The major objective of this study was to evaluate the role of APN in the development of ALI. We demonstrated a novel role for APN in that it protects against the development of ALI and limits the associated systemic response to LPS. These observations in mice suggest a potential mechanism by which human obesity is a risk factor for the development of ALI.
To characterize the mechanisms mediating APN’s protective effects we focused on the early response to lung injury in APN−/− mice. Unexpectedly, total cell counts and cytokine concentrations in BAL fluid of wt
and APN−/− mice were not significantly different at early time points. These findings led us to explore APN’s ability to modulate the ALI response through actions outside the airspace of the lung. We hypothesized that the vascular endothelium of the lung mediates APN’s protective actions in this model because vascular permeability (wet:dry ratio and BAL protein concentration) is increased in APN−/− mice. This hypothesis is supported by observations showing that lung endothelial cells express APN receptors and that APN localizes on the surface of lung endothelium (28
). In addition, previous work has shown that targeted deletion of APN promotes an activated endothelial cell phenotype under basal, non-stressed conditions, including upregulation of E-selectin (28
). In the current study, we confirmed the baseline upregulation of pro-inflammatory genes in isolated lung endothelial cells of APN−/− mice and demonstrated further upregulation of IL-6 and other pro-inflammatory genes in lung endothelial cells early after i.t.
LPS. Based on these findings, it is tempting to speculate that increased serum IL-6 levels in APN −/− mice result from endothelial secretion. This is further supported by studies demonstrating that recombinant APN effectively inhibited LPS induced IL-6 production in primary lung endothelial cells in culture. However, our study does not directly address this potential mechanism of increased circulating IL-6 in LPS challenged APN −/− mice. Taken together, our findings indicate that APN modulates the ALI response to LPS at least in part through its actions on lung endothelium. Consistent with this notion, it has been reported that circulating levels of vWF, a marker of endothelial injury, are observed in obese patients with ALI (4
). Presumably, these patients would have low APN levels at the onset of ALI.
To further test whether APN’s suppression of the early response to LPS is conferred outside the airspace of the lung, a model of relative lung APN deficiency using T-cad−/− mice was utilized. T-cad is a major APN receptor/binding protein that is expressed on lung endothelium (32
). T-cad is unique in that it lacks an intracellular signaling domain, suggesting that it serves predominantly as a docking protein (30
), and appears to play an essential role in the transport of APN into the airspace of the lung (30
). In this regard, the inflammatory response to LPS in T-cad−/− mice that lack APN in the airspace was similar to that of wt
mice. While these data support the notion that circulating APN influences the ALI response, perhaps through an effect on lung endothelium, it is important to note that higher serum APN levels did not protect against the development of ALI in T-cad −/− mice. We speculate this relates to the high, possibly saturating concentration of APN in serum of wt
mice, with further increases having marginal effects on lung endothelial responses.
Notably, results from this study effectively exclude T-cad’s participation as a “receptor” that mediates APN’s effects in lung in this model because T-cad−/− and wt
mice displayed equivalent responses to i.t.
LPS administration. These findings are in contrast to those in the heart where T-cad expression is essential for mediating APN’s protective effects in models of ischemia-reperfusion injury and pressure overload hypertrophy (31
). This contrast may be explained by the phenotypic and functional differences that have been described between the pulmonary and systemic vasculature (33
). While our data do not allow us to characterize the specific receptor responsible for APN’s actions, previous work from our laboratory demonstrated expression of APN receptors 1 and 2 on lung endothelium, pointing to these receptors as candidates (28
The current study suggests that APN may suppress inflammatory responses to LPS by targeting lung endothelium. These findings are in agreement with recent work demonstrating that APN ameliorates vascular remodeling in murine models of allergic airway inflammation and chronic hypoxia and that endothelial dysfunction might underlie the previously described emphysematous phenotype of APN−/− mice (35
). However, we acknowledge that APN’s protective effects may extend to other vascular beds as illustrated by observations of exacerbated endothelial cell activation in the systemic circulation during intra-abdominal sepsis in APN−/− mice (38
). Furthermore, our data indicate that that APN’s actions are not limited to endothelium. For example, we previously found that APN maintains alveolar macrophage quiescence (23
However, in this study, while we confirmed that unstimulated CD45+ immune cells of APN−/− mice had increased expression of pro-inflammatory cytokines, we did not find an exaggerated LPS-induced response in these cells at early time points when compared to wt mice. In fact, we found decreased induction of TNF-α and Nox2 in CD45+ cells isolated from APN −/− mice. These findings indicate that APN exerts differential effects on endothelial cells and leukocytes in lung. These effects could be mediated by actions of different APN oligomeric fractions or binding of APN to different cell surface receptors. Future studies investigating the mechanisms mediating APN’s differential effects will be important to develop targeted therapies that selectively suppress activation of endothelial or immune cells in lung.
One other important finding in this study is that APN appears to play a key role in the selective recruitment of inflammatory cells into lung. This is evident from increased neutrophil infiltration into lungs of APN −/− mice. We speculate this may also explain the observed divergence in lung IL-10 levels between wt and APN −/− mice, in that APN may preferentially recruit IL-10 producing cells into unchallenged and injured lung. Studies that evaluate APN’s direct and indirect effects on recruitment of inflammatory cells into the lung will be important.
Although our study did not evaluate the influence of APN on other cell types such as lung epithelium, the absence of an exaggerated inflammatory response in BAL fluid of APN−/− mice does not support a major effect on this population.
While the current study focused on the cellular response to LPS, APN may have other anti-inflammatory actions mediated through non-cellular mechanisms. Previous reports indicate the APN has anti-inflammatory actions that do not require direct cell interactions. For example, APN has been shown in vitro
to bind LPS (39
). However, if this mechanism played a significant role, decreased levels of BAL cytokines should have been seen in wt
vs. APN−/− mice 4 hours after i.t.
LPS. In addition, APN pre-treatment of HPAECs followed by media change prior
to LPS challenge was capable of effectively suppressing the LPS response. Both findings suggest that APN-mediated binding and/or sequestering LPS does not represent a major mechanism of action of APN’s modulation of this acute lung injury response.
Another key finding in this study was the observation that restoration of serum APN levels attenuated the response to i.t. LPS. Decreased BAL protein concentration in Ad-APN-APN−/− mice 4 hours after LPS administration suggests that APN acts, at least in part, by enhancing endothelial barrier function. This hypothesis is further supported by our in vitro studies demonstrating direct suppressive effects of APN on lung endothelial cells exposed to LPS. Although IL-6 concentration was not significantly different between LPS-injured Ad-APN and Ad-gal/APN −/− mice we speculate that higher serum APN levels may be required for longer periods to facilitate anti-inflammatory responses in lung. In addition, it is possible that untoward effects of adenoviral infection may have influenced the results of these studies since IL-6 concentration and BAL protein levels were generally higher in adenoviral exposed mice. Collectively, the rescue studies indicate APN exerts a protective effect on the lung endothelium and suggest a potential therapeutic role for this protein in individuals at risk for ALI.
It is worth mentioning that a recent clinical study from our laboratory demonstrated that high serum levels of APN were associated with increased mortality in critically ill patients with acute respiratory failure (40
). This report appears to contradict our results in mice. However, studies in APN −/− mice investigated the role of APN in the development of LPS-induced ALI, where APN was absent prior to onset of lung injury. In contrast, human studies evaluated the association between APN and mortality in a diverse population (only 21% of which had ALI) with established acute respiratory failure. We speculate that higher APN levels in these patients reflect the host response to critical illness and have little or no correlation with serum levels prior to onset of disease. Thus, to confirm findings in mice, future clinical studies will need to investigate the relationship between baseline serum APN levels and risk of developing ALI.
Although our study supports that APN deficiency may be a mechanistic link between obesity and ALI, other adipokines are altered during obesity and may contribute to the pathogenesis of ALI. For example, leptin (41
), a highly abundant adipokine, is structurally similar to the IL-6 family of cytokines and demonstrates pro-inflammatory activity on a variety of cell types. The i.t.
administration of leptin in mice has been shown to induce ALI (42
). However, clinical studies have not identified a correlation between leptin levels and ALI in human subjects, and this may be explained, in part, by the fact that chronic elevation in serum leptin levels is often associated with resistance at the level of the receptor (42
). Future studies examining the role of other adipokines in the development of ALI will be required.
In summary, we identified a previously unrecognized role for APN in limiting the development of ALI in mice. Furthermore, our findings suggest that this effect is mediated in part by APN’s direct actions on lung endothelium. Based on this work, we speculate that measuring circulating APN may be important in defining one’s risk for developing ALI.