Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D2 receptor. D2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

Interferon-ß is a potent inhibitor of cell growth and cortisol production in vitro and sensitizes human adrenocortical carcinoma cells to mitotane.

Adrenocortical carcinoma (ACC) is an aggressive tumor with very poor prognosis. Novel medical treatment opportunities are required. We investigated the effects of interferon-ß (IFN-ß), alone or in combination with mitotane, on cell growth and cortisol secretion in primary cultures of 13 human ACCs, three adrenal hyperplasias, three adrenal adenomas, and in two ACC cell lines. Moreover, the interrelationship between the effects of IGF2 and IFN-ß was evaluated. Mitotane inhibited cell total DNA content/well (representing cell number) in 7/11 (IC50: 38±9.2 µM) and cortisol secretion in 5/5 ACC cultures (IC50: 4.5±0.1 µM). IFN-ß reduced cell number in 10/11 (IC50: 83±18 IU/ml) and cortisol secretion in 5/5 ACC cultures (IC50: 7.3±1.5 IU/ml). The effect of IFN-ß on cell number included the induction of apoptosis. IFN-ß strongly inhibited mRNA expression of STAR, CYP11A1, CYP17A1, and CYP11B1. Mitotane and IFN-ß induced an additive inhibitory effect on cell number and cortisol secretion. IGF2 (10 nM) inhibited apoptosis and increased cell number and cortisol secretion. These effects were counteracted by IFN-ß treatment. Finally, IFN-ß inhibited IGF2 secretion and mRNA expression. In conclusion, IFN-ß is a potent inhibitor of ACC cell growth in human primary ACC cultures, partially mediated by an inhibition of the effects of IGF2, as well as its production. The increased sensitivity of ACC cells to mitotane induced by treatment with IFN-ß may open the opportunity for combined treatment regimens with lower mitotane doses. The inhibition of the expression of steroidogenic enzymes by IFN-ß is a novel mechanism that may explain its inhibitory effect on cortisol production.

Immunoreactivity score using an anti-sst2A receptor monoclonal antibody strongly predicts the biochemical response to adjuvant treatment with somatostatin analogs in acromegaly.

CONTEXT: Somatostatin receptor subtype 2 (sst2A) protein expression has been demonstrated to positively correlate with somatostatin analog treatment outcome in GH-secreting adenomas. Recently, a new rabbit monoclonal anti-sst2A antibody (clone UMB-1) has been validated as a reliable method to selectively detect sst2A protein levels in formalin-fixed tissues. OBJECTIVE: The aim of the study was to establish whether the evaluation of sst2A protein levels, assessed with a routine reproducible immunohistochemistry protocol using UMB-1 antibody, may predict the successful adjuvant therapy with somatostatin analogs in acromegalic patients. DESIGN, SETTING, AND PATIENTS: Thirty-six acromegalic patients from our referral hospital were evaluated retrospectively. Sst2A expression analysis was performed by immunohistochemistry in 25 patients and by quantitative RT-PCR in 26 patients. Sst2A immunoreactivity was evaluated using an immunoreactivity score (IRS), which takes into account both the percentage of positive cells and staining intensity. INTERVENTIONS: Patients with persistent disease after surgery (n = 26) were treated with somatostatin analogs for a median duration of 6 months. MAIN OUTCOME MEASURE: GH and IGF-I levels were measured before and after postoperative treatment. RESULTS: Sst2A IRS showed a significant positive correlation with both GH (P = 0.039) and IGF-I (P = 0.001) suppression by octreotide. Sst2A IRS was negatively associated with IGF-I levels reached after treatment (P = 0.001), and patients that achieved IGF-I normalization showed significantly higher sst2A IRS compared to the group that was not normalized (P = 0.002). A sst2A IRS of at least 5 showed a sensitivity of 86% and a specificity of 91% in predicting IGF-I normalization during adjuvant octreotide treatment. CONCLUSION: Sst2A IRS with the anti-sst2A antibody UMB-1 represents a valid tool in the clinical practice to identify acromegalic patients likely to be responders to adjuvant therapy with the currently available somatostatin analogs.

The role of mTOR inhibitors in the inhibition of growth and cortisol secretion in human adrenocortical carcinoma cells.

Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.

Potent inhibitory effects of type I interferons on human adrenocortical carcinoma cell growth.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare tumor with a poor prognosis. Despite efforts to develop new therapeutic regimens for metastatic ACC, surgery remains the mainstay of treatment. Interferons are known to exert tumor-suppressive effects in several types of human cancer. DESIGN: We evaluated the tumor-suppressive effects of type I interferons (IFN)-alpha2b and IFNbeta on the H295 and SW13 human ACC cell lines. RESULTS: As determined by quantitative RT-PCR analysis and immunocytochemistry, H295 and SW13 cells expressed the active type I IFN receptor (IFNAR) mRNA and protein (IFNAR-1 and IFNAR-2c subunits). Both IFNalpha2b and IFNbeta1a significantly inhibited ACC cell growth in a dose-dependent manner, but the effect of IFNbeta1a (IC50 5 IU/ml, maximal inhibition 96% in H295; IC50 18 IU/ml, maximal inhibition 85% in SW13) was significantly more potent, compared with that of IFNalpha2b (IC50 57 IU/ml, maximal inhibition 35% in H295; IC50 221 IU/ml, maximal inhibition 60% in SW13). Whereas in H295 cells both IFNs induced apoptosis and accumulation of the cells in S phase, the antitumor mechanism in SW13 cells involved cell cycle arrest only. Inhibitors of caspase-3, caspase-8, and caspase-9 counteracted the apoptosis-inducing effect by IFNbeta1a in H295 cells. In H295 cells, IFNbeta1a, but not IFNalpha2b, also strongly suppressed the IGF-II mRNA expression, an important growth factor and hallmark in ACC. CONCLUSIONS: IFNbeta1a is much more potent than IFNalpha2b to suppress ACC cell proliferation in vitro by induction of apoptosis and cell cycle arrest. Further studies are required to evaluate the potency of IFNbeta1a to inhibit tumor growth in vivo.

IFN-beta is a highly potent inhibitor of gastroenteropancreatic neuroendocrine tumor cell growth in vitro.

IFN-alpha controls hormone secretion and symptoms in human gastroenteropancreatic neuroendocrine tumors (GEP-NET) but it rarely induces a measurable tumor size reduction. The effect of other type I IFNs, e.g., IFN-beta, has not been evaluated. We compared the antitumor effects of IFN-alpha and IFN-beta in BON cells, a functioning human GEP-NET cell line. As determined by quantitative reverse transcription-PCR analysis and immunocytochemistry, BON cells expressed the active type I IFN receptor mRNA and protein (IFNAR-1 and IFNAR-2c subunits). After 3 and 6 days of treatment, IFN-beta significantly inhibited BON cell growth in a time- and dose-dependent manner. IC50 and maximal inhibitory effect on day 6 were 8 IU/mL and 98%, respectively. In contrast, the effect of IFN-alpha resulted significantly in a less potent effect (IC50: 44 IU/mL, maximal inhibition: 26%). IFN-alpha induced only cell cycle arrest, with an accumulation of the cells in S phase. IFN-beta, apart from a more potent delay in S-G2-M phase transit of the cell cycle, also induced a strong stimulation of apoptosis, evaluated by flow cytometry (Annexin V and 7-AAD) and measurement of the DNA fragmentation. Besides, only IFN-beta severely suppressed chromogranin A levels in the medium from BON cells after 6 days of treatment. In conclusion, IFN-beta is much more potent, compared with IFN-alpha, in its inhibitory effect on GEP-NET cell proliferation in vitro through the induction of apoptosis and cell cycle arrest. Further studies are required to establish whether IFN-beta has comparable potent tumor growth inhibitory effects in vivo.

The multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somatostatin receptor type 5.

OBJECTIVE: Currently, there is no effective medical treatment for patients with pituitary-dependent Cushing's disease. A novel somatostatin (SS) analogue, named SOM230, with high binding affinity to SS receptor subtypes sst(1), sst(2), sst(3) and sst(5) was recently introduced. We compared the in vitro effects of the sst(2)-preferring SS analogue octreotide (OCT) and the multi-ligand SOM230 on ACTH release by human and mouse corticotroph tumour cells. METHODS: By quantitative RT-PCR the sst subtype expression level was determined in human corticotroph adenomas. In vitro, the inhibitory effect of OCT and SOM230 on ACTH release by dispersed human corticotroph adenoma cells and mouse AtT20 corticotroph adenoma cells was determined. In addition, the influence of dexamethasone on the responsiveness to OCT and SOM230 was studied. RESULTS: Corticotroph adenomas expressed predominantly sst(5) mRNA (six out of six adenomas), whereas sst(2) mRNA expression was detected at significantly lower levels. In a 72 h incubation with 10 nmol/l SOM230, ACTH release was inhibited in three out of five cultures (range -30 to -40%). Ten nmol/l OCT slightly inhibited ACTH release in only one of five cultures (- 28%). In AtT20 cells, expressing sst(2), sst(3) and sst(5), SOM230 inhibited ACTH secretion with high potency (IC(50) 0.2 nmol/l). Dexamethasone (10 nmol/l) pre-treatment did not influence the sensitivity of the cells to the inhibitory effect of SOM230, suggesting that sst(5) is relatively resistant to negative control by glucocorticoids. CONCLUSIONS: The selective expression of sst(5) receptors in corticotroph adenomas and the preferential inhibition of ACTH release by human corticotroph adenoma cells by SOM230 in vitro, suggest that SOM230 may have potential in the treatment of patients with pituitary-dependent Cushing's disease.

Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells.

In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5'-O-(3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst(2A+2B) mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (B(max)) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas B(max) declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRH-induced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing's disease.

Dopamine receptor expression and function in human normal adrenal gland and adrenal tumors.

Dopamine is known to play a role in the modulation of aldosterone and catecholamine secretion from the adrenal gland, where dopamine receptors (DR), in particular the DR type 2 (D(2)), have been found to be expressed. DR expression has also been demonstrated in some types of benign adrenal tumors. The aims of the current study were to evaluate DR expression and D(2) localization in the normal adrenal gland and in different types of benign and malignant adrenal tumors, as well as to evaluate the in vitro effects of the dopamine agonists bromocriptine and cabergoline on hormone secretion in nontumoral adrenal cells. Adrenal tissues from 25 patients, subjected to adrenal surgery for different diseases, were studied. These included three normal adrenals; five adrenal hyperplasias; four aldosterone-secreting, two cortisol-secreting, and two clinically nonfunctioning adrenal adenomas; two aldosterone-secreting, two cortisol-secreting, and two androgen-secreting adrenal carcinomas; and three pheochromocytomas. In all tissues, DR and D(2) isoform (D(2long) and D(2short)) expression was evaluated by RT-PCR. D(2) localization was also evaluated by immunohistochemistry using a specific polyclonal antibody, whereas D(2)-like receptor expression was evaluated by receptor-ligand binding study, using the radiolabeled D(2) analog (125)I-epidepride. The effects of bromocriptine and cabergoline on baseline and ACTH and/or angiotensin II-stimulated aldosterone, cortisol, and androstenedione secretion were evaluated in cell cultures derived from five different adrenal hyperplasia. At RT-PCR, both D(1)-like and D(2)-like receptors were expressed in all normal and hyperplastic adrenals. D(2) and D(4) were expressed in aldosterone- and cortisol-secreting adenomas, cortisol-secreting carcinomas, and clinically nonfunctioning adenomas, whereas no DR was expressed in aldosterone- and androgen-secreting carcinomas. D(2), D(4), and D(5) were expressed in pheochromocytomas. In all D(2)-positive tissues, both D(2) isoforms were expressed, with the exception of one case of aldosterone-secreting adenoma and the cortisol-secreting carcinomas, in which only the D(2long) isoform was expressed. D(2)-like receptor expression was confirmed at receptor-ligand binding study. At immunohistochemistry, D(2) was mainly localized in the zona glomerulosa and reticularis of the adrenal cortex and, to a lesser extent, in the zona fasciculata and medulla of normal and hyperplastic adrenal tissue. In the positive tumors, D(2) was localized in the tumoral cells. At the in vitro study, a significant inhibition of both baseline and ACTH-stimulated aldosterone secretion was found after high-dose cabergoline, but not bromocriptine, administration; and a significant inhibition of angiotensin-II-stimulated aldosterone secretion was found after both bromocriptine and cabergoline administration in the adrenal hyperplasias. In conclusion, the current study demonstrated that both D(1)-like and D(2)-like receptors are expressed in the normal adrenal gland and in a percentage of adrenal adenomas or carcinomas. Bromocriptine and cabergoline induce only a minor inhibition of the secretion of adrenal hormones in the nontumoral adrenal gland in vitro, not excluding, however, the possible effective use of dopamine agonists in vivo in the treatment of adrenal tumors.

The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro.

To determine the inhibitory profile of the novel somatostatin (SRIF) analog SOM230 with broad SRIF receptor binding, we compared the in vitro effects of SOM230, octreotide (OCT), and SRIF-14 on hormone release by cultures of different types of secreting pituitary adenomas. OCT (10 nM) significantly inhibited GH release in seven of nine GH-secreting pituitary adenoma cultures (range, -26 to -73%), SOM230 (10 nM) in eight of nine cultures (range, -22 to -68%), and SRIF-14 (10 nM) in six of six cultures (range, -30 to -75%). The sst analysis showed predominant but variable levels of somatostatin receptor (sst)(2) and sst(5) mRNA expression. In one culture completely resistant to OCT, SOM230 and SRIF-14 significantly inhibited GH release in a dose-dependent manner with an IC(50) value in the low nanomolar range. In the other cultures, SOM230 showed a lower potency of GH release inhibition (IC(50), 0.5 nM), compared with OCT (IC(50), 0.02 nM) and SRIF-14 (IC(50), 0.02 nM). A positive correlation was found between sst(2) but not sst(5) mRNA levels in the adenoma cells and the inhibitory potency of OCT on GH release in vivo and in vitro, and the effects of SOM230 and SRIF-14 in vitro. In three prolactinoma cultures, 10 nM OCT weakly inhibited prolactin (PRL) release in only one (-28%), whereas 10 nM SOM230 significantly inhibited PRL release in three of three cultures (-23, -51, and -64.0%). The inhibition of PRL release by SOM230 was related to the expression level of sst(5) but not sst(2) mRNA. Several conclusions were reached. First, SOM230 has a broad profile of inhibition of tumoral pituitary hormone release in the low nanomolar range, probably mediated via both sst(2) and sst(5) receptors. The higher number of responders of GH-secreting pituitary adenoma cultures to SOM230, compared with OCT, suggest that SOM230 has the potency to increase the number of acromegalic patients which can be biochemically controlled. Second, compared with OCT, SOM230 is more potent in inhibiting PRL release by mixed GH/PRL-secreting adenoma and prolactinoma cells.

Somatostatin receptors in malignant lymphomas: targets for radiotherapy?

UNLABELLED: Somatostatin (SS) receptor (sst) scintigraphy is widely used in the visualization of neuroendocrine tumors expressing sst, and radiotherapy using radionuclide-labeled SS analogs has been introduced for treatment of patients with neuroendocrine tumors. Previous sst scintigraphy studies revealed that malignant lymphomas can also be visualized using this technique. The question has been addressed whether lymphomas might also be possible targets for radiotherapy using radionuclide-labeled SS analogs. Therefore, we investigated in vitro the characteristics of lymphoma tissues and lymphoid cell lines to evaluate whether lymphomas can be targets for radiotherapy. METHODS: Six orbital lymphomas, 2 Hodgkin's lymphomas, and 2 non-Hodgkin's lymphomas from the neck region were collected. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR were performed to detect and quantify the expression of sst(1-5) mRNA. Receptor autoradiography studies using [(125)I-Tyr(3)]octreotide were performed to evaluate binding to sst on cryostat sections of lymphomas. Immunohistochemistry was used to investigate expression of sst(2) and sst(3). Membrane binding studies and in vitro internalization experiments using [(125)I-Tyr(3)]octreotide were performed to study binding and uptake of [(125)I-Tyr(3)]octreotide by lymphoid cell lines (JY, TMM, APD) and primary cells derived from a B-cell-derived chronic lymphatic leukemia. RESULTS: A selective expression of sst(2) and sst(3) messenger RNA (mRNA) was demonstrated. By quantitative RT-PCR, expression levels of sst(2) and sst(3) mRNA were relatively low. Autoradiography studies revealed low binding of [(125)I-Tyr(3)]octreotide, whereas immunoreactivity could not be detected for sst(2) and sst(3) by immunohistochemistry. On the lymphoid cell lines only low numbers of high-affinity SS binding sites were found. In vitro, uptake of [(125)I-Tyr(3)]octreotide by these cells was also very low. CONCLUSION: On the basis of our findings, we conclude that lymphomas do not appear to be candidates for radiotherapy using radionuclide-labeled SS analogs. However, lymphomas are highly radiosensitive tumors and further clinical studies should be performed to evaluate whether the low receptor density is sufficient for targeting treatment in these tumors.

Crucial role for somatostatin receptor subtype 2 in determining the uptake of [111In-DTPA-D-Phe1]octreotide in somatostatin receptor-positive organs.

UNLABELLED: Human somatostatin (SS) receptor (sst)-positive tumors can be visualized by gamma camera scintigraphy after the injection of [(111)In-diethylenetriaminepentaacetic acid (DTPA)-D-Phe(1)] octreotide. Uptake of [(111)In-DTPA-D-Phe(1)]octreotide is dependent on sst-mediated internalization of the radioligand by the tumor cells. Human sst-positive tumors frequently express multiple sst subtypes. In vitro studies have demonstrated that the 5 sst subtypes (sst(1-5)) differentially internalize sst-bound ligand. The present study was performed to evaluate the role of sst(2) in vivo in determining the uptake of [(111)In-DTPA-D-Phe(1)]octreotide, as well as of the more "universal" ligand [(111)In-DTPA]SS-14, by sst-positive organs expressing multiple sst subtypes. METHODS: Wild-type and sst(2) knockout mice (n = 4 per treatment group) were injected intravenously with 1 MBq (0.1 micro g) [(111)In-DTPA-D-Phe(1)]octreotide or [(111)In-DTPA]SS-14. After 24 h, the animals were sacrificed and radioactivity in the organs under investigation was determined. In addition, the sst subtype messenger RNA (mRNA) expression pattern in these organs was determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: RT-PCR analysis demonstrated the presence of all 5 sst subtype mRNAs in the adrenals and pituitary of wild-type mice but no sst(2) in the knockout mice. The thymus expressed mRNA for sst(2) and sst(4) mRNA in wild-type mice, whereas no sst(2) was detected in knockout mice. In wild-type mice, the in vivo uptake values (in percentage injected dose per gram of tissue) of [(111)In-DTPA-D-Phe(1)]octreotide for the pituitary, adrenals, pancreas, and thymus amounted to 1.2 +/- 0.2, 0.26 +/- 0.03, 0.18 +/- 0.03, and 0.30 +/- 0.05, respectively, in wild-type mice. Compared with wild-type mice, sst(2) knockout mice had dramatically lower uptake values in these organs-lower by 97%, 83%, 96%, and 94%, respectively (P < 0.01 vs. wild type). Comparable differences in the uptake of radioactivity between wild-type and knockout mice were found using [(111)In-DTPA]SS-14 as the radiotracer. Interestingly, in some organs expressing sst(2) mRNA (liver, muscle, and peripheral blood mononuclear cells), no specific binding of [(111)In-DTPA-D-Phe(1)]octreotide or [(111)In-DTPA]SS-14 to sst in vivo was found, suggesting that the sst(2) protein expression level was very low in these tissues. CONCLUSION: The uptake of [(111)In-DTPA-D-Phe(1)]octreotide and [(111)In-DTPA]SS-14 in sst-positive organs is determined predominantly by sst(2).

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes.

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.