Figure 3.
Subsets 1 and 2 spontaneously release the antiinflammatory cytokine IL-10 and respond to LPS stimulation with an antiinflammatory signature. (A) Real-time RT-PCR analysis of IL-10 mRNA expression by freshly isolated FACS-sorted cLP subsets from C57BL/6 mice. Results are mean real amounts ± SD, normalized to GAPDH (1/2ΔCt), of at least three independent experiments with 10 pooled mice in each. (B) Spontaneous release of IL-10 measured by ELISA after overnight culture of freshly isolated FACS-sorted colonic subset 1 or 2, compared with splenic (SPL) DCs, splenic MPs, or MOs. Results are mean ± SD of three experiments with 5–10 pooled mice in each. (C and D) Representative plots (C) and quantification (D) of IL-10–GFP reporter expression by cLP subsets 1–4 freshly isolated from Vert-X mice. FACS plots present the IL-10–GFP/CD11b staining profile after pregating on each individual cLP subset. Quadrant gates were set using isotype controls. For IL-10–GFP negative controls, cells were prepared from WT mice. Results are mean ± SD of three mice analyzed separately and are representative of two experiments. (E) Real-time RT-PCR analysis of cytokine mRNA expression in FACS-sorted cLP DC/MP from C57BL/6 mice. Results are represented as real amounts, normalized to GAPDH (1/2ΔCt). Data are mean ± SEM of at least three independent sorting experiments performed in duplicate. ND, not detected. (F) Quantification of the cytokines secreted by cLP subsets 1 and 2 and control splenic MPs and DCs after 24 h of culture in media alone (open bars) or in the presence of 1 µg/ml LPS (closed bars), using the SearchLight multiplex cytokine immunoassays. Results are mean ± SD of three independent experiments with 10 pooled colons and 3 pooled spleens each. *, P < 0.05; **, P < 0.01; ***, P < 0.001.