Amnion Cells In Culture Research Articles

Infection and labor: IV. Cachectin—tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor

A growing body of evidence supports a causal link between subclinical intrauterine infection and preterm labor. The mechanisms responsible for the onset of parturition in this setting have not been elucidated. The conventional view has been that bacterial products increase prostaglandin biosynthesis by intrauterine tissues and this, in turn, leads to the onset of labor. An alternative or complementary mechanism is that microbial products activate the host monocyte-macrophage system and that cytokines released during this process signal the initiation of parturition by stimulating prostaglandin biosynthesis by intrauterine tissues. This study was conducted to determine if cachectin-tumor necrosis factor is present in the amniotic fluid of women with intraamniotic infection and whether this cytokine can alter the rate of prostaglandin biosynthesis by intrauterine tissues. Amniotic fluid from 54 women was assayed for tumor necrosis factor. Tumor necrosis factor was not detectable in the amniotic fluid of women without intraamniotic infection regardless of the presence or absence of term or preterm labor. On the other hand, the amniotic fluid of 11 of 15 women with preterm labor and intraamniotic infection had measurable tumor necrosis factor. This cytokine stimulated prostaglandin E2 biosynthesis by amnion cells in monolayer culture in a dose-dependent fashion. These data support the concept that macrophage activation is involved in the onset of human parturition in the setting of infection. We propose that the host (fetus and/or mother) signals the onset of parturition through the secretion of inflammatory cytokines released in response to bacterial invasion.

Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.

This study was conducted as part of an investigation to evaluate the hypothesis that bacterial toxins (LPS or lipoteichoic acid), acting on macrophage-like uterine decidua to cause increased formation of cytokines, may be involved in the pathogenesis of infection-associated preterm labor. We found that cachectin/tumor necrosis factor-alpha (TNF-alpha) was synthesized and secreted into the culture medium by human decidual cells and explants in response to treatment with LPS. LPS treatment also caused an increase in PGF2 alpha production by decidual cells and explants. In amnion cells in monolayer culture, TNF-alpha stimulated PGE2 formation, and TNF-alpha was cytostatic (inhibited [3H]thymidine incorporation into DNA) but not cytolytic in amnion cells. TNF-alpha was not detectable (less than 0.34 ng/ml) in the amniotic fluid of normal pregnancies at midtrimester or at term before or after the onset of labor (n = 44); but TNF-alpha was present at concentrations between 2.8 and 22.3 ng/ml in amniotic fluids of 4 of 20 pregnancies with intact membranes complicated by preterm labor (less than 34 wk gestational age). LPS was present in 10 of the 20 amniotic fluids of preterm labor pregnancies, including all four in which TNF-alpha was present. Bacteria were identified in only one of the four LPS-positive, TNF-alpha-positive fluids. Cytokine formation in macrophage-like decidua may serve a fundamental role in the pathogenesis of preterm labor, including increased prostaglandin formation and premature rupture of the membranes.

Interlukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1α and IL-1α were incubated with amnion cells in culture, and prostaglandin E 2 (PGE 2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE 2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p > 0.05). Indomethacin blocked the effectof IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.

Mechanical stretching increases prostaglandin E2 in cultured human amnion cells.

Mechanical stretching increases synthesis and release of prostaglandin E2 (PGE2) in cultured amnion cells. The maximum level of PGE2 in stretched amnion cells is three times higher than that in nonstretched amnion cells. The maximum level of PGE2 in stretched cell medium is nine times higher than the maximal level of PGE2 in nonstretched cell medium. 3H-thymidine incorporation into DNA is 211 +/- 25 cpm/10(5) cells in nonstretched groups; that in stretched groups is 582 +/- 94 cpm/10(5) cells. There is a significant difference between the two groups. These results suggest that stretch stimulation facilitates prostaglandin production in fetal membranes, which may contribute to uterine contraction in labor.

Glucocorticoids stimulate prostaglandin synthesis in human amnion cells by a receptor-mediated mechanism.

Prostaglandin E2 (PGE2) synthesis by human amnion increases with the onset of labor and is thought to participate in the initiation and maintenance of parturition. Since cortisol levels increase in amniotic fluid in late pregnancy, we studied the effects of glucocorticoids on cultured term human amnion cell PGE2 output. In 24-h studies, the synthetic glucocorticoid dexamethasone stimulated basal PGE2 output 2-fold over control levels at 16-500 nmol/L. PGE2 output was dramatically stimulated (greater than 10-fold) when, after dexamethasone pretreatment, the cells were incubated with calcium ionophore A23187 or arachidonic acid (AA) for 2 h. Maximum effects were achieved at 31 nmol/L dexamethasone. Basal PGE2 output was stimulated at 12 h of dexamethasone treatment, whereas A23187- or AA-stimulated PGE2 output was enhanced after 3-6 h of dexamethasone pretreatment. Cortisol (50 and 500 nmol/L) also enhanced basal and stimulated PGE2 output, while dehydroepiandrosterone sulfate, 17 beta-estradiol, and progesterone were ineffective. The glucocorticoid receptor antagonist RU 38486 attenuated dexamethasone-enhanced basal and stimulated PGE2 output. Dexamethasone pretreatment had no effect on basal or stimulated PGE2 output from cultured term chorion cells, suggesting tissue specificity. We conclude that glucocorticoids specifically enhance PGE2 output from cultured amnion cells via a receptor-mediated mechanism. We speculate that the action of glucocorticoids is to increase the capacity of the cells to convert AA to PGE2.

Effects of fetal urinary corticosteroids, catecholamines and kallikrein on PGE 2 synthesis in monolayer cultures of human amnion and chorion cells

We have studied the effects of several compounds isolated from fetal urine on the production of PGE 2 by amnion and chorion cells which were maintained in primary monolayer culture. We conclude that desoxycorticosterone and vanillylmandelic acid stimulate PGE 2 synthesis by amnion, but adrenaline and kallikrein increase the biosynthesis of PGE 2 by chorion. These data suggest that human fetal urine could play a major role in events of parturition.

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase.

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.

Response of human amnion cells in culture to 1,25-dihydroxycholecalciferol: Increased 25-hydroxycholecalciferol 24-hydroxylase activity and prostaglandin Esub 2 formation

Response of human amnion cells in culture to 1,25-dihydroxycholecalciferol: Increased 25-hydroxycholecalciferol 24-hydroxylase activity and prostaglandin Esub 2 formation

A product from human decidua inhibits prostaglandin production by human amnion

Human decidua was obtained from nonlaboring women after elective cesarean section. Decidua was incubated with media alone for 20 hours and this media (decidual conditioned media) was then incubated with amnion cells in monolayer culture and amnion rings. A 90% decrease in PGE2 production by amnion cells in monolayer culture was demonstrated in the presence of decidual conditioned media when compared to controls. In short term incubations with fresh amnion, decidual conditioned media decreased the production of PGE2 in amnion by greater than 25% of the control rate of production in 17 of 21 experiments. These observations suggest that human decidua produces a factor capable of inhibiting prostaglandin production by amnion.

Epidermal growth factor-stimulated prostaglandin E 2 production in human amnion cells: specificity and nonesterified arachidonic acid dependency

The production of prostaglandin E 2 (PGE 2) by human amnion cells in primary monolayer culture was stimulated 2- to 150-fold by mouse epidermal growth factor (mEGF). The effect of mEGF on amnion cell PGE 2 production was dependent on the time of treatment and the concentration of mEGF used. The rate of PGE 2 production by these cells was maximal between 2 and 4 h of treatment with mEGF; thereafter, the rate of production of PGE 2 declined. The stimulation of PGE 2 production was maximal at concentrations of mEGF of greater than 5 ng/ml. A number of other growth factors, steroid and protein hormones, and various other test agents were ineffective or only minimally effective in stimulating PGE 2 production by amnion cells. The stimulatory effect of mEGF on PGE 2 production in these cells was dependent on the presence of serum in the culture medium; alternatively, mEGF was effective in stimulating PGE 2 production by amnion cells in serum-free medium that was supplemented with arachidonic acid bound to albumin. Thus, we conclude that the marked stimulation of PGE 2 production by amnion cells treated with mEGF is not due to an action of mEGF to stimulate the release of arachidonic acid from cellular glycerophospholipid storage forms; rather, these data are supportive of the conclusion that mEGF-stimulated PGE 2 production is dependent on the presence of nonesterified i.e., free, arachidonic acid in the medium.

Preterm labor: Stimulation of arachidonic acid metabolism in human amnion cells by bacterial products

There is a strong association between preterm labor and infection. Some potentially pathogenic bacteria have phospholipase activity, and it has been suggested that release of phospholipase from these organisms may increase prostaglandin E2 synthesis in amnion cells and hence initiate preterm labor. In this study we established monolayer amnion cell cultures from tissue collected at elective cesarean section at term before labor. Cells were prelabeled with tritiated arachidonic acid and then further incubated after addition of 2%, 5%, or 10% (vol/vol) filtered medium in which either group B β-hemolytic streptococcus, Streptococcus viridans, Escherichia coli, Bacteroides fragilis, or Lactobacillus had been growing. Tritiated arachidonic acid and its metabolites released by the amnion cells in these or control incubates were extracted from culture medium and separated by high-performance liquid chromatography. Addition of conditioned medium from each of the organisms with the exception of Lactobacillus caused an increase in overall arachidonic acid metabolism. There was an increase in the ratio of cyclooxygenase to lipoxgenase metabolism and in prostaglandin E2 production in particular when compared to controls. The profile of arachidonic acid metabolism in amnion cells following addition of filtered bacterial medium resembled that obtained from amnion cells cultured following spontaneous labor. We suggest that abnormal bacterial colonization of the genital tract may lead to an increase in arachidonic acid metabolism in amnion cells with an increase in prostaglandin E2 production and the consequent initiation of preterm labor.

Response of human amnion cells in culture to 1,25-dihydroxycholecalciferol: Increased 25-hydroxycholecalciferol 24-hydroxylase activity and prostaglandin E2 formation

We investigated the possibility that 1,25-dihydroxycholecalciferol acts in human amnion as a physiologic calcium ionophore to effect increased prostaglandin E2 production in this tissue. This possibility was based on the propositions that (1) this metabolite of vitamin D3 acts in other target tissues, that is, bone, intestine, chorioallantoic membrane, kidney, and intestine, to effect increased calcium absorption, (2) that 1,25-dihydroxycholecalciferol is present in amniotic fluid, which bathes the amnion, and (3) that the mobilization of calcium in amnion is associated with stimulation of enzymes, namely, phospholipase A2 and phosphatidylinositol-specific phospholipase C that act ultimately to effect the release of arachidonic acid, which in turn leads to increased prostaglandin E2 production. We found that human amnion cells, maintained in primary monolayer culture, are responsive to 1,25-dihydroxycholecalciferol. Treatment of these cells with 1,25-dihydroxycholecalciferol for 18 hours brought about the induction, in a dose-dependent manner, of 25-hydroxycholecalciferol 24-hydroxylase, a marker of 1,25-dihydroxycholecalciferol action. In addition, treatment of human amnion cells in monolayer culture with 1,25-dihydroxycholecalciferol caused, within 12 hours, a significant increase in the synthesis of prostaglandin E2, which was maintained for the duration of treatment, that is, 48 hours. These findings may be indicative of a significant physiologic role for 1,25-dihydroxycholecalciferol in metabolic processes that are important in amnion, including transport, amniotic fluid volume homeostasis, and the initiation of parturition.

The effect of fetal urine on arachidonic acid metabolism in human amnion cells in monolayer culture

Human amnion cells in primary monolayer culture were used as a model system to evaluate the regulation of arachidonic acid metabolism and prostaglandin E2 production in amnion. Amnion cells were incubated with carbon 14—labeled arachidonic acid, and after various times the distribution of radiolabeled arachidonic acid in the lipids of these cells were determined. After incubation for 72 hours, 91% of the total radiolabeled arachidonic acid incorporated into cellular lipids was present in glycerophospholipids, and 9% was present in neutral lipids. The formation of [14C]prostaglandin E2 from [14C]arachidonic acid was maximal after 8 hours. In these studies the effect of human fetal urine on arachidonic acid metabolism in these cells was investicated (the production of prostaglandin E2 by amnion cells is increased by treatment with fetal urine). In cells incubated with [14C]arachidonic acid in the incubation medium, treatment with fetal urine for 4 hours caused a threefold to fourfold increase in [14C]prostaglandin E2 production, yet there were no detectable differences in the content of [14C]arachidonic acid in specific glycerophospholipids or neutral lipids of the cells after such treatment. In other studies, amnion cells were preincubated for 72 hours with [14C]arachidonic acid, and thereafter the cells were treated with fetal urine for 24 hours. With fetal urine treatment the amount of [14C]arachidonic acid in triacylglycerols decreased significantly compared with that in nontreated cells, but the formation of [14C]prostaglandin E2 was not increased. Thus we suggest that in response to fetal urine, prostaglandin E2 is formed from arachidonic acid that is released from a highly specific, stimulus-sensitive lipid pool or else from arachidonic acid that is derived from extracellular sources.

Incidence and significance of chromosomal translocations in prenatal diagnosis

In the study reported here the incidence and significance of translocations in antenatal diagnosis are described with reference to data of the cytogenic laboratory of Cologne University Gynecological Clinic. During the last 10 years 19 translocations were found in 4532 amnion cell cultures (0.44%), without exception in single pregnancies. In 18 cases the chromosome aberrations were balanced, i.e., the location of the genetic material was changed without loss or addition of parts of chromosomes. In one case the diagnosis of an unbalanced translocation in the form of a translocation mongolism led to termination of pregnancy. All of the other pregnancies were continued to term. Phenotypically and clinically the children were normal. The translocations were inherited in 78.9% (n = 15; 11 from the mother, 4 from the father), and de novo in 21.1% (n = 4). The results are in agreement with those of the DFG study, which identified 0.66% translocations in over 10,000 antenatal diagnoses. Of the balanced chromosome aberrations all of the newborns but one were clinically and phenotypically healthy. In cases of inherited translocations in particular, no increase in risk to the child could be determined. In cases of de novo chromosome aberrations there is a slight increase in the residual risk due to loss of minute portions of the gene. With careful technique this risk may be assumed to be very slight, so that in such cases also there is no indication for termination of pregnancy.

EFFECT OF BACTERIAL PRODUCTS ON PROSTAGLANDIN E PRODUCTION BY AMNION CELLS

An in-vitro model was set up to study a possible causal relation between bacterial colonisation of extraplacental membranes and spontaneous preterm labour. When amnion cells in tissue culture were exposed to bacterial products, their prostaglandin E output rose considerably. The degree of response varied with the organism. These findings support the theory that bacterial products may be responsible for the premature onset of labour.

Despite a massive increase in cortisol secretion in women during parturition, there is an equally massive increase in prostaglandin synthesis. A paradox?

In this investigation, we sought to resolve the apparent paradox that is posed by the fact that there is a simultaneous increase in the production of prostaglandin and cortisol in women during labor. A paradox obtains, since in most tissues, cortisol acts to inhibit prostaglandin formation. Using previously characterized model systems for the in vitro study of arachidonic acid metabolism in amnion, decidua, and myometrium, we found that prostaglandin production by amnion and endometrial stromal cells in monolayer culture was not decreased by glucocorticosteroid treatment. On the other hand, prostaglandin production by myometrial smooth muscle cells in culture was inhibited by greater than 90% in response to dexamethasone (10(-7) M) treatment. Importantly, the major prostaglandin produced by myometrium, as well as myometrial smooth muscle cells in culture, is prostacyclin, a prostaglandin that acts to cause uterine quiescence. We suggest that the immunity of amnion and decidua to the action of glucocorticosteroids may allow for the accelerated production of prostaglandins E2 and F2 alpha, which act to cause myometrial contractions; simultaneously, glucocorticosteroid produced in large quantities in women in labor may lead to decreased production of prostacyclin by myometrium, thereby reducing uterine quiescence. In this coordinated manner, the uterine contractions that culminate in delivery of the fetus may proceed uninterrupted in the face of increased cortisol production.

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E 2 is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E 2 by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E 2 than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E 2; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E 2 biosynthesis in this tissue.

Teichoic acids of Streptococcus agalactiae: chemistry, cytotoxicity, and effect on bacterial adherence to human cells in tissue culture.

The ratio of teichoic acid to lipoteichoic acid (LTA) in a strain of Streptococcus agalactiae type III was found to be 8:1, with the total amount of LTA being 0.1% of the dry weight of the organism. Purified teichoic acid contained D-alanine and possibly a small amount of D-glucose and was approximately 22 glycerol phosphate units in length. The linkage between each of these units was 1-3. In addition, LTA contained a complex lipid, more glucose, and an unusually high content of a short-chain fatty acid, tridecanoic acid. This LTA was cytotoxic for a variety of human cell monolayers in tissue culture, including one derived from the human central nervous system. Established human cells were more sensitive than primary cell monolayers to this LTA, with as little as 12.5 micrograms of LTA per ml being cytotoxic for HeLa cells. Teichoic acid (250 micrograms/ml) was nontoxic under identical conditions. These cytotoxicity results suggest an LTA involvement in group B streptococcal pathogenesis. Also, the first model system for the study of group B streptococcal adherence to primary human embryonic amnion cells in tissue culture is detailed. This system was used to quantitate pronounced differences in tissue tropism between S. agalactiae and Streptococcus pyogenes and showed enhanced binding by this group A coccus over that of S. agalactiae for amnion cell monolayers. The adherence of both streptococcal species to only a portion (40%) of these amnion cells suggested that host cell receptor expression may vary for primary cells in vitro. Finally, this strain of S. agalactiae was shown to adhere to amnion cells by a non-LTA-mediated mechanism. The possibility of an LTA-mediated versus a protein-mediated adherence mechanism for host cells that is related to the virulence of S. agalactiae is discussed.

Stimulation of prostaglandin E 2 production in amnion cells in culture by a substance(s) in human fetal and adult urine

A protein or protein-associated substance(s) in human fetal urine effects a 10- to 600-fold increase in prostaglandin E 2 (PGE 2) synthesis by human amnion cells maintained in monolayer culture; the increase in PGE 2 synthesis is maximal by 12 h. Fetal urine did not cause a similar increase in PGE 2 synthesis in cells derived from other human tissues tested. A similar PGE 2 synthesis-stimulatory activity is present in adult urine and the activity in adult urine is correlated inversely (p < 0.001) with creatinine concentration. These findings are suggestive that a substance produced in, or excreted by, kidney acts to induce PGE 2 synthesis in a tissue-specific manner.

Typing of herpes simplex virus with type-specific human immunoglobulin M in an indirect immunofluorescence assay.

Sera from nine individuals with suspected primary herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) infection were screened to identify those containing HSV type-specific immunoglobulin M (IgM). Selected sera were then utilized in an IgM-specific indirect immunofluorescent-antibody HSV-typing assay (patent pending). To evaluate the procedure, 29 HSV isolates were grown in cultures of continuous human amnion cells, fixed, and used as substrates for indirect immunofluorescence. Determination of virus type was based on intensity of fluorescence of the substrate with HSV-1- and HSV-2-specific antisera after staining with fluorescein-conjugated anti-human IgM. Typing of the isolates by restriction endonuclease digestion showed that of 29, 18 were HSV-2 and 11 were HSV-1. Results by IgM-specific indirect immunofluorescent-antibody assay were identical to those by restriction endonuclease digestion for 27 of the isolates; 2 isolates failed to replicate adequately in the test cells. The IgM-specific indirect immunofluorescent-antibody procedure appears to be a simple, rapid, and accurate technique which could be of use to clinical virology laboratories.