The production of interleukin-1beta from human fetal membranes is not obligatory for increased prostaglandin output

Abstract

Bacterial endotoxin increased the expression of mRNA (maximal after 4 hr) for interleukin-1beta (IL-1beta) and the release of mature protein from intact human fetal membranes. In contrast, the change in expression of mRNA for type 2 cyclo-oxygenase (COX-2) was biphasic, with peaks after 0.5-1 hr and after 8 hr of culture. An antibody to IL-1beta was without effect after 4 hr of culture, inhibited endotoxin-stimulated prostaglandin E2 (PGE2) production after 8 hr of culture, and caused a parallel decrease in the expression of mRNA for COX-2. We conclude that endotoxin induced the expression of COX-2 through IL-1beta-independent and IL-1beta-dependent mechanisms, and these differences are time dependent. Corticotrophin-releasing hormone (CRH) or platelet-activating factor (PAF) also increased the expression of mRNA for IL-1beta and the release of IL-1beta from some, but not all, fetal membranes. The antibody to IL-1beta did not affect CRH-stimulated or PAF-stimulated PGE2 production or COX-2 expression. We conclude that CRH and PAF can induce the expression of IL-1beta, but this is not obligatory for increased PGE2 release, and the effect of these stimuli on COX-2 expression is a direct, IL-1beta-independent effect.

Figure 1

The production of IL-1β from human fetal membrane discs cultured for the time periods shown. Tissues were cultured in medium alone, with endotoxin, with CRH, or with PAF. All data are means ± SEM from five to eight separate replicate experiments. *P < 0·05 versus medium alone.

Figure 2

(a) Expression of mRNA (after RT-PCR) for IL-1β and GAPDH by intact fetal membranes. (b) Relative expression of cDNA for IL-1β was assessed by quantitative RT-PCR. The tissues used were selected from those in Fig. 1, on the basis of their production of IL-1β. Tissues were cultured with medium, endotoxin, CRH, or PAF for 8 hr. All data are means ± SEM (n =4 tissues from separate replicate experiments). *P < 0·05 versus medium alone.

Figure 3

The production of PGE₂ from intact fetal membrane discs under different culture conditions was determined. (a) IL-1β, or IL-1β + IL-1β-neutralizing antibody. (b) endotoxin, CRH, PAF stimuli alone or with IL-1β-neutralizing antibody. All data are means ± SEM (n =8 separate replicate experiments), except for (a) in which a typical experiment is shown (n =3 discs from one fetal membrane). *P < 0·05 versus medium alone.

Figure 4

Relative expression of mRNA for COX-2 assessed by RT-PCR. Fetal membranes were selected from those shown in Fig. 3, after 8 hr of culture. Membranes were cultured with medium alone or were treated with endotoxin alone or with endotoxin + IL-1β-neutralizing antibody. (b) Membranes were treated with CRH alone, CRH + IL-1β-neutralizing antibody, PAF alone, or PAF + IL-1β-neutralizing antibody. All data are means ± SEM (n =4 tissues from separate replicate experiments). *P < 0·05 versus endotoxin alone.

Figure 5

The expression of mRNAs for (a) COX-2 (b) IL-1β and (c) COX-1 were assessed quantitatively by RT-PCR. The data are expressed as ratios to the expression of GAPDH. Tissues were cultured in medium alone, with endotoxin, or with endotoxin + cycloheximide. All data are means ± SEM (n =3 separate membrane cultures). *P < 0·05 versus medium alone. †P < 0·05 versus endotoxin alone.