Relaxin augments the inflammatory IL6 response in the choriodecidua.

UNLABELLED: Intrauterine infection frequently leads to preterm birth (PTB), with the pathophysiology involving activation of the innate immune system and its associated inflammatory response. The choriodecidua produces relaxin (RLN) and elevated levels are associated with preterm premature rupture of the fetal membranes. However, it is not increased in bacterially-mediated PTB, but may act as an endogenous sterile inflammatory mediator. Elevated systemic RLN levels from the corpus luteum are also associated with PTB, but the mechanism is unknown. In clinical obstetrics, intrauterine inflammation or infection can coexist with elevated RLN. Therefore, in this study, we further characterized the effects of RLN alone or together with an inflammatory mediator on the production of IL1B, CSF2 (GM-CSF), IL6, IL8 and TNF, from chorionic cytotrophoblasts (CyT), decidual fibroblasts (DF) and stromal cells (DSC), using interleukin-1 beta (IL1B) to mimic sterile inflammation or lipopolysaccharide (LPS) for bacterial infection. Endogenous differences between the cells showed that the CyT expressed more RLN, its receptor RXFP1 and the RXFP1 splice variant D. CyT also showed the most robust cAMP response to RLN with increased IL6 secreted after 4 h, preceded by increased transcription at 1 h, likely due to activation of RXFP1 and cAMP. When all cell types were treated with IL1B and RLN, RLN augmented secretion of IL6 and IL8 from CyT and DF, but not DSC. Similarly, RLN augmented LPS-induced IL6 secretion from CyT and DF. Despite the structural similarity between TLR4 and RXFP1, blocking TLR4 in CyT had no effect on RLN-induced IL6 secretion, suggesting specific activation of RXFP1. Thus, we have shown that in the presence of a low level of intrauterine inflammation/infection, elevated RLN could act on the CyT and DF to augment the inflammatory response, contributing to the pathophysiology of PTB. SUMMARY: RLN augments the inflammatory responses induced by IL1B or LPS in chorionic cytotrophoblasts and decidual fibroblasts.

Relaxin modulates proinflammatory cytokine secretion from human decidual macrophages.

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.

Relaxin stimulates interleukin-6 and interleukin-8 secretion from the extraplacental chorionic cytotrophoblast.

In the absence of infection, decidual relaxin (RLN) expression is increased in patients with preterm premature rupture of the membranes (PPROM) resulting in preterm birth, but it is not known whether inflammation stimulates RLN expression or vice versa. This study examined the effect of lipopolysaccharide (LPS) on the expression of RLN mRNA and secreted protein and whether RLN treatment influences secretion of proinflammatory cytokines from the fetal membranes. Explants of human fetal membranes in vitro and rhesus monkey fetal membranes in vivo were treated with LPS, which increased expression of IL-6 but had no effect on RLN. RLN treatment stimulated IL-6 and IL-8 secretion from choriodecidual explants in a subset of patients, as well as from isolated chorionic cytotrophoblast cells but not decidual cells. In vivo results obtained in rhesus monkeys after intra-amniotic infusion of RLN demonstrated increased IL-6 and IL-8 concentrations in amniotic fluid. Our results indicate that increased decidual RLN expression is independent of LPS but may induce a local sterile inflammatory process which potentially contributes to extracellular matrix degradation and weakening of the fetal membranes.

The human relaxin receptor (LGR7): expression in the fetal membranes and placenta.

The relaxin receptor has been recently described as a leucine-rich repeat G-protein coupled receptor and designated as LGR7. A closely related receptor, LGR8, is co-expressed by some cells. This study explored the expression of the genes for these receptors in the human fetal membranes and placenta by RT-PCR and the LGR7 protein by immunolocalization. The results showed that LGR7 was well expressed in the fetal membranes, with significantly more in the decidua (p<0.05) than in the amnion. On the other hand, relatively low levels were expressed in the placenta. The major splice variant of LGR7 was undetectable in either the placenta or fetal membranes. Expression of LGR8 was also below the level of detectability in either tissue. Immunostaining for LGR7 was conducted with antisera to both its endodomain and ectodomain, in order to seek evidence for a solubilized ectodomain. However, similar staining patterns were obtained with both antisera, with predominant staining in the cells of the amniotic epithelium, chorionic cytotrophoblast and decidua. Full-thickness fetal membranes from preterm deliveries, before and after labor or after preterm premature rupture of the membrane (PPROM) and labor were collected. In addition, membranes at term, both before and after spontaneous labor were used for analysis of LGR7 gene expression. There was significantly greater LGR7 expressed (p=0.01) in the preterm period compared to term, indicating a potentially important role for relaxin at this time. There was a marginal decline in LGR7 gene expression after labor and delivery both at preterm and term, which did not reach significance. Immunostaining patterns showed less inter-patient variability than did gene expression, with more intense staining for LGR7 after labor and delivery.

Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes.

Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5'-flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription. The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P<0.05) in severe infection. The PBEF protein was immunolocalized, in both normal and infected tissues, to both the normal fetal cells of the amnion and chorion and the maternal decidua of the membranes, and to the invading neutrophils. These stained strongly and were likely to contribute to the increased expression in infection. The amniotic epithelial cell line (WISH cells) has been used as a model to study PBEF gene modulation. Lipopolysaccharide, interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha and IL-6 all significantly increased the expression of PBEF in 4 h of treatment. The addition of dexamethasone to IL-1beta and TNFalpha significantly reduced the response of PBEF to these cytokines. IL-8 treatment failed to alter PBEF gene expression. Thus PBEF is a cytokine expressed in the normal fetal membranes and upregulated when they are infected. It is likely to have a central role in the mechanism of infection-induced preterm birth.

Lysyl oxidases: expression in the fetal membranes and placenta.

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term.

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection.

OBJECTIVE: This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis. STUDY DESIGN: Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis. RESULTS: In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted. CONCLUSION: A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.

Developmental regulation of the human relaxin genes in the decidua and placenta: overexpression in the preterm premature rupture of the fetal membranes.

The decidua and placenta synthesize the human relaxins, termed H1 and H2, believed to be involved in collagen remodeling in the amnion and chorion in an autocrine/paracrine manner. The developmental regulation of the relaxin genes was quantitated in normal pregnancy by in situ hybridization histochemistry with six 48-mer oligonucleotide probes that detect both relaxin genes. A significant increase in relaxin expression occurred in both decidua (p < 0.01) and placenta (p < 0.05) at 12.5-14.4 wk gestation, with the mean peak value in the placenta more than double that of the decidua, suggesting a coordinate regulation of the relaxin genes. At term after spontaneous labor and delivery, a marginal increase in both decidual and placental relaxin gene expression occurred. Given these normal data, three abnormal preterm situations were investigated: 1) premature uterine contractions without prior rupture of the membranes, 2) premature rupture of the fetal membranes (PPROM), 3) cesarean section for medical reasons with intact membranes and no uterine contractions. Tissues showing intrauterine infection were eliminated. Significantly more relaxin was expressed in the preterm decidua from patients with PPROM when compared to patients in group 1 (p < 0.02) or group 3 (p < 0.008). These data were confirmed by Northern analysis with a relaxin cRNA probe. The placental tissues after PPROM also had a significantly higher and a uniform overexpression of relaxin in the placental syncytiotrophoblast. Tissues collected at term, in comparison, showed no such increases in decidua or placenta.

Control of peripartal collagenolysis in the human chorion-decidua.

OBJECTIVE: This study was designed to observe the in vivo status of chorion-decidua gene expression for some major metalloproteinases, an inhibitor (tissue inhibitor of metalloproteinase-1), and an activator (tissue plasminogen activator) and the hormone relaxin at accessible time points in the peripartal period. STUDY DESIGN: Chorion-decidua from patients at cesarean section with and without labor and after spontaneous labor and delivery was used for preparation of poly (A)+ ribonucleic acid and quantitative Northern analyses with a series of oligo and complementary deoxyribonucleic acid probes. RESULTS: In the period designated as the period before parturition, relaxin and matrix metalloproteinase-1 (interstitial collagenase) gene expression were relatively high, reflecting the controlled loss of amniotic collagen necessary for fetal membrane expansion without rupture as the uterine volume increases. When active labor has begun, but before delivery, matrix metalloproteinase-3 (stromelysin) and matrix metalloproteinase-9 (type V collagenase) gene levels significantly increased. After normal spontaneous labor and delivery, tissue plasminogen activator and tissue inhibitor of metalloproteinase-1 messenger ribonucleic acids significantly increased, together with a marginally increased expression of the genes for matrix metalloproteinase-1, matrix metalloproteinase-2 (type IV collagenase), and relaxin. CONCLUSION: The expression of the genes for some major collagenolytic enzymes, an inhibitor, activator, and relaxin in the chorion-decidua, is different at the different stages of parturition.

Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium.

Relaxin (RLX) is a product of the human corpus luteum, pregnancy decidua and placenta, prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) are products of the cyclic endometrium and of the pregnancy decidua. All three proteins are thought to function interdependently in endometrium/decidua as local factors within the uterus without reaching the systemic circulation. In this study, the avidin-biotin immunoperoxidase method for immunolocalization with monoclonal or polyclonal antibodies has been applied to serial sections of endometria obtained from patients at different stages of the menstrual cycle and in early and late gestation. This allowed the cellular localization of the three proteins to be followed simultaneously through the reproductive stages from cyclic endometrium to term gestational decidua. The production, as opposed to sequestration of RLX from an ovarian source was demonstrated by the application in parallel of an antibody to the processed hormone and its connecting peptide. RLX was shown localized to the glandular and luminal epithelia in the proliferative and secretory phases. The decidualized stromal cells also immunostained for RLX in the late secretory phase and in early and late pregnancy. PRL was localized first to the glandular epithelium and then stroma, appearing after RLX, IGFBP-1 appeared later in the secretory phase and predominantly in the decidualized stromal cells confirming previous studies. In contrast, all three proteins were immunostained in early pregnancy and increased to term gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

The human fetal membranes: a target tissue for relaxin.

The purpose of this study was to adduce further evidence for a paracrine role for human decidual relaxin (Rlx) in the remodelling of collagen in the fetal membranes in the peripartal period. The binding of [125I]porcine Rlx to membrane-enriched fractions from fetal membranes as well as from dispersed cells from the fetal membranes was used to demonstrate the presence of specific Rlx receptors. Rlx added in vitro to cultured amnion/chorion cells increased the release of plasminogen activator and collagenase into the medium. Rlx had no effect on the release of beta-glucuronidase. An in vivo correlate of these in vitro results was obtained, the detection of plasminogen activator and collagenase in amniotic fluids. The active fraction of collagenase was increased in amniotic fluids collected after spontaneous rupture of the membranes. PRL, hCG, estrogen, and progesterone added in equimolar amounts to cultured amnion/chorion cells from elective cesarean sections and normal term deliveries also effected the release of plasminogen activator and collagenase. The greatest effects were found in cells from cesarean section tissue, in terms of the stimulation of plasminogen activator release by Rlx and PRL and of collagenase release by prostaglandin F2 alpha and, to a lesser extent, by Rlx, PRL, and hCG. We conclude that human fetal membranes are targets for a number of hormones, including the decidual paracrine hormones Rlx, PRL, and prostaglandin F2 alpha as well as estrogen, progesterone, and hCG. These hormones act to release or inhibit the enzymes involved in collagen breakdown before rupture of the fetal membranes.

Plasma relaxin levels during suckling and oxytocin stimulation in the lactating sow.

Plasma relaxin levels were measured in animals at different stages of lactation and related to the amount of nuzzling and suckling behavior exhibited by the piglets. Only in some acute suckling episodes was relaxin secreted rapidly and episodically in spite of normal piglet and sow behavior and interaction. However, when the piglets were removed from the dams 6 h before suckling, the sows were very restless and the relaxin response to suckling was delayed. Oxytocin injection in lactating but nonsuckled sows caused an episodic secretion of relaxin similar to suckling itself. The source of relaxin in the lactating sow may be the old corpus luteum, since progesterone levels increased acutely, somewhat reflecting the profile of relaxin increase over the suckling episode.