and functional responses to allergic stimuli has been demonstrated in murine models through the use of gene knockout strategies and by the administration of selective or nonselective pharmacological COX inhibitors (2
). To further study the contribution of COX-derived prostanoids to lung function and allergic airway inflammatory responses, we generated Tr mice in which human COX-1 was specifically targeted to airway epithelial cells using the murine CC10 promoter. In comparison to WT littermates, naive COX-1 Tr mice were shown to be normal in all aspects of baseline lung function and BAL fluid cellular characteristics, and histological examination did not reveal any evidence of morphological changes resulting from the presence of the transgene. Thus, constitutively increased airway COX-1 expression did not result in detectable alterations of lung function, cellularity, or structure under basal conditions. Interestingly, COX-1-deficient mice also do not demonstrate any alterations in baseline lung functional, cellular, or structural characteristics (2
), suggesting that COX-1-derived prostanoids are not crucial regulators of lung function under normal circumstances. However, naive COX-1 Tr mice had significantly elevated BAL fluid PGE2
content and demonstrated decreased airway responses to aerosol administration of the bronchoconstrictor methacholine, indicative of a beneficial effect of the transgene and its prostanoid products on airway responsiveness to cholinergic stimulation.
In healthy humans, inhalation of PGE2
results in bronchodilation (6
), whereas the analysis of Penh responses in conscious mice suggests that it exerts a bronchoconstrictive effect when administered alone in high doses and a bronchodilatory effect on airways challenged with a cholinergic agonist (20
). Interestingly, however, invasive analysis of respiratory mechanics does not support a constrictive effect of aerosolized PGE2
in naive mice (21
), but reveals that PGE2
decreases methacholine-induced airway constriction (20
). Similarly, we did not observe an alteration of basal airway tone in unchallenged naive COX-1 Tr mice compared to WT mice despite the increased airway PGE2
content, but found that methacholine-induced alterations of pulmonary mechanics were attenuated in COX-1 Tr mice. Our data support the concept that PGE2
is not a critical regulator of respiratory mechanics under basal conditions and are consistent with the results of a recent study (22
), which used genetic approaches to alter pulmonary PGE2
levels in mice via deletion or lung-specific overexpression of microsomal PGE synthase-1 and showed that neither of these manipulations altered baseline lung resistance.
Having demonstrated that COX-1 Tr mice had increased airway levels of PGE2 and a blunted response to inhalational challenge with a cholinergic bronchoconstrictor, we next examined the responses of these mice to allergic airway inflammation induced by sensitization and challenge with OVA. Significant increases of total BAL fluid cells, eosinophils, and levels of a variety of ILs implicated in allergic lung inflammation were observed in allergic WT and COX-1 Tr mice, and histological evidence of allergic inflammation was also noted. However, none of these parameters differed between WT and COX-1 Tr mice. Furthermore, invasive analysis of lung function revealed no differences between allergic WT and COX-1 Tr mice in any of the respiratory parameters measured, including responsiveness to inhaled methacholine. Thus, airway-specific overexpression of COX-1 and the resulting increase of airway PGE2 levels observed at baseline were not sufficient to alter the inflammatory and functional responses to allergen sensitization and challenge in this model.
We surmised that the lack of any measurable differences in airway inflammation and responsiveness was likely due in part to the comparable up-regulation of COX-2 that was observed in the lungs of allergic WT and COX-1 Tr mice, which augmented PG production and thereby masked any favorable effect of the COX-1 transgene. Interestingly, the BAL fluid PG profile in allergic mice differed considerably from that observed in naive animals. Levels of the proinflammatory and bronchoconstrictive PGF2α
, in addition to the anti-inflammatory and bronchodilatory PGE2
, were elevated in allergic COX-1 Tr mice compared with allergic WT mice, whereas in naive mice only PGE2
levels differed between the genotypes. Although positive feedback regulation of COX-2 expression by PGs has been demonstrated (23
), we did not observe any differences in lung COX-2, PGD synthase, or mPGES-1 protein expression between WT and COX-1 Tr allergic mice. Furthermore, studies performed with COX-2 null/COX-1 WT and COX-2 null/COX-1 Tr mice indicated that the increased levels of PGE2
, and PGD2
in allergic COX-1 Tr mice were not COX-2 dependent, since these levels did not change in the absence of COX-2. These observations suggest that mechanisms other than COX-2-mediated increases in pro and anti-inflammatory PGs were responsible for the altered airway PG profile and lack of beneficial outcome in allergic COX-1 Tr mice.
Given our observations, we propose the following scenario to explain the airway PG profiles and inflammatory and functional outcomes observed in this study (). Under nonallergic conditions, airway PGE2
was selectively increased in COX-1 Tr mice owing to the fact that PGE synthase is the predominant PG synthase in the naive airway (4
) and/or is coupled most efficiently to COX-1 to generate PGE2
from COX-1-derived PGH2
. The net effect of this was a decrease in airway responsiveness to methacholine in COX-1 Tr mice compared with WT mice. In the allergic airways of WT and COX-1 Tr mice, the availability of PGH2
was increased as a result of COX-2 up-regulation; however, PGH2
was maintained at a higher level in COX-1 Tr mice than in WT mice due to the sustained presence of the COX-1 transgene. Consequently, influx of inflammatory cells and transcellular metabolism of airway cell-derived PGH2
contributed to increased generation of PGE2
(), the absolute levels of which were greater in COX-1 Tr mice than in WT mice due to the greater availability of PGH2
in the former. Our data suggest that transcellular metabolism of PGH2
, a phenomenon believed to underlie altered PG levels in other experimental animal and human studies (24
), is the most likely explanation for the observed airway PG levels. In contrast to PG levels, the absolute levels of airway leukotrienes did not differ between allergic WT and COX-1 Tr mice; only when COX-2 was absent from the system did arachidonic acid become available for shunting to lipoxygenase-mediated metabolism, and this was partially reversed in the presence of the COX-1 transgene.
A proposed scheme to account for the airway eicosanoid profiles observed in nonallergic and allergic WT and COX-1 Tr mice
Since airway inflammation and responsiveness did not differ between WT and COX-1 Tr mice despite the observed differences in absolute airway PG levels, we speculate that the relative ratio of pro- and anti-inflammatory PGs within the airway, which were found to be similar between genotypes, was a more important determinant in the allergic response than were the absolute levels of these mediators. As such, it is doubtful that a higher level of expression or an alternate location for the COX-1 transgene within the airways would have influenced the allergic outcomes in the present study, as these PG ratios would likely have still remained similar between allergic mice of both genotypes. Additionally, it should be emphasized that airway COX-1, regardless of the level or location of expression, would not be expected to influence the sensitization to Ag administered by i.p. injection, as was the protocol used in this study. As such, the documented worsening of allergic airway responses in COX-1 null mice (2
) can likely be ascribed to defects in the initial sensitization to Ag and in the subsequent adaptive immune responses to repeated airway challenges, indicating a critical role for nonairway COX-1 in the overall process. Indeed, COX-1 expression and activity is important in areas such as local lymph nodes where Ag presentation occurs and the thymus during immune system development (26
). Thus, although we postulated that increasing COX-1 expression solely in the airways might have improved allergic responses, our data clearly indicate that this was not the case. Furthermore, whereas COX products, in particular PGE2
, have been suggested to inhibit the development of allergen-induced airway inflammation and hyperresponsiveness through inhibition of the development of a Th2-mediated adaptive immune response (12
), the fact that the selective and beneficial increase of PGE2
in the naive COX-1 Tr airway was not maintained under allergic conditions underscores the inability of airway COX-1 overexpression to limit allergic airway inflammation.
Given the anti-inflammatory potential of PGE2
in the lung (27
), it is reasonable to predict that airway inflammatory and functional outcomes in allergic airway disease may be reduced via strategies designed to selectively maintain increased airway levels of this prostanoid throughout the course of allergic inflammation. Alternatively, selective activation of certain PGE2
receptors that are thought to mediate the beneficial actions of this PG in the airways may prove beneficial. In this regard, a recent report (28
) describing the inhibition of allergic airway inflammation and hyperresponsiveness in mice by a synthetic agonist specific for the PGE2
suggests that this pathway may have therapeutic benefit. It is important to note that PG signaling is mediated by an array of receptors that are expressed on numerous cell types within the allergic airway and that the levels and expression patterns of these receptors are likely dynamically regulated during the course of an inflammatory response such as the one elicited in the model we used. Although it is beyond the scope of the present study to examine such events, it is likely that changes in the levels or pattern of expression of specific PG receptors on airway and/or inflammatory cells contributed to the inflammatory and functional outcomes observed herein.
In summary, we have demonstrated that constitutive overexpression of COX-1 in the murine airway increases PGE2
content and decreases responsiveness to cholinergic stimulation. Despite this beneficial effect on airway responsiveness in naive mice, the inflammatory and functional responses of the lung to an allergic stimulus are not altered in the presence of the COX-1 transgene, likely due in part to a balanced generation of pro- and anti-inflammatory PGs under allergic conditions. We propose that other genetic and/or pharmacologic strategies designed to alter airway levels of PGs (22
) or to specifically target downstream signaling events may result in a more robust alteration of basal lung function and provide benefit in this and other models of lung disease in which diminished or altered COX activity are implicated.