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Departments of Internal Medicine III (D.F., P.M.v.H., P.M.v.K., J.Z., D.M.M., E.G.R.L.-K.), Immunology (P.M.v.H.) and Cardiothoracic Surgery (A.J.J.C.B.), Erasmus University, Rotterdam, The Netherlands; and Department of Molecular & Clinical Endocrinology and Oncology (D.F., A.C., G.L.), "Federico II" University, Naples, Italy
Address all correspondence and requests for reprints to: Leo J. Hofland, University Hospital Dijkzigt, Department of Internal Medicine III, Room Bd277, Dr Molewaterplein 40, Rotterdam, 3015 GD, The Netherlands. E-mail: hofland{at}inw3.azr.nl
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We investigated the SS distribution and SS-R expression pattern in the normal human thymus using autoradiography, membrane homogenate binding studies, and RT-PCR. In addition, the effect of SS and octreotide on growth of cultured thymic epithelial cells (TEC) was studied.
By autoradiography, binding of [125I-Tyr0]-SS-28 and [125I-Tyr3]-octreotide was detected in all seven thymuses studied. Specific [125I-Tyr3]-octreotide binding was shown on membrane preparations from thymuses, while not from cultured thymocytes. RT-PCR showed the expression of sst1, sst2A and sst3 messenger RNA (mRNA) in the thymic tissue, whereas sst1 and sst2A mRNAs were found in isolated TEC. SS mRNA was present in thymic tissue and in isolated TEC. SS and octreotide significantly inhibited 3H-thymidine incorporation in 3 of 3 and 6 of 6 TEC cultures, respectively. The percent inhibition ranged from 38.8 to 66.8% for SS and from 19.1 to 59.5% for octreotide.
In conclusion, SS mRNA and sst1, sst2A, and sst3 mRNAs are expressed in the normal human thymus. Cultured TEC selectively express sst1 and sst2A mRNA and respond in vitro to SS and octreotide administration with an inhibition of cell proliferation. These data suggest a paracrine/autocrine role of SS and its receptors in the regulation of cell growth in thymic microenvironment.
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Introduction |
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Like other regulatory peptides found in the brain, SS has also been localized in lymphatic tissue (12). Among the lymphatic organs, the thymus from different species including the human has been shown to contain the highest amount of SS, suggesting a modulatory role of the peptide in this organ (13, 14, 15). Furthermore, SS-R have been demonstrated in vitro in various lymphatic tissues (16). Recently, a high in vivo uptake of 111In-DTPA-D-Phe1-octreotide was shown in patients bearing thymomas and thymic carcinoids (17, 18), and a successful treatment with octreotide was reported in one patient with thymoma (19). The presence of SS and SS-R in the thymus suggests their involvement in controlling the immuno- and/or neuroendocrine functions in this organ. Moreover, SS analog treatment of thymic tumors and related paraneoplastic diseases might represent a new therapeutic approach to these disorders.
The present study was designed to investigate the expression and role of SS and SS-R subtypes in the normal human thymus. The receptor expression pattern in the thymuses was studied in vitro by SS-R autoradiography, membrane homogenate binding studies, and RT-PCR to identify SS-R subtypes. In addition, the in vitro effect of SS, octreotide, and the growth factors insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) on cell proliferation was investigated in primary human thymic epithelial cell (TEC) cultures. Because fibroblasts often contaminate cultures of epithelial cells, we used a specific antibody to human fibroblasts (mAb ASO2) coupled with magnetic beads for the selective removal of fibroblasts from these cultures. This new monoclonal antibody recognizes a human fibroblast-specific antigen located on the cell surface (20).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SS receptor autoradiography
Receptor autoradiography was carried out as described by Reubi
et al. (21). Briefly, 10-micrometer thick cryostat (Jung
CM3000, Leica Corp., Germany) sections of the tissue
samples were mounted onto precleaned gelatin-coated microscope glass
slides and stored at -80 C for at least 3 days before the experiment
to improve the adhesion of the tissue to the slide. As radioligands,
the SS analogs [125I-Tyr3]-octreotide and
[125I-Tyr0]-SS-28 (ANAWA Laboratories,
Wangen, Switzerland) were used. Specific activities of the radioligands
amounted approximately 2000 Ci/mmol. To wash out endogenous SS, the
sections were preincubated at room temperature for 10 min in 170
mM Tris-HCl (pH 7.4). Thereafter, the sections were
incubated for 60 min at room temperature in 170 mM Tris-HCl
(pH 7.4), 5 mM MgCl2, 1% BSA, 40 µg/ml
bacitracin with [125I-Tyr3]-octreotide (final
concentration approximately 80–160 pmol/liter) or
[125I-Tyr0]-SS-28 (approximately 80–160
pmol/liter). Nonspecific binding was determined in a sequential section
in the presence of excess unlabeled Tyr3-octreotide (1
µM) or SS-28 (1 µM), respectively. The
incubated sections were washed twice for 5 min in incubation buffer
containing 0.25% BSA and once in incubation buffer without BSA. After
a short wash with distilled water to remove salts, the sections were
air dried and exposed to Kodak X-OMAT AR or Hyperfilm-3H
(Amersham, Houten, The Netherlands) for 3–7 days in x-ray
cassettes. Histology was performed on hematoxylin-eosin stained
sequential cryosections.
RT-PCR
Poly A+ mRNA was isolated using Dynabeads Oligo
(dT)25 (Dynal AS, Oslo, Norway) from cell
pellets containing approximately 106 cells or from
approximately 50 mg frozen powdered tissue that was grinded in a metal
homogenizer cooled by liquid nitrogen. The cells were lysed during 2
min on ice in a buffer containing 100 mM Tris-HCl (pH 8),
500 mM LiCl, 10 mM EDTA (pH 8), 1% LiDS, 5
mM DTT and 5 U/100 µl RNAsin (HT Biotechnology Ltd., Cambridge, UK). The mixture was centrifuged at 14,000 rpm
for 1 min to remove cell debris. To the supernatant 100 µl prewashed
Dynabeads Oligo (dT)25 were added, and the mixture was
incubated for 5 min on ice. Thereafter, the beads were collected with a
magnet, washed three times with 10 mM Tris-HCl (pH 8), 0.15
M LiCl, 1 mM EDTA, 0.1% LiDS, and once with a
similar buffer from which LiDS was omitted. Poly A+ mRNA
was eluted from the beads in 50 µl of a 2 mM EDTA
solution (pH 8) during 2 min at 65 C. To avoid contamination by genomic
DNA, the isolated polyA+ RNA was subjected to a second
purification by capturing the RNA on a fresh aliquot of prewashed
Dynabeads Oligo (dT)25 and washing the captured RNA as
above. Finally, the captured RNA was washed once with buffer used for
the reverse transcriptase reaction.
Complementary DNA (cDNA) was synthesized using the poly A+ mRNA captured on the Dynabeads Oligo (dT)25 in a buffer containing 50 mM Tris-HCl (pH 8.3), 100 mM KCl, 4 mM DTT, 10 mM MgCl2, 1 mM of each deoxynucleotide triphosphate, 10 U RNAsin, and 2 U AMV Super Reverse Transcriptase (HT Biotechnology Ltd., Cambridge, UK) in a final volume of 20 µl. This mixture was incubated for 1 h at 41 C.
One-tenth from each cDNA library immobilized on the paramagnetic beads
was used for each amplification. The amplification reaction mixtures
contained cDNA template, 0.5 U SuperTaq (HT Biotechnology Ltd., Cambridge, UK), 50 µM of each
deoxynucleotide triphosphate (HT Biotechnology Ltd.,
Cambridge, UK), 5 pmol of each of a pair of oligonucleotide primers
specific for the human sst1–5 receptor subtypes, the
ß-actin gene or the human SS gene (see Table 1
) in a buffer of 10 mM
Tris-HCl (pH 9), 50 mM KCl, 2 mM
MgCl2, 0.01% (wt/vol) gelatin, 0.1% Triton X-100 in a
final volume of 50 µl. The sequences of the primers for
sst1–5 are derived and/or adapted from Kubota et
al. (8) and Wulfsen et al. (22). The PCR reaction was
carried out in a DNA thermal cycler with heated lid (Perkin Elmer Cetus Instruments, Gouda, The Netherlands). After an
initial denaturation at 94 C for 5 min, the samples were subjected to
40 cycles of denaturation at 94 C for 1 min, annealing for 2 min at 59
C, and extension for 1 min at 72 C. After a final extension for 7 min
at 72 C, 10 µl aliquots of the resulting PCR products were analyzed
by electrophoresis on 1.5% agarose gels stained with ethidium
bromide.
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Cell dispersion and cell culture
After the specimens were placed in HBSS supplemented with HSA
5% (Cealb, CLB, Amsterdam, The Netherlands), penicillin
(105 U/liter), fungizone (0.5 mg/liter), blood clots, and
fibrous capsules were carefully removed and the specimens were
dissected and washed several times with the HBSS + HSA. The minced
tissues were enzymatically dissociated with collagenase (Sigma Chemical Co., St. Louis, MO; 2 mg/ml) for 1–2 h at 37 C. The
dispersed cells were precultured for 5–7 days in 15 mg/cm2
collagen type I (Collagen S, type I, Boehringer Mannheim,
Mannheim, Germany) precoated 75-cm2 flasks
(Costar, Cambridge, MA) at a density of 5 x
106 cells/flask in 10 ml culture medium. The culture
medium, the same used during the experiments, was MEM D-valine
(Gibco BRL, Life Technologies Ltd., Paisley,
Scotland, UK) supplemented with 10% FCS, penicillin (105
U/liter), fungizone (0.5 mg/ml), L-glutamine (2
mmol/liter). The pH of the medium was adjusted to 7.4. The cells were
cultured at 37 C in a humid CO2-incubator. After this
period, cell viability was detected by trypan blue exclusion and varied
between 65 and 90%. The cells, which had not attached to the flasks,
mainly thymocytes, were harvested.
The isolation of TEC was indirectly performed using sheep antimouse IgG coated magnetic beads and a ASO2 antihuman fibroblast primary antibody kit (Dianova GmbH, Hamburg, Germany). This mouse monoclonal antibody reacts specifically with membrane-bound protein of human fibroblasts of different origin (20). The magnetic beads were coated with the specific primary antibody and mixed with the target cell suspension to form bead rosetted cells. Rosetted cells (fibroblasts) were collected at the tube wall and isolated using a magnetic separator rack. For a complete negative selection, the supernatant, containing the cells which were not bound to the beads (TEC), was treated once again with the coated beads. The remaining suspension containing isolated TEC was used for the experiments.
TEC (10,000–20,000 cells per well) were seeded in 1 ml culture medium in 24-well collagen type I precoated plates (Costar) and allowed to attach for 24 h. Then, tests substances were added, and the cells were incubated for 72 h. Proliferation was measured by adding 1 µCi of [methyl-3H]-thymidine (91 Ci/mmol; Amersham) for the last 24 h in each well. Thereafter, the medium was removed and the cells were washed twice with ice cold 0.9% NaCl solution and harvested using a 0.05% NH3 solution. These samples were exposed overnight to 10% trichloroacetic acid followed by another wash with 0.9% NaCl solution. The cells were transferred to scintillation counting vials after solubilization in 1 M NaOH and incorporated radioactivity was measured, after neutralization with HCl and the addition of scintillation fluid, in a liquid scintillation counter (Betamatic, Packard, Downers Grove, IL). For RT-PCR studies, TEC were seeded in collagen-coated flasks (see above) and grown to confluence. Thereafter, the cells were harvested and mRNA was isolated as described above.
Immunocytochemical detection of keratin on cultured TEC
For keratin staining, TEC were cultured on collagen-coated glass
coverslips. The cells were fixed for 10 min with methanol at the end of
the incubation period. Cytokeratin staining was performed with a PAP
Kit System (code K518; Dako Corp., Glostrup, Denmark).
Staining for keratin was performed after each experiment in all TEC
cultures studied.
SS receptor binding studies
The method of membrane isolation and the reaction conditions
were the same as described by Reubi (23). Briefly, membrane
preparations (corresponding to 30–50 µg protein) of tissue samples,
freshly dispersed cells or cultured cells were incubated in a total
volume of 100 µl at room temperature for 60 min with increasing
concentrations of [125I-Tyr3]-octreotide with
and without excess (1 µM) of unlabeled
Tyr3-octreotide in HEPES buffer (10 mM HEPES, 5
mM MgCl2 and 0.02 g/liter bacitracin, pH 7.6)
containing 0.2% BSA. After the incubation, 1 ml ice-cold HEPES buffer
was added to the reaction mixture, and membrane-bound radioactivity was
separated from unbound by centrifugation during 2 min at 14,000 rpm in
a Eppendorf microcentrifuge. The remaining pellet was
washed twice in ice-cold HEPES buffer, and the final pellet was counted
in a 
-counter. Specific binding was taken to be total binding minus
binding in the presence of 1 µM unlabeled
Tyr3-octreotide.
Test substances
In all experiments SS-14 (Bachem, Inc., Hannover,
Germany) and octreotide (Novartis, Basle, Switzerland) were used
at a concentration of 10 nM, IGF-I (Bachem, Inc.) at a concentration of 10 nM and EGF
(Bachem, Inc.) at a concentration of 10 ng/ml.
Statistical analysis
Data are expressed as mean ± SEM, n = 4
wells per treatment group. All data were analyzed by ANOVA to determine
overall differences between treatment groups. When significant
differences were found, a comparison between treatment groups was made
using the Newman-Keuls test. SS-R binding data were analyzed by the
method of Scatchard. Receptor binding studies and RT-PCR experiments
were performed at least twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows an example of the staining for
keratin of a TEC population that was used for the functional study
(see below). Virtually no fibroblasts (keratin-negative) were
present.
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), and in agreement with a previous
study (16). Binding was not homogeneous and mainly localized in the
medulla (Fig. 2).
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). As a
control for binding, rat brain cortex membranes were used. An example
of saturation binding data with Scatchard analysis is shown in Fig. 3.
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; no. 5, Table 2
). Conversely, mRNA
encoding for sst4 and sst5 was absent (Table 2;
Fig. 4). In cultured TEC, RT-PCR analysis showed the presence of SS,
sst1 and sst2A mRNA, while in cultured
thymocytes only mRNA for ß-actin was detected (Table 4; Fig. 4).
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). The SS analog
octreotide (10 nM) significantly inhibited
3H-thymidine incorporation by 38.7, 49.8, and 40.6%,
respectively, in these three cultures (Fig. 5A), and by 19.1, 48.2, and
59.5% in three other additional cultures of TEC (Fig. 5B) derived from
the series of thymuses which showed
[125I-Tyr3]-octreotide binding at
autoradiographic and membrane binding studies. IGF-I significantly
stimulated 3H-thymidine incorporation in two out of three
cultures and EGF significantly stimulated 3H-thymidine
incorporation in all the three cultures in which were tested (Table 5). Moreover, octreotide significantly
inhibited EGF-stimulated 3H-thymidine incorporation by
25 ± 5% in one culture in which it was tested (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present report, we studied the distribution and the expression of SS-R subtypes and SS in 7 normal human thymuses. We first demonstrated specific [125I-Tyr3]-octreotide binding on cryostat sections from thymic tissue, mainly in the medulla, which is the thymic compartment where the TEC is the predominant cell type, as well as in thymic tissue homogenates. Furthermore, we characterized the SS-R subtype expression in the same thymuses that were used for receptor binding studies. In human thymic tissue, we found by RT-PCR the presence of mRNA encoding for sst1 and sst2A and sst3. The expression of the subtypes appeared heterogeneous on the different cell subsets. In fact, we found a specific binding of [125I-Tyr3]-octreotide on membrane preparations from thymic tissue, whereas no binding was found on cultured thymocytes. Interestingly, RT-PCR showed the selective expression of sst1 and sst2A on the cultured TEC, whereas neither SS-R subtype was found on cultured thymocytes with this technique. Sst1 and sst2A seem stronger expressed in the cultured TEC compared with the thymic tissue, because with the selection method used to establish primary TEC cultures, an enrichment of SS-R-expressing TEC was obtained. Again, it should be emphasized that the cultured thymocytes, which represent the major cellular component of the thymus, did not express any SS-R subtype. Moreover, the finding that SS-R are expressed on only a subset of the cells in the thymus also explains the relatively low SS-R levels measured by receptor binding studies on membrane homogenates of the whole thymic tissue. Finally, this heterogeneous SS-R expression pattern in the human thymus may also explain the apparent discrepancy between the SS-R levels in the thymic tissues and the maximal inhibitory effect of SS and octreotide on TEC proliferation.
Furthermore, TEC seem to be the major site of SS production in the normal human thymus. In fact, by RT-PCR we demonstrated the presence of mRNA for SS in isolated TEC from all the thymuses of the present series. Because our study showed the existence of a specific SS-R subtype distribution pattern as well as evidence for endogenous SS production in the human thymus, we also studied the effects of SS and the SS analog octreotide on TEC proliferation. At this purpose, we established pure epithelial cell cultures, demonstrated by staining for cytokeratin that showed 95–100% cytokeratin-positive cells in all the cultures. In these human TEC cultures, we observed a significant inhibition of cell proliferation by SS and octreotide in all cases.
The heterogeneity of distribution of SS-R subtypes on specific cells, in combination with the endogenous production of SS, suggest an important and dynamic regulatory role of this peptide in the human thymus. The data of the present study indicate that SS may exert a paracrine/autocrine inhibitory action. This effect seems to be mediated by the sst2A and/or sst1, which are selectively expressed on cultured TEC. The binding of SS or its analogs to these receptor subtypes is associated with an activation of intracellular tyrosine phosphatase activity (29, 30). Moreover, sst2A is also linked to cell membrane potassium and calcium channels in a manner that ligand binding influences the intracellular concentration of these ions and the cell membrane polarization (31).
Although we did not investigate the expression of SS-R and the effect of SS analogs on thymic tumoral cells, it can be suggested that a disturbance in this pathway may play a role in the pathogenesis of neoplastic and related autoimmune diseases in the human thymus. Recently, a high in vivo uptake of 111In-DTPA-D-Phe1-octreotide was reported in patients bearing thymic neoplasms (17, 18) and a successful treatment with octreotide was reported in one patient with thymoma (19). This effect might be mediated by SS-R. Although in a previous study no in vitro evidence of SS-R has been found in four thymomas, it is well known that there is a considerable heterogeneity between and within differentiated tumors with respect to the density and the specificity of SS-R binding sites (32).
We also demonstrated the expression of sst3 in the normal human thymic tissue, whereas sst3 mRNA was undetectable in cultured TEC. This may be explained by the fact that this receptor subtype is expressed by a different epithelial cell subset or on macrophages. Emerging data regarding signal transduction pathways linked to SS-R showed a cell cycle-dependent induction of apoptosis by octreotide (33). Apoptosis seems signaled through the sst3 and associated with dephosphorylation-dependent conformational change in wild-type p53 (34). Although controversial data about alterations of p53 expression have been reported in thymic epithelial tumors (35, 36), octreotide-induced apoptosis might represent an additional mechanism involved in the regulation of cell proliferation in human thymus.
Another important aspect involves the local production of growth factors and cytokines by TEC and thymocytes (27, 28, 37). In our study, IGF-I and EGF significantly stimulated in vitro TEC proliferation. Moreover, in a preliminary experiment, octreotide significantly inhibited EGF-stimulated cell proliferation. Although an inhibition of the production of these factors might represent an indirect mechanism of action of SS and its analogs, a direct mechanism involving the phosphoprotein phosphatase activity associated to sst2 could not be ruled out (38). Further studies are required to investigate the precise mechanism of action of SS in inhibiting growth factors-stimulated TEC proliferation.
These findings suggest that the SS-R-mediated effects of SS and octreotide on TEC growth may act via not mutually exclusive different mechanisms, supporting the physiological paracrine/autocrine role of SS in controlling cell growth in the human thymic microenvironment. A further conceptual aspect might be the implication of the role of the intrathymic production of SS in the main function of the thymus, namely the maturation and differentiation of T-lymphocytes. Moreover, a disturbance in these loops may be important in the pathogenesis of autoimmune and neoplastic diseases involving this organ. In this respect, a better understanding of the functional significance of the presence of SS and SS-R subtypes in the human thymus might lead to new approaches in the medical management of these disorders.
In conclusion, we have demonstrated a heterogeneous expression of SS-R subtypes within the human thymus. Furthermore, TEC respond in vitro to SS and octreotide administration with an inhibition of cell proliferation. These data, together with the evidence of other peptide hormones, cytokines and their receptors in the thymus, support the concept of the thymus as the organ where the interactions between the endocrine and the immune systems are mostly represented. Future studies should investigate the influence of SS and its analogs on local cytokine production, which is known to represent the other arm of the immuno-endocrine pathway.
Received June 22, 1998.
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, total binding; 
,
nonspecific binding in presence of 1 µM of
Tyr3-octreotide; •, specific binding (total minus
nonspecific binding). Inset: Scatchard analysis of the binding data
(Kd, 0.5 nM and Bmax, 15 fmol/mg
membrane protein; no. 4, Table 3
, octreotide;