Biomedical subjects
Publications and source records attributed to E L Hooghe-Peters.
An alternative splice acceptor site in intron 1 of the human GHF-1/PIT-1 gene was sequenced. The use of this splice site is responsible for a 78-bp in-frame insertion upstream from exon 2 and leads to the hGHF-2/PIT-2 cDNA detected in normal human pituitary.
It is at present well established that prolactin exerts a non-specific immunoactivating function. In this work we tested whether the immunosuppressant drugs cyclosporin A, FK506 and rapamycin influence prolactin release from rat pituitary cells in primary culture. The tested drugs had no effect on the prolactin release measured during a 2h incubation period, indicating that they do not influence the secretion of prolactin from intracellular stores into the culture medium. During longer incubation times (48h), however, prolactin release was diminished to 56% +/- 18 (10 microM cyclosporin A), 64% +/- 14 (1 microM rapamycin) or 64% +/- 7 (1 microM FK506), suggesting an effect on prolactin production. At these drug concentrations no toxic effects were observed. The data indicate that inhibition of pituitary prolactin synthesis might contribute to the immunosuppressant action of cyclosporin A, rapamycin and FK506.
Cells expressing IL-6 mRNA were detected by in situ hybridization in normal pituitaries. In normal untreated rat pituitary the expression was very low. Within hours after IP administration of liposaccharide, IL-6 mRNA accumulated in the anterior lobe of the pituitary. Production of IL-6 was monitored after dissociation and culture of pituitary cells. High levels (8000 pg/ml) were recovered after 72 hr in culture. In normal human pituitaries, less than 1% of cells expressed IL-6 mRNA or IL-6 receptor mRNA (IL-6-R mRNA). In gonadotropinomas, prolactinomas, and non-functioning adenomas, only rare, scattered positive cells were found for either IL-6 or IL-6-R. In contrast, both genes were highly expressed in ACTH- and GH-secreting tumors at the junction of adenoma and infiltrating fibrous tissue and around blood vessels. The combined expression of IL-6 and IL-6-R suggests that IL-6 acts in an autocrine or in a paracrine way in ACTH and GH adenomas.
The effects of oestradiol on prolactin gene expression were studied by quantitative in situ hybridization histochemistry in different prolactin pituitary cell (sub)populations, which had been obtained by separation on a discontinuous Percoll gradient. When cells were incubated in vitro in the presence of oestradiol (10(-8) M) for a period of 4, 24, 48 and 72 h, there was an increase in the amount of prolactin mRNA, from 24 h on, only in high-density prolactin cells and lactotrophs of the total cell suspension. In contrast, the amount of prolactin mRNA in lactotrophs of low density did not change upon treatment with oestradiol. Pharmacological treatment with 50 micrograms oestradiol/day (s.c.) of random cycling female rats in vivo for 14 days increased the total number of prolactin gene-expressing cells and more lactotrophs were recovered at high density after Percoll gradient centrifugation. These results suggest a preferential stimulatory effect of oestradiol on prolactin gene transcription on a subpopulation of lactotrophs. Changes observed in prolactin cell layers after oestradiol treatment in vivo may represent a preferential effect in situ on a particular mammotroph cell subpopulation.
Time- and dose-dependent effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin (PRL) and growth hormone (GH) release were examined in static and dynamic rat pituitary cell incubations and on different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Quantitative in situ hybridization histochemistry allowed us to examine in parallel the effects of PACAP on PRL and GH gene expression. PACAP did not alter GH or PRL secretion in a dynamic superfusion system, in any cell population tested. Static incubations (30 min, 2-36 h) with PACAP 38 resulted in a significant increase in GH release and stimulated GH synthesis, as measured by the cytoplasmic accumulation of GH mRNA in the somatotrophs. These effects on synthesis and release were also observed after the enrichment of GH cells on Percoll gradients. PRL release was not altered by longer periods of incubation. Although no significant changes were observed in PRL secretion after 38 h, accumulation of cytoplasmic PRL mRNA was significantly stimulated in total pituitary cell suspension. After fractioning lactotrophs on Percoll gradients, the stimulatory effect of PACAP on PRL synthesis was lost. These results suggest that PACAP stimulates GH release and synthesis, and that it may act as a physiological regulator of this cell type. The PRL cell is not the most likely target cell type for PACAP. Effects observed on PRL synthesis in the total cell population may involve paracrine action of other hormone- or non-hormone-secreting cell types.
BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.
The expression of the Pit-1/GHF-1 transcription factor (hereafter Pit-1), which controls the expression of growth hormone (GH) and prolactin (PRL) in the pituitary gland, has been documented in human and rat hemopoietic and lymphoid tissues and cell lines. Pit-1 mRNA was detected by in situ hybridization in about 1% of rat bone marrow cells and in the spleen red pulp and marginal zone. Pit-1 was also expressed in human tonsils (mantle zone), in the thymus (rat and human, non-lymphoid cells), in lipopolysaccharide-stimulated rat peritoneal cells and in non-hepatocyte cells in the liver (rat and human). A detailed investigation of the rat spleen showed a very similar distribution for Pit-1, GH and PRL mRNA and Pit-1, GH and PRL proteins (detected by immunocytochemistry). Using polymerase chain reaction followed by Southern hybridization, the expression of Pit-1 could be confirmed in human and rat spleen, bone marrow and thymus. HL60 and RAJI leukemic cells were also positive. The sequence of fragments amplified from rat spleen and from human bone marrow completely matched published sequences of rat and human pituitary Pit-1, respectively. Expression of GH and PRL in lymphoid tissues has been documented. The straightforward hypothesis would therefore be that Pit-1's main function in lymphoid tissues is controlling GH and PRL expression, as in the pituitary gland. GH and PRL may be hemopoietic and lymphoid growth and differentiation factors.
The pituitary secretory proteins growth hormone (GH) and prolactin (PRL) do not, as yet, have major roles in haematology or immunology. Recent evidence indicates that these hormones are haemopoietic growth factors and exert immunomodulatory functions at physiological concentrations. Here Robert Hooghe and colleagues discuss the significance of these hormones on different aspects of the immune system.
The hGH/hCS genes, clustered on chromosome 17 in the 5' to 3' order GH-N, CS-L, CS-A, GH-V and CS-B, show a high degree of sequence identity. The expression product of the GH-V gene is the placental growth hormone, which replaces pituitary GH in maternal blood throughout pregnancy. By means of mRNA competitive hybridization using 32P-labelled and unlabelled 30 bases long oligonucleotides, we first optimized specific hybridization conditions. In situ hybridization was then performed to locate the GH-V mRNA encoding placental growth hormone. The hGH-V gene appears expressed in the placental syncytiotrophoblast. Unlike the CS-A and CS-B genes (both encoding hPL) which are expressed uniformly in the syncytiotrophoblast, the GH-V mRNA is located in a few syncytiotrophoblast cells only.
The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).
Adenomas can develop from each cell type of the anterior pituitary. In the normal pituitary, three of these cells types, the GH-, prolactin- and TSH-secreting cells, express the transcription factor Pit-1/GHF-1 which is responsible for prolactin and GH (and probably TSH) cell commitment, differentiation, probably proliferation and gene expression. We have analysed the expression of Pit-1/GHF-1 in a panel of human pituitary adenomas. All GH-, prolactin- and TSH-expressing adenomas studied expressed the Pit-1/GHF-1 factor, as demonstrated by in-situ hybridization and immunocytochemistry. The expression was higher in adenomas than in normal human pituitary. In contrast, ACTH- and LH-FSH-secreting and non-secreting adenomas were negative. Seven transplants of the spontaneous rat prolactinoma SMtTW were also investigated and all were found to be positive. This further stresses the analogy between these tumours and human prolactinomas. Taken together, the data confirm that Pit-1/GHF-1 expression is restricted to GH-, prolactin- and TSH-expressing cells, and the increased expression in adenomas is compatible with a role of Pit-1/GHF-1 in cell proliferation.
The expression of interleukin (IL)-1 beta, IL-6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL-1 beta, IL-6 and IL-6 receptor (IL-6R). The expression of IL-1R was more widespread. Within hours after injury, an increased expression of IL-1 beta, and thereafter of IL-6 was documented. The expression of IL-1R and IL-6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL-6 (detected by immunocytochemistry) agreed with the ISH patterns for IL-6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL-1 and IL-6. Increases in IL-1 and later in IL-6 levels were detected. The rapid and concomitant increased expression of IL-1 beta, IL-6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.
OBJECTIVE: We studied the effects of long-term in-vitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messenger ribonucleic acid (mRNA) levels. DESIGN: Human GH-secreting pituitary adenoma cells were cultured for periods from 4 days up to 3 weeks without or with octreotide (10 nM) and/or bromocriptine (10 nM). The effects of these drugs were measured on GH release, intracellular GH concentrations and intracellular GH mRNA levels. PATIENTS: Thirteen patients with GH-secreting pituitary adenomas were studied. Twelve patients were untreated, one had been pretreated with octreotide (12 weeks, 3 x 100 micrograms daily). MEASUREMENTS: GH, PRL, alpha-subunit and IGF-I concentrations in plasma, media and cell extracts were determined by immunoradiometric or radioimmuno-assays. GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. RESULTS: Incubation of the adenoma cells for 4 days with 10 nM octreotide induced a dose-dependent inhibition of GH release and a parallel increase (increase varying between 124 and 617% of control) in the intracellular GH levels was observed in six of seven adenomas. In addition, bromocriptine, when effective in inhibiting GH release by the adenomas, also induced an increase in intracellular GH levels. Even after 3 weeks of exposure to 10 nM octreotide in vitro there was a statistically significant increase in intracellular GH levels (between 191 and 923% of control). Withdrawal of octreotide after 6 days of incubation resulted in a lowering of intracellular GH levels to control values, showing that the octreotide-induced increase in intracellular GH is reversible. In a 96-hour incubation with 10 nM octreotide, GH mRNA levels were increased in two, and slightly decreased in one of the three adenomas tested. This effect was time dependent in that there was no significant effect of 10 nM octreotide on GH mRNA levels in a 24-hour incubation. CONCLUSIONS: (1) Long-term in-vitro exposure of GH-adenoma cells to octreotide causes an increase in intracellular GH levels in the majority of the adenomas, probably because of an increase in GH mRNA levels in the adenoma cells; and (2) this considerable increase in intracellular GH levels may be one of the explanations for the relatively poor effect of octreotide on tumour shrinkage in patients with GH-secreting pituitary adenomas.
We have examined the effects of dopamine on prolactin gene expression using quantitative in-situ hybridization histochemistry in different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Administration of dopamine resulted in a drastic reduction in hybridization of 35S-labelled DNA probe complementary to prolactin mRNA in total pituitary cells and in lactotrophs with low density. In contrast, dopamine significantly stimulated mRNA accumulation in prolactin-secreting cells with high density compared with other cell layers. The combined use of Percoll gradient and quantitative in-situ hybridization is a valuable and sensitive method with which to examine prolactin-secreting cell response to a given stimulation. Prolactin-secreting cells with high and low density clearly show functional heterogeneity in their response to dopamine.
In situ hybridization has become one of the most powerful tools for localizing specific gene products in a tissue. Labelled molecular probes are used on tissue sections or on cells for localization of their hybridization sites. In situ hybridization makes it possible to define the spatial and temporal distribution of specific genes. The use of probes dually labelled, or the combined use of a molecular probe and labelled antibodies provides unprecedented opportunities to examine gene expression in individual cells. In addition, the quantification of gene products can shed light on regulation of gene expression at the cellular level.
GH mRNA expression and GH release by individual cells derived from four GH-secreting pituitary adenomas were studied by in-situ hybridization and the reverse haemolytic plaque assay, respectively. In addition the percentage of PRL mRNA-containing cells was determined in these cell suspensions. The percentages of GH mRNA-containing cells varied between 52 and 89 while the percentages of GH plaque forming cells varied between 25 and 77. Frequency distributions of GH mRNA levels in individual cells and of individual GH plaque areas showed a majority of the cells having low GH mRNA levels and secreting low amounts of GH respectively, while there is a low proportion of cells expressing high GH mRNA levels and forming large GH plaques. There was a significant correlation between the GH mRNA levels and the GH plaque areas of individual cells from the four adenomas (P less than 0.001). The percentages of PRL mRNA-containing cells in the four different adenomas amounted to less than 1, 5, 2 and 18. Cultured cells from the adenomas consisting of 5 and 18% PRL mRNA-containing cells also contained and released measurable amounts of PRL. Our data show that individual cells from GH-secreting pituitary adenomas are heterogeneous with respect to GH mRNA expression, a small proportion of the cells expressing a high amount of GH mRNA. The heterogeneity in GH mRNA expression is correlated with the heterogeneity in GH release. These observations suggest that a considerable part of GH secreted from a GH-secreting pituitary adenoma is produced by a minority of the GH-secreting tumour cell population.(ABSTRACT TRUNCATED AT 250 WORDS)
We have investigated which areas produce interleukin 6 (IL 6) in human tonsils. This growth factor is required for the terminal differentiation of B lymphocytes into plasmocytes. Using 35S-labeled IL 6 cDNA we demonstrated IL 6 gene expression over various areas of the tonsils, with consistent exception of the follicles, by in situ hybridization. It is, therefore, proposed that B cells are stimulated during their migration out of the follicles.
An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18-21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80-95% of the cells were immunoreactive.