title: The novel antimicrobial peptide beta3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity.
abstract: 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 beta-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 beta-defensins has not been explored.
  
  
  OBJECTIVE
  This study was undertaken to test the hypothesis that amnion cells synthesize beta-defensins under either basal or stimulated conditions.
  
  
  METHODS
  Amnion epithelial FL cells (ATCC CCL 62) were cultured in Ham's F12 and Dulbecco's 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 microg/mL or peptidoglycan [PG]: 10 microg/mL). Reverse transcription real-time polymerase chain reaction was performed with the use of human-specific primers for beta1, beta2, beta3, and beta4 defensins to compare basal messenger RNA (mRNA) levels of defensins and in response to treatment. beta-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 beta-defensins with beta3-defensin mRNA levels significantly higher compared with others ( P < .001, 1-way analysis of variance [ANOVA]). beta3 was the only beta-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 beta3-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 beta3-defensin protein by immunohistochemistry. Conspicuous beta3-defensin staining was also detected in the chorio-decidua.
  
  
  CONCLUSIONS
  Amnion cells have the ability to produce beta-defensins. The beta3-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.
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University, New Haven, Conn, USA.
authors.forename: Irina A
authors.initials: IA
authors.lastname: Buhimschi
authors.forename: Maher
authors.initials: M
authors.lastname: Jabr
authors.forename: Catalin S
authors.initials: CS
authors.lastname: Buhimschi
authors.forename: Anelia P
authors.initials: AP
authors.lastname: Petkova
authors.forename: Carl P
authors.initials: CP
authors.lastname: Weiner
authors.forename: Ghassan M
authors.initials: GM
authors.lastname: Saed
mesh_terms: D000650:Amnion / Q000276:immunology*; D017931:DNA Primers; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D023083:beta-Defensins