The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia

Figure 1

Reduced cyclic AMP levels in EOC2 microglia following TNF-α are rescued by PDE4, MEK, m-PKI, or NF-κB p65 inhibition. Unstimulated, resting EOC2 microglia express very little ED1 ((a) red) and exhibit significant intracellular immunoreactivity for cyclic AMP ((b) green). Upon activation with TNF-α, there was a marked induction of ED1 (c) and a dramatic reduction in cyclic AMP (d) within 3 h. Inhibiting PDE4 activity with Rolipram in TNF-α treated microglial cells did not alter ED1 expression (e) but restored cyclic AMP (f). The antagonism of PKA with m-PKI did not alter the production of either ED1 (g) or cyclic AMP (h). Inhibiting MEK with PD98059 or NF-kB nuclear translocation with JSH-23 reduced ED1 ((i) and (k), resp.) and also restored cyclic AMP ((j) and (l), resp.). Scale bar = 20 μm. Measurements of ED1 (red) and cyclic AMP (green) immunoreactive (IR) signal intensity were obtained in microglia using fluorescent microscopy and Image J software for the different imaged conditions (m). Quantitation of cyclic AMP concentrations in TNF-α challenged microglial cells (i) showed the restoration of cyclic AMP to resting levels following treatment with inhibitors of PKA, ERK, and NF-κB and partially with PDE4. Results shown are the average from three independent culture plate replicates for each treatment condition examined. Cell nuclei are visualized using the nuclear stain Hoechst (blue). Errors are given as SEMs. Statistical significance relative to naive controls is indicated at , , or and versus TNF-α stimulated cells at .