Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-α generation

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNFα) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists.
2. In human monocytes that were adherent to plastic, neither prostaglandin D₂ (PGD₂), prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNFα-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells.
3. Exposure of human monocytes to LPS (3 ng ml⁻¹, ∼ EC₈₄) resulted in a time-dependent elaboration of TNFα which was suppressed in cells pretreated with prostaglandin E₁ (PGE₁), PGE₂ and cicaprost. This effect was concentration-dependent with mean pIC₅₀ values of 7.14, 7.34 and 8.00 for PGE₁, PGE₂ and cicaprost, respectively. PGD₂, PGF_2α and U-46619 failed to inhibit the generation of TNFα at concentrations up to 10 μM.
4. With respect to PGE₂, the EP-receptor agonists, 16,16-dimethyl PGE₂ (non-selective), misoprostol (EP₂/EP₃-selective), 11-deoxy PGE₁ (EP₂-selective) and butaprost (EP₂-selective) were essentially full agonists as inhibitors of LPS-induced TNFα generation with mean pIC₅₀ values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE₁, another EP₂-selective agonist, AH 13205, inhibited TNFα generation by only 21% at the highest concentration (10 μM) examined. EP-receptor agonists which have selectivity for the EP₁- (17-phenyl-ω-trinor PGE₂) and EP₃-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNFα generation.
5. Pretreatment of human monocytes with the TP/EP₄-receptor antagonist, AH 23848B, at 10, 30 and 100 μM suppressed LPS-induced TNFα generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves.
6. Given that AH 13205 was a poor inhibitor of TNFα generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 and 30 μM AH 13205 inhibited the generation of TNFα by 31% and 53%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves at either concentration examined.
7. Since PGD₂ and 17-phenyl-ω-trinor PGE₂ (EP₁-agonist) did not suppress TNFα generation, the EP₁/EP₂/DP-receptor antagonist, AH 6809, was employed to assess if EP₂-receptors mediated the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 μM AH 6809 did not affect LPS-induced TNFα generation but produced a parallel 3.5 fold rightwards shift of the PGE₂ concentration-response curve.
8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNFα generation. One of these probably represents IP receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank order of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP₂- and/or EP₄- and/or IP) or novel EP-receptors mediate the inhibitory effect of PGE₂ on TNFα generation.

     

Stimulation of PGE receptors EP2 and EP4 protects cultured neurons against oxidative stress and cell death following beta-amyloid exposure

Alzheimer's disease (AD) is associated with gliosis, neuroinflammation and higher levels of prostaglandins. Conflicting roles for cyclooxygenases and prostaglandins in the etiopathology of AD have been reported. We hypothesized that PGE2 signaling through EP2 and EP4 G-protein-coupled receptors could protect against amyloid beta-peptide (Abeta) neurotoxicity by increasing the cAMP signaling cascade. Using primary neuronal cultures, we investigated the presence of EP receptors (EP1-4) and the action of PGE2 and EP receptor agonists on neuronal susceptibility to Abeta1-42 toxicity. Low concentrations (1 microm) of PGE2, butaprost (EP2 agonist), and 1-hydroxy-PGE1 (EP4/EP3 agonist) were neuroprotective against Abeta1-42 toxicity, while sulprostone (EP3/EP1 agonist) at similar doses had no detectable effects. EP2 and EP4 receptor-mediated neuroprotection would involve changes in cAMP levels, as both EP2 and EP4 agonists increased intracellular cAMP concentration by approximately doubling basal levels, and both exhibited neuroprotective actions against Abeta-induced toxicity. The protein kinase A (PKA) inhibitor RpcAMPS significantly attenuated the neuroprotection by butaprost, but not that by 1-hydroxy-PGE1, implying differences between EP2 and EP4 receptor protective mechanisms. Additionally, the increase in reactive oxygen species generated following exposure to Abeta was reduced by stimulation of both EP2 and EP4 receptors. Together, these results indicate that PGE2 can protect neurons against Abeta toxicity by acting on given receptors and stimulating a cascade of intracellular events, including the cAMP-PKA pathway. We propose that development and testing of specific PGE2 receptor agonists downstream of cyclooxygenase could lead to therapeutic applications.