Lipopolysaccharide Regulates Cytokine Expression In Cytotrophoblast And Its Relationship With Pre-eclampsia

IntroductionPre-eclampsia is a pregnant-specific disease, which seriously endangers health ofpregnancy and perinatal fetus, and is characterized by hypertension, proteinuria andedema. The study of preeclampsia mechanism is the focus in the researching field ofperinatal medicine. Systemic inflammatory response syndrome (SIRS) is the pivotal inpreeclampsia. Pro-inflammatory cytokines increase in circulation in SIRS. It isindicated that these cytokines present in pre-eclampsia as well. In further study, it isconfirmed that placenta is the resource of those cytokines. But the cause that results incytokine releasing in placenta remains unclear. It is suggested that decreased invasionof cytotrophoblast lead to shallow placentation and hypoxia, which may be the reasonfor cytokine releasing in placenta. However, recent studies indicate thatcytotrophoblast has some properties of innate immune cells. Molecular patternrecognition in cytotrophoblast may be the cause of cytokine releasing in placenta. Tolllike receptor-4 is expressed in cytotrophoblast. Toll like receptor -4 is a type of patternrecognition receptor in innate immune system. Its ligand is lipopolysachharide, a kindof toxin from gram-negative bacterial cell wall. Lipopolysachharide enter body wheninfections occur. Innate immune cell such as mononuclear cell receiveslipopolysachharide stimulation and product pro-inflammation cytokines. It is themechanism by which system inflammatory response syndrome occurs in septicaemiaand pyamia. Lipopolysachharide is found to be associated with pre-eclampsia. It is speculated that LPS can stimulate cytotrophoblast to release cytokines, which play animportant role in pre-eclampsia.Cytokines such as tumor necrosis factor-α,interleukin-6,interleukin-10 andinterferon-γwere found to be produced in cytotrophoblast. However, cytokine has agreat variety and complicated mechanism. It is insufficient to put up conclusionsaccording to investigating the role of one or a few cytokines in pre-eclampsia.Therefore, we will preliminarily explore the regulation effects of lipopolysachhadde oncytokine releasing in cytotrophoblast and its relationship with pre-eclampsia. Weconduct this study in two aspects. Firstly, in the macro-aspect, we use a new proteomeinvestigation tool, cytokine antibody arrays, to study the regulation effect of LPS oncytokine releasing in cytotrophoblast. Cytokine antibody arrays can simultaneouslydetermine a variety of cytokines. In this study, we will use Raybiotech HumanCytokine Antibody ArrayⅢ, which can determine 42 kinds of cytokine at the sametime. Secondly, in the micro-aspect, we will explore the effects of lipopolysachharideon tumor necrosis factor-a releasing and apoptosis in cytotrophoblast and observe theexpression of tumor necrosis factor-αin placenta.Tumor necrosis factor-α, which mostly derived from mononuclear phagocyte, hasgeneral biologic activity. Tumor necrosis factor-a is an important pro-inflammatoryand immune-regulation media. It plays roles in pathophysiological mechanism indiseases. A low level of tumor necrosis factor-αis found in epithelium and basemembrane in endometrium and ovarian stroma in normal conditions. In normalpregnancy, tumor necrosis factor-αis expressed in fetus and deciduas, and has immuneregulation effects between mother and fetus. However, tumor necrosis factor-αismarkly elevated in pre-eclampsia. The levels of tumor necrosis factor-αand tumornecrosis factor-αreceptor have increase before clinical symptom and sign occur inpre-eclampsia. It is indicated that the level of tumor necrosis factor-αis positivecorrelation with the condition of pre-eclampsia. The mechanism of tumor necrosis factor-αresults in pre-eclampsia is that it injuries vascular endothelial cells, inhibits theproduction and releasing of nitrogen monoxidum and induced apoptosis.ObjectiveCytokine antibody arrays were employed to determine the levels of cytokinesreleased by cytotrophoblast with LPS treatment. Further, the effect of LPS on apoptosisof cytotrophoblast and the role of tumor necrosis factor-αin the procession wereinvestigated. At last, the tumor necrosis factor-at expression in placenta inpre-eclampsia was observed. On the base of the above studies, we explore therelationship between cytokine releasing induced by LPS in cytotrophoblast andpre-eclampsia.MethodsCytotrophoblast was isolated from early pregnant villous and cultured inserum-free media. It was treated with LPS at variety concentrations. RaybiotechHuman Cytokine Antibody ArrayⅢwas used to determine expression of cytokinesproduced in cytotrophoblast with treatment of lipopolysachharide. Hoechst DNAstaining, flow cytometry, and electron transmission microscope were used to determinethe apoptotic-inducing effect of lipopolysachharide on cytotrophoblast. To explore thetumor necrosis factor-αexpression in cytotrophoblast, confocal microscope andenzyme-linked immunosorbent assay were used. At the end, immunochemistry wasemployed to determine the expression of tumor necrosis factor-αin placenta frompre-eclampsia patients.ResultsResults of Raybiotech Human Cytokine Antibody ArrayⅢindicated thatlipopolysachharide could regulate cytokine expression in cytotrophoblast. It increasedthe expression of CXC chemokines such as growth related gene production and growthrelated gene production-α, and inhibited the expressions of CC chemokines expressionsuch as monocyte chemoattractant protein-2, macrophage inflammatory protein 1 δ,and reduced upon activation, normal T cell expressed and secreted. It promoted theexpression of interleukin-1β, interleukin-6, interleukin-12, interferon-γand of tumornecrosis factor-αand oncostatin-M, which were pro-inflammatory cytokines, andinhibited the expression of interleukin-4 and interleukin-13, which wereanti-inflammatory cytokines. But, it increased the expression of anti-inflammatorycytokine, interleukin-10. About growth factor, lipopolysachharide had a promotingeffect on the expression of interleukin-2, interleukin-7, vascular endothelial growthfactor, and platelet derived growth factor. However, it down-regulated the expressionof epidermal growth factor, interleukin-15, and angiogenin, lipopolysachhafidecould not alter the expressions of monocyte chemoattractant protein-2, monocytechemoattractant protein-3, macrophage derived chemokines, epthelial-neutrophilactivating peptide, tumor necrosis factor-β, insulin-like growth factor-1,macrophagecolony stimulating factor, interleukin-5, monokine induced by interferon gamma,granulocyte colony stimulating factor, stem cell factor, Leptin,stromal-dereivedfactor-1, I-309, TRAC and transforming growth factor-β1 incytotrophoblast.The results of Hoechst DNA staining, electron transmission microscope and flowcytometry showed that lipopolysachharide induced apoptosis of cytotrophoblast in adose-dependent manner. The results of enzyme-linked immunosorbent assay andconfocal microscope showed that lipopolysachharide induced tumor necrosis factor-αexpression in cytotrophoblast in a dose-dependent manner, too. A positivecorrelationship was found between apoptosis and expression of tumor necrosisfactor-α.In immunochemistry study, it was revealed that tumor necrosis factor-αexpression in pre-eclampsia was increased, as contrast to normal pregnancy. ConclusionsLipopolysachharide regulated cytokine expression in cytotrophoblast. Thesecytokines played important role in inflammatory reaction, vascular endothelial injuryand immune imbalance, which were important mechanism in pre-eclampsia. Tumornecrosis factor-a induced apoptosis in cytotrophoblast and was tightly connected withpre-eclampsia. All these revealed that infection in pregnancy was predisposing factorof pre-eclampsia and antibiotic treatment had great value in prevention and treatmentfor pre-eclampsia.