Figure 5.

Interaction of d7EB progenitor cells with CD11b+ cells decreases iNOS-derived NO production by CD11b+ cells. A: NO production by d7EB cells. d7EB cells (1 × 10⁶) in 1 ml of medium were placed per well of a 12-well plate and stimulated or not with LPS, 1 μg/ml. NO was measured (see Methods) in the collected supernatant at the indicated times. Results are representative of 3 similar experiments. Error bars represent SEM. B: d7EB cells express eNOS. Western blot for eNOS protein in cell lysates from human d7EB cells. The specificity of the depicted band is compared with lysates from eNOS knockout mouse splenocytes. Blot is representative of 3 separate experiments. C: NO was measured (see Methods) in supernatants of directly co-cultured human d7EB and mouse CD11b+ cells (ratio, 1:5) isolated from septic mice. The co-cultured cells were stimulated with LPS, 1 μg/ml, for either 6 or 12 hours. At both times, NO production was significantly lower in the co-cultured wells compared with CD11b+ cells alone. N = 3 for each experiment. *P < 0.05; error bars represent SEM. D: Decreased iNOS expression in CD11b+ cells co-cultured with human d7EB cells. After 12 hours of co-culture, CD11b+ cells were selected using CD11b magnetic beads, and lysed. Western blot for iNOS protein was performed. Blot is representative of 3 separate experiments. E: Decreased intracellular iNOS expression in CD11b+ cells isolated from lungs of mice transplanted or not with d7EB cells 12 hours after CLP. CD11b+ cells were selected using magnetic beads, and permeabilized for intracellular staining for iNOS. Plot representative of 3 separate experiments. Open lines represent isotype-matched control. F: Exposure of CD11b+ cells to NO alters subsequent inflammatory profile. CD11b+ cells isolated from septic mice were stimulated with LPS, 1 mg/ml, and the indicated amounts of the NO donor diethylenetriamine NONOate (DETA NO) for 12 hours. Supernatants were collected, and TNF-α and IFN-γ concentrations were measured using an ELISA. N = 3 for each experiment. *P < 0.05 for each DETA NO concentration compared with 0; error bars represent SEM. G: Western blot for iNOS from lysates of CD11b+ cells stimulated with LPS, 1 mg/ml, and the indicated amounts of NO donor diethylenetriamine NONOate (DETA NO) for 12 hours. Density measurements of three western blots are quantified in the bar graph. *P < 0.05 for each condition compared with CD11b+ cells cultured without NO donor or d7EB cells; error bars represent SEM.