Elevated levels of TNF-alpha and IL-6 may contribute to the putative endothelial dysfunction of preeclampsia.

The authors explore the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and possibly other inflammatory cytokines are overproduced by the placenta in response to local ischemia/hypoxia contributing to increased plasma levels, and subsequent endothelial activation and dysfunction in the pregnancy disorder, preeclampsia. It is widely held that inadequate trophoblast invasion and physiologic remodeling of spiral arteries initiate placental ischemia/hypoxia in preeclampsia. Furthermore, focal areas of placental hypoxia have been implicated in the production of "toxic" factor(s) by the placenta, which circulate and cause maternal disease. Placental trophoblast cells and fetoplacental macrophages normally produce TNF-alpha and interleukin-1 (IL-1), which are capable of producing endothelial cell activation and dysfunction. Hypoxia has recently been reported to increase TNF-alpha and IL-1 production by term villous explants from the human placenta. Placental cells also express erythropoietin (EPO), which is the prototype molecule for transcriptional regulation by hypoxia in mammals. Interestingly, TNF-alpha and IL-1 have DNA sequence homologous or nearly homologous to the hypoxia-responsive enhancer element of the EPO gene, thus providing a potential, but as of yet, untested molecular link between placental hypoxia and stimulation of cytokine production. Inflammatory cytokines overproduced by the placenta in response to hypoxia may then lead to increased plasma levels and endothelial activation and dysfunction in preeclampsia. The purpose of this short review is to critically evaluate the hypothesis that placental cytokines contribute to the pathogenesis of preeclampsia. Of note, the etiology of the disease presumably related to deficient trophoblast invasion is beyond the scope of this work.

It has been hypothesized that inadequate placentation in the hypertensive disorder of pregnancy known as preeclampsia creates foci of placental ischemia/hypoxia leading to the elaboration of factors that compromise systemic endothelial function to produce disease sequelae. As tumor necrosis factor-alpha (TNF alpha) and interleukin-1 (IL-1) are inflammatory cytokines capable of eliciting endothelial cell dysfunction, we investigated whether the production of these inflammatory cytokines by cultured villous explants from the human placenta was affected by incubation in reduced oxygen (2% O2). The term placenta produced TNF alpha, IL-6, and low levels of IL-1alpha and IL-1beta under standard tissue culture conditions. Hypoxia significantly increased TNF alpha, IL-1alpha, and IL-1beta production by 2-, 6-, and 23-fold, respectively, but did not affect IL-6 production. Further, cytokines were immunolocalized to the syncytiotrophoblast layer as well as to some villous core cells. Hypoxic regulation of placental TNF alpha and IL-1beta production also appeared to differ based on gestational age. Finally, treatment with either cobalt chloride or an iron chelator mimicked the hypoxic response, suggesting that stimulation of placental cytokine production may involve a heme-containing, O2-sensing protein. These results suggest that placental hypoxia can lead to the elaboration of inflammatory cytokines, which may contribute to the pathophysiology of preeclampsia.

Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived cytokine that is also reportedly produced by granulosal cells and is localized in luteal cells. The present study employed serum-free culture of midcycle bovine luteal cells to investigate the effects of TNF-alpha, alone and with other cytokines, on luteal function. TNF-alpha (1-1000 ng/ml) produced a dose-dependent increase in prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha synthesis on all days of culture, but had no effect on basal progesterone (P4) production. TNF-alpha, in combination with other known stimulators of luteal PG synthesis, interleukin-1 beta (2.5 ng/ml) or interferon-gamma (IFN-gamma, 100 U/ml), had synergistic effects on PGF2 alpha production (greater than 50-fold above control, P less than 0.05) whereas interferon-alpha (1000 U/ml) significantly suppressed TNF-alpha-stimulated PGF2 alpha production. By day 7 of culture, TNF-alpha inhibited LH-stimulated P4 production (P less than 0.05). Luteal cell numbers were significantly reduced by IFN-gamma but not by TNF-alpha alone. However, the combination of TNF-alpha + IFN-gamma was extremely cytotoxic (only 20% of cells maintained as compared to control). Finally, TNF-alpha (100 ng/ml) enhanced the expression of Class I major histocompatibility complex antigens on cultured bovine luteal cells but did not alter IFN-gamma induction of Class II major histocompatibility complex antigens. In light of these findings, it appears that TNF-alpha, in conjunction with other cytokines, is a modulator of luteal cell function in vitro. The stimulation of PG synthesis, as well as cytotoxic effects of TNF-alpha, may suggest a role in luteolysis.

Circulating levels of maternal erythropoietin (EPO) rise during gestation due to increased biosynthesis of the hormone. Our objective was to investigate the human placenta as a potential extrarenal site of EPO production. Using two monoclonal antibodies recognizing different antigenic determinants, we identified immunoreactive EPO associated with villous cytotrophoblast, endovascular and intravascular cytotrophoblast, cytotrophoblast cell columns, and syncytiotrophoblast of first and second-trimester placenta as well as syncytiotrophoblast and extravillous cytotrophoblast of normal third-trimester and preeclamptic placenta. In addition, cultured JAR (trophoblast-derived) choriocarcinoma cells, cytotrophoblasts isolated from term placenta, villous core cells, and possibly other nontrophoblast cells within the decidual basal plate expressed immunoreactive EPO. Using reverse transcription-polymerase chain reaction and EPO-specific primers, a 378 bp DNA product was amplified from placental tissues of various gestational ages, cytotrophoblasts isolated from term placenta, and JAR choriocarcinoma cells. The amplified product yielded restriction enzyme fragments of predicted sizes. On Southern analysis, hybridization was observed for two of these fragments in which the radiolabeled EPO cDNA probe did not overlap with the primer sequences. Finally, the JAR choriocarcinoma cells elaborated EPO into the culture medium as determined by enzyme-linked immunosorbant assay and expressed EPO mRNA as determined by Northern analysis, both of which were stimulated by hypoxia (15-20 torr). Taken together, these results suggest a new site of EPO expression: the trophoblast cell of the human placenta.

It is postulated that inadequate remodeling of the uterine spiral arteries in preeclampsia leads to focal ischemia and generation of inflammatory cytokines, such as tumor necrosis factor (TNF alpha) and interleukins (ILs), by the placenta. Our objective was to compare TNF alpha, IL-1 alpha, IL-1 beta, and IL-6 levels in placentas from patients with preeclampsia and normal term pregnancies. Because the placenta is a large heterogeneous organ, we analyzed multiple sites per placenta. On the average, there was a 3-fold variation in cytokine protein levels across the eight sites analyzed for each placenta. However, there were no significant overall differences among the normal term, preeclamptic, and preterm placentas from women without preeclampsia. There were also no significant differences in TNF alpha messenger ribonucleic acid between the normal term and preeclamptic placentas, although TNF alpha messenger ribonucleic acid levels were lower in placentas from preterm patients without diagnosis of preeclampsia than in the normal term placentas. In vitro, hypoxia stimulated the production of TNF alpha, IL-1 alpha and IL-1 beta, but not that of IL-6, by placental villous explants from both groups of patients, and this was not exaggerated in preeclampsia. Finally, although peripheral and uterine venous levels of TNF alpha were elevated in preeclamptic women compared with normal term patients, the ratio of uterine to peripheral venous TNF alpha levels was not significantly different from 1.0 for either patient group. Taken together, these results suggest that sources other than the placenta contribute to the elevated concentrations of TNF alpha and IL-6 found in the circulation of preeclamptic women.

In nonfertile cycles, the absolute steroidogenic capacity of the primate corpus luteum, as reflected in the expression of messenger RNA (mRNA) for the progesterone biosynthetic enzymes cytochrome P450 cholesterol side-chain cleavage (P450SCC) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), progressively declines until luteal regression. Despite this progressive loss in luteal cell function, the elaboration of CG by the implanted blastocyst is able to prolong the functional lifespan of the corpus luteum. It was the purpose of this study to investigate the relationship between aging of the primate corpus luteum and the cellular mechanisms by which the decline in luteal cell function is arrested by CG. Corpora lutea were obtained from cynomolgus monkeys on days 11 or 16 of the luteal phase after a 7-day treatment period with increasing doses of human CG (hCG) given intramuscularly beginning on days 5 or 10. Corpora lutea were also obtained from control animals on days 5, 10, 11, and 16 of the luteal phase. Human CG treatment significantly (P < 0.05) elevated both serum progesterone and estradiol levels throughout the treatment period; however, progesterone production in animals treated with hCG late in the luteal phase (days 10-16) steadily declined after the third treatment day. Expression of mRNA for P450SCC and 3 beta-HSD was markedly stimulated (P < 0.05) by hCG treatment early in the luteal phase. However, 3 beta-HSD message levels in corpora lutea from animals treated with hCG on days 10-16 were not different from those of day-16 control corpora lutea, whereas P450SCC mRNA was only minimally stimulated. There was a dramatic (P < 0.05) increase in mRNA levels for the aromatase enzyme and low density lipoprotein receptor in animals given hCG in both the early and the late luteal phase. In conclusion, there appears to be a differential responsiveness of the primate corpus luteum to hCG stimulation dependent upon luteal age. The loss in responsiveness to hCG in terms of maintenance of mRNA levels is reflective of the inability of the late luteal phase corpus luteum for continued progesterone biosynthesis in the face of heightened luteotropic stimulation.

It is well established that LH has an obligatory role in the acute production of progesterone by the primate corpus luteum in vivo because interruption of LH support to the corpus luteum at any time during the luteal phase is accompanied by an immediate and sustained fall in serum progesterone concentrations. However, recent studies have demonstrated that maximal steroidogenic capacity of cultured human luteal cells and maximal levels of messenger RNAs (mRNAs) for cholesterol side chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase, delta 5-4 isomerase (3 beta-HSD) in luteal tissue are observed shortly after luteinization and decline thereafter throughout the remainder of the luteal phase. These findings would suggest that the role of LH in the acute regulation of progesterone production may differ from its role in the expression of mRNAs for steroidogenic enzymes. We initiated the current studies to define the role of LH upon the expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum. For this purpose, we treated cynomolgus monkeys with a potent GnRH antagonist for 1, 2, and 3 days during the luteal phase of the menstrual cycle and measured levels of mRNAs for P450scc and 3 beta-HSD in corpora lutea. Treatment of monkeys with the GnRH antagonist reduced bioactive LH concentrations to less than 5 ng/ml by 48 h of treatment, and LH concentrations remained less than 5 ng/ml thereafter. Serum progesterone concentrations were reduced by 74% after 1 day of antagonist treatment, 88% after 2 days of antagonist treatment, and by more than 95% after 3 days of GnRH antagonist treatment. Although progesterone secretion was markedly diminished after 24 h of antagonist treatment, there were no differences in mRNAs for P450scc and 3 beta-HSD between antagonist-treated and control animals. However, mRNAs for P450scc and 3 beta-HSD were significantly (P < 0.05) reduced after 2 days of antagonist treatment and were nearly nondetectable after 3 days of antagonist treatment. These results demonstrate a temporal dissociation of the effects of LH on the acute regulation of progesterone secretion and the maintenance of specific mRNAs involved in progesterone production. Nonetheless, the results clearly show that LH is required for the continued expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum.