Leukocytes modulate 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in human granulosa-lutein cell cultures.

It is well established that there are interactions between the immune and reproductive systems. The ovary contains indigenous macrophages, as well as other classes of leukocytes in smaller numbers. Cytokines secreted by these cells have been shown to have the ability to regulate ovarian steroidogenesis. In the present study, the effect of leukocytes on 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in human granulosa-lutein cells was examined. In addition, individual cytokines were also tested for their ability to regulate this enzyme. The follicular aspirates of patients undergoing IVF treatment were used as a source of granulosa cells. Cells isolated from these aspirates were found to contain between 15 and 60% leukocytes as assessed by flow cytometry (FACS). Leukocytes were removed from the sample preparations by the use of immunomagnetic beads coated with CD45 antibody, which recognises a surface antigen on all classes of leukocyte. Removal of leukocytes significantly decreased the 11beta-HSD activity in the granulosa cells, assayed after 3 days of culture, from 7.3 (2-20) to 3.5 (1-10) pmol cortisone formed/50000 cells/4 h (medians and ranges, n = 15). Addition of IL-5 and IL-6 significantly increased the 11beta-HSD activity in granulosa cell cultures both in the presence and absence of leukocytes. Addition of IL-4 and IFN-gamma increased 11beta-HSD activity only in the leukocyte-depleted granulosa cell cultures, whereas IL-2 had no effect on either of the cultures. The data suggests that leukocytes interact with the ovarian cells through cytokine secretion and/or cell-cell contact to increase the 11beta-HSD activity in human granulosa cells.

Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells.

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.

Effect of oestradiol-17 beta on the expression of oestrogen receptor mRNA in human tonsillar cells.

We have investigated the expression of oestrogen receptor (ER) mRNA in Ficoll-separated tonsillar cells and the changes that occur with the addition of oestradiol (OE2) both in the presence and the absence of the T cell mitogen phytohaemagglutinin (PHA). The amounts of ER mRNA and beta-actin mRNA in the samples were determined by slot blotting and hybridization and quantified by densitometry. The levels of ER mRNA were normalized against the beta-actin mRNA content. In the presence of OE2 (7 x 10(-8) M) after a 10-h culture there was a significant decrease (P < 0.05) to about 66% of the control (0-h culture) ER mRNA levels. Stimulating the cultures with PHA (1 microgram/ml), without the presence of OE2, had no effect on the expression of ER mRNA. However, when OE2 was present in a 10-h culture of PHA-stimulated cells, the ER mRNA level was significantly decreased (P < 0.05) to about 60% of control levels. In 24-h cultures, the presence of OE2 and/or PHA had no effect. When separated T cell preparations from the tonsils were used, no significant effects of OE2 were seen in either the 10-h or 24-h cultures. In conclusion, OE2 downregulates the ER mRNA content in a tonsillar mononuclear cell system in vitro as it does in many primary oestrogen target cells.

Effect of 17ß-oestradiol on expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar cells.

Oestradiol (E(2)) alters lymphocyte functionin vitro including T cell DNA synthesis and B cell immunoglobulin production in human tonsillar, splenic and peripheral blood cells. We have investigated whether one mechanism for this effect is that E(2) modifies the expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar mononuclear cells. Without E(2), addition of PHA (1 µg ml(-1)) for 10 h increased the expression of IL-2 and IL-6 mRNA but had no significant effect on IFN-γ mRNA. In separated T cells after 24 h incubation, E(2) (7×10(-8) M: ) increased only the IFN-γ mRNA levels. However, when E(2) was present in PHA-stimulated T cell cultures, mRNA levels from all cytokines were suppressed. E(2) decreased IL-2 mRNA levels in the T cell preparation after 24 h culture. For IL-6, E(2) decreased mRNA both in mononuclear cells and T cells after 10 h incubation. For IFN-γ, E(2) decreased mRNA levels in the mononuclear cell preparation after 24 h culture. Stimulation of the T cell preparation with PHA after 24 h incubation with E(2) decreased the IFN-γ mRNA levels compared to the cultures incubated with E(2) only. One part of the action of E(2) may be through a block in the up-regulation of the mRNA of IL-2, IL-6 and IFN-γ in activated cells.

Effects of 17 beta-oestradiol on function of lymphocytes from normal donors and patients with common variable immunodeficiency (CVID).

Effects of oestradiol (E2) have been studied on the in vitro T cell-dependent differentiation of B cells from peripheral blood and spleen using normal donors and patients with the antibody deficiency disease CVID. We also studied whether it modifies T cell DNA synthesis. The effect of E2 was examined on cultures of B cells with T cells for IL-2-driven immunoglobulin secretion or of T cells for phytohaemagglutinin (PHA)-driven DNA synthesis. Interestingly, in control experiments without E2, the normal sex difference in immunoglobulin production is reversed in CVID. The data show that for normal individuals there is no major difference between male and female donors in the in vitro actions of E2 on blood B and T lymphocytes. With normal blood B cells, E2 failed to affect IgM production, but it did inhibit IgG. In normal splenic cells, E2 increased both IgM and IgG secretion in a similar way to the tonsillar cell data previously reported. E2 on normal blood T cell DNA synthesis was stimulatory. With blood cells from CVID patients an interesting contrast was seen. As with normal B cells, E2 had no effect on IgM secretion by those CVID blood B cells able to secrete IgM. However, a difference between patients and normals was that E2 did not inhibit the IL-2-driven IgG production by those CVID B cells able to secrete IgG. For T cell function, the stimulatory effect of E2 on CVID T cell DNA was as in normal T cells. However, E2 failed to restore CVID B and T cell function to normal levels. These data suggest that there may be subtle defects in the pathway of action of E2 in CVID lymphocytes.

Effect of 17 beta-estradiol on immunoglobulin secretion by human tonsillar lymphocytes in vitro.

Sex steroid hormones play a role in the complex network of immune responses but the mechanism of their action is still unclear. Effects of a wide range of doses of 17 beta-estradiol (E2: 0.2-100 ng/ml) on human tonsillar lymphocyte cultures were examined. B and T lymphocyte enriched preparations were stimulated with various concentrations of interleukin-2 and the production of immunoglobulin was measured. Addition of E2 increased B cell immunoglobulin production in a T cell dependent way with intact T cells being obligatory. The effects of E2 were also examined on DNA synthesis by tonsillar T cells. E2 alone caused a significant increase in T cell DNA synthesis. With phytohaemagglutinin-stimulated T cell cultures there was a significant increase in DNA synthesis with E2 at pharmacological doses. Different cell surface and activation markers (including CD25, p75, HLA-DR, CD28) on tonsillar lymphocytes were also studied after exposure to E2. The presence of E2 made no significant difference in the expression of the markers either alone or when the activation antigens were induced by other stimuli. We have shown that intact T cells are needed for the action of E2 on tonsillar B lymphocyte differentiation and have excluded several mechanisms of action of E2 since common activation antigens are unaffected.