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. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: J Immunol. 2012 Apr 9;188(10):5086–5093. doi: 10.4049/jimmunol.1102914

Figure 7.

Figure 7

C5a suppression of LPS-induced IL-27(p28) is related to differential roles of the NFκB and PI3K-Akt signaling pathways. (A) Effects of different doses of the IκB-inhibitor Bay11-7082 on IL-27(p28) production from Wt macrophages after LPS, 6h, ELISA.(B) Wt macrophages were stimulated for 2 h with LPS alone, the combination of LPS and C5a (100 nM) or left as untreated controls (Ctrl). Nuclear lysates were prepared an analyzed for NFκB activity for binding of its DNA consensus sequence (5’-GGGACTTTCC-3’) by immunodetection. (C) Bead-based assay for phospho-Akt (threonine 308). Wt macrophages were stimulated for 30 min with LPS and C5a (100 nM) alone or in combination, or left as un stimulated controls. (D) Macrophages were stimulated with either LPS, Poly (I:C) or left as un stimulated controls. Cells were fixated after 60 min and phosphorylation of Akt (threonine 308) was analyzed by flow cytometry. Histograms were gated to only show F4/80+ cells. (E) RT-PCR of mRNA levels for IL-27(p28) from macrophages treated with LPS alone or in combination with wortmannin (1 µM) or Bay11-7082 (10 µM), 6h.(F) Effects of specific inhibition of the PI3K-Akt pathway by wortmannin (1–0.1 µM) for the release of IL-27(p28) from LPS-activated Wt macrophages, ELISA, 6 h. (G) Differential effects of wortmannin (1 µM) on production of IL-27(p28) when induced by either LPS or by Poly (I:C), 20 h. (H) PI3K-Akt blockade by wortmannin reverses C5a-mediated suppression of IL-27(p28) release from macrophages after LPS, 8 h, ELISA.(I) Plasma levels of IL-27(p28) in C57BL/6J Wt mice 8 h after endotoxemia. Mice had been injected with wortmannin (1 mg/kg BW i.p., n=4) or vehicle (n=5) 1 h before LPS (10 mg/kg BW i.p.). All data shown except (I) were done with thioglycollate elicited macrophages from C57BL/6J (Wt) mice. Wortmannin and Bay11-7082 were added 1 h before LPS (1 µg/ml), Poly (I:C) (1 µg/ml) or C5a (100 nM). Data are representative of 3 independent experiments or were performed with the numbers of mice as indicated. *p < 0.05, error bars represent s.e.m.