Evidence suggests that NO supplementation in the form of NO donors exerts an anti-inflammatory effect in animal models of endotoxemia through inhibition of nu-clear factor B activation and iNOS expression, and decreased production of proinflammatory cytokines. 26, 27 Therefore, whether the NO-producing d7EB cells exerted a similar anti-inflammatory effect on CD11bϩ cells was determined. d7EBs were co-cultured with CD11bϩ cells, and nitrite concentration was measured in the supernatant after LPS stimulation. Supernatant NO concentrations at 6 and 12 hours after LPS stimulation of cocultured d7EB and CD11bϩ cells were significantly lower than the supernatant NO concentration from CD11bϩ cells alone ( Figure 5C ). This effect could not be attributed to increased apoptosis because apoptosis was not significantly different in the co-cultured CD11bϩ cells compared with the CD11bϩ cells cultured alone (see Supplemental Figure S1 at http://ajpamjpathol.org). Analysis of iNOS expression in co-cultured CD11bϩ cells showed decreased protein expression in LPS-stimulated CD11bϩ cells interacting with d7EB cells (Figure 5D ). This finding was supported by measurement of iNOS expression Co-culture of d7EB cells with CD11bϩ lung cells reduces production of inflammatory cytokines. Lung CD11bϩ cells from septic mice were co-cultured with d7EB cells (ratio, 5:1) either directly or separated by a 0.4-m pore size Transwell filter. Non-co-cultured CD11bϩ mouse cells from septic mice were used as controls. The cells were stimulated with LPS, 1 mg/ml, for 6 hours, and supernatant was collected for cytokine measurement using an ELISA specific for mouse cytokines. Directly co-cultured cells demonstrated a decrease in TNF-␣ and IFN-␥ production, whereas production of these cytokines was not significantly different from that in controls when the cells were separated using Transwell filters. N ϭ 3 experiments. *P Ͻ 0.05; error bars represent SEM.