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Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN’s role in the early development of ALI to lipopolysaccharide (LPS) was investigated. Intra-tracheal (i.t.) LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN−/−) mice compared to wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN−/− mice as early as 4 hours after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 hours after LPS administration showed increased pro-inflammatory gene expression (e.g. IL-6) only in endothelial cells of APN−/− mice when compared to wt mice. Direct effects on lung endothelium were demonstrated by APN’s ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient (T-cad−/−) mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after i.t. LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g. obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium.
Acute lung injury (ALI) is a life-threatening condition whose annual incidence in the United States has steadily increased over the last several decades. Recent data indicate that approximately 200,000 individuals are affected by this condition each year, and current estimates demonstrate a 30–40% mortality with even greater numbers left with temporary or permanent disabilities (1).
While the pathogenesis of ALI is complex, hallmark features include immune and endothelial cell activation, loss of vascular integrity, and accumulation of protein-rich fluid in the airspaces of the lung (2–3). Specific predisposing risk factors for the development of ALI in the face of systemic stress are incompletely defined. However, recent epidemiological data point to obesity as an important risk factor for development of ALI (4–7). In fact, clinical prediction scores utilizing obesity have stratified obese patients as being at higher risk for developing this condition (8). While certain adipocyte-derived hormones called adipokines contribute to a number of chronic inflammatory conditions including diabetes and cardiovascular disease, the impact of these hormones on the development of acute critical illness such as ALI is not well understood (9–13).
Adiponectin (APN), a highly abundant adipocyte-derived adipokine (with microgram/milliliter concentrations in serum) circulates as low, middle, and high molecular weight complexes. Paradoxically higher serum levels are present in lean, healthy individuals compared to obese and diabetic patients (14). Although APN was initially described as an insulin-sensitizing agent, more recent work has defined its pleiotropic anti-inflammatory and vascular protective role (15–18). For example, clinical studies show an inverse relationship between APN levels and circulating concentrations of pro-inflammatory markers such as C-reactive protein, IL-6, and TNF-α (19–22). Moreover, experimental studies in mice demonstrate that APN contributes to immune homeostasis in the lung (23) and protects against inflammatory and post-ischemic injury in other tissue beds, including liver, muscle, brain, and heart (24–27). The immune and vascular protective properties of APN led to speculation that hypoadiponectinemia may play a role in the development of ALI. To evaluate APN’s role in ALI, we utilized a well-established model of LPS-induced lung injury in mice with targeted APN gene deletion.
All studies were performed using two-month old, gender-matched mice. This time point was selected because it precedes the development of the vascular phenotype in APN−/− mice (28). C57BL/6 APN−/− mice were provided by N. Maeda, T. Funahashi, and Y. Matsuzawa (Osaka University, Osaka, Japan). Wt C57BL/6 mice were obtained from Charles River laboratories. C57BL/6 T-cad−/− mice were provided by B. Ranscht. All animal experiments were reviewed and approved by Boston University’s Institutional Animal Care and Use Committee (IACUC). Mice were maintained in a 12-h light, 12-h dark schedule and given food and water ad libitum.
ALI was induced by the administration of a one-time i.t. injection of 100 μg LPS (1 mg/ml). Delivery of LPS was performed using the tongue-pull maneuver in anesthetized mice. At select time points after LPS administration lung tissue, serum, and BAL fluid were obtained for analysis. Lung wet:dry weights were performed as previously described (28), and BAL protein concentration was measured by Bradford Assay and Pierce bicinchoninic acid assay.
Following i.t. injection, mice were observed and scored by the mouse assessment score (MAS) every four hours (with strict, IACUC-approved guidelines for euthanasia). Points were assigned based on four categories of physical appearance: a) Coat, 1 = smooth, 2 = mild ruffling, 3 = significant ruffling, b) Activity level, 1 = normal (exploring cage), 2 = lethargic (moves slowly without stimulation), 3 = sedentary (moves only with stimulation), 4 = immobile (minimal movement with stimulation), c) Respiratory effort, 1 = normal, 2 = labored, 3 = labored, irregular, d) Posture, 1 = moving or resting normally, 2 = huddled. Scores ranged from 4 (normal) to 12 (most abnormal).
Immuno-staining was performed on lung sections after antigen retrieval using Retrievagen A (Zymed, South San Francisco, CA) at 100°C for 20 minutes, and quenching endogenous peroxidases with 3% H2O2. Sections were blocked with 2% BSA in PBS followed by staining with primary anti-CD45, anti-B220, anti-CD3, anti-F4/80 (BD Pharmingen, San Jose, CA), or anti-Gr-1 antibody (R&D Systems, Minneapolis, MN) at RT for 1 hour. Sections were washed, and after application of secondary antibody (R&D Systems) tissues were developed using Vectastain ABC (Vector Labs, Burlingame, CA) and 3,3′-Diaminobenzidine (Vector Labs).
Lung sections were evaluated and scored independently by 2 members of the lab trained in histological assessment and use of the scoring system. For each mouse, 3 different lobes were examined for the following features: interstitial edema, hemorrhage, and neutrophil infiltration. Each feature could receive a score of 0 (no injury), 1 (minimal injury), 2 (moderate injury), or 3 (severe injury). This was totaled for a given lobe’s score, and the 3 lobes averaged to generate a score for each mouse, giving a minimum score of 0 and a maximum of 9.
Nalgene Nunc Maxisorp plates were coated with primary antibody against either IL-6, TNF-α, or IL-10 (R&D systems) for one hour at room temperature (RT) then washed with PBS and 0.5% TWEEN (PBS-T). After blocking with casein, samples were added to plates for one hour at RT. Following washing, biotinylated secondary antibody was applied for one hour followed by streptavidin-HRP conjugate (Jackson ImmunoResearch, West Grove, PA) diluted at 1:20,000. Reaction was developed with 0.01% tetramethylbenzidine (TMB) dissolved in dimethyl sulfoxide (DMSO) and 0.5% hydrogen peroxide and measured using endpoint spectrometry.
Lung tissue from 2 month old wt, APN−/−, and T-cad−/− mice were digested with intermittent mechanical disruption using a razor blade and with the enzymes dispase (BD Biosciences, Bedford, MA) and collagenase A (Roche, Basel, Switzerland) for 60 minutes at 37°C. Following digestion, single cell suspensions were passed through a 70 μm filter to remove excess debris.
Prior to flow cytometry analysis, cell suspensions were immuno-stained with FITC-labeled rat anti-mouse CD45 and PE-labeled rat anti-mouse CD31 or isotype controls (BD Pharmingen, Franklin Lakes, NJ). Immuno-stained cells were then subjected to flow cytometry cell sorting (MoFlo, Beckman-Coulter, Brea, CA) to separate endothelial (CD31+45−) and immune cell (CD45+31−) populations. Cells were sorted into PBS solution and immediately transferred to RLT buffer and RNA isolation was performed using the Qiagen RNeasy Plus Mini kit (Qiagen, Valencia, CA).
Real-time quantitative PCR (qRT-PCR) was performed on cDNA amplified using the WT-Ovation kit (NuGen Technologies Inc., San Carlos, CA). Applied Biosystems StepOne apparatus was used to measure gene expression for IL-1a, IL-6, Nox2, E-selectin. 18s ribosomal RNA was used to normalize RNA concentration for each sample.
Human pulmonary artery endothelial cells (HPAECs) were obtained from Lonza (Walkersville, MD) and grown in EGM complete media. Cells were pre-treated for 24 hours with full-length human recombinant APN produced in a mouse myeloma cell line (R&D Systems) at 0, 2 or 10 μg/ml. Following pre-treatment, cells were washed, then exposed to either PBS or LPS (E. Coli 055:B5, List Biological Laboratories, Campbell, CA) at 100 ng/ml for 24 hours and supernatant was collected for cytokine analysis.
Homogenized lung tissue was preserved in protease-inhibitor (Roche Complete Mini) solution. Western blot for RelA and serum APN was performed using 20 μg of protein; BAL APN was loaded by equal volume, 5 μl/lane. RelA specimens were separated by electrophoresis in a 10% Bis-Tris gel, APN specimens on a 3–8% Tris-acetate gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Primary antibody for β-actin, RelA, phospho-RelA (CellSignaling, Danvers, MA) or APN (R&D Systems) was applied overnight at 4°C in TBS followed by secondary antibody staining with anti-rabbit horseradish peroxidase [HRP] (Millipore, Billerica, MA) for one hour at RT. Chemiluminescence was performed with Western Lightning detection reagent (PerkinElmer, Waltham, MA).
Adenoviral vectors containing the gene for beta-galactosidase (Ad-gal) and full-length mouse APN (Ad-APN) were prepared as previously described (29). Adenovirus was injected (200 μl) into the jugular vein 5 days prior to administration of i.t. LPS. Adiponectin levels were measured by ELISA (B-Bridge International, Cupertino, CA.) in serum samples obtained 4 hours after i.t. administration of LPS.
Statistical significance for difference between groups was assessed using Student’s T-test. 1-way ANOVA and Student’s T-test were used for figure 9a. A cut-off value of p < 0.05 was used to determine significance.
ALI was induced in wt and APN−/− mice by administering a one-time i.t. injection of LPS (100 μg). The ability of APN to modulate the physiological response to LPS was examined using a 12-point assessment score based on mouse physical characteristics (fig. 1a). Results showed that within 4 hours of LPS administration, APN−/− mice appeared more ill with increased piloerection and decreased mobility. Assessment scores remained elevated for APN−/− mice at 8 and 24 hours after injection (fig. 1a). This exaggerated systemic response in APN−/− mice was associated with increased serum levels of the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 at the 4 hour time point (fig 1b).
To assess local injury response to LPS, detailed lung histological examination was performed. As early as 4 hours after LPS administration, marked differences in lung injury were observed between wt and APN−/− mice. As shown in Figure 2, peri-vascular exudates, thickened alveolar septa, and airspace edema were exhibited in APN−/− mice, but were noticeably attenuated in wt mice at both 4 and 24 hours (fig 2b). Consistent with this injury pattern, BAL fluid protein concentration (fig. 2c) and lung wet:dry ratios (fig. 2d) were increased in APN−/− mice. Taken together, these findings indicate APN attenuates both the systemic and local pulmonary response to i.t. LPS.
Since tissue damage in ALI is coupled to the inflammatory response, we speculated that lung injury in APN−/− mice would be associated with an exaggerated infiltration of immune cells into the airspace compartment. To test this, detailed assessment of lung inflammation was performed 4 hours after LPS administration. This time point was selected based on its association with the histological onset of lung injury in APN−/− mice. Surprisingly, analysis of BAL fluid at this early time point did not detect differences in total cell counts or in inflammatory cytokine concentrations (IL-6, TNF-α, IL-10) in wt and APN−/− mice (fig. 3). These findings led to speculation that APN’s influence on the early injury response is mediated through anti-inflammatory actions occurring outside the airspace of the lung. To explore this further, cellular infiltration at 4 hours was examined in lung sections after immuno-staining for the pan-hematopoietic marker CD45. CD45+ cells accumulated along vascular endothelium in wt mice. In contrast, CD45+ cells had already infiltrated into lungs of APN−/− mice and were found in clusters scattered throughout the lung parenchyma (fig. 4a). This observed increase in cellular infiltration was confirmed by morphometric analyses demonstrating a 2.5-fold increase in CD45+ cells in lungs of APN−/− mice at 4 hours (fig. 4b). The infiltrating cells in APN −/− mice were predominately neutrophils based on their staining positive for Gr-1 (fig. 4c) and negative for B220, CD3 and F4/80 (data not shown). Cellular infiltration was associated with increased activation of the pro-inflammatory transcription factor NF-κB’s RelA subunit (fig. 5a), higher levels of pro-inflammatory cytokines TNF-α and IL-6, and decreased concentration of the anti-inflammatory cytokine IL-10 in lung homogenates of APN−/− mice (Fig. 5b). Taken together, these findings suggest that APN protects against development of ALI at least in part through its ability to suppress inflammation outside the airspace of the lung.
Since APN is known to inhibit immune and endothelial cell activation under basal, non-stressed conditions, we speculated that its ability to protect against the development of ALI is mediated through inhibitory effects on these cell populations. To test this possibility, gene expression was evaluated in primary immune and endothelial cells isolated from lung digests of wt and APN−/− mice 4 hours after LPS administration. As expected, baseline expression of targeted pro-inflammatory genes was increased in immune and endothelial cells of APN−/− mice. However, in response to LPS, pro-inflammatory gene expression was relatively down-regulated in immune cells. In contrast, increased expression of IL-6, E-selectin, and NOX2, a regulatory enzyme during oxidative stress, was observed in endothelial cells of APN−/− mice (fig. 6). These findings suggest that early after LPS administration, endothelial cells are an important target of APN’s anti-inflammatory properties.
To establish APN’s ability to suppress endothelial cell activation to LPS, primary human lung endothelial cells (HPAECs) were cultured for 18 hours in the presence or absence of APN. The state of endothelial cell activation was determined by measuring IL-6 concentration in cell supernatants after exposure to LPS (100 ng/ml) for 24 hours. Consistent with an inhibitory effect, APN demonstrated a dose-dependent suppression of LPS-induced IL-6 production in lung endothelial cells (fig. 7).
To investigate the hypothesis that APN protects against ALI through suppressing endothelial cell activation, we utilized a unique mouse model of compartmentalized APN deficiency. Mice that are deficient in the APN receptor T-cad display a defect in transport of APN into tissues (30–31). As depicted in Figure 8, T-cad−/− mice have elevated serum levels of APN with low concentration in the lung. Thus, to determine whether relative lung APN deficiency is associated with an exaggerated ALI phenotype, we compared the i.t. LPS response in wt, APN−/−, and T-cad−/− mice. Mouse assessment scores measured at 4 hours were significantly decreased in T-cad−/− mice when compared to APN−/− mice and were comparable to wt mice (fig. 9a). Moreover, lung injury, measured by BAL protein concentration and assessed by histological examination was decreased in T-cad−/− compared to APN−/− mice. In addition, these mice showed decreased lung inflammation as evident by lower IL-6 concentrations and decreased CD45+ cell recruitment into the lung (fig 9b–e). These findings suggest that maintenance of serum APN is important in controlling lung inflammation through interactions with pulmonary endothelium.
To evaluate whether increasing serum APN could rescue APN deficiency, we pre-treated APN −/− mice with adenoviral vectors containing genes for APN (Ad-APN) or the control gene beta-galactosidase (Ad-gal). Serum APN concentrations measured 5 days after i.v administration of adenovirus were 19.5 +/− 11.1 mcg/ml in Ad-APN/APN −/− mice, < 0.01 in Ad-gal/APN −/− mice and 21.2 +/− 2.6 mcg/ml in Ad-gal/wt mice. Results of reconstitution studies demonstrated that mouse assessment scores were significantly decreased 4 hours after i.t. LPS in Ad-APN/APN −/− mice when compared to Ad-gal/APN−/− mice (fig 10). In addition, adenoviral mediated rescue was associated with improved endothelial barrier function as evidenced by decreased BAL protein concentration in Ad-APN/APN −/− mice. Serum and lung IL-6 concentrations were lower in Ad-APN/APN −/− mice when compared to Ad-gal/APN −/− mice but this did not reach statistical significance Taken together, these findings further illustrate the importance of APN in the lung and suggest a potential therapeutic role for this protein in enhancing endothelial barrier function during ALI.
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 (30–31), 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 (33–34). 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 (35–37). 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 (42–43). 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.
1This work was supported by National Institute of Health GrantsK08 HL077138, AG15052, and HL-59215, HL-105490.