Cytokines and Pregnancy

Evidence shows that cytokines are produced in reproductive and embryonic tissues also and are involved in the invasion of trophoblasts and that invasion is important for regulation and maintenance of pregnancy. Also, cytokines play crucial roles in implantation of embryo during early stages of pregnancy and spiral artery remodelling, regulation of trophoblast invasion, immunoregulation and initiation of labour during later stages of pregnancy. More recently it is being shown that cytokines are produced by cells in the decidua and placenta also apart from immune cells. Cytokines and chemokines are secreted from cytotrophoblasts as well as from the cells of decidua predominantly from macrophages and fibroblasts. Also their receptors have been identified on cytotrophoblasts. It has been seen that chemokines such as CX3CL1, CCL4, CCL14, and CXCL16 increase trophoblast migration or invasion. Similarly, interleukins like IL-1, IL-6, IL-8 and IL-11 which are secreted by cytotrophoblast and decidual natural killer cells promote gelatinase activity and/or promote trophoblast invasion. More and more growth factors have been identified playing key roles in trophoblast invasion but, it is still not understood fully whether some play prominent role than others or not.

Interleukin-1 (IL-1) is a polypeptide which is generally produced after inflammation, injury or an antigenic challenge by mononuclear phagocytes. IL-1is also synthesized in epidermal, epithelial, lymphoid and vascular tissues by keratinocytes, fibroblasts, B lymphocytes, natural killer cells, astrocytes, microglial cells of brain etc. Initially IL-1 was called as endogenous pyrogen as it was thought to cause fever but, later it was found that it does more than just causing fever[Dinarello 1988]. IL-1 family is a group of pro-inflammatory cytokines which consists of two ligands or agonist proteins IL-1α and IL-1β, their cell surface receptors interleukin-1 receptor type 1 (IL-1R1) and interleukin-1 receptor type 2 (IL-1R2), a non-binding receptor accessory protein (IL1RAcp) and the receptor antagonist (IL-1ra). This naturally occurring IL-1ra competes with IL-1 for receptor binding. Evidence shows that all components of the IL-1 family have been identified in the human endometrium and also, that IL-1 is present at the feto-maternal interphase. IL-1 is produced by cells of trophoblast as well as decidua and IL-1 is known to have an important role in implantation. Research on mice has shown that IL-1 has a potential role in embryo implantation as IL-1 receptor antagonist given before implantation leads to the reduction in the number of implanted embryos. IL-1 plays an essential role in onset and development of invasion which is required for blastocyst implantation and placentation and this is highly regulated by a complex system of inhibitors consisting of receptor antagonist IL-1ra and IL-1 receptor (IL-1R_I). Evidence shows that IL-1 is essential for initiating as well as maintaining adequate invasion. IL-1 when produced at the site of invasion causes stimulation of surrounding cells which leads to the synthesis and release of chemokines as well as other cytokines e.g. IL-2, IL-4 etc. causing activation of cells. During early pregnancy, IL-1 is mainly produced and secreted by maternal decidua. In the endometrium, IL-1 is produced by macrophages, glandular epithelium and stromal cells in endometrium. Invasive properties of trophoblastic cells are related to their capacity of secreting proteolytic enzymes such as matrix metalloproteinases (MMPs). IL-1 is thought to promote trophoblast invasion by stimulating MMP-9 release by human cytotrophoblast.

Interleukin-6 (IL-6) is a glycosylated polypeptide consisting of 184 amino-acids with a molecular weight of 21-28 KDa approximately.IL-6 has a four helix bundle type tertiary structure and is manufactured by a diverse number of cells in different tissues like adipose tissue, skeletal muscle, liver etc. IL-6 is a cytokine that performs multiple functions and plays an important role in immune response, host defence mechanism, regulation of haematopoiesis, acute phase reaction as well as both anti and pro inflammatory events. IL-6 exhibits its activity by binding to its membrane bound receptor known as IL-6 receptor (IL-6R). This binding of IL-6 to IL-6R causes dimerization of the signal transducing receptor subunits glycoprotein 130 (gp130). This is followed by recruitment of IL-6R complex which initiates a series of cascades. Evidence shows that mice deficient in IL-6 have reduced fertility and decrease in viable implantation sites which shows that IL-6 plays an important role in implantation. IL-6 mRNA and protein are localized in human endometrium as well and IL-6 is expressed maximum during mid-secretory phase, thus, showing importance at the time of implantation. Moreover, the secretion of IL-6 by endometrial stromal cells is accelerated by IFN-γ. It has been seen that during first trimester IL-6 mRNA and protein are present in cells of cytotrophoblast as well as syncytiotrophoblast and also, the concentration of IL-6 is higher in decidua when compared to placental tissue. As IL-6 is present at the feto-maternal interphase, it point towards its involvement in the process of trophoblast invasion which is proved also. Tumour invasion and invasion of trophoblasts share the common biochemical mediators which are MMPs and their tissue inhibitors (TIMPs). For implantation and placentation to take place massive invasion by the cells of trophoblast is essential as well as required and the invasive activities of trophoblasts are related to their ability to secrete proteolytic enzymes such as MMPs. The process of invasion is controlled very well in terms of location as well as developmental stage. Furthermore, IL-6 is known to have positive effect on the production of these MMPs by regulating their production. 

Tumour necrosis factor alpha (TNF-α) also known as cachectin is a member of TNF superfamily which consists of 8 ligands and 29 different receptors that are involved in various cellular processes produced mainly by activated macrophages and to a lesser extent by activated mast cells and natural killer (NK) cells. It is also considered as an inflammatory cytokine due to its similar activities to IL-1.TNF-α exhibits a wide range of its biological properties via two distinct receptors termed as TNFR1 and TNFR2 which are members of the TNF receptor superfamily. TNF is known to control the expression of other cytokines, immune receptors, proteases, growth factors and cell cycle genes which in turn regulate inflammation, cellular homeostasis, apoptosis, cell survival, proliferation, differentiation and migration. In situ hybridization studies and immunohistochemistry analysis has shown that TNF mRNA proteins are expressed in the endometrium as well as the smooth muscle cells of the myometrium. Furthermore, different cell types of the endometrium viz. glandular epithelial cells, macrophages, vascular cells and fibroblasts express TNF. TNF mRNA is seen to be expressed in all cell types of trophoblastic lineage which includes villous cytotrophoblasts, syncytiotrophoblasts, proliferating cytotrophoblasts of cell islands and invasive trophoblasts during the first third of gestation but, as pregnancy proceeds the expression of mRNA is switched from the population of trophoblastic cells to villous stromal cells. This is maintained even when extravillous trophoblasts cause the endothelial cells displacement of spiral arteries. However, in the later stages of pregnancy, the expression of TNF is decreased in the invasive cells and in the trophoblast giant cells; expression of TNF is totally absent. However, some researchers have shown that TNF specifically inhibits trophoblast migration and invasion but, despite its negative effects on invasion; TNF was shown to stimulate the production of MMPs specifically MMP-9 in the first trimester trophoblast explant cultures and decidual cells.

Lipopolysaccharide (LPS) is commonly found as major constituent of cell walls of gram negative bacteria. The outer layer of gram negative bacteria is phospholipoprotein bilayer of which the outer leaflet is lipopolysaccharide. LPS is extremely toxic and is termed as endotoxin. LPS consists of a toxic lipid A, a core polysaccharide and O antigen polysaccharide side chains. Lipopolysaccharide helps in maintaining the membrane structure and protection from various types of chemicals. Evidence shows that LPS can increase the production of MMPs in human foetal membranes. Also, it causes preterm birth by effecting the stimulation of cytokines and prostaglandins by host cells. Almost all MMPs are released as pro MMPs; the inactive precursor form. Hence, activation of these pro MMPs is an important factor for MMP activity to occur. The most common mechanism that has been recognised for activation of MMPs is proteolytic removal of a propeptide domain of pro MMPs which is done by a number of endogenous proteinases such as, plasmin, trypsin and other active MMP family membranes. These endogenous proteinases then form MMP-activating cascades in vivo. At the site of infection, various types of proteinases are released by pathogenic bacteria such as gram negative bacteria and LPS is a component of gram negative bacteria. Therefore, at the site of infection, bacteria induced inflammation may cause the activation of pro MMPs and bacteria by means of LPS causing degradation of matrix by upregulating MMP production and converting pro MMPs into active MMPs. The presence of infection during pregnancy is attributed to gram negative and gram positive bacteria which leads to some serious consequences such as preterm premature rupture of membranes (pPROM), preterm labour (PTM) etc. and the molecular mechanisms of PROM involves the activation of MMPs in the amniochrion. Hence, knowing how and in what way LPS stimulates MMP production will be useful.

Matrix metalloproteinases (MMPs) are proteolytic zinc requiring enzymes which consists of many different types such as Collagenases viz. MMP-1, 8, 13, 14 and 18; the Stromelysins viz. MMP-3, 10 and 11; Gelatinasesviz. MMP-2 and 9 and Matrilysins viz. MMP-7 and 26 and are responsible for degradation of extracellular matrix. During placentation gelatinases play an important role in the process of invasion by the trophoblast cells into the maternal endometrium. Development of placenta is a complex process which requires a perfect coordination between the maternal endometrium and foetal-derived trophoblast cells. The process of invasion begins when blastocyst adheres to the endometrial epithelium and during invasion the properties of invasive placental cells are similar to malignant cells but, placental cells are highly regulated. The mechanism of invasion has been studied a lot by researchers and it has been found that the ability of cytotrophoblast cells to cross the basement membrane and interstitial matrices point towards the presence of some matrix degrading enzymes which play an important role in invasion which are none other than MMPs. There is evidence that human placental trophoblast cells produce matrix proteins such as collagen IV, laminin and fibronectin in first trimester. MMP-2 which is a 72KDa and MMP-9 which is a 92KDa type IV collagenase; are the main enzymes that cause the degradation of the basement membrane which consists of type IV collagen chiefly. The activity of gelatinases also relies on the activity of other proteases which cause the transformation of pro MMPs to active MMPs. After blastocyst implantation and before the completion of placentation in first trimester trophoblast cells produce both MMP-2 and 9 but, MMP-2 dominates while MMP-9 is produced in minor amount whereas, in the third trimester cells of trophoblast secrete MMP-9 predominantly when compared to MMP-2. Physiologically, the activity of these MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs). Three tissue inhibitors viz. TIMP-1, TIMP-2 and TIMP-3 regulate proteinase activity. TIMPs inhibit the activity of different types of MMPs by binding to the zinc binding site of active MMP. TIMP-1 forms complexes and mainly regulates MMP-9 whereas, TIMP-2 with MMP-2. TIMP-3 is known as main regulator of MMPs in vivo as it supports the activation and inhibition of MMP-2. Evidence shows that TIMPs are produced throughout the pregnancy by decidual and trophoblast cells. Hence, trophoblast invasion requires MMP-2 and MMP-9 synthesis as well as activation.

Cytokines in pregnancy disorders

The process of invasion begins when the EVT starts to invade the decidualized endometrium and inner myometrium. This is followed by transformation of maternal spiral arteries into low resistance vessels as described before. Any failure in the remodelling of spiral arteries causes a decrease in uteroplacental blood flow. This poor perfusion is thought to be responsible for the release of cytokines and products of oxidative stress into maternal circulation, which may cause endothelial dysfunction. Growth factors and cytokines are responsible for blastocyst implantation, trophoblast invasion and spiral artery remodelling. Research shows that cytokines play an important role in the process of invasion of trophoblast cells by regulating the secretion of gelatinases. Furthermore, this poor or incomplete transformation of vessels has been detected in placental bed of patients having pre-eclampsia and IUGR which indicates that EVT differentiation may be disturbed in these patients. Hence, decreased invasion leads to malformation, IUGR, pre-eclampsia, spontaneous abortion, still birth etc. whereas, increased invasion is known to cause invasive mole, placenta accreta and choriocarcinoma. Therefore, the processes of invasion are of great importance for achieving normal pregnancy and as cytokines are known to regulate trophoblast invasion by regulating MMP-2 and MMP-9, any finding on how cytokines regulate invasion and what effect cytokines have on the process of invasion will be very helpful.

                                                     Dr.Bharati Sood