Modulation of macrophage activation by prostaglandins

The effect of prostaglandtn E2, iloprost and cAMP on both nitric oxide and tumour necrosis factor-α release in J774 macrophages has been studied. Both prostaglandin E2 and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-α. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E2 and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-α generation, which in turn is likely to be mediated by cAMP.


Introduction
Macrophages activated with bacterial lipopolysaccharide release a variety of mediators including nitric oxide (NO), tumour necrosis factor (TNF-a) and prostaglandins, namely prostaglandin E2 (PGE2) and prostacyclin (PGI2). [1][2][3] The cytotoxic properties of activated macrophages depend, at least in part, on the biological activities of these mediators. Thus the synthesis of NO from the amino acid t-arginine has been shown to be a major cytotoxic mechanism of activated macrophages. 1 Moreover NO has been identified as an effector molecule of the cytotoxic effects produced by TNF-ot in bovine endothelial cells. 4 Conversely, PGE2 and PGI2 inhibit the cytotoxic properties of activated macrophages. 3 Furthermore, increasing evidence suggests that the interaction existing within the biological actions of NO, prostaglandins and TNF-ot may result in a mutual regulation of their synthesis and/or release. In fact TNF-a has been reported to induce NO synthase in murine peritoneal macrophages 5 and bovine endothelial cells. 4 TNF-cg increases PGE2 production in human synovial 6 cells and human dermal fibroblasts, mouse peri- 7 8 toneal macrophages and rat Kupffer cells, as well as PGb generation in human endothelial cells. 9 Conversely, PGE2 has been shown to down-regulate the generation of TNFin macrophages of different origin i'm and HL-60 cells. 1 Moreover it has been reported that the release of TNF-0t by macrophages is inhibited by cAMP, a second messenger for both PGE2 and PGI2 .1-2 We have shown that PGE2 and the stable analogue of PGI2, iloprost, inhibited the induction of NO synthase in lipopolysaccharide-activated J774 murine macrophages. 13 We hypothesized that the inhibition of NO synthase induction could be secondary to an increase in cAMP levels in the activated macrophages. This hypothesis was also supported by the inability of both prostaglandin F2= (PGF2) and the stable analogue of thromboxane A2 (TXA2), U46619, which do not enhance cAMP levels, to modify NO generation. However, since TNF-0t induces No synthase in murine macrophages 7 and PGE2 has been shown to inhibit TNF-0t release from these cells, 2 the inhibition of NO synthase induction by PGE2 could also be secondary to the down-regulation of TNF-a in J774 cells.
In the light of the above considerations we have studied the effect of PGE2, PGI2 and cAMP on the production of NO and TNF-a by lipopolysaccharide-stimulated J774 macrophages.

Materials and Methods
The murine monocyte macrophage cell line J774 (American Tissue Culture Catalogue TIB 67, page 231) was grown in Dulbecco's modified Eagle's medium (Gibco) at 37C as described previously. 3 The cells were plated in 24-well culture plates (Falcon) at a density of 2.5 x 105 cells/ml and allowed to adhere at 37C in 5% COz/95% air for 2h. Thereafter the medium was replaced with fresh medium, cells were activated with lipopolysaccharide (0.1 I.tg/ml) from Salmo- The level of TNF<z (U/ml) in the cell medium, 3h after lipopolysacchafide challenge, was assessed in WEHI-164 cells by a biological assay 0 using recombinant human TNF-cz (Sigma) as reference standard and rabbit antimurine TNF<z (Genzyme) antiserum which cross-reacts with rat TNF-cz in order to assess the specificity of TNF-0tdependent cytolytic activity.

Discussion
We have shown that PGE2 and PG12, and its stable analogue iloprost, which are known to activate adenylate cyclase, inhibit the induction of NO synthase in lipopolysaccharide-activated J774 macrophages. We also demonstrated that this action was not shared by PGF2= and the stable analogue of TXA2, U46619, which do not enhance cAMP levels. The results of the present study show that cAMP, as its permeable form dibutytryl cAMP, inhibits lipopolysaccharideinduced NO release by J774 macrophages, and confirm that this release is inhibited by PGE2 and iloprost but not by PGF2=. The selective type IV phosphodiesterase inhibitor, RO-201724,16 potentiated not only the inhibitory effect of cAMP but also the effect of the two prostanoids. In the light of these findings our results strongly suggest that the inhibitory action of the two prostanoids on NO synthase induction in lipopolysaccharide-activated J774 macrophages is mediated by cAMP. This evidence is also supported by recent results showing that in murine peritoneal macrophages, cAMP is an intermediate in the down-regulation of NO synthase by prostaglandins. 7 Our results also show that PGE2 and iloprost, but not PGF2=, inhibit in a concentration-dependent fashion of lipopolysaccharide-induced TNFz production by J774 macrophages. This effect was exhibited also by cAMP. The phosphodiesterase inhibitor RO-201724 potentiated this action. PGF2z was unable to inhibit lipopolysaccharide-induced TNF-0t release by J774 macrophages. These data suggest that, as obseeced for NO generation, the inhibitory action of the two prostanoids on TNF-cz release is likely to be mediated by cAMP. In this respect it is interesting to note that all the tested compounds were more potent in inhibiting TNF-cz than NO2-production.
Since NO seems to be the effector molecule of the cytotoxic activity of TNF-0t, 5 and PGE2 and iloprost modulate the production of TNF-cz and NO, 17 it could be hypothesized that the inhibitory action of the two prostanoids on NO synthase induction might be secondary to the inhibition of TNF-0t generation which seems to be mediated by cAMP. This hypothesis appears conceivable considering that in macrophages lipopolysaccharide stimulates not only TNF-cz and NO but also a sustained production of the prostaglandins, following the expression of inducible cyclooxygenase. In this light the downregulation of TNF-a and consequendy of NO production by endogenous prostaglandins may represent a relevant feed-back mechanism in modulating the cytotoxic effects of a sustained NO production following immunological stimulation.