Molecular Human Reproduction, Vol. 6, No. 6, 555-560,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
ß-endorphin inhibits the production of interleukin-8 by human chorio–decidual cells in culture
Perinatal and Maternal Studies Group, Division of Biomedical Sciences, School of Health Sciences, University of Wolverhampton, Wolverhampton, UK
Abstract
Interleukin-8 (IL-8) is produced by human decidual cells in culture, and may play a role in the initiation of parturition. ß-endorphin is released in significant amounts into the maternal and fetal circulation during labour. The effect of ß-endorphin on IL-8 production by human chorio–decidual cells in culture was investigated. Mixed cells were obtained from the decidual surfaces of 35 term placentas. The cells were plated out at 10x106 cells per well in Roswell Park Memorial Institute 1640 culture medium. After 48 h the cells were washed and incubated with either plain culture medium (control), 1 µmol/l progesterone, 1–100 nmol/l ß-endorphin or 1 nmol/l N-acetyl ß-endorphin. After 48 h, IL-8 concentrations were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Experiments were repeated in the presence of naloxone (1 µmol/l) and using calcium-deficient culture medium. Progesterone (P < 0.0002) and ß-endorphin (P < 0.0005) significantly inhibited the production of IL-8. The inhibitory effect of ß-endorphin was blocked by naloxone and by using calcium-deficient medium. N-acetyl ß-endorphin had no significant effect on IL-8 production. These findings suggest that ß-endorphin has an inhibitory effect on IL-8 production by decidual cells, and that the effect is mediated via opioid receptors and is calcium-dependent.
ß-endorphin/decidua/interleukin-8/parturition
Introduction
The mechanisms involved in the initiation and control of progress of human parturition have not been fully elucidated. Pregnancy has previously been thought to be an immunosuppressive state (Medawar, 1953
), however it has recently been proposed that the maternal adaptive immune system may be suppressed, but the innate system is stimulated (Sacks et al., 1999). The involvement of inflammatory mechanisms, such as the production of prostaglandins and neutrophil infiltration in the uterus and cervix, in the initiation of parturition is well established. Interleukin-8 (IL-8) is a pro-inflammatory cytokine, which is involved in the initiation of parturition (Kelly et al., 1992, 1994; Kelly, 1996). It is a neutrophil attractant, causing infiltration into tissues and degranulation. It has been shown that human decidual cells obtained at term produce IL-8 in culture (Kelly et al., 1992, 1994; Dudley et al., 1996). Progesterone has been found to significantly inhibit the production of IL-8 by these cells, suggesting that progesterone may be partly responsible for local immunosuppression and maintaining pregnancy. A decrease in the local concentration of progesterone or the blockade of its effects (Karalis et al., 1996) could be involved in the initiation of parturition.
The control of neutrophil infiltration in the inflammatory process in the initiation of labour is not clearly understood. There is evidence that IL-8 is released from the amnion, chorio–decidua and the placenta at term (Osmers et al., 1995a; Denison et al., 1998; Elliott et al., 1998; Laham et al., 1999). Specific mRNA for IL-8 has been detected in decidua, chorion and amnion at term (Dudley et al., 1996). IL-8 is also involved in cervical ripening, via infiltration of neutrophils and release of proteinases (Osmers et al., 1995b; Sennstrom et al., 1997; Winkler et al., 1999). IL-8 production may also have a role in preterm labour caused by intrauterine infection (Trautman et al., 1992; Keelan et al., 1997; Hsu et al., 1998).
The decidual layer represents the interface between the fetal and maternal tissues, and local production of inflammatory mediators in the decidua could be very important in the initiation of parturition. It has been demonstrated that the human decidua contains stromal cells and also cells of the immune system, such as large granular lymphocytes, macrophages, T-lymphocytes, fibroblasts, and polymorphonuclear leukocytes, particularly at term (Bulmer et al., 1988; Casey and McDonald, 1988; Vince et al., 1990; Khan et al., 1991). It has been estimated that 47% of the term decidual tissue may be bone-marrow derived cells (Vince et al., 1990).
ß-endorphin is a peptide hormone released from the anterior pituitary gland in response to stress. Stressful or painful stimuli cause the release of corticotrophin-releasing hormone (CRH) from the hypothalamus, which stimulates the release of ß-endorphin, adrenocorticotrophic hormone (ACTH) and 
-melanocyte stimulating hormone (-MSH) from the anterior pituitary gland. ß-endorphin is cleaved enzymatically from a larger precursor molecule, pro-opiomelanocortin (POMC), which also gives rise to ACTH and -MSH. ß-endorphin is released in relatively large amounts into both the maternal and fetal circulation during labour, in response to stress and pain (Goland et al., 1981; Thomas et al., 1982; Bacigalupo et al., 1987; Chan and Smith, 1992). Mean peak plasma concentrations of 113 pg/ml (32.6 pmol/l) (Goland et al., 1981), 153 pg/ml (44.2 pmol/l) (Bacigalupo, 1987) and 50 fmol/ml (50 pmol/l) (Chan and Smith, 1992) of ß-endorphin have been reported in maternal blood during delivery. During pregnancy the placenta also produces ß-endorphin (Chan and Smith, 1992) and POMC (Cooper et al., 1996; Raffin-Sanson et al., 1999). Mean total ß-endorphin concentrations of 201 fmol/g wet weight of term placenta have been reported (Chan and Smith, 1992). CRH and POMC-derived peptides have also been identified in human myometrium (Clifton et al., 1998). There is some debate as to whether the placenta produces ß-endorphin or the N-acetylated form of the hormone, N-acetyl ß-endorphin. The placenta is able to N-acetylate compounds via N-acetyltransferase enzymes (Derewlany and Koren, 1994). Chan and Smith (1992) have reported that up to 60% of the ß-endorphin in placental extracts was the N-acetylated form. The N-acetylated form is non-bioactive as it cannot interact with opioid receptors. POMC mRNA has been identified in leukocytes (Blalock, 1989; Lyons and Blalock, 1997) and these cells have been shown to produce endorphins (Smith et al., 1987).
ß-endorphin is an agonist at predominantly µ and 
opioid receptors (Barnea et al., 1991). Binding of opioid agonists to µ and receptors on the surface of target cells causes a decrease in the intracellular concentration of free Ca2+ ions. Classically the role of ß-endorphin in labour is thought to be purely one of providing endogenous analgaesia (Raisanen et al., 1984). However, ß-endorphin is known to be immunosuppressive (Pedersen et al., 1997), and may have other physiological roles during labour. ß-endorphin has been found to inhibit human chorionic gonadotrophin (HCG) secretion by placental explants in culture in early pregnancy (Barnea et al., 1991) and to inhibit the uptake of Ca2+ ions and the release of prostaglandins in isolated strips of rat uterus (Faletti et al., 1992).
The aims of this study were to investigate the effect of ß-endorphin and N-acetyl ß-endorphin on the production of IL-8 by human chorio–decidual cells in culture and to investigate whether any effects were mediated via opioid receptors and were dependent on extracellular Ca2+.
Materials and methods
Reagents
All reagents and culture media were obtained from Sigma (Poole, UK), unless otherwise indicated. A stock solution of progesterone was made up in ethanol and then diluted with complete medium (the final concentration of ethanol was 0.016%). The control medium for the progesterone treatment contained 0.016% ethanol to match the ethanol content of the progesterone solution, otherwise the controls were complete medium. ß-endorphin (human, synthetic, non-acetylated), N-acetyl ß-endorphin and naloxone were made up in sterile saline before being diluted with complete medium.
Experimental procedure
Placentae were obtained from 35 elective Caesarean sections at term, with ethical approval of the South Birmingham Area Health Authority Ethical Committee. The pregnancies and deliveries were singletons and were free of complications. The placentas were washed to remove excess blood and transported at room temperature before use. A method for decidual cell culture was adapted from Kelly et al. (1992). Decidual tissue was dissected from the maternal surface of the placenta. The tissue was macerated into small pieces (~1 mm3) and then digested in RPMI 1640 medium containing trypsin (0.5%) and DNAase (20 mg/l) at 37°C for 40 min with constant mechanical stirring. The digested mixture was filtered through nylon mesh (500 µm pore size) and 10% fetal calf serum (FCS) was added to inactivate the trypsin. The filtrate was centrifuged at 500 g for 10 min. The pellet of mixed cells was washed twice and resuspended in complete medium, Roswell Park Memorial Institute (RPMI) 1640 containing 10% FCS, penicillin (50 µg/ml), streptomycin (50 µg/ml) and gentamycin (5 µg/ml). Following a cell count, the cell suspension was diluted with medium to produce a cell concentration of 107 cells per ml. The mixed cells were plated out at 107 cells per well in 24-well plates and incubated in an atmosphere of 5% CO2 in air at 37°C. The viability of the cells harvested was >95%, using the Trypan Blue exclusion test. The cells were incubated for 48 h prior to treatment with each agent, to allow the cells to recover from the trypsin digestion. After the first 48 h incubation the cells were washed twice in complete medium.
In the first series of experiments, the cells were resuspended in either control complete medium (containing 0.016% ethanol) or complete medium containing progesterone (1 µmol/l). The cells in five wells on each plate were incubated for a further 48 h with each agent for each of the placentas used for this series of experiments. The medium was then removed from each well, centrifuged at 500 g for 10 minutes and the supernatant stored at –70° C until assayed. IL-8 concentrations in the supernatants were measured using a specific human IL-8 ELISA kit (Quantikine; R & D Systems, Abingdon, UK), with a detection limit of 10 pg/ml of IL-8. The cell culture supernatants were diluted by a factor of 10 before assay and the IL-8 production was expressed as ng/ml/106 cells per 48 h culture.
Using the same method, a dose–response study for ß-endorphin was conducted. The effect of concentrations of 1, 10 and 100 nmol/l ß-endorphin on IL-8 release were compared. The effect of N-acetyl ß-endorphin (1 nmol/l) on IL-8 release was investigated. Experiments were repeated with the inclusion of naloxone (1 µmol/l), an opioid receptor antagonist, to investigate whether any effects observed were opioid receptor mediated. All experiments were repeated using calcium-deficient medium (Life Technologies, Paisley, UK) to investigate the requirement of extracellular Ca2+ for any observed effects. The concentrations of Ca2+ in the media were not measured, but there was a concentration difference in Ca2+ of 0.423 mmol/l between the complete medium and the Ca2+-deficient medium.
Statistical analysis
The measured concentrations of IL-8 were analysed using two-way analysis of variance. Statistical significance was taken as P < 0.05.
Results
The coefficient of variation for intra-assay reproducibility for the IL-8 assay method was 6.8% at an IL-8 concentration of 0.18 ng/ml. The mixed chorio–decidual cells produced IL-8 in culture (pooled mean concentration 13.7 ng/ml/106 cells, range 0.18–70.0). There was a high degree of variability in production between cells in separate wells and between placentas. Two-way analysis of variance consistently showed significant (P < 0.0002) variation in production between the cells obtained from separate placentas. Basal IL-8 production (pooled mean concentration 13.7 ng/ml/106 cells) was significantly (P < 0.001) lower when the cells were cultured in Ca2+-deficient medium (pooled mean concentration 0.25 ng/ml/106 cells). IL-8 concentrations in progesterone (1 µmol/l) treated wells were significantly (P < 0.0002) lower than those in the control wells, suggesting that progesterone significantly inhibited the release of IL-8 by these cells in culture (Figure 1). IL-8 concentrations in the ß-endorphin (1, 10 and 100 nmol/l) treated wells were significantly (P < 0.0005) lower than those in the control wells, suggesting that ß-endorphin significantly inhibited the release of IL-8 by these cells in culture (Figure 2). Naloxone (1 µmol/l) appeared to block the inhibitory effect of ß-endorphin on IL-8 release, although the effect of ß-endorphin was not significant in this experiment (Figure 3). In Ca2+ deficient medium the inhibitory effect of ß-endorphin was not observed. Under these conditions, ß-endorphin at 1 nmol/l (P < 0.05) and 10 nmol/l (P < 0.001), caused a significant increase in IL-8 release (Figure 4a), which was blocked by naloxone (Figure 4b). N-acetyl ß-endorphin (1 nmol/l) had no significant effect on IL-8 production [control pooled mean concentration 3.6 ng/ml/106 cells (SEM = 1.1, n = 19) versus pooled mean concentration 2.6 ng/ml/106 cells (SEM = 0.7, n = 21) in the presence of N-acetyl ß-endorphin].
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Discussion
A preliminary report of these findings has been published previously (Nandhra et al., 1995). The basal concentrations of IL-8 produced by the control chorio–decidual cells were similar to those reported by Kelly (control mean 11.0 ng/ml) (Kelly et al., 1992, 1994). Variation in the production of IL-8 between cells has previously been reported with this cell culture technique. The bone marrow-derived cells of the term decidual tissue are the likely source of IL-8. Various types of cell can produce IL-8, including macrophages, monocytes, neutrophils and fibroblasts. The local release of IL-8 in the decidua would attract neutrophils into the tissue and cause them to degranulate, releasing lytic enzymes. This would initiate an inflammatory reaction, involving the production of prostaglandins. Increased local production of prostaglandin (PG) E2 and PGF2 in the decidua, leading to contraction of the myometrium, is known to be an early step in the initiation of human parturition (Gibb, 1998). A local inflammatory reaction in the decidua could facilitate separation of the placenta from the endometrium during the third stage of labour. The production and release of IL-8 is known to be Ca2+-dependent (Kuhns et al., 1998) and culturing the cells in Ca2+-deficient medium significantly inhibited the release of IL-8 from the cells.
Progesterone significantly inhibited the production of IL-8, confirming the previous findings of Kelly et al. (1992). Progesterone is known to be immunosuppressive, and it is possible that relatively high local concentrations during pregnancy inhibit the maternal immune response to the placenta. A decrease in the local concentration of progesterone or blockade of its activity (Karalis et al., 1996) would increase IL-8 production, which could be an important step in the initiation of parturition.
A novel finding of this study was that ß-endorphin significantly inhibited the production of IL-8 by human chorio–decidual cells in culture. ß-endorphin inhibited the production of IL-8 to a similar extent at a much lower concentration than progesterone, therefore ß-endorphin is more potent in this respect. ß-endorphin is produced by the placenta during pregnancy (Chan and Smith, 1992) and might be involved in maintaining local immunosuppression. As ß-endorphin has been shown to be produced by leukocytes (Smith et al., 1987), this suggests that autocrine or paracrine control may be active in the leukocyte population of the decidua. The importance of interactions between the neuroendocrine system and the immune system for reproductive physiology has been previously emphasised (Blalock and Costa, 1989).
High circulating concentrations of ß-endorphin as a result of maternal stress could delay the onset of labour at term. Delay in labour and tocolysis in response to stress has been observed in other mammals (Naaktgeboren, 1979). Once active labour has been initiated, ß-endorphin could be involved in the control of progression of labour. It is released from the anterior pituitary gland in response to stress and pain, so the more stressful and painful the labour the more will be released. Increased blood concentrations could have the effect of slowing down the progress of labour. It is well known that maternal plasma concentrations of ß-endorphin are correlated with perceived pain scores during labour (Raisanen et al., 1984). However, maternal stress is also associated with preterm labour ( Lockwood, 1995). It appears that in preterm labour the roles of the stress hormones CRH (Hobel et al., 1999; Lockwood, 1999) and cortisol (Mazor et al., 1994) are more important than that of ß-endorphin. It is possible that the effects of ß-endorphin are different at term compared with preterm or that the local decidual concentrations of ß-endorphin are higher at term.
Naloxone blocked the inhibitory effect of ß-endorphin, suggesting that it was mediated via opioid receptors. Cells of the immune system have been shown to express µ receptors (Chuang et al., 1995a), 
receptors (Carr et al., 1989; Chuang et al., 1995b) and opioid receptors (Carr et al., 1989; Gaveriaux et al., 1995). As naloxone is a non-specific opioid antagonist, it was not possible to determine which opioid receptor was responsible, but ß-endorphin is more potent at µ and receptors (Barnea et al., 1991). When extracellular Ca2+ was reduced, the inhibitory effect of ß-endorphin was not observed, suggesting that the mechanism was dependent on extracellular Ca2+ and involved the inhibition of Ca2+ influx. The significant increase in IL-8 release caused by ß-endorphin in Ca2+-deficient medium was an unexpected finding. It was blocked by naloxone, suggesting mediation via opioid receptors, stimulation of which normally decreases intracellular Ca2+. There may have been some stimulation of intracellular Ca2+ release in response to a reduced Ca2+ gradient across the cell membrane. Many cellular processes are Ca2+-dependent, so culturing the cells in Ca2+-deficient medium may have affected their normal physiology and altered their responses to stimuli.
The lack of effect of N-acetyl ß-endorphin on IL-8 release in normal medium suggests that it is not biologically active at opioid receptors and agrees with previous findings. It is possible that N-acetyl ß-endorphin could exert biological effects via pathways other than those involving opioid receptors. Placental microsomes are able to deacetylate compounds (Derewlany and Koren, 1994), so N-acetyl ß-endorphin could be converted to ß-endorphin in vivo in the placenta.
It has been shown previously that the enzymatic digestion of placental tissue and separation of cells technique used in cell culture can itself stimulate the production of cytokines (Lonsdale et al., 1996). In this study the cultured isolated cells were always used as their own controls, in order to compensate for this effect.
In conclusion, these findings suggest that ß-endorphin inhibits IL-8 production by human chorio–decidual cells, and that this effect is mediated via opioid receptors and is calcium dependent.
Acknowledgments
We would like to thank Reena Sidhu for preparing the figures and for statistical analysis, Rodney Kelly for his help in establishing the cell culture method for human chorio–decidual cells and Harry Gee for arranging the collection of tissue.
Notes
1 To whom correspondence should be addressed at: School of Health Sciences, University of Wolverhampton, 62–68 Lichfield Street, Wolverhampton, WV1 1DJ, UK. E-mail: R.J.Carson{at}wlv.ac.uk 
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Submitted on December 7, 1999; accepted on March 10, 2000.
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