American Journal of Respiratory and Critical Care Medicine

Retraction: The authors of this article, in conjunction with the Journal editors, have decided to retract the article because of problems with the reliability of its data. The Journal had previously published an Expression of Concern regarding this article. All the authors have agreed to this retraction except for one, whom we have been unable to contact. The authors regret the inconvenience to the Journal and its readers.

Rationale: Obesity is associated with increased prevalence and severity of asthma. Adipose tissue macrophages can contribute to the systemic proinflammatory state associated with obesity. However, it remains unknown whether alveolar macrophages have a unique phenotype in overweight/obese patients with asthma.

Objectives: We hypothesized that leptin levels would be increased in the bronchoalveolar lavage fluid from overweight/obese subjects and, furthermore, that leptin would alter the response of alveolar macrophages to bacterial LPS.

Methods: Forty-two subjects with asthma and 46 healthy control subjects underwent research bronchoscopy. Bronchoalveolar lavage fluid from 66 was analyzed for the level of cellular inflammation, cytokines, and soluble leptin. Cultured primary macrophages from 22 subjects were exposed to LPS, leptin, or leptin plus LPS. Cytokines were measured in the supernatants.

Measurements and Main Results: Leptin levels were increased in overweight/obese subjects, regardless of asthma status (P = 0.013), but were significantly higher in overweight/obese subjects with asthma. Observed levels of tumor necrosis factor-α were highest in overweight/obese subjects with asthma. Ex vivo studies of primary alveolar macrophages indicated that the response to LPS was most robust in alveolar macrophages from overweight/obese subjects with asthma and that preexposure to high-dose leptin enhanced the proinflammatory response. Leptin alone was sufficient to induce production of proinflammatory cytokines from macrophages derived from overweight/obese subjects with asthma.

Conclusions: Ex vivo studies indicate that alveolar macrophages derived from overweight/obese subjects with asthma are uniquely sensitive to leptin. This macrophage phenotype, in the context of higher levels of soluble leptin, may contribute to the pathogenesis of airway disease associated with obesity.

Scientific Knowledge on the Subject

Obesity is associated with an increased risk of developing asthma, and obese patients with asthma have more severe disease with altered response to standard therapies.

What This Study Adds to the Field

We show that, similar to adipocyte tissue macrophages that exhibit an increased proinflammatory response in obesity, primary alveolar macrophages derived from overweight/obese subjects with asthma demonstrate enhanced proinflammatory responses. Our data suggest an interaction between asthma and obesity, which results in a unique population of alveolar macrophages with a proinflammatory response to leptin stimulation.

There is an increased risk of developing asthma in individuals who are overweight and obese (subsequently referred to as overweight/obese) (15). The risk of asthma increases steadily with increasing body mass index (BMI) and is higher in women (1, 6). Furthermore, BMI negatively impacts asthma quality of life scores, asthma control scores, severity of exacerbations, and asthma-related hospitalizations (69). Together, these studies suggest a role of obesity in the pathogenesis of asthma. However, the mechanisms by which obesity may contribute to airway disease remain poorly understood.

Evidence suggests that adipose tissue is metabolically active, participating not only in energy homeostasis but also in inflammation (12). Adipose tissue secretes biologically active cytokines, including tumor necrosis factor (TNF)-α and IL-6, as well as adipokines, including leptin, adiponectin, plasminogen activator inhibitor (PAI)-1, and resistin (10, 11). Obesity is associated with systemic inflammation, which is characterized by elevated serum levels of these adipokines, chemokines, and acute-phase proteins (12). Adipose tissue is infiltrated by a significant number of macrophages that contribute to inflammation (13). These adipose tissue macrophages can secrete TNF-α, which contributes to insulin resistance in overweight/obese individuals (14). It remains unclear whether obesity is associated with an altered macrophage phenotype in the lung.

Obesity is also associated with altered levels of serum leptin, and the effects of leptin are believed to be proinflammatory in diseases outside of the lung (15). Leptin is associated with increased airway hyperresponsiveness and serum IgE in murine models (16). In humans, higher serum leptin levels are associated with the presence of asthma, an effect that is more prominent in women. However, serum leptin levels do not entirely account for the increased risk of asthma associated with obesity (17). Leptin levels are elevated in bronchoalveolar lavage fluid (BALF) obtained from overweight/obese subjects and is correlated to increased BMI. BALF leptin content is correlated with serum leptin content, leading to the conclusion that leptin diffuses into the lung from the circulation (18). Interestingly, overweight/obese individuals with asthma demonstrated the highest levels of both BALF and serum leptin. However, no differences in inflammatory markers or oxidative stress were noted in this cohort (18). In spite of these preliminary findings the role of leptin in the pathogenesis of airway disease remains poorly understood.

The aim of this study was to determine whether the level of leptin in the airspace was associated with either BMI or lung function, and whether leptin contributes to the pathobiology of asthma in overweight/obese subjects. Inclusion of both overweight/obese and lean asthmatic and nonasthmatic (normal control) subjects facilitated identification of a unique macrophage phenotype dependent on both asthma and obesity.

Some of the results of these studies have been previously reported in the form of an abstract (19).

Study Subjects

The Duke University (Durham, NC) Institutional Review Board approved the protocol. Subjects were recruited from the population in Durham County and the surrounding areas. Informed consent was obtained from each subject (18–65 yr of age). Obese (BMI, >30) and overweight (BMI, 25–30) subjects were pooled into one group denoted as “OA” for overweight/obese asthma and “ON” for normal control subjects (20). Subjects with asthma met National Asthma Education and Prevention Program criteria for mild asthma; had physician-diagnosed asthma, the presence of reversible airflow obstruction, and a methacholine PC20 (provocative concentration of methacholine causing a 20% fall in FEV1) not greater than 8 mg/ml; and used only rescue bronchodilators. Healthy subjects had a methacholine PC20 greater than 16 mg/ml, no evidence of airflow obstruction, and no history of pulmonary disease. Exclusion criteria included use of maintenance medication for treatment of asthma, antibiotics, or oral corticosteroids within 4 weeks of the study. In general, subjects who used inhaled corticosteroids within 4 weeks of study enrollment were excluded.

Bronchoscopy, bronchoalveolar lavage fluid measurements, and ELISA

Research bronchoscopy was performed on 88 subjects and cell counts/differentials were determined as previously described (21). BALF from 66 subjects was concentrated 20-fold (Millipore, Billerica, MA). Adiponectin and leptin were quantified by ELISA (R&D Systems, Minneapolis, MN). A multiplex cytokine assay was used to measure IL-1β, IL-5, IL-6, IL-8, IL-10, IL-12 (p70), IL-13, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1β, IFN-γ–inducible protein-10 (IP10), vascular endothelial growth factor (VEGF), and TNF-α (Millipore, Billerica, MA). BALF protein levels were analyzed (Pierce, Rockford, IL). Cytokine levels were then normalized to protein and recorded as picograms per milligram.

Macrophage Ob-Rb receptor expression and functional response

Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine expression of the long form of the leptin receptor (Ob-Rb) (Hs00174497_m1 [cat. no. 4331182]; Applied Biosystems/Life Technologies, Carlsbad, CA). Ex vivo macrophage experiments were performed on samples obtained from 22 subjects. Briefly, 5 × 105 cells per well were cultured for 2 hours, and nonadherent cells were removed. Cells were cultured in triplicate in the presence/absence of leptin (250 ng/ml, 20 h) followed by ultrapure Escherichia coli 0111:B4 LPS (InvivoGen, Carlsbad, CA) (100 ng/ml, 90 min). Dose and duration of LPS exposure were based on studies with macrophages obtained from overweight subjects with asthma (see Figure E1 in the online supplement) and prior publications that used leptin at 500 ng/ml in experiments assessing the peritoneal macrophage response to LPS (22). The cell-free supernatants were stored at –80°C and cells were stored in TRIzol (Invitrogen, Carlsbad, CA). Multiplex ELISA (Millipore, Billerica, MA) was used to measure cytokine levels (IL-5, IL-6, IL-8, IL-10, IFN-γ, and TNF-α) in supernatants obtained from these experiments.

Statistical Analysis

Statistical analyses were performed with JMP statistical software (SAS, Cary, NC). The distribution of cytokine levels was not a normal Gaussian distribution and therefore the data were log-transformed with base 10. Two-way analysis of variance (ANOVA) with an interaction term was used to compare demographics and cytokine and adipokine levels. Cell counts, BALF leptin, adiponectin, and cytokine levels were dependent variables; sex, weight, and asthma status were independent variables. We performed analyses for two- and three-way interactions using the independent variables. We adjusted the analysis for BMI. We also adjusted for multiple comparisons using the Bonferroni method with familywise error rate controlled at 10% and an unadjusted P value less than 0.003 was required to achieve significance.

To further characterize the differences in BALF leptin levels, BALF TNF-α levels, and Ob-Rb expression, we performed one-way ANOVA with nonparametric Wilcoxon rank sum test and Tukey-Kramer test for group comparisons.

Linear regression was used to determine the relationship between log-transformed normalized BALF leptin and log BMI and FEV1. Two-way ANOVA was used to determine the effects of BALF leptin levels on FEV1 after adjusting for BMI. Methacholine PC20 was not normally distributed and therefore Spearman’s ρ was used to determine the correlation between methacholine PC20 and BALF leptin levels.

The cytokine levels obtained from macrophage culture supernatants were normalized to unexposed control and analyzed by one-way ANOVA with paired comparisons. Paired t tests were used to compare the pretreatment and posttreatment cytokine levels within each group as data were normally distributed. Two-way ANOVA was used to determine the effects of weight status, asthma status, and a combination of these factors on the production of various cytokines in macrophage supernatants. Statistical significance was defined as two-sided P < 0.05 for all analyses.

Subject Characteristics

Subject characteristics are shown in Table 1. Of the 88 subjects enrolled, 42 had asthma and 46 were normal control subjects. Obesity was associated with an older age group regardless of asthma status (P = 0.004). There were no differences in sex between groups. Overweight/obese subjects were more likely to be black (P = 0.0012). The mean BMI was not significantly affected by asthma status. Lung function as quantified by FEV1 (liters) was lowest in the overweight/obese subjects with asthma (P = 0.022 compared with lean asthma; P = 0.034 compared with obese normal). No subjects took statins or thiazolidinediones, which could have a potential antiinflammatory effect (2325). Twenty-two subjects were included in the ex vivo experiments and the characteristics of this cohort are included in Table 2. The subjects for ex vivo experiments were relatively well matched in the four groups except for the lean asthma group, which was composed only of female subjects.


Normal Subjects
Subjects with Asthma
Weight/Asthma StatusLean (n = 23)Overweight/Obese (n = 23)Lean (n = 17)Overweight/Obese (n = 25)
P ValueMean (SD)Mean (SD)Mean (SD)Mean (SD)
Age, yrO0.00426.9 (6.6)33.2 (11.1)25.82 (8.6)32.8 (12.1)
Sex, male:femaleO0.5510M:13F7M:16F6M:11F9M:16F
RaceO0.001216 White8 White13 White16 White
A0.395 Black15 Black2 Black9 Black
O–A0.992 Asian2 Asian
BMI, kg/m2O0.000122.8 (1.4)32.4 (4.4)21.4 (1.86)33.9 (9.4)
FEV1, LO0.0343.69 (0.76)3.24 (0.64)3.21 (0.77)2.97 (0.82)
FEV1, %O0.22102.2 (14.0)98.4 (9.1)88.7 (15.6)85.3 (15.1)
FEV1/FVC ratioO0.3982.479.87473.7
Methacholine PC20O0.81>16>160.97 (1.4)0.99 (1.7)
Number of subjects with allergic rhinitisO0.483/235/2317/1722/25
Number of subjects receiving ICSON/AN/AN/A0/171/25
Number of subjects with GERDO0.111/233/232/177/25

Definition of abbreviations: BMI = body mass index; F = female; GERD = gastroesophageal reflux disease; ICS = inhaled corticosteroids; M = male; N/A = not applicable; methacholine PC20 = provocative concentration of methacholine causing a 20% fall in FEV1.

Variables included in the analysis include weight (O) and asthma (A) with an interaction term (O–A). Bold type represents statistical significance.


Normal Subjects
Subjects with Asthma
Weight/Asthma StatusP ValueLean (n = 5)Overweight/Obese (n = 4)Lean (n = 5)Overweight/Obese (n = 8)
Mean (SD)Mean (SD)Mean (SD)Mean (SD)
Age, yrO0.5328.6 (8.6)28 (2.0)24.33 (2.5)30 (10.6)
Sex, male:femaleO0.952M:3F1M:3F0M:5F3M:5F
RaceO0.974 White2 White3 White3 White
A0.981 Asian2 Asian2 Black5 Black
BMI, kg/m2O0.000422.6 (1.4)29.94 (3.7)20.8 (1.21)35.1 (8.6)
FEV1, LO0.743.61 (0.76)3.41 (0.2)3.1 (0.48)3.13 (0.56)
FEV1, %O0.82102.3 (12.7)101.5 (9.1)93.3 (6.12)93.4 (10.5)
FEV1/FVC ratioO0.6188.984.276.778.3
Methacholine PC20O0.14>16>163.02 (2.7)1.1 (0.9)

Definition of abbreviations: BMI = body mass index; methacholine PC20 = provocative concentration of methacholine causing a 20% fall in FEV1.

Variables included in the analysis include weight (O) and asthma (A) with an interaction term (O–A). Bold type represents statistical significance.

Effects of Obesity on BALF Differential Cell Counts

To determine the effects of obesity on asthma phenotype, we obtained BALF and performed differential cell counts. No group differences were appreciated in BALF differentials (Table E1). In this cohort, 13% of lean subjects with asthma and 15% of overweight/obese subjects with asthma had greater than 2% eosinophils in BALF (data not shown).

Effect of Obesity and Asthma on Adipokine Levels in BALF

Previous work has shown increased levels of leptin in both serum and BALF from overweight/obese subjects (17, 18, 26). We also identified a significant correlation between log BALF leptin levels and log BMI (Figure E2). In our cohort, BALF leptin levels were significantly higher in overweight/obese subjects with asthma (Figure 1A), an effect that was more prominent in females (Figure 1B). There were no detectable differences in leptin levels between groups of male subjects (Figure 1C). Despite the association between adiponectin and asthma (27), we did not identify any differences in BALF adiponectin levels between groups. There were no significant differences in the mean BMI of male and female subjects (26.2 ± 0.88 vs. 28.78 ± 1.16, P = 0.15, respectively), suggesting that sex differences in BALF leptin levels are not related primarily to higher BMI in females. In spite of this, the range of BMI values appeared quite different between males and females (21.32–38.87 in males and 18.0–60.9 in females).

We noted an inverse correlation between FEV1 and log BALF leptin levels (r2 = 0.17, P = 0.005) that was present after adjusting for BMI. We did not observe a significant correlation between leptin levels and bronchial hyperresponsiveness.

Effect of Obesity and Asthma on BALF Cytokine Profiles

Previous work supports the hypothesis that a systemic proinflammatory phenotype exists in obesity. Therefore, we measured the levels of IL-1β, IL-5, IL-6, IL-8, IL-10, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, IP10, VEGF, and TNF-α in BALF. Using a Bonferroni correction, a significant P value in this analysis is considered P < 0.003. Although we did not observe significant differences in many of these factors in the BALF, we did observe several trends. The presence of asthma had an effect on BALF IL-5 (P = 0.011) and IL-10 (P = 0.04) levels. The interaction between asthma and weight status resulted in higher BALF IFN-γ (P = 0.02) and TNF-α (P = 0.03) levels in overweight/obese subjects with asthma. There was an effect of the sex × weight interaction on MCP-1 levels, with overweight/obese females with asthma having the highest levels of MCP-1 in the BALF (P = 0.04). Interestingly, VEGF levels were low in overweight/obese subjects regardless of asthma status (P = 0.049). Obesity and female sex had effects on BALF leptin levels (P = 0.011 and P < 0.0001, respectively) (Table 3).


Normal Subjects
Subjects with Asthma
Weight/Asthma StatusP ValueLean (n = 17)Overweight/Obese (n = 17)Lean (n = 12)Overweight/Obese (n = 20)
BAL BiomarkerMean (95% CI)Mean (95% CI)Mean (95% CI)Mean (95% CI)
IFN-γ, pg/mlO0.918.4 (4.1–17.3)3.31 (1.5–7.7)4.70 (2.0–10.7)10.2 (6.9–15.1)
IL-10, pg/mlO0.632.36 (1.02–5.37)1.26 (0.59–2.75)2.52 (0.92–6.98)3.71 (2.47–5.57)
IL-5, pg/mlO0.740.83 (0.4–1.9)0.49 (0.2–1.0)1.03 (0.5–2.2)1.60 (1.0–2.5)
TNF-α, pg/mlO0.417.03 (2.2–10.9)4.56 (2.9–7.3)3.79 (2.0–7.1)9.82 (6.6–14.4)
MCP-1, pg/mlO0.63691.83 (467.7–1,023.3)724.43 (524.81–1,000)630.95 (363.1–1,096.5)616.59 (446.68–851.14)
VEGF, pg/mlO0.051,000 (575.44–1,737.8)549.5 (371.53–812.83)1,122 (602.55–2,137.9)676.08 (416.87–1,122.1)
Leptin, pg/mlO0.01126.97 (13.19–40.76)49.32 (22.1–76.54)15.35 (5.91–24.79)142.45 (67.78–217.11)

Definition of abbreviations: BAL = bronchoalveolar lavage; CI = confidence interval; MCP-1 = monocyte chemotactic protein-1; TNF-α = tumor necrosis factor-α; VEGF = vascular endothelial growth factor.

Independent factors include weight (O), asthma (A), and the interaction term (O–A). Bold type represents statistical significance.

Prior studies demonstrate that leptin can increase TNF-α production in adipocyte macrophages (13); however, the effects of leptin on lung macrophages are unknown. We aimed to determine whether BALF TNF-α levels differed between groups and noted higher levels in overweight/obese subjects with asthma in comparison with lean subjects with asthma (P = 0.03) and overweight/obese normal subjects (P = 0.04) (Figure 2A), an effect that was more prominent in females (Figure 2B). There were no observed differences between groups of male subjects (Figure 2C). Furthermore, we determined that BALF leptin and TNF-α are significantly correlated, but in female subjects only (r2 = 0.36, P = 0.0008) (Figures 3A–3C).

Leptin Ob-Rb Receptor Expression on Alveolar Macrophages

It is recognized that the Ob-Rb receptor binds the long form of leptin and is the dominant receptor that mediates intracellular signaling. We determined that Ob-Rb is expressed in alveolar macrophages; however, no significant differences in Ob-Rb mRNA expression were noted between the four groups (data not shown). This finding does not necessarily address the functional consequences of soluble leptin on alveolar macrophages.

Functional Response of Alveolar Macrophages in a Manner Dependent on Obesity and Asthma

To determine whether the LPS and leptin response in macrophages is different, based on the presence or absence of asthma and obesity, we exposed primary alveolar macrophages to bacterial LPS ex vivo. For the purpose of this article, we have focused on the biological response to the bacterial toxin LPS as a marker of innate immune recognition. Similar to LPS, we hypothesized that there would be an immediate recognition of endogenous leptin in lung macrophages resulting in a proinflammatory signaling response.

We exposed lung macrophages to leptin, LPS, and leptin pretreatment followed by LPS exposure. Macrophages obtained from overweight/obese subjects with asthma produced significantly higher levels of IL-5, IFN-γ, TNF-α, and IL-10 in response to leptin, LPS, and leptin followed by LPS when compared with their respective unexposed control state (P < 0.05). We observed no increases in IL-6 production in response to leptin. However, LPS and leptin–LPS exposure resulted in significant increases in IL-6 production in overweight/obese subjects with asthma (P = 0.047 and P = 0.031, respectively). IL-8 levels were increased in response to both leptin (P = 0.016) and leptin–LPS (P = 0.047) but not LPS alone. There were no differences in cytokine production in the other three subject groups. Interestingly, only macrophages from overweight/obese subjects with asthma respond in a proinflammatory manner after the 90 minutes of exposure to LPS. This suggests that the response in these macrophages may be the result of secretion of preformed cytokines. The lack of response in the three other subject groups may be related to the duration of LPS exposure.

We then focused on determining whether differential cytokine production occurred between groups of subjects. There was significant variability in baseline cytokine levels and therefore we performed fold change comparisons, as we believe correction for constitutive levels of cytokines most accurately reflects the biological response to leptin and LPS. Leptin increased IL-5 in overweight/obese (P = 0.006) and lean subjects with asthma (P = 0.009) as compared with lean normal subjects (Figure 4A). IFN-γ levels were significantly higher in overweight/obese subjects with asthma when compared with lean normal (P = 0.005) subjects (Figure 4B). In addition, TNF-α and IL-8 levels were higher in overweight/obese subjects with asthma as compared with all the other groups (Figures 4C and 4D). The proinflammatory response to leptin appears most prominent in alveolar macrophages obtained from overweight/obese subjects with asthma.

We then aimed to determine the effects of LPS on lung macrophages in the absence of leptin treatment. LPS significantly increased IL-5 levels in overweight/obese subjects with asthma when compared with overweight/obese (P = 0.01) and lean (P = 0.005) normal subjects (Figure 4A). There were no differences in IFN-γ (Figure 4B) and TNF-α (Figure 4C) between groups. IL-8 levels were significantly higher in overweight/obese subjects with asthma when compared with lean (P = 0.01) and overweight/obese normal subjects (P = 0.003) (Figure 4D).

Previous studies provide evidence that leptin can prime subsequent response to LPS in cultured macrophages (22). Therefore, we pretreated macrophages with leptin followed by exposure to LPS. We noted that leptin–LPS exposure significantly increased IL-5 levels in overweight/obese subjects with asthma as compared with the other groups (Figure 4A). In addition, there were significantly higher IFN-γ and TNF-α levels in overweight/obese subjects with asthma when compared with overweight/obese normal (P = 0.016 and P = 0.003, respectively) and lean asthma (P = 0.031 and P = 0.017, respectively) subjects (Figures 4B and 4C). IL-8 levels were also higher in overweight/obese subjects with asthma in comparison with lean (P = 0.013) and overweight/obese (P = 0.001) normal control subjects (Figure 4D). These data indicate that pretreatment of macrophages with leptin before LPS exposure results in an enhanced proinflammatory response and that overweight/obese subjects with asthma have the greatest response. We did not observe lower baseline cytokine levels in overweight/obese subjects with asthma and therefore we attribute the increased fold change in cytokine levels to a more robust response to stimulation with leptin and LPS.

Given the significant response to leptin–LPS exposure noted in overweight/obese subjects with asthma, we aimed to determine whether the effects of leptin and LPS were additive or synergistic in this group of subjects. We noted a significant difference in IL-5 (P = 0.01) and TNF-α (P = 0.02) levels when comparing leptin and leptin–LPS exposure (Figures 4A and 4C). In addition, we noted a significant difference in IL-5 (P = 0.02) and IL-8 (P = 0.03) levels in response to leptin–LPS as compared with LPS only (Figures 4A and 4D). The effects of leptin pretreatment followed by LPS exposure appear additive, but we cannot rule out the possibility of leptin priming the macrophage response to LPS.

Last, we performed analyses to determine the effects of weight status, asthma status, and the interaction of these two factors on the difference in cytokine levels after exposure to leptin, LPS, and leptin–LPS. The obesity × asthma interaction resulted in increased IFN-γ, TNF-α, IL-8, and IL-10 in response to LPS. In addition, TNF-α, IL-8, and IL-6 levels were altered by the interaction between obesity and asthma in response to leptin pretreatment followed by LPS exposure (Tables E2A–E2C).

This study supports that obesity and asthma intersect to create unique inflammatory responses in the lung. We provide further support for the existing evidence that leptin levels are increased in the lungs of overweight/obese subjects (18, 28) and that female sex impacts the levels of leptin in the lungs. Furthermore, we demonstrate that increased levels of BALF TNF-α may be related to increased leptin levels in the lungs. The findings in this study demonstrate that overweight/obese subjects with asthma have increased levels of leptin and an increased alveolar macrophage response to high-dose leptin. This microenvironment would be anticipated to generate elevated levels of proinflammatory cytokines in the airspace. It has been previously reported that adipose tissue macrophages exhibit a proinflammatory state (29, 30). Our data provide evidence that alveolar macrophages from overweight/obese subjects with asthma similarly demonstrate a proinflammatory state.

Primary alveolar macrophages derived from overweight/obese subjects with asthma have an enhanced response to high-dose leptin resulting in increased levels of proinflammatory cytokines (IL-8 and TNF-α). The augmented response to leptin is not likely to be related to an increase in Ob-Rb expression, because we observed no differences in receptor expression as determined by mRNA levels. In fact, the augmented response is surprising given the fact that obesity is typically associated with leptin resistance, particularly in the brain (31, 32). The altered response of macrophages may be related to either activation of resident cells or recruitment of a new population of circulating cells. Alternatively, selective leptin resistance may be present in these subjects with central leptin resistance promoting obesity due to lack of appropriate responses to satiety, whereas peripheral cellular responses to leptin remain intact (33). Characterization of macrophage populations derived from the lungs of overweight/obese subjects with asthma could provide improved understanding of this differential response. These observations could have important implications given the leptin-rich environment in the lung and the functional consequences of leptin stimulation on macrophage populations.

This study also demonstrates important interactions between leptin and LPS. Leptin pretreatment resulted in further increases in the levels of IL-5, IFN-γ, and TNF-α in overweight/obese in comparison with lean subjects with asthma. Initially, we hypothesized that leptin primes the subsequent response to LPS as observed in mouse models (22). Our data suggest an additive effect of leptin on the biological response to LPS. Because obesity is associated with increased leptin levels, this could result in increased proinflammatory responses via various environmental stimuli that activate the innate immune system. Increased circulating free fatty acids in overweight/obese subjects could result in increased Toll-like receptor (TLR)-4 expression on macrophages (34), presenting an opportunity for increased LPS binding and the subsequent production of proinflammatory cytokines. Future studies will focus on understanding the distribution of TLRs on macrophages from obese subjects. Interestingly, obesity alone is not sufficient to elicit enhanced proinflammatory response as demonstrated by similar macrophage responses between obese and normal weight individuals without asthma. Overweight/obese subjects with asthma demonstrate an increased macrophage inflammatory response that appears to be a consequence of the interaction between asthma and obesity.

The current study has both strengths and limitations. We have a carefully characterized cohort of subjects with asthma stratified on the basis of BMI. This represents the first report to our knowledge of primary alveolar macrophages from obese subjects with asthma that includes subjects not receiving inhaled corticosteroid therapy. Our study is limited by the use of high doses of leptin, but is similar to previous work performed with murine peritoneal macrophages and response to LPS (22). Of note, serum leptin levels average 75 ng/ml (18). The physiological relevance of our dose of leptin will be determined in future studies. The dose and duration of LPS were based on our kinetic studies of alveolar macrophages obtained from obese/asthmatic subjects. We were surprised by the limited response to leptin and LPS in both the lean normal and obese normal groups. Future studies will require additional doses of leptin and dose–response assessment to LPS. Cell availability limits studies of primary human alveolar macrophages. The effect of atopic status on inflammatory responses and bronchial hyperresponsiveness remains unclear at this time.

Lack of atopy may play a role in alterations in airway hyperresponsiveness in overweight/obese subjects with asthma (35); however, the underlying mechanisms are poorly understood. Our study was underpowered to detect any differences in macrophage responses based on the presence or absence of atopy.

Adipose tissue macrophages play an important role in mediating systemic inflammation and the development of insulin resistance in obesity (36). Macrophages play an important role in the pathogenesis of diseases related to the metabolic syndrome. Prior studies aimed at identifying possible mechanisms that mediate the overweight/obese asthma phenotype have determined that CD4+ T cells do not appear to play a key role in this association (35). We now propose that alterations in macrophage responses create a unique inflammatory environment in the lungs of overweight/obese subjects with asthma. We suspect that macrophages are the key regulatory cells in the lung, but further studies are clearly warranted. It will be crucial to determine whether macrophage responses are altered by significant weight loss. We suggest a paradigm in which adipocyte production of leptin can amplify alveolar macrophage–derived inflammation in the overweight/obese patient with asthma. Our findings provide novel insights into the interaction that exists between obesity and asthma. Continued studies in this area could lead to more effective therapeutic approaches for overweight/obese patients with asthma.

The authors thank Denise Beaver, R.R.T., Donna Jinwright, C.R., Rhonda Webb, M.S., Cathy Hathcock, R.R.T., and Molly Huggins, B.Sc.

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Correspondence and requests for reprints should be addressed to Njira Lugogo, M.D., Duke University Medical Center, P.O. Box 2629, Durham, NC 27710. E-mail:

Supported by American Thoracic Society grant 07-012 and by grants P50-HL-084917, HL-05-009, HL086887, ES016126, and AI081672.

Author Contributions: N.L.L. participated in the conception of the project, performance of the experiments, data analysis, and writing of the manuscript. J.W.H. assisted in study design and data interpretation, and was substantially involved in writing the manuscript. D.L.H. recruited study subjects and assisted with experiments. L.G.Q. was instrumental in assisting with data interpretation and manuscript revision. D.F., T.D.C., and E.N.P.-K. performed the majority of the experiments and generated the data presented in this article. Drs. Ingram and Wang participated in data interpretation and revision of the manuscript. Prof. Jung offered statistical guidance and provided feedback on statistical analyses. Dr. Kraft provided the scientific guidance for the project and participated in study design, data interpretation, and revision of the manuscript.

This article has an online supplement, which is available from this issue’s table of contents at

Originally Published in Press as DOI: 10.1164/rccm.201109-1671OC on July 5, 2012

Author disclosures

American Journal of Respiratory and Critical Care Medicine

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