Recent studies have shown that the hormonal form of vitamin D, 1,25-dihydroxyvitamin D₃ (calcitriol), can affect both tissues and cells that are not directly involved in calcium homeostasis. In particular, a role for calcitriol as a regulator of immune cell differentiation and proliferation has been proposed. Specific high-affinity intracellular receptors for calcitriol (VDR) are detectable in activated T cells, and activated macrophages are able to synthesize calcitriol. A possible paracrine mechanism of action has been postulated. Vitamin D may therefore have a similar role to that of other immune regulatory molecules such as cytokines. The precise interaction of calcitriol with the cytokine network is not yet fully defined, but its ability to modulate immune cells in vitro and its association with inflammatory diseases are now well documented. These findings are outlined in this review with particular reference to effects on macrophages and lymphocytes.

During pregnancy, immune activity is tightly regulated so that antimicrobial protection of the mother and fetus is balanced with the need for immune tolerance to prevent fetal rejection. In this setting, the maternal–fetal interface, in the form of the uterine decidua, provides a heterogeneous immune cell population with the potential to mediate diverse activities throughout pregnancy. Recent studies have suggested that vitamin D may be a key regulator of immune function during pregnancy, with the fetal–maternal interface representing a prominent target. Among its non-classical actions are potent immunomodulatory effects, including induction of antibacterial responses and modulation of T-lymphocytes to suppress inflammation and promote tolerogenesis. Thus, vitamin D may play a pivotal role in normal decidual immune function by promoting innate responses to infection, while simultaneously preventing an over-elaboration of inflammatory adaptive immunity. Research to date has focused upon the potential role of vitamin D in preventing infectious diseases such as tuberculosis, as well as possibly suppressing of autoimmune disease. Nevertheless, vitamin D may also influence facets of immune function not immediately associated with primary innate responses. This review summarises our current understanding of decidual immune function with respect to the vitamin D metabolism and signalling, and as to how this may be affected by variations in maternal vitamin D status. There has recently been much interest in vitamin D supplementation of pregnant women, but our knowledge of how this may influence the function of decidua remains limited. Further insight into the immunomodulatory actions of vitamin D during pregnancy will help shed light upon this.

Two isozymes of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) are responsible for the interconversion of the active glucocorticoid, cortisol in man, (corticosterone in the rodent), to the inactive 11-keto metabolite, cortisone (11-dehydrocorticosterone). We have examined the regulation of type 1 11 beta-HSD (11 beta-HSD1) using primary cultures of rat and human hepatocytes, both of which express only 11 beta-HSD1. Only 11 oxo-reductase activity could be demonstrated in cultured hepatocytes (apparent Km for cortisone 382 +/- 43 nM in human hepatocytes, apparent Km for 11-dehydrocorticosterone 14.6 +/- 1.5 microM in rat hepatocytes). There exists a marked discrepancy between 11 beta-HSD oxo-reductase activity and 11 beta-HSD1 mRNA levels in cultured human hepatocytes and human liver. Thus oxo-reductase specific activity is much higher in the cultured hepatocytes (7.2 +/- 0.01 nmoles cortisol/mg/h vs 0.89 +/- 0.06 for whole liver homogenates) whilst the converse is true for steady state 11 beta-HSD1 mRNA levels (0.78 +/- 0.02 vs 1.94 +/- 0.07 in whole liver, 11 beta-HSD1/18S expressed as arbitrary units). Carbenoxolone has a significant inhibitory effect on 11 oxo-reductase activity in both rat and human hepatocytes. However, there is clear species-specific regulation of 11 oxo-reductase activity by thyroid hormone (tri-iodothyronine (T3)), which increases 11 oxo-reductase activity in rat hepatocytes but has no effect on activity in human hepatocytes, and progesterone which inhibits activity in human hepatocytes, but has no effect on activity in rat hepatocytes. Neither T3 nor progesterone altered 11 beta-HSD1 mRNA levels. A series of growth factors (hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor beta 1) were without effect on 11 oxo-reductase activity in cultured rat hepatocytes. In contrast to homogenates of human liver, cultured hepatocytes express only 11 beta-HSD oxo-reductase activity. This is inhibited by carbenoxolone and shows species-specific regulation by T3 and progesterone. Growth factors do not appear to regulate activity or expression of 11 beta-HSD1. The discrepant enzyme activity data and 11 beta-HSD1 mRNA expression in hepatocytes and whole liver could reflect unstable 11 beta-HSD1 oxo-reductase activity or, alternatively, an additional 11 beta-HSD oxo-reductase isoform in cultured hepatocytes.

ABSTRACT

Incubation of bovine parathyroid cells with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) decreased both preproparathyroid mRNA levels and parathyroid hormone (PTH) secretion. There was a fall to 56·6 ± 13·7% (mean ± s.e.m.) and 65·1 ± 9·3% in mRNA levels and PTH secretion respectively at 1 nmol 1,25-(OH)₂D₃/l, and 41·1 ± 13·6% and 42·0 ± 12·1% at 10 nmol 1,25-(OH)₂D₃/l after 24 h. After 48 h in 0·1 nmol 1,25-(OH)₂D₃/l, mRNA levels had fallen to 35·3 ± 12·6% and PTH secretion to 32·1 ± 5·0%. In human adenomatous cells, however, incubation with 1,25-(OH)₂D₃ (10 nmol/l) had no effect on either mRNA levels or PTH secretion even after 48 h. This lack of sensitivity of adenomatous cells to 1,25-(OH)₂D₃ was not due to an absence of receptors (3847 ± 39 receptors/ng cytosolic protein in adenomatous cells compared with 4068 ± 371 in bovine cells) or receptors being of low affinity.

Cortisol (1 μmol/l) caused a reduction in the number of receptors for 1,25-(OH)₂D₃ in bovine parathyroid cells of approximately 20% within 24 h of incubation, but no change in affinity. This decrease was accompanied by abolition of the response to 1,25-(OH)₂D₃ and was reversible, in that withdrawal of cortisol for the final 24 h of incubation was sufficient for the response to return, the number of receptors having returned to control values.

These results suggest that only a small percentage of receptors for 1,25-(OH)₂D₃ in bovine parathyroid cells may be functional at any one time. Furthermore, the insensitivity of human adenomatous cells to 1,25-(OH)₂D₃ does not seem to be due to a lack of receptors but may be due to a defect in the interaction between the receptor protein and the PTH gene.

Journal of Endocrinology (1989) 123, 137–142

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

ABSTRACT

Receptors for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5′-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning − 700 to + 50 bp with 200 μg cytosolic protein gave 288±63% (mean ± s.d.) of binding in the absence of protein. In contrast, there was no significant reaction with the −1350 to − 700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the − 700 to + 50 bp fragment. The receptor-binding site was further defined to −700 to − 100 bp as deletion of the − 100 to + 50 bp did not reduce receptor binding.

Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50 000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the −700 to + 50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)₂D₃ (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay.

Autoradiography following incubation of immobilized receptors with the −700 to +50 bp, −485 to −50 bp, −700 to −100 bp and −700 to −485 bp fragments revealed a binding site for the receptor for 1,25-(OH)₂D₃ between −485 and −100 bp upstream of the initiation site in the bovine PTH gene.

Journal of Endocrinology (1990) 126, 355–359

Glucocorticoids play a fundamental role in the endocrinology of pregnancy but excess glucocorticoids in utero may lead to abnormalities of fetal growth. Protection against fetal exposure to cortisol is provided by the enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) located in the human placental trophoblast. By contrast, relatively little is known concerning the function of glucocorticoid-activating 11β-HSD1, which is strongly expressed within human maternal decidua. To address this we have assessed: i) changes in decidual 11β-HSD1 expression across gestation and ii) the functional role of glucocorticoids in decidua. Human decidua was collected from women undergoing surgical termination of pregnancy in first (n = 32) and second (n = 10) trimesters, and elective caesarean sections in the third trimester (n = 9). Analysis of mRNA for 11β-HSD1 by real-time RT-PCR showed increased expression in second (9.3-fold, P < 0.01) and third (210-fold, P < 0.001) trimesters. Studies using primary cultures of decidual cells also revealed higher levels of cortisol generation in the third trimester. Changes in decidual 11β-HSD1 with gestation were paralleled by increased expression of the apoptosis markers caspase-3 and annexin-V, particularly in cluster designation (CD)10^−VE non-stromal cells (20-fold in third trimester relative to first trimester). Apoptosis was also readily induced in primary cultures of third trimester decidual cells when treated with cortisol, cortisone, or dexamethasone (all 100 nM for 24 h). The effect of cortisone but not cortisol or dexamethasone was blocked by an 11β-HSD inhibitor confirming the functional significance of endogenous cortisol generation. These data show that autocrine metabolism of glucocorticoids is an important facet of the feto-placental unit in late gestation and we propose that a possible effect of this is to stimulate programmed cell death in human decidua.