Macrophage‐Adipocyte Interaction: Marked Interleukin‐6 Production by Lipopolysaccharide

Abstract

Objective: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low-grade infection by gram-negative bacteria appears to promote atherogenesis. We hypothesized a ligand for tolllike receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage-adipocyte interaction. Research Methods and Procedures: RAW264.7 macrophage cell line and differentiated 3T3-L1 preadipocytes were co-cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 production was evaluated. Results: Co-culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up-regulated IL-6 production (nearly 100-fold higher than that of adipocyte culture alone, p Ͻ 0.01), whereas TNF-␣ production was not significantly influenced. This increase was partially inhibited by anti-TNF-␣ neutralizing antibody. Recombinant TNF-␣ and LPS synergistically upregulated IL-6 production in adipocytes. However, this increase did not reach the level of production observed in co-cultures stimulated with LPS. Discussion: A ligand for TLR-4 stimulates macrophages to produce TNF-␣. TNF-␣, thus produced, cooperatively upregulates IL-6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up-regulated IL-6 would greatly influence the pathophysiology of diabetes and its vascular complications.

DNA microarray analyses of genes expressed differentially in 3T3-L1 adipocytes co-cultured with murine macrophage cell line RAW264.7 in the presence of the toll-like receptor 4 ligand bacterial endotoxin

“…We suggested in our previous study that TNF-a secreted from RAW macrophages plays important roles in markedly upregulating IL-6 translation in 3T3-L1 adipocytes, as the neutralizing TNF-a action with antibodies partially abolished the enhanced IL-6 production. 9 Therefore, we speculate that, at least in this situation, these RAW macrophages have the M1 phenotype. In fact, it has already been demonstrated that NF-kB is activated, and that cyclooxygenase-II, TNF-a, inducible nitric oxide synthase and IL-12 are expressed in response to LPS in RAW macrophages.…”

“…8 We previously reported that co-culturing adipocytes and macrophages in the presence of endotoxin resulted in markedly enhanced (more than 100-fold increase) production of IL-6 as compared with culturing each cell type alone, and suggested that co-existence of adipocytes and macrophages in the presence of TLR-4 ligands further exacerbates obesity-associated inflammatory conditions. 9 These data also suggest that gene expressions other than that of IL-6 may also be greatly influenced, in adipocytes co-cultured with macrophages, by the presence of TLR-4 ligand. Therefore, in this study, we analyzed gene expression profiles in adipocytes co-cultured with macrophages in the presence of endotoxin and attempted to detect genes with differential expressions under such conditions to further understand dynamic changes in gene expressions in adipocytes.…”

“…Pre-adipocytes were maintained in a Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and were differentiated into mature adipocytes with 4.5 mM glucose, 1 mM insulin, 1 mM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine, as described earlier. 9 Mature adipocytes and RAW264.7 were co-cultured in a transwell system (Corning Inc., Acton, MA, USA) with a 0.4-mm porous membrane to separate the upper and lower chambers. Then, 1 Â 10 5 differentiated 3T3-L1 cells were cultured in the lower chamber, whereas 5 Â 10 4 RAW cells were cultured in the upper chamber.…”

“…Then, 1 Â 10 5 differentiated 3T3-L1 cells were cultured in the lower chamber, whereas 5 Â 10 4 RAW cells were cultured in the upper chamber. 9 …”

Intracellular Infections Enhance Interleukin-6 and Plasminogen Activator Inhibitor 1 Production by Cocultivated Human Adipocytes and THP-1 Monocytes

“…Also, the duration of coculture influences the increase in TNF-␣ mRNA expression in cocultures of 3T3-L1 and THP-1 (31). Incubation with bacterial lipopolysaccharide in a coculture of 3T3-L1 and RAW264 did not influence TNF-␣ production but increased IL-6 production 100-fold, suggesting an exaggerated biological interaction between macrophages and adipocytes in persistent low-grade infection by gram-negative bacteria (35).…”