Figure 5. T. serrulatus venom (TsV)-induced macrophages IL-1β production is amplified by PGE₂ via the cAMP pathway.

(a) The relative mRNA expression levels for Ptger1, Ptger2, Ptger3 and Ptger4 are related to the expression level of Ptger3 in resting macrophages of C57BL/6 mice. The fold increase in mRNA expression after TsV stimulation for 2 and 6 h was determined in comparison with that in non-stimulated cells. (b) Macrophages pre-treated with PGE₂ (0.01–10 μM, 10 min), were stimulated with TsV and 24 h later supernatants were collected for IL-1β quantification. (c) J774.1 macrophages were pre-treated or not with EP2 (AH6809 1 μM), or EP4 (AH23848 1 μM) antagonists, or indomethacin (Indo 10 μM), for 30 min, followed or not by PGE₂ (10 μM, 10 min), before TsV addition. Supernatants were collected 24 h later for IL-1β quantification. (d) cAMP in J774.1 macrophage pre-treatment or not with EP2 antagonist (AH6809 1 μM), or indomethacin (Indo 10 μM) for 30 min; followed or not by PGE₂ (10 μM, 3 min), before TsV addition for 5 min. (e) IL-1β production by J774.1 macrophages pre-treated or not with PKA inhibitor (H89 25 μM, 2 h); or forskolin (FSK 25 μM, 10 min), before TsV addition for 24 h. (f) J774.1 macrophages were pre-treated or not with PGE₂ (10 μM), or forskolin (FSK 25 μM) for 10 min; or with PKA-inhibitor (H89 25 μM, 2 h), and then stimulated or not with TsV. Cell lysates were obtained 2 h later for phospho-NFκB p65 (Ser536) and total NFκB p65 quantification. (g) J774.1 were pre-treated or not with NFκB inhibitor (20 nM, 30 min), before TsV and the supernatant was collected 24 h later for IL-1β quantification. The data are representative of two independent experiments performed in triplicate. Macrophages were stimulated with 50 μg ml⁻¹ of TsV. (h) A schematic representation of the cell signalling pathway for IL-1β production induced by TsV is shown. (a–g) Significant differences P<0.05 are marked with symbols and the error bars denote s.d. *Medium only versus stimulus or treatments, and ^#TsV versus treatments, according to one-way ANOVA with Bonferroni's post-test.