Tight association between macrophages and adipocytes in obesity: Implications for adipocyte preparation

OBJECTIVE: To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level. METHODS: Adipose tissue from diet-induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte-macrophage interaction in both intact white adipose tissue (WAT) and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi-quantitative real time PCR and immunohistochemical analysis. RESULTS: Three dimensional image analysis of WAT and the separated "adipocyte" floating layer revealed lipid-engorged macrophages, macrophages in contact with lipid droplets and sheath-like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL-10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer. CONCLUSION: Macrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages.