The regulation of prostaglandin output from term intact fetal membranes by anti-inflammatory cytokines

Abstract

Prostaglandins are some of the main mediators which control parturition, and their production by intrauterine tissues can be up-regulated by pro-inflammatory cytokines. Anti-inflammatory cytokines may oppose these effects, and in this study we have investigated how two such cytokines affected fetal membrane function. Interleukin-10 (IL-10) inhibited the output of prostaglandin E2 (PGE2) from intact fetal membranes under basal and lipopolysaccharide (LPS)-stimulated conditions, and there was a parallel decrease in the expression of mRNA for COX-2. IL-10 also inhibited the production of interleukin-1beta (IL-1beta) and the expression of mRNA for IL-1beta, indicating that this cytokine has a broad anti-inflammatory effect. Transforming growth factor-beta1 (TGF-beta1), which is generally considered to be anti-inflammatory had opposite effects on PGE2 production, in that it increased the output of PGE2 for up to 8 hr. TGF-beta1 increased levels of type-2 cyclo-oxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) protein, and also activated the cPLA2 enzyme present; the profile of effects is similar to that of the pro-inflammatory cytokine IL-1beta, and was not expected. Combinations of TGF-beta1 with IL-1beta also increased PGE2 output and caused appropriate changes in prostaglandin pathway enzymes, whereas TGF-beta1 and IL-1alpha had more limited effects. Further studies are needed to establish the physiological significance of these findings, but TGF-beta1 does not seem to act as an inhibitory cytokine in intact fetal membranes at term.

Figure 1

(a) The effects of IL‐10 and LPS on the production of PGE₂ from human fetal membranes. All data are means± SEM (n = 5). (b) The presence of mRNA for COX‐2 in typical samples. (c) Quantification of the changes in levels of mRNA for COX‐2 in samples after 8 hr of culture. All data are means± SEM (n = 3). * P < 0·05 versus corresponding samples not containing IL‐10.

Figure 2

(a) The effects of IL‐10 and LPS on the production of IL‐1β from human fetal membranes. All data are means± SEM (n = 6). (b) The presence of mRNA for COX‐2 in typical samples. (c) Quantification of the changes in levels of mRNA for COX‐2 in samples after 8 hr of culture. All data are means± SEM (n = 3). * P < 0·05 versus control; ** P < 0·05 versus LPS alone.

Figure 3

The effects of TGF‐β1 alone, or in combination with IL‐1β or IL‐1α on (a) the output of PGE₂ from intact fetal membranes during 4 hr of culture or (b) the expression of mRNA for COX‐2. All cytokines were present at 1 ng/ml. PGE₂ output was compared to control from triplicate determinations from five separate replicate experiments (means± SEM). Basal PGE₂ output was 20–80 pg PGE₂/ml/4 hr, with a maximum stimulated output of 50–200 pg/ml/4 hr. COX‐2 mRNA levels were assessed in fetal membranes from four separate experiments (means± SEM). * P < 0·05 versus control.

Figure 4

Studies on the presence of COX‐2 protein in intact fetal membranes under the culture conditions shown. (a) Immunoblot for COX‐2 protein from different membranes. (b) Quantification of changes in COX‐2 protein normalized to β‐actin after 4 hr stimulation with the cytokines shown (means± SEM n = 3). * P < 0·05 versus control.

Figure 5

The effects of TGF‐β1 alone, or in combination with IL‐1β or IL‐1α on (a) levels of mRNA for cPLA₂ or (b) levels of cPLA₂ protein. In (a) the expression of mRNA was normalized to GAPDH prior to expression relative to control tissue samples. In (b) cPLA₂ levels were normalized to β‐actin levels and expressed as a fold change (means ± SEM n = 4). * P < 0·05 versus control.

Figure 6

(a) The separation of phosphorylated (P‐cPLA₂) and dephosphorylated (DeP‐cPLA₂) forms of cPLA₂ by PAGE, followed by immunoblotting. Time 0 samples were cut from the tissues and frozen immediately at –70°. (b) Quantification of the levels of P‐cPLA₂ from replicate experiments, after 2 hr incubation with the cytokines shown. Data were corrected for β‐actin levels and expressed as a fold change (means± SEM n = 4). * P < 0·05 versus control.

Figure 7

(a) The separation of P‐cPLA₂ and DeP‐cPLA₂ from cytosolic (C) and membrane (M) fractions of intact fetal membranes, followed by immunoblotting. (b) Distribution of total cPLA₂ after correction for β‐actin levels (means±SEM n = 3). * P < 0·05 for difference between cytosol and membrane fractions for each treatment group.