Quantitative and functional expression of somatostatin
receptor subtypes in human thymocytes
Diego
Ferone1,3,
Rosario
Pivonello1,4,
P. Martin
van Hagen1,2,
Virgil A. S. H.
Dalm1,
Elgin G. R.
Lichtenauer-Kaligis1,
Marlijn
Waaijers1,
Peter M.
van Koetsveld1,
Diana M.
Mooy1,
Annamaria
Colao4,
Francesco
Minuto3,
Steven W. J.
Lamberts1, and
Leo J.
Hofland1
1 Departments of Internal Medicine and
2 Immunology, Erasmus Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands; 3 Department
of Endocrinological and Metabolic Sciences, University of Genova,
16132 Genoa; and 4 Department of Molecular and
Clinical Endocrinology and Oncology, "Federico II" University,
80131 Naples, Italy
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ABSTRACT |
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-Tyr11-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.
thymus; immune system; ligand binding; reverse
transcriptase-polymerase chain reaction
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INTRODUCTION |
THE IMMUNE AND NEUROENDOCRINE SYSTEMS
cross talk by using similar ligands and receptors. Neurohormones
modulate the function of lymphoid organs and are produced by immune
cells as well, thereby exerting a paracrine/autocrine action in
immunoregulation (4). Receptors for different
neurohormones, such as hypothalamic-pituitary and gastrointestinal
hormones, are expressed by immune and lymphoid accessory cells
(4, 29). These neuroendocrine circuits seem to exert a
pleiotropic control on the physiology of the thymus, the main lymphoid
organ (32). Particularly, the intrathymic production of classical neurohormones suggests that paracrine and
autocrine interactions, mediated by these compounds and their respective receptors, influence both thymic lymphoid and stromal compartments (31, 32).
Somatostatin (SS) represents one of the most relevant neuropeptides
involved in neuroimmunoendocrine interaction (19, 45). The
wide spectrum of actions of SS and the presence of SS receptor (SSR) in
lymphoid organs imply a broad functional role of this peptide in the
immune system (17, 27, 29, 45).
We (14) have recently demonstrated the expression of SS
itself and of three different SSR subtypes (sst) within the human thymus. Messenger RNAs encoding for sst1,
sst2A, and sst3 were found in a series of
normal thymic tissues. sst1 and sst2A were selectively expressed on cultured thymic epithelial cells (TEC), and
both SS and its analog octreotide inhibited in vitro TEC proliferation. No SSR subtype mRNA was detectable in 7- to 14-day-cultured thymocytes (14), whereas our preliminary data have recently
demonstrated a low number of SS-binding sites on freshly isolated human
thymocytes (11). On the other hand, SSR are expressed on
thymocytes of different animal species (9, 33, 39), and in
humans SS is known to modulate different functions of T lymphocytes,
which directly derive from thymocytes (45). Moreover,
sst3 mRNA has recently been demonstrated to be
constitutively expressed in human resting peripheral T lymphocytes
(16). Thymocytes are a heterogeneous cell population. In
fact, when progenitors enter the thymus from bone marrow, they lack
most of the specific T cell markers. The interactions with the thymic
microenvironment trigger the expression of T cell-specific surface
molecules. First, CD2 is the marker of immature thymocytes when they do
not express the TCR-CD3 complex or the co-receptors CD8 and CD4
(42). These cells are called "double-negative"
thymocytes and are a highly heterogeneous pool of cells that include
several early stages in T cell development (44). Thus
thymocytes undergo maturation through a series of stages that can be
distinguished by the differential expression of the TCR-CD3 complex,
CD8, and CD4. CD3
CD4+CD8
represents an intermediate thymocyte subset before the
"double-positive" CD4+CD8+ thymocytes
stages (20). Finally, the
CD3+CD4+CD8+ subset further
differentiates into mature CD4+ or CD8+
single-positive thymocytes (15, 44).
The present study was designed to investigate the presence and
potential role of SSR in human thymocytes. The receptor expression pattern was evaluated in vitro in freshly isolated thymocytes by
SSR-binding studies on membrane homogenate and by RT-PCR to identify
and quantify SSR subtypes on different thymocyte subsets. In addition,
the in vitro effect of SS and octreotide on cell proliferation was
investigated in isolated human thymocytes.
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MATERIALS AND METHODS |
Samples.
Thymic tissues were removed from 13 patients (age range between 3 mo
and 5 yr) to allow adequate exposure of the heart during cardiovascular
surgery. Samples from these thymuses were used in the present study.
The protocol was in accordance with the Helsinki Doctrine on Human
Experimentation, and informed consent was obtained from the patients'
parents. All samples were histopathologically normal and were taken
fresh at the operation.
Protocol of the study.
Thymocytes were freshly isolated from the thymic samples and used for
binding studies on membrane homogenates with iodinated SS-14 (Table
1). Thymocytes derived from four samples
were separated in subpopulations for RT-PCR studies (sample
nos. 5-8, Table 1; see RT-PCR studies).
Thymocytes from five samples of the same series (nos. 5-7,
10, and 11, Table 1) were used for the in vitro primary
cell cultures. Thymocytes from three samples were used to study the
induction of apoptosis (nos. 11-13, Table 1;
see Study of apoptosis).
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Table 1.
SS receptor expression in human thymocytes determined by Scatchard
analysis of 125I-Tyr11-labeled SS-14 binding on
membrane homogenates
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Cell dispersion, cell separation, and cell culture.
Thymocytes were collected using a filter chamber (NPBI,
Emmer-Compascuum, The Netherlands) and placed in RPMI 1640 (GIBCO-BRL-Life Technologies, Paisley, Scotland) supplemented with 10%
heat-inactivated FCS, penicillin (105 U/l), and fungizone
(0.5 mg/l). The pH of the medium was adjusted to 7.4. Cell viability
was determined before each study and was >95%. These thymic cell
suspensions generally contain >95% thymocytes, as has been
demonstrated by flow cytometry (FACS) and anti-CD2 antibodies, which
selectively bind to thymocytes, in a series of normal pediatric
thymuses (36). To confirm this, we performed FACS analysis
using a FACScan cytometer (Becton-Dickinson, Erembodegem, Belgium) and
anti-CD2 antibodies (Becton-Dickinson). Cytometry and additional
fluoconjugated antibodies were used to determine the proportion of the
intermediate/mature CD3+ thymocyte subset and the
CD14+ monocyte-macrophage fraction. Thymic cells
(106) were sorted by setting appropriate electronic gates
with the dual-laser FACS system (Becton-Dickinson). For RT-PCR analysis and for functional studies, thymic cells were first depleted from the
monocyte fraction (see below) and subsequently separated into subpopulations by use of magnetic beads coated with specific antibodies (Dynal, Oslo, Norway). The cells were suspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and incubated with the coated beads in plastic tubes kept on ice for 30 min. By
continuous rotation of the tubes, the cells and beads were kept in
suspension. The tubes were then placed in a magnetic rack to separate
the supernatant from the bead-captured cells. The nonselected cells in
the supernatant were used for the subsequent rounds of selection with
appropriate antibody-coated beads. The bead-captured cells were washed
five times with PBS containing 0.5% BSA, counted, and evaluated for
specificity by determining the percentage of cells rosetted by the
beads, which was >98% in all cases. The thymocyte suspension was
depleted from the monocyte fraction by using beads coated with CD14
antibodies (CD14+). To isolate intermediate/mature
thymocytes (CD3+), anti-CD3-coated beads were used. The
remaining cells (after a second round of depletion with anti-CD3-coated
beads) were further incubated with anti-CD2-coated beads to obtain the
immature thymocyte fraction (CD2+CD3
).
Additional freshly isolated thymocytes (from 5 cases), which did not
undergo bead separation, as well as negatively selected CD3+ and CD2+CD3
cell fractions
(from 2 cases), were seeded (5 × 106 cells/well) in 1 ml of culture medium in 24-well plates (Costar, Cambridge, MA). Then,
test substances were added, and the cells were incubated for 24 h
for functional experiments. Cell viability was constantly tested during
the separation procedure as well as before and after functional studies
and was satisfactory.
SSR membrane-binding studies.
The method of membrane isolation and the reaction conditions were
previously described (14).
125I-Tyr11-labeled SS-14 (Amersham, Houten, The
Netherlands) binding to the thymocyte membranes was analyzed. Briefly,
membrane preparations (corresponding to 30-50 µg of protein) of
freshly dispersed cells were incubated in a total volume of 100 µl at
room temperature for 30 min with increasing concentration of
125I-Tyr11-SS-14 with and without excess (1 µM) of unlabeled SS-14 in HEPES buffer (10 mM HEPES, 5 mM
MgCl2, and 0.02 g/l bacitracin, pH 7.6) containing 0.2%
BSA. After the incubation, 1 ml of ice-cold HEPES buffer was added to
the reaction mixture, and membrane-bound radioactivity was separated
from unbound by centrifugation for 2 min at 14,000 rpm in an Eppendorf
microcentrifuge. The remaining pellet was washed twice in ice-cold
HEPES buffer, and the final pellet was counted in a
-counter (1470 Wizard, Wallac, Turku, Finland). Specific binding was taken to be total
binding minus binding in the presence of 1 µM unlabeled SS-14
and ranged between 12.7 and 45.8% of the total binding
(32.4 ± 3.3).
Functional studies.
In all experiments, SS-14 (Bachem, Hannover, Germany) and octreotide
(Novartis Pharma, Basel, Switzerland), dissolved in the culture medium
(RPMI 1640 supplemented with 10% heat-inactivated FCS, penicillin, and
fungizone), were used at a concentration of 10
13,
10
12, 10
10, 10
8, and
10
6 M. The culture medium was added to the control wells
to evaluate the possible effects of the vehicle. After 24 h,
proliferation was measured by adding 1 µCi of
[methyl-3H]thymidine (91 Ci/mmol; Amersham)
for the last 6 h in each well of the 24-well plates. Thereafter,
the cell suspension was transferred to 5-ml tubes and precipitated with
10% trichloroacetic acid, and the pellet was washed once again in
trichloroacetic acid. After solubilization in 1 M NaOH, the cells were
transferred to scintillation-counting vials, 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).
RT-PCR studies.
RT-PCR was performed as previously described (14).
Briefly, poly(A)+ mRNA was isolated using Dynabeads
oligo(dT)25 (Dynal) from cell pellets containing
0.5-1 × 106 cells per sample. cDNA was
synthesized using the poly(A)+ mRNA captured on the
Dynabeads oligo(dT)25 as solid-phase and first-strand
primer. One-tenth of the cDNA was used for each amplification by PCR
with the use of primer sets specific for human sst1-5, SS, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Table
2). Several controls were included in the
RT-PCR experiments. To ascertain that no detectable genomic DNA was
present in the poly(A)+ mRNA preparation, the cDNA
reactions were also performed without reverse transcriptase and
amplified with each primer pair (no introns have yet been found in the
coding region of these genes, which include the oligonucleotide
amplimers used in this study). Amplification of the cDNA samples with
the HPRT-specific primers served as a positive control for the quality
of the cDNA. To exclude contamination of the PCR reaction mixtures, the
reactions were also performed in the absence of DNA template in
parallel with cDNA samples. As a positive control for the PCR reactions
of SSR receptor subtypes, 0.001-0.1 µg of human genomic DNA,
representing ~300 to 30,000 copies of sst template, was amplified in
parallel with the cDNA samples. As a positive control for the PCR of
HPRT and SS, aliquots of a cDNA sample were amplified, because these primer pairs did enclose introns in the genomic DNA. In the thymocyte preparation, only sst2 and sst3 mRNAs were
detectable. To quantify sst2 and sst3 mRNAs, a
quantitative RT-PCR was performed by the TaqMan Gold nuclease assay
(Perkin-Elmer, Foster City, CA) and the ABI PRISM 7700 Sequence
Detection System (Perkin-Elmer) for real-time amplification according
to the manufacturer's instructions. The specific primer and probe
sequences that were used for the quantitative RT-PCR are reported in
Table 3. The amount of sst2 and sst3 mRNA was determined by means of a standard curve
generated in each experiment from known amounts of human genomic DNA.
For the determination of the amount of HPRT mRNA, the standard curve was obtained by including dilutions of a pool of cDNAs known to contain
HPRT. The amount of sst2 and sst3 mRNA was
calculated relative to the amount of HPRT and is given in arbitrary
units.
Study of apoptosis.
To study induction of apoptosis, thymocytes were incubated for
24 h without or with different concentrations of SS-14 or
octreotide. After 24 h, the cells were collected and pelleted by
centrifugation. Thereafter, the medium was carefully removed, and the
cells were lysed in lysis buffer (Roche Diagnostics, Mannheim, Germany)
for 30 min at room temperature. Subsequently, the lysate was
centrifuged at 200 g for 10 min, and 20 µl of each lysate
were used for photometric measurement of cytoplasmic histone-associated
DNA fragments (mono- and oligonucleosomes) by means of a Cell Death
Detection ELISAPLUS assay (Roche Diagnostics). Values are
expressed as absorbance units (A405 nm
A490 nm).
Statistical analysis.
Data are expressed as means ± SE. In functional
studies, n = four 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.
SSR-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 |
SSR-binding study.
Using membrane homogenate binding, specific
125I-Tyr11-SS-14 binding was demonstrated on
freshly isolated thymocytes in all cases. Binding of
125I-Tyr11-SS-14 could be displaced with excess
unlabeled SS-14. Scatchard analysis of the binding data revealed a
single class of high-affinity binding sites, with an apparent
Kd ranging between 0.3 ± 0.1 and 3.1 ± 1.0 nM and a low maximum binding capacity (Bmax) ranging between 11 ± 1.1 and 59 ± 5.1 fmol/mg membrane protein
(Table 1). As a control for binding, rat brain cortex membranes were used. An example of saturation binding data with Scatchard analysis is
shown in Fig. 1.

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Fig. 1.
Binding of 125I-Tyr11-labeled
somatostatin (SS)-14 to a membrane homogenate preparation of human
thymocytes. , Specific binding (total minus nonspecific
binding in presence of 1 µM SS-14). Inset: Scatchard
analysis of the binding data [Kd, 0.4 nM and
maximal binding capacity (Bmax), 18.4 fmol/mg membrane
protein; no. 9, Table 1].
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SS and SSR subtype expression.
The bead separation method showed that thymocytes after cell counting
were >95% (
98% of rosetted cells), whereas CD14+ cells
were <5% among the filtered thymic cells, in all of the cases
examined. This finding was confirmed by FACS analysis (data not shown).
Moreover, the data are in agreement with other authors, who performed
this evaluation on a larger series of age- and sex-matched pediatric
thymuses (41). The percentage of thymocytes in each subgroup after bead separation ranged from 95.5 to 99.2%. By RT-PCR, sst2A and sst3 mRNA expression was detected in
freshly isolated thymocytes from four of four cases tested (nos.
5-8, Table 1), whereas mRNA encoding for SS,
sst1, sst4, and sst5 was absent (Fig. 2B). No mRNA encoding
for any SSR subtype was detectable in thymocytes after 7-14 days
of culture (data not shown), confirming our previous observation
(14). In CD14+ cells, the presence of only
sst2A mRNA was detected (Fig. 2C). RT-PCR of
thymocytes after separation into immature
CD2+CD3
and intermediate/mature
CD3+ fractions revealed sst2A and
sst3 mRNA expression in both subsets. Table
4 summarizes the results of RT-PCR
analysis, and an example is shown in Fig. 2. Quantitative
RT-PCR analysis revealed a higher number of sst3 mRNA
copies in the intermediate/mature CD3+ thymocyte fraction
compared with the immature CD2+CD3
one (Fig.
3A). Conversely, the number of
sst2A mRNA copies was higher in the immature
CD2+CD3
fraction compared with the mature
CD3+ thymocytes in three of four cases (Fig.
3B). The sst3-to-sst2A ratio
increased with the level of thymocyte maturation (Fig. 3C).

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Fig. 2.
Heterogeneous expression of SS and SS receptor (SSR)
subtype (sst1-5) mRNAs in human thymocytes and CD14
cells. Poly(A)+ mRNA was reverse transcribed, and cDNA was
amplified by PCR. PCR products of the sst1-5 were
separated on 1% agarose gel and stained with ethidium bromide. M,
100-bp ladder. A: control; B: freshly isolated
thymocytes; C: CD14+ cells (sample
no. 5, Table 1). RT-PCR analysis of each tissue was
performed at least twice with identical results.
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Table 4.
Heterogeneity of SS and SS receptor subtype mRNA expression in
different fractions of human thymocytes and CD14+ thymic
cells as determined by RT-PCR
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Fig. 3.
Quantitative SSR RT-PCR in human thymocytes. Quantitative
analysis of RT-PCR data showed a different amount of sst2A
and sst3 mRNA in immature CD2+CD3
and intermediate/mature CD3+ thymocytes calculated relative
to the amount of hypoxanthine-guanine phosphoribosyltransferase (HPRT)
and given in arbitrary units. A:
sst3/HPRT mRNA ratio; B:
sst2A/HPRT mRNA ratio; C:
sst3/sst2A mRNA ratio. Data derived from
4 different thymuses. sst2A assay: correlation coefficient
standard curve (R2) 0.980 ± 0.005, slope
of the curve 3.47 ± 0.11, (n = 3);
sst3 assay: correlation coefficient standard curve
(R2) 0.971 ± 0.0045, slope of the curve:
3.27 ± 0.15, (n = 3). Open bars, immature
CD2+CD3 ; filled bars, intermediate/mature
CD3+ thymocytes.
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Effect of SS and octreotide on [3H]thymidine
incorporation in thymocytes.
SS-14 significantly inhibited [3H]thymidine incorporation
in all five cultures (nos. 5-7, 10, and 11,
Table 1) of freshly isolated thymocytes (whole population) in a
dose-dependent manner. The inhibition was statistically significant at
a concentration of 10
6 (ranging between 45 and 77%) and
10
8 M (ranging between 27 and 41%) in all five cases at
a concentration of 10
10 M in four of five cases (ranging
between 21 and 64%) and at a concentration of 10
12 M in
3 of 5 (ranging between 26 and 42%) cases (Fig.
4, A-E). The SS analog
octreotide significantly inhibited [3H]thymidine
incorporation in only one culture of thymocytes (whole population) at
concentrations of 10
6 (43%) and 10
8 M
(23%) (Fig. 4, F-L). In the
CD2+CD3
thymocyte cultures, both SS-14 and
octreotide significantly inhibited [3H]thymidine
incorporation in the two cases evaluated (Fig.
5, A-D). The inhibition was
dose dependent and statistically significant at all concentrations
(except at the concentration of 10
13 M in one case).
Conversely, in the CD3+ thymocyte cultures, only SS-14
significantly inhibited [3H]thymidine incorporation in a
dose-dependent manner in both cases evaluated, whereas octreotide was
ineffective (Fig. 6, A-D).

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Fig. 4.
Effects of somatostatin (A-E) and octreotide
(F-J) on [3H]thymidine incorporation in
thymocyte cultures from 5 different thymuses (whole population).
Thymocytes were incubated in RPMI 1640 supplemented with 10%
heat-inactivated FCS, penicillin, and fungizone for 24 h in
quadruplicate without or with the drugs indicated at the concentrations
of 10 13, 10 12, 10 10,
10 8, and 10 6 M. Values are expressed as
counts/min and are means ± SE (n = 4 wells/treatment group). *P < 0.01 vs. control;
**P < 0.05 vs. control.
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Fig. 5.
Effects of somatostatin (A and B) and
octreotide (C and D) on
[3H]thymidine incorporation in isolated
CD2+CD3 thymocyte cultures from 2 different
thymuses. For culture conditions and legends see Fig. 4. Values are
means ± SE (n = 4 wells/treatment group; nos. 10 and 11, Table 1). *P < 0.01 vs. control; **P < 0.05 vs. control.
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Fig. 6.
Effects of somatostatin (A and B) and
octreotide (C and D) on
[3H]thymidine incorporation in isolated CD3+
thymocyte cultures from 2 different thymuses. For culture conditions
and legends see Fig. 4. Values are means ± SE
(n = 4 wells/treatment group; nos.
10 and 11, Table 1). *P < 0.01 vs.
control; **P < 0.05 vs. control.
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Study of apoptosis.
SS-14, but not octreotide, significantly increased the amount of
histone-associated DNA fragments, which are measurable after induced
cell death (apoptosis), in all of the early cultures of freshly
isolated and purified thymocytes (whole population). The number of
apoptotic cells was significantly higher at all concentrations in
one case (Fig. 7C) and at the
concentrations of 10
6, 10
7, and
10
8 M and at the concentrations of 10
6 and
10
7 M in the remaining two, respectively (Fig. 7,
A and B).

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Fig. 7.
Effects of somatostatin (A-C) and octreotide
(D-F) on apoptosis in early thymocyte cultures
(whole population). Thymocytes were incubated for 24 h without or
with the drugs indicated at the concentrations of 10 10,
10 9, 10 8, 10 7, and
10 6 M. Values are absorbance units and are expressed as
percent control (n = 3, triplicate determination on
cells obtained from nos. 11-13, Table 1).
*P < 0.05 vs. control.
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DISCUSSION |
The thymus is responsible for promoting the differentiation and
maturation of lymphoid precursor cells into mature T lymphocytes. The
developing T cells are embedded in an epithelial network known as the
thymic stroma. Other cells of hematopoietic origin participate in
constituting the complex architecture of this organ; these cells
include dendritic cells and thymic macrophages (8, 24). Interactions between thymic stromal cells and thymocytes are mediated by direct contact and via soluble factors and play a crucial role in T
cell development (2, 25). Among the soluble factors, neuropeptides have been demonstrated to be involved in the regulation of thymic functions. The intrathymic production of classical
hormones suggests that, in addition to the endocrine circuits,
paracrine/autocrine interactions may exist in the thymus, influencing
both the lymphoid and the stromal compartments of the organ
(32).
SS is a neuropeptide with a wide spectrum of actions (19).
The biological effects of SS are mediated via five specific, high-affinity, G protein-coupled receptors (20). The
presence of the neuropeptide and its receptors has been demonstrated in the human thymus (14, 29, 30). We (14) have
recently described the expression of sst1,
sst2A, and sst3 mRNAs in human thymic tissue.
Cultured TEC selectively expressed sst1 and
sst2A mRNA (14). sst2 mRNA has
been detected in murine resting thymocytes (9), in
contrast with the expression in the rat where these cells selectively
express sst3 and sst4 mRNAs (39).
These differences pointed to species-specific expression of SSR
subtypes in immune cells. Moreover, another study showed the presence
of sst2 mRNA in fresh rat thymocytes and demonstrated that
the activation of these cells upregulates the expression of
sst1 (33). It should be mentioned that many
extrinsic factors and changes in the microenvironmental conditions
might regulate the expression of SSR (3, 46). SS itself
could be involved in the regulation of receptor expression (5). This might explain why, although we found SS-binding
sites on freshly isolated thymocytes (11), in long-term
cultured thymocytes SSR mRNA was lost (14).
In the present study, using freshly isolated thymocytes, we first
demonstrated specific 125I-Tyr11-SS-14 binding
on thymocyte membrane homogenates. The number of SS-binding sites was
very low on these thymic cells, which are a heterogeneous population
mainly formed by intermediate/mature thymocytes (41, 44).
Subsequently, we characterized the SSR subtype expression by RT-PCR. In
the whole population of freshly isolated thymocytes, sst2A
and sst3 mRNA expression was detected, whereas in
thymocytes after 7-14 days of culture, no mRNA encoding for SSR
subtypes was detectable, confirming our previous findings (14). Because in freshly isolated thymocytes cells are
present at different levels of maturation, we investigated whether
sst2A and sst3 mRNA could be differentially
expressed in the diverse stages of maturation. We separated the whole
thymocyte population into intermediate/mature CD3+ and
immature CD2+CD3
fractions, and by RT-PCR we
detected sst2A and sst3 mRNA in both thymocyte
subpopulations. However, by quantitative RT-PCR analysis, we
demonstrated the predominant expression of sst3 mRNA in
CD3+ thymocytes. These cells represent the subset of
thymocytes that have reached a higher level of maturation during the
complex cascade of events occurring in the thymic network
(15). Interestingly, sst3 mRNA has been found
constitutively expressed in peripheral T lymphocytes, which directly
derive from mature thymocytes (16). Conversely, a
predominant expression of sst2A mRNA was found in the
CD2+CD3
thymocytes, which are the immature
fraction. The CD2+CD3
thymocytes form in the
developed thymus a very small but highly heterogeneous pool of cells,
whereas the CD3+ intermediate/mature cells represent the
major proportion of thymocytes (1). Most of these cells
are destined to die as a consequence of failing selection
(26). Cell death in the thymus occurs by a process known
as programmed cell death, or apoptosis, which is a common
feature in many developmental pathways (26). The sst3 expressed on these cells might be involved in
SS-mediated apoptosis (34). In fact, we have found
that SS-14, but not octreotide, which has a lower affinity for
sst3, may increase the amount of apoptotic cells
when incubated in human thymocyte cultures as a whole population.
Moreover, preliminary data indicated that the number of apoptotic
cells is significantly higher in the intermediate/mature CD3+ fraction, compared with the
CD2+CD3
one, when tested separately (Ferone
D, van Hagan PM, Lamberts SWJ, and Hofland LJ, unpublished
observations). It is intriguing that another synthetic SS analog has
been found to induce apoptosis in cultured human lymphocytes as
well (42). Moreover, it has recently been shown that
octreotide has a modulatory effect on anti-CD3 and
dexamethasone-induced apoptosis of murine thymocytes (43). However, because the immature
CD2+CD3
thymocytes are intensively
proliferating cells undergoing a rearrangement process, the predominant
presence of the sst2A on this very small subset suggests
the involvement of this SSR subtype in the early phase of thymocyte
development within the thymus. Furthermore, the data presented in this
study demonstrate that both SSR subtypes on human thymocyte subsets may
be activated upon binding with their own ligands. In fact, after the
administration of SS-14, we found an inhibition of
[3H]thymidine incorporation in all of the early cultures
of either whole population or isolated
CD2+CD3
and CD3+ thymocyte
fractions. Conversely, the inhibition of [3H]thymidine
incorporation by the SS analog octreotide occurred in only one of the
five cultures of whole thymocyte population and only at high
concentrations. On the contrary, octreotide administered in the
CD2+CD3
isolated thymocytes induced a
significant inhibition of [3H]thymidine incorporation in
a dose-dependent manner, whereas this SS analog was ineffective in
cultures of CD3+ isolated thymocyte fractions. This
evidence is in line with the higher affinity of this SS analog for
sst2A compared with that for sst3
(20). The possibility cannot be fully ruled out
that, at high concentration, octreotide may act via the
sst3. On the other hand, emerging data pointed to the role
of either sst2A or sst3 in promoting
apoptosis by p53-dependent and -independent mechanisms,
respectively (37). However, the intracellular pathways mediating the SS-dependent activities regulating cell growth, proliferation, and death might still be partially inactive in the
immature thymocyte fraction.
Finally, it is also noteworthy to mention that, in CD14+
cells, which are cells belonging to the monocyte-macrophage lineage, the presence of only sst2A mRNA was detected. This finding
is in agreement with our previous reports on the selective expression of this SSR subtype on human macrophages and monocytes (21, 38,
40). SSR are widely distributed within the human thymus on the
different cell subsets forming this organ. The stromal compartment
preferentially expresses sst1 and sst2A mRNA,
whereas lymphoid cells express mainly sst3 and, to a lesser
extent, sst2A mRNA. Preliminary observations seem to
confirm this evidence at the protein level. In fact, using polyclonal
antibodies, we have recently studied SSR subtype expression by
immunohistochemistry in the normal as well as in neoplastic thymic
tissues, where this pattern of receptor distribution is basically
maintained (12). However, in thymic tumor,
sst2A immunoreactivity has been found on the endothelium of
intratumoral vessel as well, whereas sst3 immunoreactivity
has been clearly observed on normal reactive thymocytes but also on
some tumor cells (10, 13). These data support the evidence
of strong compartmentalization of neuroendocrine peptide receptors in
lymphoid tissue (29), as it has also been shown for the
expression of vasoactive intestinal peptide (VIP) receptors on murine
and rat thymocytes (7). In fact, the two VIP receptors
display a distinct distribution in different thymocyte subsets,
suggesting that the expression of neuropeptide receptors could be
developmentally regulated and vice versa (7).
We have previously demonstrated (14) that SS mRNA is
present in the human thymus in TEC, whereas, as is shown in the present study, SS mRNA was undetectable in thymocytes. However, preliminary data from our group have shown that the SS-like peptide cortistatin-17 is highly expressed in human lymphoid cells, including thymocytes (6). This evidence suggests that SS produced by a subset
of TEC, but perhaps endogenous cortistatin produced by thymocytes as
well, may affect thymic cell populations in a paracrine and/or autocrine manner. Therefore, SS and SS-like peptides may participate in
regulating T cell differentiation and selection in the thymus.
In conclusion, the heterogeneous expression of SSR in different cell
subsets within the human thymus, together with the endogenous production of SS, SS-like peptides, and other neurohormones, emphasizes once more the pivotal role played by neuropeptide hormones in this
organ. The maturation and selection of the T cell repertoire are two of
the most intriguing processes and involve a number of factors. SS,
likely produced by TEC (14), seems to affect both the
lymphoid and the microenviromental compartments of the thymus. TEC are
known to drive the most important phases of T cell maturation and
differentiation; however, thymocytes might affect TEC functions as well
(23). Thus a bidirectional interaction pathway exists
between the two main cell components of the thymus, and SS might be
part of this complex circuit. Moreover, SS is known to affect the
production of immunoglobulins and interleukins, which are well
recognized factors participating in the sophisticated and elegant
process leading to the maturation of cellular immunity (28,
36). In this light, SS represents an important molecule involved
in the chain of events that results in the generation of the T cell repertoire.
 |
ACKNOWLEDGEMENTS |
We are grateful to A. J. J. C. Bogers (Dept. of
Cardiothoracic Surgery, Dijkzigt University Hospital, Rotterdam) for
providing thymic specimens, and to Piet Smaal (Audio-Visual Center) for assistance in preparing the illustrations.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. Ferone, Dept. of Endocrinological and Metabolic Sciences, Univ. of
Genova, Viale Benedetto XV, 6, 16132 Genoa, Italy (E-mail: ferone{at}unige.it).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 17, 2002;10.1152/ajpendo.00205.2001
Received 14 May 2001; accepted in final form 9 July 2002.
 |
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