Chemokine profiles of human visceral adipocytes from cryopreserved preadipocytes: Neutrophil activation and induction of nuclear factor-kappa B repressing factor
Introduction
Obesity significantly increases the risk of life-threatening conditions, such as heart disease, stroke, high blood pressure, type 2 diabetes, and cancer, and obese people are also more likely to develop infections. Obesity may influence humoral and cell-mediated immune responses [15]. Macrophages and neutrophils play a critical role in obesity-related inflammation [33].
Morbid obesity is associated with activation of innate immunity and neutrophil activation is fundamental to the innate immune response. Human neutrophil elastase (HNE) is a serine protease stored in the azurophilic granules of neutrophils that is linked to inflammatory diseases, including idiopathic pulmonary fibrosis, rheumatoid arthritis, and adult respiratory distress syndrome [12]. Alpha-1 antitrypsin (A1AT) is a major circulating inhibitor of neutrophil proteases, with HNE and A1AT forming a protease/protease inhibitor pair. An imbalance between them might cause tissue damage, promoting carcinogenesis and tumor progression [39], [44]. Imbalance between A1AT and HNE also contributes to development of obesity-related inflammation, insulin resistance, and hepatic steatosis [28]. Furthermore, elevation of serum HNE levels is related to prehypertension and airflow limitation in obese women [13].
Excess adiposity is an established risk factor for metabolic diseases. Visceral adipose tissue plays a major role in the low-grade inflammation associated with obesity and leukocyte infiltration into adipose tissue is critical for obesity-related inflammation. Chemokines induce chemotaxis (migration) of target cells along concentration gradients. Serum levels of some chemokines are dramatically increased in obese versus lean individuals, and expression of chemokine receptors by inflammatory cells is elevated in the omental and subcutaneous adipose tissue of obese persons [1], [40]. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) is a CC chemokine that guides monocytes to leave the blood and become tissue macrophages, which is the first step toward inflammation. Obese adipose tissue is characterized by enhanced infiltration of macrophages [18]. Obese people have higher circulating levels of many inflammatory markers than lean people [7], including elevation of MCP-1 and interleukin (IL)-8 [23]. MCP-1 is overexpressed in obese mice compared with lean controls, and white adipose tissue is a major source of MCP-1 [36]. The MCP-1 gene is a target of nuclear factor-kappa B (NF-kB). Interestingly, HNE promotes synthesis of IL-8 by human airway smooth muscle cells through induction of NF-kB repressing factor [19]. These reports suggest that activation of neutrophils may modulate chemokine expression in the adipose tissue of obese individuals, while the differing chemokine profiles of obese and non-obese persons indicate a shift from innate immunity to adaptive immunity in obesity. Accordingly, we investigated the chemokine profile of human visceral adipocytes derived from cryopreserved preadipocytes and the effect of HNE on adipocyte chemokine production.
Section snippets
Human visceral adipocytes
Human preadipocytes were obtained from DS Pharma Biomedical Co., Ltd. (Suita, Japan) and were cultured according to a standard protocol [17]. Human visceral preadipocytes were isolated from omental adipose tissue obtained with the informed consent of the donors (Zen-Bio, Inc., Research Triangle Park, NC). Donor information is described in Table 1.
Extraction of RNA and reverse transcription polymerase chain reaction (RT-PCR)
Monocytes (1 × 106 cells) were extracted with 1 mL of Isogen RNA kit (Nippon Gene, Toyama, Japan). Total RNA was isolated and precipitated according to
Results
Human preadipocytes that were cryopreserved at the end of primary culture could be propagated for two passages prior to differentiation into adipocytes. Fig. 1 shows the histological appearance of fibroblast-like preadipocytes before differentiation (a and c), and that of mature adipocytes stained with Oil red O to detect neutral fat (triglycerides) and lipids (b and d).
Chemokine responses associated with obesity-related inflammation were well preserved in cultured human adipocytes derived from
Discussion
In obese persons, the expanding adipose tissue is characterized by infiltration of proinflammatory immune cells that cause chronic low-grade inflammation. Phenotypic switching of macrophages is an important mechanism of adipose tissue inflammation, and cells from the adaptive immune system are involved in this process [37]. The present study demonstrated that the chemokine responses associated with obesity-related inflammation were well preserved in cultured human adipocytes derived from
Conclusion
Adipocytes obtained from persons with a BMI > 30 kg/m2 were found to have a different chemokine profile to adipocytes from persons with a BMI < 30 kg/m2. Only adipocytes from persons with a BMI > 30 kg/m2 expressed lymphotactin-β and fractalkine mRNA. In addition, HNE induced NF-kB repressing factor, suggesting that it may modulate adaptive immunity in obesity-related diseases.
Conflict of interest statement
All authors have no conflicts of interest concerning this work.
Acknowledgment
This study was supported by Kumamoto Health Science University special fellowship grant No. 27-A-01.
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