4-1BB/4-1BBL Interaction Promotes Obesity-Induced Adipose Inflammation by Triggering Bidirectional Inflammatory Signaling in Adipocytes/Macrophages
Figure 4
Disruption of the 4-1BB/4-1BBL interaction suppresses expression of inflammatory cytokines in direct contact cocultures. Raw264.7 macrophages were seeded onto 3T3-L1 adipocytes with/without pretreated with neutralizing anti-4-1BBL antibody (TKS-1) or Rat IgG (5 μg/mL) in serum-free medium for 24 h. Total RNAs were isolated and analyzed the level of IL-6, MCP-1, and TNF-α by qRT-PCR (a). The protein levels of IL-6, MCP-1, and TNF-α in cell-free supernatants were collected from 3T3-L1 adipocytes and Raw264.7 macrophages coculture (b) and from cocultures of 3T3-L1 adipocytes and peritoneal macrophages (c). Illustration of coculture system with adipocytes/macrophages (d). After 24 h coculture of Raw264.7 macrophages and 3T3-L1 adipocytes, these cells were separated using the CD11b MicroBead system. Levels of inflammatory cytokine mRNAs were detected in the adipocytes (e) and macrophages (f). Co, coculture. Data are the mean ± SEM of three independent experiments performed in duplicate. ; ; ; (compared with rat IgG-treated cells).
