Characterization of the macrophage-stimulating activity from Ureaplasma urealyticum.

PROBLEM: 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. METHOD OF STUDY: 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. CONCLUSION: 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.

Characterization and partial purification of a macrophage-stimulating factor from Mycoplasma hominis.

PROBLEM: Mycoplasma hominis is one of the most common pathogens of the genital tract and is associated with increased production of proinflammatory cytokines in reproductive tissues during preterm labor. The mechanism by which M. hominis, an organism lacking cell walls, increases the production of proinflammatory cytokines is unknown. METHOD OF STUDY: We characterized and purified a macrophage-activating factor from this organism. RESULTS: Extraction of whole organisms with Triton-X-114 demonstrated that the activity was primarily associated with the detergent phase. Macrophage-stimulating activity (MSA) of detergent extracts of M. hominis was not inhibited by polymyxin B or heating but was completely abrogated by alkaline hydrolysis and partially reduced by proteinase K digestion. Further experiments that utilized Toll-like receptor (TLR)-2- and TLR-4-transfected cells, revealed that the detergent extracts activate TLR-2 but not TLR-4 signal transduction. Purification of the activity using preparative SDS-PAGE and reverse phase chromatography experiments led to the isolation of a 29-kDa protein. CONCLUSIONS: These experiments suggest that the MSA of M. hominis is due to a lipophillic factor that interacts with TLR-2 rather than TLR-4 (as does lipopolysaccharide), to increase tumor necrosis factor (TNF)-alpha by macrophages. It is known that TNF-alpha can cause preterm labor and intrauterine fetal death and that it is upregulated in amniotic fluid samples infected with M. hominis.