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Original Article

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Ik-Hwan Han1, Sung Young Goo2, Soon-Jung Park2, Se-Jin Hwang3, Yong-Seok Kim4, Michael Sungwoo Yang5, Myoung-Hee Ahn1, Jae-Sook Ryu1
The Korean Journal of Parasitology 2009;47(3):205-212.
Published online: August 28, 2009

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Corresponding author (jsryu@hanyang.ac.kr)
• Received: June 28, 2009   • Revised: August 3, 2009   • Accepted: August 5, 2009

Copyright © 2009 by The Korean Society for Parasitology

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Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis
Korean J Parasitol. 2009;47(3):205-212.   Published online August 28, 2009
DOI: https://doi.org/10.3347/kjp.2009.47.3.205
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Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis
Korean J Parasitol. 2009;47(3):205-212.   Published online August 28, 2009
DOI: https://doi.org/10.3347/kjp.2009.47.3.205
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Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis
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Fig. 1 Trichomonas vaginalis stimulates human monocyte-derived macrophages (HMDM) to produce proinflammatory cytokines. Human macrophages (1.25 × 106/well of 96-well culture plate) were incubated with live trichomonads (1, 5, and 10 × 104), opsonized trichomonads (1, 5, and 10 × 104) and T. vaginalis lysate (10 and 100 µg/ml). Cell culture supernatant was harvested after 6 hr and 24 hr of incubation, and analyzed for levels of TNF-α, IL-1β, and IL-6 using cytokine-specific ELISA. *P < 0.05 versus untreated macrophages.
Fig. 2 NF-κB activation induced by Trichomonas vaginalis (T) in human macrophages (M). (A) Phosphorylation of NF-κB p65 expression in human macrophages reacted with T. vaginalis was determined by western blot. (B) Nuclear translocation of NF-κB p65 in human macrophages stimulated with T. vaginalis (low) compared with medium alone (up). Human macrophages (5 × 106/cover glass) were incubated with live T. vaginalis (1 × 106) for 30 min. Cells were then fixed and subjected to immunofluorescent microscopy after staining with anti-NF-κB p65 Ab (Red) and DAPI (Green) to visualize nuclei.
Fig. 3 NO production (A) and iNOS expression (B) by HMDM stimulated with T. vaginalis (T). Human macrophages (1.25 × 106/well of 96-well culture plate) were incubated with live trichomonads (1.25 × 105), opsonized trichomonads (1.25 × 105), or T. vaginalis lysate (100 µg/ml). NO was determined by the Griess reagent after 1 hr, 4 hr, and 24 hr of incubation (A). For identification of iNOS expression, human macrophages (1.25 × 106/well) were incubated with live trichomonads (1.25 × 105) for 1 hr. The iNOS expression was examined by western blot with anti-iNOS Ab (B). *P < 0.05 versus untreated macrophages.
Fig. 4 Effects of L-NMMA (an iNOS inhibitor) on NF-κB activity of human macrophages induced by T. vaginalis. L-NMMA-preteated HMDM or THP-1 cells was stimulated with live T. vaginalis. NF-κB activities were determined by western blot (A) or luciferase assay (B). Human macrophages were coincubated with live trichomonads (macrophage : trichomonads = 5 : 1) for 2 hr, and then western blot was done with rabbit anti-phospho NF-κB p65 monoclonal Ab (A). The PCH110 and PGL3 NF-κB promoter constructs were transfected into THP-1 cells with LipofectAmine 2000. Luciferase activity was assayed with a luciferase assay kit. Cell extracts obtained from THP-1 cells stimulated with live trichomonads for 1 hr were prepared in 500 µl of 1 × reporter lysis buffer. The luciferase activity was measured with a Luminometer (B).
Fig. 5 Effects of L-NMMA on TNF-α production in HMDM cells induced by live T. vaginalis. L-NMMA-pretreated macrophage suppressed T. vaginalis-triggered TNF-α expression, measured by RT-PCR (A) and ELISA (B), respectively. *P < 0.05 versus macrophages stimulated with T. vaginalis.
Fig. 6 Effects of iNOS inhibitor or NF-κB inhibitor on TNF-α production in macrophages induced by T. vaginalis. Pretreatment of human macrophages (THP-1 or HMDM) with L-NMMA or NF-κB inhibitor (PDTC or Bay11-7082) showed significant reduction of trichomonad-induced TNF-α production in THP-1 (A) and HMDM (B). Protein levels of TNF-α were measured by ELISA. *P < 0.05 versus untreated macrophages. †P < 0.05 versus T. vaginalis-stimulated macrophages.

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Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis
PHD : Parasites, Hosts and Diseases

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