The expression of interleukin-15 and interleukin-18 by human term placenta is not affected by lipopolysaccharide

The aim of the study was to examine the stimulatory effect of the inflammatory agent lipopolysaccharide (LPS) on the capacity of human term placenta to secrete interleukin (IL)-15 and IL-18. Isolated placental cotyledons from normal human term deliveries were dually perfused for ten hours with perfusion medium alone (n=5) or with perfusion medium containing LPS (1 microg/kg perfused placental tissue) (n=5). Placental tissue was collected from three different placental compartments (amnion, chorion, and placenta) before and after perfusion. The placental tissues collected were homogenized and examined for IL-15 and IL-18 by ELISA. In addition, formalin-fixed and paraffin-embedded sections from term placentas before perfusion were stained by immunohistochemistry to characterize the cellular origin of placental IL-15 and IL-18. Statistical significance was determined using paired/unpaired t-test. p<0.05 was considered significant. Our results show that IL-15 and IL-18 are produced more by chorionic tissue, as compared to the amnion and placental tissues. Moreover, we show that IL-15 and IL-18 are expressed by epithelial cells of the amnion, chorionic cells of the chorion and decidual cells of the decidua. However, IL-15, but not IL-18, was expressed also by syncytiotrophoblasts of the villi. Perfusion of LPS did not affect the capacity of amnion, chorion and placental tissues to secrete IL-15 and IL-18, as compared to control. The expression of IL-15 and IL-18 in the different compartments of the human placenta suggests a possible role for these two cytokines in normal placental development, pregnancy and labor. Moreover, our results indicate that IL-15 and IL-18 are not part of the mechanism of the response of human placenta to LPS.     

Mechanism of zinc uptake by microvilli isolated from human term placenta.

The mechanism of zinc (Zn) uptake by microvillous membrane vesicles prepared from human term placenta has been studied. The uptake was complex, with two processes being identified. In the first process, uptake was rapid, reaching equilibrium within 2 min, and was temperature dependent, with a Q10 of 1.5. Equilibrium Zn levels were sensitive to osmotic pressure, with Zn binding at infinite osmolarity being 69% iso-osmotic value. The uptake was saturable, with a Vmax of 58 +/- 2 nmol/mg protein/min and an apparent Kt of 128 +/- 13 microM. Uptake was inhibited by increasing extravesicular K+ concentration, dropping from 0.91 +/- 0.03 nmol/mg/min at 0 extravesicular K+ to 0.47 +/- 0.03 at an extravesicular K+ concentration of 150 mM ([Zn] = 1.0 microM). In the presence of both valinomycin, an electrogenic ionophore, and nigericin, an electroneutral exchanger, an outwardly directed K+ gradient stimulated Zn uptake. Similarly, preloading vesicles with Zn and imposing an inward gradient resulted in a temperature dependent efflux of Zn. The data suggest that there is a K+ dependent Zn transporter in vesicle membranes, and we suggest that the evidence is biased in favour of a Zn/K+ exchanger rather than Zn being dependent on the membrane potential.     

Release of cytotoxin by macrophages on treatment with human placenta lectin

Human lectin purified from placenta induced release of cytotoxin from a murine macrophage cell line and human peritoneal monocytes. This activity was not due to contamination of the lectin preparation with lipopolysaccharide.     

In vivo release of non-neuronal acetylcholine from human skin by dermal microdialysis: effects of sunlight, UV-A and tactile stimulus

Non-neuronal acetylcholine (ACh) is expressed in epithelial, endothelial and immune cells. For example, the in vivo release of ACh from the human skin pretreated with botulinum toxin has recently been demonstrated. In the present experiments the effects of light (sunlight and solar radiation by a commercial UV-A applier) and of a tactile stimulus on the release of non-neuronal ACh were investigated. Release of ACh from the proximal and distal shin, i.e. anterior tibial region, was measured by dermal microdialysis in 20 min samples over a time period of at least 140 min. Control experiments were performed in a dark room throughout. In some experiments volunteers were exposed to sunshine (80-140 min) or the shin region was illuminated (80-95 min) by a commercial UV-A lamp (400 W at a distance of 50 cm). In control experiments ACh release between 20 and 80 min (B1) amounted to 118+/-32 pmol (n=17) and gradually declined between 80 and 140 min (B2) to 112+/-34 pmol, resulting in a B2/B1 ratio of 0.95. When the skin was exposed to sunlight ACh release increased from 205+/-58 pmol (B1) to 349+/-122 pmol resulting in a B2/B1 ratio of 1.70. UV-A radiation, however, had no significant effect on the B2/B1 ratio. When very smooth tactile stimuli were applied by a cotton wool tip for 20 min to the skin close to the microdialysis membranes in a dark room, ACh release was increased from 9+/-2 pmol/20 min to 52+/-36 (n=7). In conclusion, the in vivo release of ACh from the human skin appears to be regulated by external stimuli like sunlight and tactile stimuli.     

Functional significance of non-neuronal acetylcholine in skin epithelia

In recent years, the physiological role of non-neuronal acetylcholine (ACh) and its receptors (AChR) in epidermal physiology has been under intense investigation. However, little is known about the role of the non-neuronal cholinergic system in inflammatory skin diseases. We chose the clinically nicotine-dependent skin disease hidradenitis suppurativa (HS) as model to study the influence of long term nicotine ingestion on epidermal morphology and AChR expression. HS is a chronic inflammatory, disabling disease of unknown pathogenesis emerging from the pilosebaceous unit of the intertriginous areas. In order to correlate our findings to specific nicotine effects, we used the organotypical coculture system (OTC) and raised artificial epidermis in the presence of nicotine. After 12 days in culture control OTC showed a mature epithelium, while nicotine treated OTCs were significantly thicker. Using immunofluorescence analysis, nicotine treated OTCs produced significantly stronger immunoreactivity (IR) for the alpha3, M(3) and M(5) AChR antisera than control. In contrast, the alpha7 nAChR antiserum showed a slightly reduced IR in the granular layer and the alpha9 nAChR IR retracted to the lower suprabasal layers. In HS epidermis we found the strongest IR for all AChR around the follicular infundibulum while in the sinus epithelia it was only weak. In contrast to the nicotine treated OTC, the alpha7 nAChR IR in the hyperplastic HS epidermis was clearly extended to all living layers. Altogether we provide first hints for a causative role of the non-neuronal cholinergic system in the pathogenesis of HS by promoting infundibular epithelial hyperplasia and thus follicular plugging.     

Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis

Angiogenesis is a critical physiological process for cell survival and development. Endothelial cells, necessary for the course of angiogenesis, express several non-neuronal nicotinic acetylcholine receptors (AChRs). The most important functional non-neuronal AChRs are homomeric α7 AChRs and several heteromeric AChRs formed by a combination of α3, α5, β2, and β4 subunits, including α3β4-containing AChRs. In endothelial cells, α7 AChR stimulation indirectly triggers the activation of the integrin αvβ3 receptor and an intracellular MAP kinase (ERK) pathway that mediates angiogenesis. Non-selective cholinergic agonists such as nicotine have been shown to induce angiogenesis, enhancing tumor progression. Moreover, α7 AChR selective antagonists such as α-bungarotoxin and methyllycaconitine as well as the non-specific antagonist mecamylamine have been shown to inhibit endothelial cell proliferation and ultimately blood vessel formation. Exploitation of such pharmacologic properties can lead to the discovery of new specific cholinergic antagonists as anti-cancer therapies. Conversely, the pro-angiogenic effect elicited by specific agonists can be used to treat diseases that respond to revascularization such as diabetic ischemia and atherosclerosis, as well as to accelerate wound healing. In this mini-review we discuss the pharmacological evidence supporting the importance of non-neuronal AChRs in angiogenesis. We also explore potential intracellular mechanisms by which α7 AChR activation mediates this vital cellular process.     

Structure and binding properties of collagen type XIV isolated from human placenta

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN- collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.     

Urea and amino acid transport by an isolated human placenta]

A method of bilateral perfusion of the isolated human placenta was used to study the urea transport from the fetal placental stream into the maternal one, and of amino acid transport in the opposite direction. Experiments demonstrated that the method provided a sufficiently full perfusion of the intervillous space and offered possibilities for studying the placental transport. With equal amino nitrogen concentration in both circulations, its content in the fetal stream increased during the experiment. This elevation was more expressed when amino acid was added to the maternal circulation. The idea of amino acid "secretion" by the trophoblast cell elements into the fetal circulation was confirmed by the above experiments.     

Release of cytochrome c from isolated mitochondria by etoposide

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.     

Annexin 6 modulates the maxi-chloride channel of the apical membrane of syncytiotrophoblast isolated from human placenta

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.     

Gangliosides from human placenta

Gangliosides of human placenta were studied, using biochemical methods and specific antibodies. The placenta was found to contain three types of gangliosides with oligosaccharide chains Lac, GgOse4 and nLcOse4.     

Release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from perifused human placenta

The release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from the normal human placenta and the effect of some stimulatory agents on their release were studied in vitro using a perfusion system. Each perfusate was assayed for hCG and hCG-alpha in its own homologous radioimmunoassay systems. Both hCG and hCG-alpha were released from the placenta at any stage of gestation in our perfusion system. Much more hCG than hCG-alpha was released from the placenta in early gestation. By comparison, however, hCG-alpha increased gradually with the gestational age. The amount of hCG-alpha released was almost equal to that of hCG in the placenta in the 17th gestational week. After the 22nd gestational week, hCG-alpha was released in larger quantities than hCG, and about 10 times more hCG-alpha than hCG was released from the term placenta. These results were also confirmed by gel filtration of perfusates on a Sephadex G-100 column. hCG-alpha, compared with hCG, was present in excess in gel filtrated perfusates in the last two trimesters. By adding 1 mM dibutyryl cyclic AMP to the perifusion medium, the release of both hCG and hCG-alpha was stimulated significantly. Synthetic luteinizing hormone releasing hormone (LH-RH) at concentrations of 10 ng/ml and 100 ng/ml had no effect, but at a high concentration (1 microgram/ml), LH-RH stimulated the release of them. Moreover, mouse epidermal growth factor (EGF) stimulated not only the release of hCG and hCG-alpha but also their production, because both hCG and hCG-alpha levels rose progressively with the time course in the presence of EGF. The present studies demonstrate that the perifusion system of chorionic tissues is a useful method for investigating the release of hCG and its subunits in vitro.