Release of C-reactive protein in response to inflammatory cytokines by human adipocytes: linking obesity to vascular inflammation.

To the Editor: Obesity, the most common nutritional disorder in industrialized countries, is associated with increased cardiovascular mortality and morbidity (1). C-reactive protein (CRP), an acutephase protein and an important predictor of future cardiovascular events in apparently healthy men and women (2), has been thought to be synthesized in the liver following stimulation by cytokines, such as interleukin (IL)-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha (3). Recently, however, the extrahepatic synthesis of CRP was found to occur under similar proinflammatory conditions (4). There is also some evidence for the presence of the CRP message in human adipose tissue (5). In the present study, we investigated whether CRP is produced by cells in adipose tissue in response to inflammatory stimuli using an in vitro model and whether this phenomenon might be modulated using antiinflammatory drugs. Primary cultures of human adipocytes from adipose tissue were performed as previously described (6), and the isolated adipocytes were incubated under the conditions required for each particular experiment. C-reactive protein levels in the cell supernatants were measured using an enzyme-linked immunoadsorbent assay specific for human CRP. The minimum concentration detected by the assay was 1.6 ng/ml. All experiments were performed in duplicate. Adipocytes were cultured in tubes, and cells from different donors were used for each experiment. The cells were incubated with recombinant human IL-1-beta (25 ng/ml), IL-6 (10 ng/ml), resistin (100 ng/ml), adiponectin (1 g/ml), or leptin (40 ng/ml). For the modulation experiments, at the same time of stimulation, cells were incubated with vehicle (dimethylsulfoxide), aspirin (5 mol/l), troglitazone (10 mol/l), or fluvastatin (5 mol/l). Doses and timing were chosen on the basis of findings from previous experiments. After 48 h, the culture supernatants were concentrated and assayed for CRP levels. Data are presented as the mean value SD and were analyzed using one-way analysis of variance followed by the Scheffe test for multiple comparisons. Statistical significance was indicated at the level p 0.05. Figure 1A shows a representative experiment (n 4) of CRP production by adipocytes following treatment with inflammatory cytokines. The incubation of adipocytes with IL-1-beta or IL-6 resulted in more than doubling of CRP production compared with the production in unstimulated cells (p 0.05). Adipocytes treated with either adiponectin and leptin did not produce CRP compared with unstimulated cells. Finally, resistin induced an almost three-fold increase in CRP production (p 0.01). In order to mimic more pathophysiological conditions, we used combinations of the active stimuli together (n 3), and the results are shown in Figure 1B. Combination of IL-1-beta and IL-6 induced an almost three-fold increase in CRP production (p 0.01). The addition of resistin led to an even larger increase in CRP production (p 0.01). Figure 2 shows the effect of fluvastatin, troglitazone, and aspirin on the production of CRP. Treatment with fluvastatin or troglitazone led to a significant, but not complete, inhibition of CRP release from adipocytes (p 0.05). Finally, a larger, but still not complete, modulation of CRP release from adipocytes was observed after treatment with aspirin (p 0.05). In the present study, we showed for the first time the production of a major acute-phase protein, CRP, by adipocytes isolated from human adipose tissue in response to inflammatory cytokines, thereby suggesting a new link between obesity and vascular inflammation. Adipose tissue secretes various bioactive substances, generally referred to as adipocytokines, including IL-6, TNF-alpha, leptin, adiponectin, and resistin, that may contribute to obesity-linked metabolic and vascular diseases (7). In addition, obese individuals have high circulating levels of a range of inflammatory markers produced by adipose tissue, including IL-1-beta and IL-6 (8), cytokines responsible for both hepatic and extrahepatic production of CRP (4). In several studies, high plasma levels of this acutephase protein were strongly associated with obesity and obesityrelated diseases (9). There is recent evidence of CRP expression in human adipose tissue as well. Ouchi et al. (5) showed CRP mRNA expression in human adipose. However, they made no attempt to investigate the stimuli able to induce CRP. Here, we found that human adipocytes cultured in vitro produced CRP after exposure for 48 h to inflammatory cytokines, such as IL-1-beta, IL-6, and