Disorders of surfactant homeostasis play prominent roles in many lung diseases. Surfactant deficiency is central to the pathogenesis of newborn respiratory distress syndrome and is also important in adult respiratory distress syndrome (40
). At the other extreme, excessive surfactant accumulation is found in patients with pulmonary alveolar proteinosis (6
). In addition, the collectin family members, SP-A and SP-D, are increasingly recognized to play important roles in the pulmonary innate immune response (41
) by opsonizing pathogens and facilitating cellular bacteriocidal activity. However, the factors that regulate levels of surfactant in the alveolar space are incompletely understood. In this study, we identified a novel mediator involved in homeostasis of the collectins and phospholipids in the adult mouse lung: the epithelial integrin αvβ6. We also determined that the mechanism by which integrin αvβ6 mediates this homeostasis is through regulation of TGF-β activation.
We found that active TGF-β was both necessary and sufficient to regulate surfactant phospholipid and collectin homeostasis in adult mouse lungs. This was evidenced by an increase in phospholipids and collectins in the absence of active TGF-β and alternatively an almost complete normalization of these surfactant constituents after induced expression of the active form of TGF-β1 in the lungs of Itgb6−/−
mice. This regulatory effect of active TGF-β on surfactant components in Itgb6−/−
mice was observed even in mice several months old, well after postnatal lung development was complete. Thus, our findings extend the observations of others by demonstrating the in vivo
role of active TGF-β on the regulation of these surfactant components. In addition, these findings illustrate how critical TGF-β is to the homeostasis of surfactant in adult mouse lungs. This may have significant implications for treatment approaches that inhibit active TGF-β in fibrotic lung diseases, such as idiopathic pulmonary fibrosis. Furthermore, the identification of a role for TGF-β in regulating surfactant in adult lungs may aid the understanding of certain aspects of phospholipid derangements noted in patients with idiopathic pulmonary fibrosis (42
), a condition found to be associated with increased levels of TGF-β (43
While active TGF-β was required for homeostasis of lung phospholipids and collectins, SMAD3, a key intracellular signaling molecule for TGF-β, was not. We speculate there are several possible explanations for this. First, this finding is not unprecedented in light of the accumulating evidence that regulatory SMAD2 and 3 can mediate different cellular functions in vivo
and in vitro
). Second, there is the possibility that compensatory pathways are activated in Smad3
-deficient mice, and thus are able to bypass the deficiency of SMAD3. While this explanation may account for the normal levels of surfactant components in Smad3
-deficient mice, it is clear that other aspects of pulmonary homeostasis, such as control over inflammatory cell recruitment, requires signaling by SMAD3. We excluded the possibility that our findings might be the result of genetic strain differences between the TGF-β–deficient mouse models and Smad3
-deficient mice by analyzing the alveolar content of phospholipids and collectins in Itgb6−/−
mice on the same genetic background as Smad3
-deficient mice. We speculate that SMAD3-independent TGF-β signaling pathways allow for normal surfactant homeostasis in Smad3
-deficient mice, and that failure to activate these SMAD3-independent TGF-β pathways accounts for the surfactant abnormalities in Itgb6−/−
Additional evidence to support the idea that active TGF-β can regulate pulmonary homeostasis through signaling pathways independent of SMAD3 comes from alveolar macrophages from Smad3
-deficient mice. Compared with alveolar macrophages from Itgb6−/−
macrophages show less dramatic morphologic changes (, , and ). In addition, we previously found that alveolar macrophages from Itgb6−/−
mice expressed mRNA transcripts for matrix metalloproteinase-12 at levels approximately 100- to 200-fold higher than that from control mice, which was regulated by active TGF-β (18
). In contrast, alveolar macrophages from Smad3−/−
mice have barely detectable increases of matrix metalloproteinase-12 mRNA (Figure E8), indicating that there are additional signaling pathways that can respond to active TGF-β despite a total deficiency of SMAD3. Overall, these differences in alveolar macrophage homeostasis between Smad3
-deficient mice and Itgb6−/−
mice identify an important area for further research to understand the various signaling pathways used by active TGF-β to regulate macrophage function.
Our results suggest that the mechanism of TGF-β–mediated surfactant regulation is not through GM-CSF. Mice lacking GM-CSF (Csf2−/−
) also demonstrate elevated levels of surfactant constituents (12
). In these GM-CSF–deficient mice, the primary cause for surfactant dysregulation was found to be a defect in catabolism of surfactant (47
). Specifically, the majority of alveolar macrophages from these mice were unable to effectively catabolize surfactant components due to an immature state of differentiation (11
). Reconstitution of GM-CSF–deficient mice with GM-CSF protein was able to restore surfactant homeostasis (38
). Additional studies identified that GM-CSF stimulates induction of the PU.1 transcription factor, which causes alveolar macrophage differentiation and enables surfactant clearance (11
). In support of these data, the majority of alveolar macrophages from GM-CSF–deficient mice lacked staining for fluoride-resistant α-naphthyl acetate esterase activity, a marker of fully differentiated alveolar macrophages (27
). While our findings from Itgb6−/−
mice also suggest a defect in lipid processing by alveolar macrophages, the majority of macrophages from these mice were positive for fluoride-resistant α-naphthyl acetate esterase activity, suggesting that they are fully differentiated. Furthermore, we found increased, not decreased, levels of GM-CSF mRNA in lung homogenate from Itgb6−/−
mice compared with wild-type littermates, as well as similar mRNA levels of PU.1 in alveolar macrophages from wild-type and Itgb6−/−
mice. Taken together, these data suggest that TGF-β may directly or indirectly influence alternative catabolic pathways in macrophages that are not dependent on GM-CSF.
We also considered the possibility that a deficiency of active TGF-β in the lung leads to macrophage apoptosis, since alveolar macrophages from Itgb6−/− mice appear to contain pycnotic nuclei (data not shown). We did find that alveolar macrophages from Itgb6−/− mice were highly fluorescent without staining, and with annexin V and acridine orange staining these macrophages showed a staining pattern that suggested apoptosis (data not shown). However, we were unable to measure additional markers of apoptosis or senescence using multiple methods (Western blots for caspase 3, p21, tunnel staining, immunocytochemistry for caspase 3, propidium iodine staining, and DNA laddering). Thus, we cannot convincingly conclude that macrophage apoptosis contributes to the surfactant abnormalities that we measured.
Despite differences in macrophage maturation states between GM-CSF–deficient mice and Itgb6−/−
mice, there appear to be several lung phenotypic features shared between these two models that we speculate may represent a common phenotype observed when clearance of surfactant is defective. Both models have features consistent with an elevation of surfactant in the airspace, notably the accumulation of eosinophilic material in the alveolar spaces and foamy lipid–laden macrophages. These two mouse models also share the feature of spontaneous development of increased inflammation in the lung—specifically, the presence of peribronchovascular aggregates of lymphoid cells, which include B and CD4+ T cells (10
). Another feature shared by these two models is the fact that the deletion of GM-CSF or integrin β6 subunit had no detectable influence on mRNA levels for surfactant proteins even though alveolar surfactant protein and lipid levels were elevated. This supports the idea that impaired clearance rather than increased surfactant synthesis is likely responsible for the accumulation of phospholipids and collectins in Itgb6−/−
mice as it is in GM-CSF–deficient mice.
Although addition of exogenous TGF-β1 has been shown to reduce surfactant protein A mRNA and protein in ex vivo
human fetal lung tissue (32
), our in vivo
data show that a depletion of TGF-β results in elevated BAL levels of surfactant protein A without influencing protein or transcript levels within epithelial cells. This apparent discrepancy may be attributable to important differences in experimental design. The in vivo
models that we studied represent the consequences of interfering with normal levels of active TGF-β in the lung, whereas the fetal lung tissue model represents the effects of exogenous TGF-β1, which may or may not represent normal levels that are realized in vivo
. In any case, our results suggest that in vivo,
an increase in transcript levels is not the mechanism responsible for the elevation of SP-A in mice lacking active TGF-β. Rather, our findings suggest that a depletion of active TGF-β leads to a decrease in clearance of SP-A in the lung.
In contrast to SP-A, we found that depletion of TGF-β led to increased SP-D levels in both BAL fluid and alveolar epithelial cells, without an increase in transcript levels. The disproportionate increase of protein levels for SP-D compared with SP-A in Itgb6−/−
alveolar epithelial cells was also observed in the BAL fluid, where we measured a 100% and 200% increase in SP-A and -D, respectively. This finding is consistent with observations in humans with pulmonary alveolar proteinosis as well as other mouse models of defective surfactant clearance, where SP-D was found to be disproportionately increased compared with other surfactant proteins (39
). The reason for this is not known, but it has been speculated that an increase in SP-D may be a compensatory response to increased phospholipid levels (39
). Mouse models have also demonstrated the important role of SP-D in maintaining surfactant homeostasis (24
). Thus, SP-D may play an accessory role in regulating aspects of pulmonary homeostasis and in turn, its levels may be differentially regulated by many other local signals from resident lung cells.
In summary, our data indicate that the activation of latent TGF-β1 by the αvβ6 integrin is central to maintaining normal basal levels of phospholipids and collectins in the alveolar space. Based on the sum of alveolar epithelial cell and macrophage data from Itgb6−/− mice, we speculate that a depletion of active TGF-β leads to decreased catabolism of phospholipids and collectins in the lung. These in vivo findings in adult mice call attention to the importance of αvβ6 integrin and active TGF-β in maintaining normal levels of phospholipids and SP-A and -D, and identify new potential targets in diseases involving surfactant dysregulation. In addition, our findings have implications for treatments of fibrotic lung diseases, such as idiopathic pulmonary fibrosis, where therapeutics may target and inhibit TGF-β1 activity in the lung.