Chandana Das

Regulation of Trophoblast Invasion by IL‐1β and TGF‐ β1

Karmakar S, Das C. Regulation of trophoblast invasion by IL‐1β and TGF‐β1. AJRI 2002; 48:210–219

Network of cytokines, integrins and hormones in human trophoblast cells

Trophoblast cells of the developing embryo are unique not only for transporting oxygen and nutrients from the mother to the fetus but also for their array of other functions throughout the pregnancy beginning from the attachment of the blastocyst to the endometrium during the process of implantation, its well regulated invasion in the uterine tissue, proliferation, differentiation and immuno-endocrine functions which subsequently maintain the pregnancy. Using human trophoblast cells in culture, we have tried to understand the molecular mechanisms which regulate such a variety of functions for trophoblast cells. Our RT-PCR studies show that trophoblast cells express the laminin and collagen receptors: integrins alpha1 and alpha2, which are both stimulated by IL-1 and IL-6. These two cytokines, also synthesised by the trophoblast cells themselves, act in an autocrine/paracrine manner to induce their own expression. In addition, IL-1 expression seems to be modulated by a large variety of cytokines and growth factors usually present in the uterine milieu whereas IL-6 expression appears to be significantly stimulated by growth factors like EGF and bFGF only. Hormones like estrogen, progesterone and hCG exhibit a general negative modulation in the expression of IL-1 and IL-6. Since, both IL-1 and IL-6 are known to be involved in the proliferation, invasion and differentiation of trophoblast cells, they might be the key factors involved in trophoblast functions.

Expression of Pro-inflammatory Cytokines in Mouse Blastocysts During Implantation: Modulation by Steroid Hormones

PROBLEM: Expression and hormonal regulation of pro‐inflammatory cytokines and their role in blastocyst activation and implantation is poorly known. The present study is aimed at analysing the expression and hormonal modulation of two pro‐inflammatory cytokines [interleukin‐1α (IL‐1α) and IL‐6] in mouse blastocysts during implantation.

Inhibition of Cytotrophoblastic (JEG-3) Cell Invasion by Interleukin 12 Involves an Interferon γ-mediated Pathway

Trophoblast invasion, like tumor invasion, shares common biochemical mechanisms. However, in contrast to tumor invasion of a host tissue, trophoblastic invasion during implantation is strictly regulated, temporospatially. Factors responsible for these important regulatory processes are presently unknown; however, studies indicate that cytokines and growth factors represent in the peri-implantation uterine milieu as the possible candidates. In this study we investigated the role of interleukin (IL) 12 in regulating trophoblast invasion and the expression of trophoblast proteases (matrix metalloprotease (MMP)-2, MMP-9, and urokinase-type plasminogen activators) and their inhibitors (tissue inhibitors of metalloprotease (TIMP) 1, TIMP-2, and plasminogen activator inhibitor (PAI)-1) using an in vitro tissue culture system of human choriocarcinoma cell line JEG-3. Our major findings show an anti-invasive role of IL-12, associated with an inhibitory effect on the proteases but with an opposite up-regulating influence on the protease inhibitor, TIMP-1, whereas TIMP-2 and plasminogen activator inhibitor 1 remained unaltered. Stimulation of JEG-3 cells with IL-12 also induced interferon (IFN)-γ production, which when neutralized using a monoclonal anti-IFN-γ antibody, F12, abrogates its ability to down-regulate the MMPs. IL-12 also mediates an IFN-γ-dependent up-regulation of E-cadherin, thereby implying that alteration in cell-cell adhesion besides regulating the proteases and the inhibitors possibly contributes to the observed anti-invasive role of this cytokine. TIMP-1, although stimulated by IL-12, was found to be unaltered by antibody F12, thereby implying a possibility of an IL-12-dependent-IFN-γ independent regulation. These findings thereby suggest an important role of IL-12 in modulation of trophoblast proteases and their inhibitors besides regulating cell-cell interactions and invasion during implantation, with far reaching possibilities for understanding the mechanism(s) and regulations of invasion and metastasis.

Immunogenicity of three C-terminal synthetic peptides of the beta subunit of human chorionic gonadotropin and properties of the antibodies raised against 45-amino acid C-terminal peptide

Immunological studies were carried out in rhesus monkeys and rabbits on three C-terminal synthetic peptides of beta-hCG (115-145; 111-145 and 101-145) after conjugating these to tetanus toxoid (TT). The immunogenicity of the peptide conjugates was comparatively poorer with reference to Pr-beta-hCG-TT conjugates at similar doses and immunization schedule. Amongst the three peptides, the best response was obtained with the 45-amino acid c-terminal peptide (45-CTP; 101-145). The anti-45-CTP recognized native hCG and was devoid of cross-reaction with hLH. hCG-induced testosterone production by mouse Leydig cells was inhibited by anti-45-CTP antiserum, although its neutralization capacity decreased more rapidly upon dilution than anti-beta-hCG sera of comparable titres. Immune complexes formed by the anti-45-CTP with hCG had a lower sedimentation value than those formed by anti-beta-hCG antisera with hCG, suggesting the presence of a limited number of immuno-determinant regions in the 45-amino acid C-terminal synthetic peptide.

Steroids Modulate the Expression of α4 Integrin in Mouse Blastocysts and Uterus During Implantation

Abstract Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of α4β1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of α4 and β1 integrin subunits indicate that steroids modulate the expression of α4β1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of α4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that α4β1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of α4 integrin in the process of implantation.

Localization of Nitric Oxide Synthase in Human Trophoblast Cells: Role of Nitric Oxide in Trophoblast Proliferation and Differentiation

PROBLEM: There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation. METHOD OF STUDY: NOS isoforms in primary‐term trophoblast and JEG‐3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG‐3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal‐type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG‐3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone‐secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG‐3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation.

Investigations on the ability of antisera produced by Pr-β-HCG-TT to neutralize the biological activity of HCG

Abstract The ability of antibodies generated by the vaccine Pr-β-HCG-TT in two species of animals to neutralize the biological activity of HCG has been investigated. The antisera from an immunized goat and serveral rhesus monkeys (Macaca mulatta) abolished the HCG induced rise in ventral prostate weight of immature male rats and of uterine weight in prepubertal mice. These were also found to be effective in preventing the binding of 125 I-HCG to receptors in rat testes and goat corpora lutea preparations. The antisera also blocked the HCG induced synthesis and secretion of progesterone in corpus luteum slices in vitro. These investigations show that the antibodies elicited by the conjugate (Pr-β-HCG-TT) form a complex with HCG, which is biologically inactive.

Clinical and immunological responses with Pr-β-hCG-TT vaccine

Abstract Six different batches of Pr-β-hCG-TT vaccine have been evaluated in 23 women for the antibody response. The anti-sera formed against these conjugates were capable of reacting immunologically with the whole hCG in radioimmunoassays and also neutralized the biological activity of hCG in radioreceptor assays. The antibody titres attained peak levels 4–6 months after the first injection. The peak titres could not be sustained and most of the subjects showed a spiky pattern. One subject was considered as non-responsive, two others had fairly low titres. Amongst the conjugates tested, batch 108 with SPLPS (sodium pthalate denatured with lipo-polysaccharides from S. entritides ) seemed more promising, but the addition of the adjuvant was found to be pyrogenic and unlikely to be accepted clinically.

Pregnancy-terminating action of a luteinizing hormone-releasing hormone agonist D-Ser(But)6desGly10ProEA in baboons.

Subcutaneous injections of luteinizing hormone-releasing hormone agonist D-Ser(But)6desGly10ProEA given to six pregnant baboons at 8-hour or 12-hour intervals over a period of 4 to 6 days around the time of the appearance of chorionic gonadotropin in the circulation diminished the plasma progesterone (P) to very low levels, leading to termination of pregnancy in all animals. The same amount of the agonist, given in a similar manner in the early luteal phase, although succeeding in reducing the plasma P levels by more than half, failed to abrogate pregnancy. The time and frequency of administration of the agonist appear to be important for interception of pregnancy.