Co-culture with tolerant cells induces tolerance in naïve macrophages <i>in vitro</i>.

<p>(<b>A, B</b>) Peritoneal cells from naïve <i>Tlr4^+/+</i> or <i>Tlr4^−/−</i> mice were co-cultured for 18 hours at 37°C with washed peritoneal cells from <i>Aoah^−/−</i> mice that had been injected with 10 µg LPS i.p. 10 days earlier. IL-6 and IL-10 were measured in culture medium. Only <i>Tlr4^+/+</i> cells co-cultured with tolerant peritoneal cells released IL-6 and IL-10. Separation of naïve <i>Tlr4^+/+</i> cells from LPS-exposed <i>Aoah^−/−</i> cells in Transwell cultures significantly decreased cytokine production, suggesting that cell-cell contact is important for delivery of bioactive LPS. (<b>C–E</b>) In the same experiments, after incubation for 18 hours the <i>Tlr4^+/+</i> cells that had been co-cultured with tolerant cells were washed with cRPMI twice and then treated with 1 µg/ml LPS for 6 hours before TNF, IL-6 and IL-10 were measured in the culture medium. Naïve macrophages co-cultured with tolerant cells became reprogrammed. Blue bars, <i>Tlr4^+/+</i> naïve macrophages; light blue bars, <i>Tlr4^−/−</i> naïve macrophages; diagonal markings indicate co-culture with tolerant cells; checked markings represent Transwell cultures. Data were combined from 3 experiments. n = 6–10. ***, P<0.001; NS, No significant difference.