title: Characterization of the macrophage-stimulating activity from Ureaplasma urealyticum.
abstract: OBJECTIVE
  Intra-amniotic infection is the most common cause of preterm labor. Infections are thought to cause preterm labor by increasing the production of proinflammatory cytokines at the maternal-fetal interface. Experiments with cell culture and animal models have indicated that bacterial lipopolysaccharide (LPS) increases the production of proinflammatory cytokines in reproductive tissues. The majority of intrauterine infections, however, are associated with Ureaplasma urealyticum, which does not contain LPS. Therefore, we performed a series of experiments to understand better the bacterial factor(s) that are responsible for the proinflammatory effects of U. urealyticum.
  
  
  METHODS
  U. urealyticum was cultivated in 3-4 L 10B broth, harvested by centrifugation, washed with saline and frozen at -85 degrees C until use. Cells were then extracted with Triton X-114 and the macrophage-stimulating activity (MSA) of the preparations was studied by evaluating their ability to stimulate tumor necrosis factor-alpha production by a monocytic cell line (THP-1 cells). Additional studies involved testing the sensitivity of the detergent extracts to heating, alkaline hydrolysis and proteinase K digestion. Interaction of Triton X-114 extracts with Toll-like receptor (TLR)-2 and TLR-4 was evaluated using cell lines transfected with one of these receptors, CD14 and a reporter gene.
  
  
  RESULTS
  Extraction of U. urealyticum with Triton X-114 demonstrated that the MSA preferentially partitioned to the detergent phase. The MSA of the detergent extracts was abrogated by proteinase K digestion or alkaline hydrolysis but only partially inhibited by heating. Further studies suggested that the detergent extracts could activate both TLR-2 and TLR-4.
  
  
  CONCLUSIONS
  These experiments suggest that the MSA of U. urealyticum is lipophilic, sensitive to alkaline hydrolysis and proteinase K digestion, partially sensitive to heating. These properties are consistent with the activity being due to a lipoprotein. Unlike other Mycoplasma species, the MSA of U. urealyticum appears to interact with both TLR-2 and TLR-4. Purification of the molecule(s) that regulate this activity may provide good therapeutic targets for anti-inflammatory strategies to prevent preterm labor caused by intrauterine infection with U. urealyticum.
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Medicine and Dentistry-Robert Wood Johnson Medical School, 125 Patterson Street, New Brunswick, NJ 08901, USA.
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Peltier
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Freeman
authors.forename: Hong H
authors.initials: HH
authors.lastname: Mu
authors.forename: Barry C
authors.initials: BC
authors.lastname: Cole
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D019286:Endopeptidase K / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D055666:Lipopeptides; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D017830:Octoxynol; D009842:Oligopeptides / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D012972:Sodium Hydroxide / Q000737:chemistry; D051195:Toll-Like Receptor 2 / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D016990:Ureaplasma urealyticum / Q000737:chemistry* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D053645:Interleukin-2 Receptor alpha Subunit; D055666:Lipopeptides; D008070:Lipopolysaccharides; D009842:Oligopeptides; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D012972:Sodium Hydroxide; D017830:Octoxynol; C010615:Nonidet P-40; C106335:macrophage stimulatory lipopeptide 2; D019286:Endopeptidase K