Antonette T Dulay1,2, Catalin S Buhimschi1,2,3, Guomao Zhao2, Emily A Oliver2, Sonya S Abdel-Razeq4, Lydia L Shook4, Mert O Bahtiyar4, Irina A Buhimschi1,2,3. 1. Department of Obstetrics & Gynecology, The Ohio State University College of Medicine, Columbus, OH, USA. 2. Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA. 3. Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA. 4. Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.
Abstract
PROBLEM: TLR4 mediates host responses to pathogens through a mechanism that involves protein myeloid differentiation-2 (MD-2) and its soluble form sMD-2. The role of sMD2 in intra-amniotic inflammation-induced preterm birth has not been previously explored. METHOD OF STUDY: Human amniotic fluid (AF) sMD-2 was studied by Western blotting in 152 AF samples of patients who had an amniocentesis to rule-out infection (yes infection, n = 50; no infection, n = 50) or women with normal pregnancy outcome (second trimester genetic karyotyping, n = 26; third trimester lung maturity testing, n = 26). Histological localization and mRNA expression of MD2 in fetal membranes were studied by immunohistochemistry and RT-PCR. The ability of fetal membrane to release sMD-2 and inflammatory cytokines was studied in vitro. RESULTS: Human AF contains three sMD-2 proteoforms whose levels of expression were lower at term. Intra-amniotic infection upregulated sMD-2. MD-2 mRNA and immunohistochemistry findings concurred. In vitro, LPS and monensin increased, while cycloheximide decreased sMD-2 production. Recombinant sMD-2 modulated TNF-α and IL-6 levels in a dose- and time-dependent fashion. CONCLUSION: sMD2 proteoforms are constitutively present in human AF. The intensity of the intra-amniotic inflammatory response to bacteria or perhaps to other TLR4 ligands may be facilitated through synthesis and release of sMD2 by the amniochorion.
PROBLEM: <span class="Gene">TLR4 mediates host responses to pathogens through a mechanism that involves protein myeloid differentiation-2 (<span class="Gene">MD-2) and its soluble form sMD-2. The role of sMD2 in intra-amniotic inflammation-induced preterm birth has not been previously explored. METHOD OF STUDY: Human amniotic fluid (AF) sMD-2 was studied by Western blotting in 152 AF samples of patients who had an amniocentesis to rule-out infection (yes infection, n = 50; no infection, n = 50) or women with normal pregnancy outcome (second trimester genetic karyotyping, n = 26; third trimester lung maturity testing, n = 26). Histological localization and mRNA expression of MD2 in fetal membranes were studied by immunohistochemistry and RT-PCR. The ability of fetal membrane to release sMD-2 and inflammatory cytokines was studied in vitro. RESULTS:Human AF contains three sMD-2 proteoforms whose levels of expression were lower at term. Intra-amniotic infection upregulated sMD-2. MD-2 mRNA and immunohistochemistry findings concurred. In vitro, LPS and monensin increased, while cycloheximide decreased sMD-2 production. Recombinant sMD-2 modulated TNF-α and IL-6 levels in a dose- and time-dependent fashion. CONCLUSION:sMD2 proteoforms are constitutively present in human AF. The intensity of the intra-amniotic inflammatory response to bacteria or perhaps to other TLR4 ligands may be facilitated through synthesis and release of sMD2 by the amniochorion.
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