The novel antimicrobial peptide β3-defensin is produced by the amnion: A possible role of the fetal membranes in innate immunity of the amniotic cavity

Background Innate immunity evolved to eliminate microorganisms before, or after their entry into the tissues, but before enough antigen is available to activate an adaptive, immune response. Innate immunity is so successful that the majority of encountered microbes are neutralized. The β-defensins are antimicrobial peptides produced by skin and mucosal surfaces and are an integral part of the innate immune system. The ability of the amnion cells, which are epithelial derivatives, to produce antimicrobial β-defensins has not been explored. Objective This study was undertaken to test the hypothesis that amnion cells synthesize β-defensins under either basal or stimulated conditions. Methods Amnion epithelial FL cells (ATCC CCL 62) were cultured in Hamʹs F12 and Dulbeccoʹs modified Eagle medium plus 10% fetal calf serum until confluence, then replated into 24-well plates at 1.5 million cells per well. Cells from triplicate wells were harvested after 1, 3, 6, and 24 hours of exposure to microbial wall components (lipopolysaccharide [LPS]: 1 μg/mL or peptidoglycan [PG]: 10 μg/mL). Reverse transcription real-time polymerase chain reaction was performed with the use of human-specific primers for β1, β2, β3, and β4 defensins to compare basal messenger RNA (mRNA) levels of defensins and in response to treatment. β-actin was used for standardization. Protein expression was investigated by immunofluorescence of the cells in culture, and by immunohistochemistry in paraffin sections of human fetal membranes from pregnancies with or without histologic chorioamnionitis. Results Amnion FL cells expressed mRNA for all known β-defensins with β3-defensin mRNA levels significantly higher compared with others (P< .001, 1-way analysis of variance [ANOVA]). β3 was the only β-defensin whose mRNA was upregulated in response to the microbial mimics LPS (1-way ANOVA, P = .019) and PG (1-way ANOVA, P = .011). Immunofluorescence confirmed that β3-defensin protein was present in cultured amnion cells, and upregulated in response to PG and LPS in distinct cells. Similarly, in tissue sections of human fetal membranes amnion epithelium was intensely positive for β3-defensin protein by immunohistochemistry. Conspicuous β3-defensin staining was also detected in the chorio-decidua. Conclusion Amnion cells have the ability to produce β-defensins. The β3-defensin appears to be the predominant epithelial defensin expressed. Its induction by microbial mimics suggests that the amniotic epithelium may play a role in the innate immunity of the amniotic cavity.