Figure 8.
Prostaglandin E2 receptor subtype activation (EP2-A) amplified IgG-induced microglia activation and triggered neuroprotection dependent on TNF-α. Butaprost, an EP2 receptor agonist, was used as a surrogate of PGE2 release from increased neuronal activity to begin exploring whether paracrine signaling from neuronal activity might influence microglial recycling endocytosis and TNF-α change from neuroprotective IgG. A, IgG significantly increased endocytosis from control and this change was further and significantly enhanced by coincubation with EP2-A, whereas EP2-A exposure alone had no impact. B, Recycling endocytosis, measured via RAb11a immunostaining, showed a similar significant increase by IgG over control that was significantly enhanced by inclusion of EP2-A. Again, EP2-A alone had no impact. C, Furthermore, IgG did not prompt a change in late endosome/early lysosome trafficking, measured via LAMP1 immunostaining. However, inclusion of EP2-A triggered the trend increase from IgG alone to a significant increase, consistent with the change seen from high [i.e., 2000 μg/ml (Fig. 3)] IgG alone, further suggesting that prostaglandins can amplify monomeric IgG-mediated endocytotic vesicle trafficking. D, Production of TNF-α from primary microglia further supported this conclusion. IgG exposure alone prompted a significant increase in TNF-α production that was further significantly (p = 0.01) amplified by EP2-A. Here, however, the amplification of cytokine secretion is tied to recycling endocytosis (and not lysosomal trafficking) because high dose IgG alone, which enhanced ED-1 (Fig. 2) and LAMP1 immunostaining (Fig. 3), did not trigger a significant increase in TNF-α (Fig. 4). E, Neuroprotection from EP2-A is caused by TNF-α because abrogation of signaling from this innate cytokine via sTNFR1 removed the neuroprotective effect to a level of injury significantly greater than with EP2-A alone. [Specific significant “p” values (unless otherwise noted above) were (A) p < 0.001; α = 1.00 with relative levels of 1.34 ± 0.03 (n = 182 cells), 1.71 ± 0.03 (n = 262), and 1.06 ± 0.03 (n = 147) vs control (1.00 ± 0.01; n = 686); (B) p < 0.001; α = 1.00 with relative levels of 1.56 ± 0.09 (n = 135 cells), 2.06 ± 0.13 (n = 110), and 0.79 ± 0.03 (n = 160) vs control (1.00 ± 0.09; n = 168); (C) p < 0.001; α = 1.00 with relative levels of 1.19 ± 0.12 (n = 33 cells), 3.12 ± 0.29 (n = 73), and 0.60 ± 0.08 (n = 47) vs control (1.00 ± 0.07; n = 67); (D) p < 0.001; α = 1.00 with relative levels of 11.90 ± 1.3, 16.00 ± 1.69, and 0.69 ± 0.04 vs control (1.00 ± 0.04) and n = 9/group; (E) p < 0.001; α = 1.00 with relative injury levels of 0.64 ± 0.02 (n = 8) and 0.95 ± 0.03 (n = 7) vs control (1.00 ± 0.03; n = 7)]. Data represent mean ± SEM and significance (*p < 0.05).