Volume 3, Issue 4 , Pages 203-207, November 2009
Proinflammatory and phagocytic functions of alveolar macrophages in obesity
Article Outline
Summary
Background
Recent epidemiologic investigations have pointed to an increase susceptibility of obese individuals to lower respiratory tract infections. The cellular mechanism responsible for this phenomenon has not been identified.
Objectives
To assess whether obesity per se impairs the proinflammatory and antimicrobial functions of alveolar macrophages (AM).
Methods
Six obese (BMI
≥30<40kg/m2) and six morbidly obese (BMI≥40kg/m2) subjects free of comorbid diseases participated in the study. A control group (BMI<25kg/m2) matched for age and gender was included. Alveolar macrophages collected by bronchoalveolar lavage were tested for lipopolysaccharide (LPS) stimulated production of TNF-α. Phagocytosis was measured by assessing the degree of ingested opsonized and unopsonized particles. Microbicidal activity was determined by the ability of AM to kill Listeria monocytogenes.
Results
The percentage of AM in the bronchoalveolar lavage was comparable among the three groups. There was no significant difference of TNF-α levels at baseline and after LPS-stimulated production between obese, morbidly obese, and nonobese subjects. Opsonized and unopsonized phagocytosis and microbicidal activity remained intact and was not affected by increasing BMI.
Conclusion
Our data suggest that in the absence of underlying comorbidities, the increased frequency of respiratory infections in obesity cannot be explained by impairment of alveolar macrophages. Further work is required to delineate the relationship between obesity and the noncellular aspects of innate immunity.
Keywords: Obesity, Alveolar macrophages, Phagocytosis, TNF-α, Microbicidal activity
Introduction
Obesity is an increasing health problem worldwide. Currently, 30–50% of the adult population in developed countries is overweight (body mass index [BMI])>25kg/m2, with 10–20% of all adults being at least moderately obese (BMI>30kg/m2) [1]. Latest estimates in the United States indicate that 64% of adults are overweight and 31% are clinically obese [2]. According to a study of national costs attributed to both overweight and obesity, medical expenses accounted for 9.1% of total U.S. medical expenditures in 1998 and may have reached as high as $78.5 billion ($92.6 billion in 2002 dollars) [3]. These costs are directly related to the increased risk for morbidity and mortality from acute and chronic medical conditions, including hypertension, dyslipidemia, coronary heart disease, diabetes mellitus, gallbladder disease, gout, arthritis, and respiratory disease [4], [5].
Less well known, but not less important, are the respiratory tract infections associated with morbid obesity. This positive association has been suggested from a cohort study of more than 100,000 participants of two large US health surveys [6]. The risk of pneumonia was nearly 2-fold higher among individuals who gained 40lb or more during adulthood. A similar increase in frequency of nosocomial pneumonia was reported in critically ill morbidly obese patients [7]. Yet, the exact cellular mechanism underlying this phenomenon is not known. Hence, we hypothesized that obesity impairs the proinflammatory and antimicrobial function of alveolar macrophages. To test this hypothesis, we conducted a prospective controlled study to evaluate a number of alveolar macrophages functions including: (1) the effect of lipopolysaccharide-mediated TNF-α release; (2) opsonized and nonopsonized phagocytosis; and (3) microbicidal activity.
Methods
Study population
After obtaining approval from the Institutional Review Board at the University at Buffalo, an informed consent was obtained from all participants. Six obese (six obese (BMI>30 and<40kg/m2) and six morbidly obese subjects (BMI≥40kg/m2) requiring mechanical ventilation for airway protection were enrolled during a 32-month period. A control group of six patients (BMI≤25kg/m2)-matched for age and gender were enrolled during the same study period. Patients with diabetes mellitus, cardiac failure, pulmonary disease, and/or history of smoking were excluded from participation. Patients receiving chronic anti-inflammatory agents, immunosuppressive treatment or steroid therapy (inhaled or systemic) were also excluded.
Isolation of alveolar macrophages
Non-bronchoscopic bronchoalveolar lavage was performed within 4h of intubation according to previously described methodology [8]. Three aliquots (20ml each) of sterile saline were instilled and aspirated. A volume of >50% was retrieved. The collected samples were strained through a single layer of loose cotton and were subsequently centrifuged at 250×g for 10min at 4°C. Total cell number was determined by a hemocytometer. The viability of the cells was assessed by Trypan blue. Differential cell counts were performed on cytopsin preparations stained with a modified Giemsa-based Diff-Quick stain (Baxter Scientific Products, McGraw Park, IL).
Alveolar macrophages cultures were performed as previously described. After washing with phosphate-buffered saline solution, the BAL cells were resuspended in RPMI 1640 (Gibco, Eragny, France), supplemented with 10% heat inactivated fetal calf serum, 2mM l-glutamine, 105U/l penicillin, 0.25mg/l amphotericin B, and 100mg/l streptomycin to a final concentration of 1×106cells/ml. The cell suspension was plated at 1×106cells/well in 24-well culture plates (Falcon Laboratories, McLean, VA) and allowed to adhere for 1h at 37°C in a 5% CO2 humidified atmosphere. Nonadherent cells were removed by three washes with warmed RPMI. The adherent cell population was >98% alveolar macrophages.
LPS-stimulated alveolar macrophages
Plated macrophages were incubated without or with 1ml of RPMI 1640 medium and lipopolysaccharide (LPS) [10μg/ml Escherichia coli (strain 026: B6)]; Sigma Chemical Co., St Louis, MO] for 1h. The culture supernatants were harvested and stored in fractions at −80°C until analysis. The concentrations of TNF-α in culture supernatants were quantified using commercially available human enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN). All assays were conducted in duplicate.
Phagocytic and bactericidal activity
Nonopsonized and opsonized (1.0μm diameter) particles were added with particle-to-cell ratio of 15:1. The tubes were incubated for 15min, and the phagocytosis was then stopped by addition of 2ml ice-cold balanced saline solution. The cell suspension was placed on a glass slide, fixed, and stained. The proportion of macrophages that ingested at least one particle and the number of fluorescent particles per positive phagocytic alveolar macrophage were noted. Bactericidal ability of the alveolar macrophages was determined by their ability to kill Listeria monocytogenes. Listeria were stored at a concentration of 1×109 colony-forming unit/ml in the RPMI-1640 medium (Gibco BRL, Life Tech Inc., Rockville, MD) and stored at −80°C until use. Separated alveolar macrophages were resuspended in 0.5ml RPMI containing 10% normal human serum. The bacteria were resuspended in the same medium at a concentration of 2×106colony-formingunit/ml and incubated for 30, 60, and 90min in 5% CO2/air. The pellets of alveolar macrophages were lysed by adding 10ml of sterilized distilled water. Release of bacteria was done by vortexing for 30s. The viable fraction of Listeria bacteria was determined by plating serial 10-fold dilutions on agar plates. The number of colonies of Listeria was counted after 48h of incubation. The rate at which alveolar macrophages killed Listeria was calculated by dividing the fraction of the initial inoculum of Listeria killed by the fraction of the initial inoculum surviving in the control (cell-free) tubes.
Statistical analysis
Data are expressed as the mean±SD. Within each group, the data were analyzed using Kruskal–Wallis one-way analysis of variance on ranks. A p value of <0.05 was considered statistically significant.
Results
The characteristics of the study population are shown in Table 1. There was no difference in the severity of illness, PaO2/FIO2, or peak airway pressures among the participants. None of the patients had radiographic abnormalities on presentation. Table 2 depicts the total cell number and composition of alveolar cells in the lavage fluid of each group. The percentage of alveolar macrophages was comparable among the three categories. Although the basal rate production of TNF-α was higher for the obese (544±120pg/ml) and morbidly obese (565±105pg/ml) compared to the nonobese group (477±142pg/ml), the difference was not statistically significant (ANOVA; p=0.4). In vitro stimulation of alveolar macrophages with LPS resulted in expected increases in the production of TNF-α among all groups (Fig. 1). The degree of stimulation was however comparable in the obese (5973±947pg/ml), the morbidly obese (6175±1186pg/ml) and the nonobese (6465±1064pg/ml) (ANOVA; p=0.7).
Table 1. Characteristics of the study population.
| Nonobese (n=6) | Obese (n=6) | Morbidly obese (n=6) | p value | |
|---|---|---|---|---|
| Age (years) | 40.8±7.9 | 38.3±5.1 | 43.5±5.8 | 0.8 |
| BMI (kg/m2) | 23.3±1.6 | 35.2±2.8 | 47.8±3.3 | <0.001 |
| APACHE II | 16.4±2.4 | 17.5±2.3 | 17.2±2.2 | 0.5 |
| PaO2/FIO2 | 467±32 | 440±23 | 431±26 | 0.08 |
| Peak airway pressure (cm H2O) | 15.8±2.7 | 17.5±2.6 | 19.2±3.3 | 0.1 |
| Reasons for mechanical ventilation | ||||
| Drug overdose | 6 | 5 | 4 | |
| Intracranial hemorrhage | 0 | 0 | 1 | |
| Seizure | 0 | 1 | 0 | |
| Head trauma | 0 | 0 | 1 | |
Table 2. Cellular composition of bronchoalveolar lavage.
| Nonobese (n=6) | Obese (n=6) | Morbidly obese (n=6) | |
|---|---|---|---|
| Total cell (×106) | 202.8±34.1 | 209.7±35.9 | 189.3±43.5 |
| Macrophage (%) | 90.8±2.6 | 88.2±3.4 | 89.5±3.3 |
| Lymphocyte (%) | 8.5±1.9 | 7.5±2.0 | 7.1±1.4 |
| Neutrophil (%) | 1.3±0.5 | 1.5±0.8 | 1.4±0.5 |
| Viability (%) | 97.3±1.0 | 97.3±1.2 | 97.8±1.6 |
Figure 1.
Alveolar macrophages production of TNF-α at baseline and after LPS stimulation in nonobese (n
=6), obese (n=6), and morbidly obese subjects (n=6).
The fraction of alveolar macrophages ingesting opsonized and nonopsonized particles were similar in each of the three groups (ANOVA; p=0.6 and p=0.9, respectively) (Fig. 2). Similarly, microbicidal activity at 30-, 60-, and 90-min incubations did not reveal a significant variation among the various groups at each of the time point selected (Fig. 3).
Figure 2.
Comparison of the percentage of alveolar macrophages ingesting both opsonized (white bars) and unopsonized particles (gray bars) between nonobese (n
=6), obese (n=6), and morbidly obese subjects (n=6).
Figure 3.
Comparison of the percentage of Listeria monocytogenes killed by alveolar macrophages at 30, 60, and 90
min incubation between nonobese (n=6), obese (n=6), and morbidly obese subjects (n=6).
Discussion
The results of our study indicate that the ex vivo anti-inflammatory and antimicrobial responses of alveolar macrophages in obese and morbidly obese patients are preserved when compared to age and gender matched nonobese individuals.
Obesity has been viewed as a low grade inflammatory state characterized by increased levels of pro-inflammatory cytokines and acute phase reactants [9], [10], [11]. The over expression of TNF-α and leptin induces the production of IL-6, CRP which are thought to contribute to the increased risk of diabetes and cardiovascular diseases. These markers can also upregulate the expression of specific receptors responsible for adherence of bacteria to endothelial and alveolar epithelial cells [12], [13]. In addition, systemic inflammatory mediators may also reduce the ability of the host to mount an adequate immune response by reducing neutrophil recruitment and impairing alveolar macrophage phagocytosis [14]. Animal studies conducted in genetically obese animals seem to confirm this hypothesis. Alveolar macrophages from leptin-deficient (ob/ob) mice and mice with dysfunctional leptin receptors (db/db) have an impaired ability to eliminate Candida albicans and to produce proinflammatory cytokines [15]. The repletion of leptin restores the phagocytic function in ob/ob mice but not from db/db mice suggesting that leptin regulates macrophage phagocytosis via direct interaction with its receptors [16].
Our results demonstrate no significant impairment in TNF-α response of alveolar macrophages upon LPS stimulation when comparing obese, morbidly obese, and nonobese subjects. In contrast to leptin-deficient animals, the phagocytic and the microbicidal activities remained relatively unaffected with increasing BMI. Since obesity in humans is related to excess calories and reduced energy expenditure rather than leptin deficiency [17], the association between obesity and increased respiratory infections might reflect a change in the local alveolar environment attributed to various comorbidities linked to excessive weight like asthma or diabetes mellitus. Although systemic mediators like leptin could potentially influence the functions of alveolar macrophages, the compartmentalization of these mediators would argue against this possibility. Our findings are corroborated by a recent study in mice which found no significant difference in the LPS-stimulated production of TNF-α by alveolar macrophages between animals fed on a high fat diet and those receiving a standard laboratory diet [18].
A potential confounding variable is the presence of sleep apnea in our study population that may contribute to the release of inflammatory markers. While none of the controls or the obese patients had prior history of sleep apnea, two of the morbidly obese patients were being treated with continuous positive airway pressure. To our knowledge, there have been no human studies that have assessed the expression of inflammatory markers in the alveolar space in patients with sleep apnea. Hence we cannot determine whether CPAP had any effect on our results.
In conclusion, the pro-inflammatory response and phagocytic functions of alveolar macrophages are preserved in obese individuals. Whether the increased rate of respiratory infections observed in cohort studies are simply a reflection of comorbid conditions associated with obesity or actually results from a disturbance in the noncellular components of innate immunity will require further investigations.
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PII: S1871-403X(09)00039-8
doi:10.1016/j.orcp.2009.05.001
Published by Elsevier Inc.
Volume 3, Issue 4 , Pages 203-207, November 2009
