• Milica Jovanović-Krivokuća Laboratory for Biology of Reproduction, Institute INEP, University of Belgrade, Banatska 31b, 11080 Belgrade-Zemun
  • Ivana Stefanoska Laboratory for Biology of Reproduction, Institute INEP, University of Belgrade, Banatska 31b, 11080 Belgrade-Zemun
  • Tamara Abu Rabi Laboratory for Biology of Reproduction, Institute INEP, University of Belgrade, Banatska 31b, 11080 Belgrade-Zemun
  • Aleksandra Vilotić Laboratory for Biology of Reproduction, Institute INEP, University of Belgrade, Banatska 31b, 11080 Belgrade-Zemun
  • Miloš Petronijević Medical Faculty, University of Belgrade, Doktora Subotića 8, 11000 Belgrade
  • Svetlana Vrzić-Petronijević Medical Faculty, University of Belgrade, Doktora Subotića 8, 11000 Belgrade
  • Ljiljana Radojčić Military Medical Academy, Crnotravska 17, 11000 Belgrade
  • Ljiljana Vićovac Military Medical Academy, Crnotravska 17, 11000 Belgrade


Infection is increasingly considered to contribute to pathological conditions in pregnancy. The placenta acts as a protective immunological fetomaternal barrier which recognizes microbes by pattern recognition receptors on the trophoblast. Lipopolysaccharide (LPS) is a cell wall constituent of Gram-negative bacteria that elicits a strong immune response. In this study, LPS from E. coli was used to treat the HTR-8/SVneo trophoblast cell line and examine its influence on cytokines IL-6, IL-8 and MIF using real-time PCR, metalloproteinases (MMP)-2 and -9 by gelatin zymography, and Western analysis of integrin subunits α1 and β1, all known to contribute to migration of human trophoblasts in vitro. The results described herein for the first time, show that MIF mRNA and secreted MIF protein were significantly elevated (2.5-3- and 2-fold, respectively) in LPS-treated cells. MMP-2 and MMP-9 levels were increased, as well as cell migration, as judged by a wound-healing test, however, no changes in the studied integrin subunits, cell viability or cell numbers were observed. The data obtained furthers our understanding of LPS actions on the trophoblast in vitro, additionally implicate MIF, and suggest that infection in vivo could indeed alter the functional characteristics of the trophoblast.

DOI: 10.2298/ABS151123012J

Key words: trophoblast; HTR-8/SVneo; cytokines; MIF; LPS

Received: November 23, 2015; Revised: December 21, 2015; Accepted: December 22, 2015; Published online: January 25, 2016

How to cite this article: Jovanović-Krivokuća M, Stefanoska I, Rabi Abu T, Vilotić A, Petronijević M, Vrzić-Petronijević S, Radojčić Lj, Vićovac Lj. MIF is among the proinflammatory cytokines increased by LPS in the human trophoblast line. Arch Biol Sci. 2016;68(4):715-22.


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Pijnenborg R, Bland JM, Robertson WB, Brosens I. Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy. Placenta. 1983;4(4):397-413.

Vićovac L, Aplin JD. Epithelial-mesenchymal transition during trophoblast differentiation. Acta Anat (Basel). 1996;156(3):202-16.

Damsky CH, Fitzgerald ML, Fisher SJ. Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo. J Clin Invest. 1992;89(1):210-22.

Lala PK, Graham CH. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev. 1990;9(4):369-79.

Librach CL, Werb Z, Fitzgerald ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH, Fisher SJ. 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol. 1991;13(2):437-49.

Jovanović M, Vićovac L. Interleukin-6 stimulates cell migration, invasion and integrin expression in HTR-8/SVneo cell line. Placenta. 2009;30(4):320-8.

Jovanović M, Stefanoska I, Radojcić L, Vićovac L. Interleukin- 8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins alpha5 and beta1. Reproduction. 2010;139(4):789-98.

Soares MJ, Chakraborty D, Kubota K, Renaud SJ, Rumi MA. Adaptive mechanisms controlling uterine spiral artery remodeling during the establishment of pregnancy. Int J Dev Biol. 2014;58(2-4):247-59.

Fritz R, Jain C, Armant DR. Cell signaling in trophoblastuterine communication. Int J Dev Biol. 2014;58(2-4):261-71.

Jovanović Krivokuća M, Stefanoska I, Abu Rabi T, Al-Abed Y, Stošić-Grujičić S, Vićovac Lj. Pharmacological inhibition of MIF interferes with trophoblast cell migration and invasiveness. Placenta. 2015;36(2):150-9.

Guleria I, Pollard JW. The trophoblast is a component of the innate immune system during pregnancy. Nat Med. 2000;6(5):589-93.

Abrahams VM, Mor G. Toll-like receptors and their role in the trophoblast. Placenta. 2005;26(7):540-7.

Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700.

Beutler B, Cerami A. Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annu Rev Biochem. 1988;57:505-18.

Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood. 1991;77(8):1627-52.

Holmlund U, Cebers G, Dahlfors AR, Sandstedt B, Bremme K, Ekström ES, Scheynius A. Expression and regulation of the pattern recognition receptors Toll-like receptor-2 and Toll-like receptor-4 in the human placenta. Immunology. 2002;107(1):145-51.

Ma Y, Krikun G, Abrahams VM, Mor G, Guller S. Cell typespecific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection. Placenta. 2007;28(10):1024-31.

Abrahams VM, Bole-Aldo P, Kim YM, Straszewski-Chavez SL, Chaiworapongsa T, Romero R, Mor G. Divergent trophoblast responses to bacterial products mediated by TLRs. J Immunol. 2004;173(7):4286-96.

Klaffenbach D, Rascher W, Röllinghoff M, Dötsch J, Meissner U, Schnare M. Regulation and signal transduction of toll-like receptors in human choriocarcinoma cell lines. Am J Reprod Immunol. 2005;53(2):77-84.

Kim YM, Romero R, Oh SY, Kim CJ, Kilburn BA, Armant DR, Nien JK, Gomez R, Mazor M, Saito S, Abrahams VM, Mor G. Toll-like receptor 4: a potential link between “danger signals,” the innate immune system, and preeclampsia? Am J Obstet Gynecol. 2005;193(3 Pt 2):921-7.

LaMarca BD, Ryan MJ, Gilbert JS, Murphy SR, Granger JP. Inflammatory cytokines in the pathophysiology of hypertension during preeclampsia. Curr Hypertens Rep. 2007;9(6):480-5.

Szarka A, Rigó J Jr, Lázár L, Beko G, Molvarec A. Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array. BMC Immunol. 2010 Dec 2;11:59.

Anton L, Brown AG, Parry S, Elovitz MA. Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasion through a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction. Hum Reprod. 2012;27(1):61-72.

Koh YQ, Chan HW, Nitert MD, Vaswani K, Mitchell MD, Rice GE. Differential response to lipopolysaccharide by JEG-3 and BeWo human choriocarcinoma cell lines. Eur J Obstet Gynecol Reprod Biol. 2014;175:129-33.

Riewe SD, Mans JJ, Hirano T, Katz J, Shiverick KT, Brown TA, Lamont RJ. Human trophoblast responses to Porphyromonas gingivalis infection. Mol Oral Microbiol. 2010;25(4):252-9.

Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, Lala PK. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res. 1993;206(2):204-11.

Irving JA, Lysiak JJ, Graham CH, Hearn S, Han VK, Lala PK. Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture. Placenta. 1995 Jul;16(5):413-33.

Hanisch FG., Dressen F, Uhlenbruck G. Quantitative microadhesion assay on polystyrene matrices. In: Gabius HJ, Gabius S, editors. Lectins and Glycobiology. Berlin: Springer-Verlag, Heidelberg; 1993. p. 411–7.

Stefanoska I, Jovanović Krivokuća M, Vasilijić S, Ćujić D, Vićovac L. Prolactin stimulates cell migration and invasion by human trophoblast in vitro. Placenta. 2013;34(9):775-83.

Lash GE, Otun HA, Innes BA, Bulmer JN, Searle RF, Robson SC. Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases. Biol Reprod. 2005;73(2):374-81.

Livak KJ, Schmittgen TD. (). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC T Method. Methods. 2001;25: 402-8.

Romero R, Roslansky P, Oyarzun E, Wan M, Emamian M, Novitsky TJ, Gould MJ, Hobbins JC. Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol. 1988;158(5):1044-9.

Nakatsuka M, Asagiri K, Noguchi S, Habara T, Kudo T. Nafamostat mesilate, a serine protease inhibitor, suppresses lipopolysaccharide-induced nitric oxide synthesis and apoptosis in cultured human trophoblasts. Life Sci. 2000;67(10):1243-50.

Li Y, Shibata Y, Zhang L, Kuboyama N, Abiko Y. Periodontal pathogen Aggregatibacter actinomycetemcomitans LPS induces mitochondria-dependent-apoptosis in human placental trophoblasts. Placenta. 2011;32(1):11-9.

Okada T, Matsuzaki N, Sawai K, Nobunaga T, Shimoya K, Suzuki K, Taniguchi N, Saji F, Murata Y. Chorioamnionitis reduces placental endocrine functions: the role of bacterial lipopolysaccharide and superoxide anion. J Endocrinol. 1997;155(3):401-10.

Mei YB, Zhou WQ, Zhang XY, Wei XJ, Feng ZC. Lipopolysaccharides shapes the human Wharton’s jelly-derived mesenchymal stem cells in vitro. Cell Physiol Biochem. 2013;32(2):390-401.

Liu Q, Qian Y, Chen F, Chen X, Chen Z, Zheng M. EGCG attenuates pro-inflammatory cytokines and chemokines production in LPS-stimulated L02 hepatocyte. Acta Biochim Biophys Sin (Shanghai). 2014;46(1):31-9.

Bauer S, Pollheimer J, Hartmann J, Husslein P, Aplin JD, Knöfler M. Tumor necrosis factor-alpha inhibits trophoblast migration through elevation of plasminogen activator inhibitor-1 in first-trimester villous explant cultures. J Clin Endocrinol Metab. 2004;89(2):812-22.

Lash GE, Otun HA, Innes BA, Kirkley M, De Oliveira L, Searle RF, Robson SC, Bulmer JN. Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels. FASEB J. 2006;20(14):2512-8.

Librach CL, Feigenbaum SL, Bass KE, Cui TY, Verastas N, Sadovsky Y, Quigley JP, French DL, Fisher SJ. Interleukin-1 beta regulates human cytotrophoblast metalloproteinase activity and invasion in vitro. J Biol Chem. 1994;269(25):17125-31.

Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med. 2006;12(9):1065-74.

Pontillo A, Girardelli M, Agostinis C, Masat E, Bulla R, Crovella S. Bacterial LPS differently modulates inflammasome gene expression and IL-1β secretion in trophoblast cells, decidual stromal cells, and decidual endothelial cells. Reprod Sci. 2013;20(5):563-6.

Shimoya K, Moriyama A, Matsuzaki N, Ogata I, Koyama M, Azuma C, Saji F, Murata Y. Human placental cells show enhanced production of interleukin (IL)-8 in response to lipopolysaccharide (LPS), IL-1 and tumour necrosis factor (TNF)-alpha, but not to IL-6. Mol Hum Reprod. 1999;5(9):885.

Svinarich DM, Bitonti OM, Araneda H, Romero R, Gonik B. Induction and posttranslational expression of G-CSF and RANTES in a first trimester trophoblast cell line by lipopolysaccharide. Am J Reprod Immunol. 1996;36(5):256-9.

Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hültner L, Heumann D, Männel D, Bucala R, Glauser MP. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat Med. 2000;6(2):164-70.

Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, Bucala R. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature. 1993;365(6448):756-9.

Komine-Aizawa S, Hirohata N, Aizawa S, Abiko Y, Hayakawa S. Porphyromonas gingivalis lipopolysaccharide inhibits trophoblast invasion in the presence of nicotine. Placenta. 2015;36(1):27-33.

Gröbner S, Lukowski R, Autenrieth IB, Ruth P. Lipopolysaccharide induces cell volume increase and migration of dendritic cells. Microbiol Immunol. 2014;58(1):61-7.

Zhang D, Li YH, Mi M, Jiang FL, Yue ZG, Sun Y, Fan L, Meng J, Zhang X, Liu L, Mei QB. Modified apple polysaccharides suppress the migration and invasion of colorectal cancer cells induced by lipopolysaccharide. Nutr Res. 2013;33(10):839-48.

Yang G, Zhou X, Chen T, Deng Y, Yu D, Pan S, Song Y. Hydroxysafflor yellow A inhibits lipopolysaccharide-induced proliferation and migration of vascular smooth muscle cells via Toll-like receptor-4 pathway. Int J Clin Exp Med. 2015;8(4):5295-302.

Li D, Fu L, Zhang Y, Yu Q, Ma F, Wang Z, Luo Z, Zhou Z, Cooper PR, He W. The effects of LPS on adhesion and migration of human dental pulp stem cells in vitro. J Dent. 2014;42(10):1327-34.

Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogenesis. Semin Reprod Endocrinol. 1999;17(3):275-90.

Li W, Unlugedik E, Bocking AD, Challis JR. The role of prostaglandins in the mechanism of lipopolysaccharide-induced proMMP9 secretion from human placenta and fetal membrane cells. Biol Reprod. 2007;76(4):654-9.

Shin MR, Kang SK, Kim YS, Lee SY, Hong SC, Kim EC. TNF-α and LPS activate angiogenesis via VEGF and SIRT1 signalling in human dental pulp cells. Int Endod J. 2015;48(7):705-16.

Roomi MW, Kalinovsky T, Rath M, Niedzwiecki A. In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors. Int J Oncol. 2014;44(1):27-34.

Renaud SJ, Macdonald-Goodfellow SK, Graham CH. Coordinated regulation of human trophoblast invasiveness by macrophages and interleukin 10. Biol Reprod. 2007;76(3):448-54.

Fest S, Aldo PB, Abrahams VM, Visintin I, Alvero A, Chen R, Chavez SL, Romero R, Mor G. Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy. Am J Reprod Immunol. 2007;57(1):55-66.

Grasso E, Paparini D, Hauk V, Salamone G, Leiros CP, Ramhorst R. Differential migration and activation profile of monocytes after trophoblast interaction. PLoS One. 2014;9(5):e97147. Available from: http://www.plosone.org/article/citationList.action?articleURI=info%3Adoi%2F10.1371/journal.pone.0097147

Ma Y, Kadner SS, Guller S. Differential effects of lipopolysaccharide and thrombin on interleukin-8 expression in syncytiotrophoblasts and endothelial cells: implications for fetal survival. Ann N Y Acad Sci. 2004;1034:236-44.




How to Cite

Jovanović-Krivokuća M, Stefanoska I, Abu Rabi T, Vilotić A, Petronijević M, Vrzić-Petronijević S, Radojčić L, Vićovac L. MIF IS AMONG THE PROINFLAMMATORY CYTOKINES INCREASED BY LPS IN THE HUMAN TROPHOBLAST LINE. Arch Biol Sci [Internet]. 2016Nov.24 [cited 2021Jan.28];68(4):715-22. Available from: http://www.serbiosoc.org.rs/arch/index.php/abs/article/view/1242