The main objective of this study was to clarify the effects of COX-2 deficiency on pulmonary fibrosis and associated lung functional changes. To do this, we employed an established model of fibrosis induced by administration of bleomycin and examined the biochemical and histologic indices of fibrosis and changes in lung mechanics that occurred in COX-2 null and wild-type littermate mice. Our data suggest that COX-2 deficiency did not exacerbate fibrosis in this model, but that it did result in a worsening of lung function decline as determined by invasive analysis of Cst, E, and H. Further, we demonstrated that fibrosis-associated decrements in lung function, but not fibrosis itself, could be prevented by airway COX-1 overexpression, indicating a complex interaction of eicosanoids in regulating various aspects of the lung injury in this model.
The role of COX-2 in the pathogenesis of pulmonary fibrosis has been examined in mouse models of vanadium pentoxide- and bleomycin-induced lung injury, with seemingly disparate results (3
). COX-2 null mice were shown to develop fibrosis after exposure to vanadium pentoxide, whereas COX-1 null and wild-type mice did not (5
). Moreover, while an initial histologic assessment (four to six mice per genotype) suggested exacerbation of pulmonary fibrosis induced by 1 mg/kg bleomycin in COX-2 null mice (3
), a more thorough analysis by the same research group later demonstrated no increase in the fibrotic outcomes in these mice relative to wild-type mice in response to this dose of bleomycin (7
). A more recent study reported enhanced bleomycin-induced fibrosis in COX-2 null mice (6
), although comparison of these findings with those of the previous studies is difficult due to the fact that the dose of bleomycin used in the latter study (0.05 units/mouse) was not adjusted for animal body weight and, according to our calculations, appears to have been greater in magnitude (~ 1.72 mg/kg for a 20-g mouse). Thus, the observation of exaggerated fibrosis in COX-2 null mice in response to bleomycin in the latter study (6
) was likely due, at least in part, to the higher dose of bleomycin that was used in comparison to that used by others and in the present study (1 mg/kg). Dissimilar genetic makeup of the mice used in the various studies may also contribute to the inconsistent findings. We acknowledge that it was impossible to control for segregation of parental alleles other than those for murine COX-2 and the transgenic human COX-1 in the F2 generation experimental groups created with our breeding scheme. However, we attempted to minimize potential experimental variability resulting from this by using large numbers of littermate mice of a single sex for all experimental outcomes, an approach similar to that used by others studying mice with a mixed genetic background (14
). As a result, we feel confident that the outcomes observed here can be attributed to the presence or absence of COX-1 and/or COX-2 and not to some other, unidentified allelic difference(s) among the experimental groups.
A decline in respiratory function is associated with poor prognosis in humans with pulmonary fibrosis and may be a more useful predictor of morbidity and mortality than traditional pathological endpoints assessed by biopsy (18
). Inclusion of lung function evaluation in murine models of pulmonary fibrosis is uncommon, yet this important analytical endpoint helps to more closely correlate these models to the human condition. In this regard, our observation of an exaggerated functional defect in the lungs of bleomycin-treated COX-2 null mice in the absence of biochemical or histologic evidence of enhanced fibrosis supports a critical role for this enzyme in regulating lung function in the setting of fibrosis. Decreased Cst
and increased E
were observed in bleomycin-treated mice, indicative of the characteristic increase in lung stiffness that leads to inefficient gas exchange and respiratory insufficiency in the fibrotic lung. These observations are consistent with other reports of decreased respiratory system compliance in bleomycin-treated experimental animals (6
). The reason for exaggerated lung function decline in COX-2 null mice compared with COX-2 WT mice in the absence of a measurable difference in fibrosis may be related to events not quantifiable by traditional means of determining fibrotic endpoints such as differences in collagen maturity, cell-specific injury, effects on smooth muscle, and regional differences in collagen deposition. Differences in fibrosis-associated changes in other extracellular matrix components such as elastin may also have contributed to this phenomenon. With regard to regional differences in collagen deposition, our analysis of fibrosis associated with small- and medium-sized airways did not reveal an increased level of airway collagen in COX-2 null mice, suggesting that this possibility was not likely contributing to the more severe lung function decline in these mice. Regardless, the fact that functional alterations were more severe in COX-2 null mice than in COX-2 WT mice indicates that absence of this enzyme renders the lung prone to enhanced functional deficits that could not be predicted based on assessment of fibrosis alone, and underscores the necessity of including lung function endpoints in the overall appraisal of injury in lung fibrosis models.
As we have documented an improvement of airway responsiveness via transgenic airway overexpression of COX-1 in COX-2 null mice in a model of allergic airway inflammation (8
), we sought to determine if similar beneficial effects on lung function might be afforded by this approach in our fibrosis model. Whereas COX-1 overexpression was found to not influence the extent or severity of lung or airway-specific fibrosis in COX-2 WT or COX-2 null mice, functional alterations induced by bleomycin were attenuated. Specifically, decreased Cst
and increased E
were not as severe in COX-2 null mice harboring the COX-1 transgene. Beneficial effects of the COX-1 transgene were not as readily apparent in COX-2 WT mice, although the bleomycin-induced increases in E
were alleviated somewhat. These findings suggest that airway overexpression of COX-1 was capable of rescuing the fibrosis-associated impairment of lung function in COX-2 null mice, an outcome that may have been related to effects on airway eicosanoid levels.
Airway PG levels did not differ but airway cysLT levels were elevated in bleomycin-treated COX-2 null mice compared with COX-2 WT mice, coincident with their more severe impairment of lung function. Moreover, airway cysLT levels were lower and lung function decrements were less severe in bleomcyin-treated COX-2 null/COX-1 Tr mice compared with COX-2 null mice, further suggestive of a causal relationship between cysLTs and lung function decline. While their role in fibrosis-associated lung function decline has not been studied, the cysLTs have been shown to be essential to the pathogenesis of experimental lung fibrosis as mice either deficient in their production or treated with pharmacologic inhibitors of 5-lipoxygenase or cysLT1 receptor antagonists are protected from bleomycin-induced fibrosis (22
). Further, the potential for cysLTs to impair dynamic pulmonary compliance in various species is well established (25
) and suggests that reduction of airway cysLT levels or antagonism of cysLT receptors may improve lung function in a variety of disease states in which these pro-inflammatory eicosanoids are implicated. The most obvious of these is asthma, for which effective inhibitors of cysLT synthesis and antagonists of cysLT receptors have been developed and are currently in use. It remains to be determined whether pharmacologic inhibition of cysLT synthesis and/or receptor signaling might benefit fibrosis-associated lung function decline in the setting of clinical or experimental fibrosis. In our model of lung fibrosis, it appears as though the improvements in dynamic (measured as E
) and static (measured as Cst
) compliance afforded by airway overexpression of COX-1 in COX-2 null mice may have been mediated by the reduction of airway cysLT levels that occurred independent of changes in airway PG levels. These are interesting observations, given that the COX-1 transgene was expressed in airway Clara cells and therefore might not have been expected to influence Cst
, a measurement normally associated with the mechanical properties of the distal airways and lung parenchyma. On this note, it is possible that expression in a more distal location such as alveolar type II cells (with expression driven by the surfactant protein A or C promoter) may have altered airway eicosanoid levels to a greater extent and/or in a region of the lung more relevant to the fibrotic and functional outcomes, and had an even more pronounced effect on fibrosis and associated functional alterations as a result. These possibilities remain to be tested and represent interesting avenues of future investigation.
In the present study, shunting of arachidonic acid metabolism toward the 5-lipoxygenase pathway likely occurred in the lungs of bleomycin-treated COX-2 null mice, leading to the increased airway cysLT levels in these animals. It is interesting to note that this increase in cysLT levels occurred in the absence of a corresponding change in airway PG levels relative to those observed in COX-2 WT mice. These observations imply the likely involvement of transcellular biosynthetic processes in the resulting airway eicosanoid levels (28
), similar to what we proposed as underlying the improved lung function resulting from COX-1 overexpression in COX-2 null mice in a model of allergic airway inflammation (8
). Moreover, airway overexpression of COX-1 in COX-2 null mice resulted in decreased cysLT levels and a corresponding increase in PG levels after bleomycin treatment. These findings, together with the lung function outcomes, highlight the intricate interaction of eicosanoid-mediated effects in the regulation of lung injury and corresponding lung dysfunction. Confounding the situation is the recognition that an array of specific eicosanoid receptors expressed on a variety of cell types contributed to the outcomes and that eicosanoid levels and receptor expression patterns are likely dynamically regulated throughout the course of injury. That said, the airway eicosanoid profiles, determined at the same time point that lung function and fibrosis assessments were conducted, suggest that cysLTs promote lung dysfunction in the setting of fibrosis in COX-2 null mice.
Finally, it is prudent to discuss the results of the present study in the context of recently published data concerning the influence of COX-2 deficiency in the bleomycin-induced lung fibrosis model. Lovgren and colleagues (6
) observed that absence of the prostacyclin receptor rendered mice as susceptible to bleomycin-induced lung fibrosis and dysfunction as did the absence of COX-2. Based on this data, these investigators postulated that prostacyclin is protective against fibrosis. However, prostacyclin levels in lung or BAL fluid of naïve or bleomycin-treated mice were not reported, thus precluding firm establishment of a role for prostacyclin in limiting the development of lung fibrosis and associated functional alterations. In the present study, airway prostacyclin levels (measured as the stable metabolite 6-keto-PGF1α
) did not differ between saline- or bleomycin-treated COX-2 WT and COX-2 null mice, suggesting that prostacyclin may not be crucial to limiting the functional alterations resulting from bleomycin administration observed herein. Similar to our findings, Hodges and coworkers (7
) reported that lung fibrosis in COX-2 WT and COX-2 null mice did not differ after administration of 1 mg/kg bleomycin. The limited fibrosis in COX-2 null mice was attributed, at least in part, to the unexpected increase in airway PGE2
levels in bleomycin-treated COX-2 null mice compared with COX-2 WT mice, an effect believed to be the result of compensatory up-regulation of COX-1 in macrophages and monocytes (7
). Furthermore, BAL fluid LTC4
levels did not differ among the genotypes 7 d after bleomycin administration, while levels at later time points were not reported (7
). In contrast to these findings, we did not observe differences in airway PGE2
levels between saline- or bleomycin-treated COX-2 WT and COX-2 null mice at the time of fibrosis assessment, but differences in cysLT levels that likely contributed to the functional outcomes were readily apparent. It is unclear why Hodges and colleagues (7
) observed increased airway PGE2
in bleomycin-treated COX-2 null mice compared with COX-2 WT mice whereas we did not, although factors including different PG quantification protocols and/or mouse genetic backgrounds may have contributed to these disparate findings. Regardless, our data are consistent with theirs in terms of the overall fibrotic outcome, namely a similar response to bleomycin in COX-2 WT and COX-2 null mice.
Collectively, our data indicate that COX-2 deficiency does not influence the development of bleomycin-induced pulmonary fibrosis, but that functional impairments associated with fibrosis are exaggerated in the absence of this enzyme. Further, we show that the adverse functional effects associated with pulmonary fibrosis in COX-2 null mice are decreased by airway COX-1 overexpression and the ensuing alteration of airway eicosanoid levels. Based on our observations, we propose that targeted alteration of pulmonary eicosanoid levels may afford benefit in the treatment of fibrosis-associated lung function decline.