Interleukin-10 and transforming growth factor-beta inhibit amniochorion tumor necrosis factor-alpha production by contrasting mechanisms of action: therapeutic implications in prematurity.
OBJECTIVE
This study was designed to detect the regulatory effect of the immunoinhibitory cytokines interleukin-10 and transforming growth factor-beta on the amniochorion production of tumor necrosis factor-alpha.


METHODS
Amniochorionic membranes were collected from women undergoing elective repeat cesarean section with no history of infection. Membranes were placed in organ explant culture for 48 hours and then stimulated with lipopolysaccharide (50 ng/ml), lipopolysaccharide plus interleukin-10 (50/ 50, 50/100 ng/ml), interleukin-10 (50 and 100 ng/ml), lipopolysaccharide plus transforming growth factor-beta (50/50 and 50/100 ng/ml), and transforming growth factor-beta (50 and 100 ng/ml). At the end of a 24-hour stimulation tissue samples were frozen for ribonucleic acid analysis and media samples were frozen for enzyme-linked immunosorbent assay. Quantitation of the messenger ribonucleic acid was accomplished by quantitative competitive polymerase chain reaction, and tumor necrosis factor-alpha protein was assayed by use of enzyme-linked immunosorbent assay.


RESULTS
Lipopolysaccharide stimulation of fetal membranes produced approximately 60,000 molecules of tumor necrosis factor-alpha messenger ribonucleic acid, whereas control tissue produced none. Lipopolysaccharide plus interleukin-10 stimulation resulted in a dose-dependent decrease in tumor necrosis factor-alpha messenger ribonucleic production (transcriptional regulation) to 6000 (50/50) and 600 (50/100) molecules. Enzyme-linked immunosorbent assay performed on media samples from these experiments demonstrated a dose-dependent reduction in tumor necrosis factor-alpha peptide release. Stimulation of membranes with lipopolysaccharide plus transforming growth factor-beta had minimal effects on tumor necrosis factor-alpha messenger ribonucleic acid and protein production compared with lipopolysaccharide-treated samples. Membranes stimulated with interleukin-10 alone showed no effect on messenger ribonucleic acid or protein levels and remained similar to the levels seen in control tissues. In the absence of lipopolysaccharide, transforming growth factor-beta treatment produced a dramatic decrease in tumor necrosis factor-alpha peptide levels without affecting messenger ribonucleic acid levels.


CONCLUSIONS
In the presence of a stimulatory agent, interleukin-10 down-regulates tumor necrosis factor-alpha release from cultured human amniochorionic membranes. Transforming growth factor-beta seems to have some stimulatory effect on transcription, and no effect on translation was seen with concurrent lipopolysaccharide stimulation. However, down-regulation of tumor necrosis factor-alpha peptide by transforming growth factor was seen in fetal membranes when not overridden by an inflammatory stimulant. This study suggests that interleukin-10 and transforming growth factor-beta can regulate tumor necrosis factor-alpha release from amniochorion under different conditions and by a different mechanism.
Fortunato
S J
SJ
Maternal-Fetal Group, Women's Health Research and Education Foundation, Nashville, TN 37203, USA.
Menon
R
R
Lombardi
S J
SJ
D000650:Amnion / Q000378:metabolism*; D001667:Binding, Competitive; D002823:Chorion / Q000378:metabolism*; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D007752:Obstetric Labor, Premature / Q000628:therapy*; D009924:Organ Culture Techniques; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10