Figure 5. PI3K inhibitors partially rescue the age-associated defect in TLR-4 induced cytokine production by inhibiting phosphorylation of Akt and GSK-3.

The graphs show cytokine secretion by purified aged and young adult splenic macrophages pretreated with either 2 or 5 µM LY294002 or 5 or 50 nM wortmannin (panels A and B) for 60 minutes and then stimulated with LPS (1µg/ml) for 24 hours. Supernatants were collected and assayed for IL-10 (panel A) and IL-12 (p40) (panel B) by sandwich ELISA. Data are presented as mean+/−SE values of 6–10 determinations and are representative of three independent experiments. The symbols * and ** indicate statistical significance of differences in responses in groups treated with LPS alone compared to groups treated with LPS + PI3K inhibitor.

Purified young adult splenic macrophages were pre-treated with either LY294002 or wortmannin for 1 hour and then stimulated with LPS (Panel C) for 15 or 30 minutes. The blots were probed for p-Akt and later probed for total Akt and actin after stripping. The numbers represent densities of bands normalized to total AKT with the values for unstimulated aged macrophages set to 1. Panel D shows relative changes in phosphorylated GSK-3 in young splenic macrophages stimulated with LPS in the presence of PI3K inhibitors. The numbers represent densities of both GSK-3alpha and GSK-3beta bands normalized to total GSK-3β with the values for unstimulated aged macrophages set to one. Results are representative of two independent experiments.

The final figure in panel C is a composite of blots for P-Akt, Akt and actin for samples run on the same membrane that was stripped and reprobed. There was a lane 6 for inhibition with LY294002 at 15 minutes, but this was deleted for consistency between wortmannin and LY294002 at 30 minutes. The same method was employed to generate the Panel D; except that membrane was probed for GSK-3 instead of Akt.