Figure 1.

PKC selectively regulates macrophage TNF-α production without affecting IL-10. Human monocyte-derived macrophages were plated out at 1 × 10⁵ cells per well in a flat-bottomed 96-well plate and pretreated with PKC inhibitors Go6983 (specificity for α, β, γ, δ, ζ), Go6976 (specificity for α and β₁) or RO-32-0432 (specificity for α,β₁ and ε) (a, b, c) for 1 hr prior to stimulation with 1 ng/ml LPS (a, b) or 50 ng/ml PMA/0·5 µg/ml ionomycin (c) and incubated for 24 hr at 37°/5% CO₂, after which time supernatants were harvested and assayed for TNF-α and IL-10 by ELISA. Data are mean cytokine levels in pg/ml of triplicate culture supernatants ± SD, showing a representative of n = 4 replicate experiments. Western blot analysis of activated phospho-PKC (d) shows PKC activation by LPS (lane 2) and PMA/ionomycin (lane 3). In addition, phospho-Western blot analysis of PKCζ (e) demonstrates LPS activation (lane 2), whereas PMA/ionomycin fails to activate this PKC isoform (lane 3). Loading controls are presented as total PKC and PKCζ blots below the corresponding phospho-Westerns. Data are representative of three replicate experiments. *P≤0·05, **P≤0·01, ***P≤0·001.