Figure 1.

LPS induces growth inhibitory capacity of macrophages and production of both NO and type I IFNs. (A) Timeline for the growth inhibition assay used to assess cytotoxic and cytostatic activity of BMDMs toward cancer cells. Mitomycin C-treated BMDMs were seeded out (6 × 10⁴ in 200 μL) and, after 24 h, stimulated with IFNs and/or TLR agonists. At 48 h, half of the cell culture supernatant (SN) was removed for analysis of nitric oxide (NO) and interferon (IFN)-α/β production, before LLC tumor cells (3 × 10³) were added to the BMDM cultures, resulting in a 20:1 BMDM effector to LLC target cell ratio. Control wells with LLC cancer cells alone, were treated correspondingly. At 72 h, radiolabeled [³H]thymidine was added to all wells, and at 90 h, cells were harvested. Growth of LLC cancer cells was measured by the incorporation of [³H]thymidine (counts per minute; cpm). (B) Growth inhibition of LLC cells co-cultured with BMDMs, which had been left untreated or stimulated with various concentrations of LPS for 24 h. Cultures of untreated LLC cells alone and mitomycin C-treated BMDMs alone were used as controls. (C) Inhibition of growth of LLC cells in co-cultures with BMDMs stimulated for 24 h with 40 ng/mL IFN-γ alone or 40 ng/mL IFN-γ in combination with various concentrations of LPS for 24 h. (D) Production of NO by the BMDMs plated for use in analysis of LLC growth inhibition presented in (B,C). The Griess assay was used to measure NO indirectly as nitrite (${\text{NO}}_2^{-}$) in the supernatants of BMDMs. (E,F) Luminex technology was used to measure the levels of IFN-α (E) and IFN-β (F) in supernatants from BMDMs plated for analysis of LLC growth inhibition shown in (B,C). The BMDMs were stimulated 24 h with LPS alone or LPS together with IFN-γ. Data are presented as means ± SD of triplicate wells from one representative experiment out of three. nd, not detectable.