Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells
Virgil A. S. H. Dalm,1
P. Martin van Hagen,1,2
Peter M. van Koetsveld,1
Sam Achilefu,3
Adriaan B. Houtsmuller,4
David H. J. Pols,2
Aart-Jan van der Lely,1
Steven W. J. Lamberts,1 and
Leo J. Hofland1
Departments of 1Internal Medicine,
2Immunology, and 4Pathology,
Josephine Nefkens Institute, Erasmus MC, 3015 GD Rotterdam, The Netherlands;
and 3Radiology Department, Washington University
School of Medicine, St. Louis, Missouri 63110
Submitted 3 February 2003
; accepted in final form 6 April 2003
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ABSTRACT
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Increasing evidence suggests that neuropeptides play a role in the
regulatory mechanisms between the neuroendocrine and immune systems. A
differential expression of the five known somatostatin (SS) receptors
(sst15) has been demonstrated in human immune cells and
tissues. However, little is known concerning regulation and expression of
sst15 and the peptide SS. Therefore, we investigated the
expression and the time-dependent regulation of sst15, SS,
and cortistatin (CST), a novel SS-like peptide, in human monocytes (MO),
monocyte-derived macrophages (MP), and dendritic cells (DC) in the basal and
lipopolysaccharide (LPS)-activated state. MO, MP, and DC selectively expressed
sst2 mRNA. SS mRNA was not detectable, whereas all samples
expressed CST mRNA. Expression levels of sst2 and CST mRNA showed
marked differences and were in the rank order of MP>>DC>>>MO.
LPS stimulation did not induce expression of SS or sst1,3,4,5.
However, sst2 mRNA expression was upregulated significantly by
stimulation with LPS. CST mRNA was upregulated as well. During differentiation
of MO in MP or DC, time-dependent, significantly increasing sst2
and CST mRNA levels were found. By confocal microscopy, the presence of
sst2 receptors was demonstrated on MP, but not on DC. This study
demonstrates for the first time a selective and inducible expression of the
recently discovered CST, as well as sst2, in human monocyte-derived
cells, suggesting a role for a CST-sst2 system rather than a
SS-sst2 system in these immune cell types.
cellular differentiation; cellular activation; neuropeptides; messenger ribonucleic acid
SOMATOSTATIN-14 AND somatostatin-28 are 14- and
28-amino acid neuropeptides, which are mainly produced in the central nervous
system, gastrointestinal tract, and endocrine glands
(40,
41). In these systems,
somatostatin (SS) has a predominant inhibitory action, especially with regard
to the release of mediators, such as hormones
(12,
13,
36). SS acts via G
protein-coupled seven-transmembrane receptors of which five different subtypes
have been cloned, named sst15
(35). In addition to their
role in endocrine tissues, it is hypothesized that neuropeptides might play a
regulatory role in the human immune system as well
(30,
32,
48). As a neuropeptide, a role
in the immune system might be ascribed to SS and its receptors
(30). In contrast to the human
immune system, the role of SS and its receptors has been studied extensively
in granulomas induced by Schistosoma Mansoni infection in the murine immune
system (57). Murine T
lymphocytes selectively expressed sst2
(11) and macrophages expressed
SS mRNA (56). Treatment of
Schistosoma Mansoni-infected mice with SS resulted in granuloma growth
inhibition (24). After these
studies in the murine immune system, the question was addressed whether SS and
sst might play a regulatory role in cells of the human immune system as well.
Monocytes and its functionally derived cells, i.e., macrophages and dendritic
cells, are important components in different pathways in the human immune
system. They play important roles in inflammatory responses by production of
both proinflammatory and immunosuppressive cytokines, antigen presentation,
and phagocytosis (8,
17,
29,
54). Previous studies have
demonstrated binding sites for SS on lymphocytes and monocytes
(9,
10), suggesting a role for sst
in these cells of the human immune system. Immunohistochemistry studies showed
that CD68-positive macrophages in human sarcoid granulomas expressed
sst2 (50).
Equivocal studies have reported on functions of SS on cells of the monocyte
lineage. Both inhibitory (7,
38) and stimulatory
(28) effects have been
described on cytokine production by macrophages. SS increased cytotoxicity of
macrophages against tumor cells as well
(37), and SS can act as an
antiangiogenic factor by inhibiting endothelial cell growth and monocyte
migration (4). However, little
is known about the expression and regulation of the different sst subtypes in
cells of the monocyte lineage, i.e., monocytes, macrophages, and dendritic
cells. Moreover, it is not known whether the natural ligand for the sst, e.g.,
SS, is expressed itself in cells of the monocyte lineage.
Therefore, we investigated in the present study mRNA expression of
sst15 and SS in monocytes and monocyte-derived macrophages
and dendritic cells. Moreover, we investigated the regulation of the
expression of the mRNA encoding an SS-like peptide, cortistatin (CST), that
was detected previously in cells and tissues from the human immune system
(19) and binds with high
affinity to all five sst (26)
and, therefore, might serve as an alternative ligand to sst. The sst and CST
mRNA levels were studied in monocytes, macrophages, and dendritic cells in
both basal and lipopolysaccharide (LPS)-stimulated conditions using a
TaqMan assay. To investigate possible regulation of mRNA levels,
expression was measured from day 1 to 6 of differentiation
of monocytes in both macrophages and dendritic cells. Expression of the sst
protein on cell membranes of macrophages and dendritic cells was studied using
FITC-labeled octreotate, a fluorescence-labeled sst2-selective
agonist, and visualized by confocal microscopy.
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MATERIALS AND METHODS
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Isolation of blood mononuclear cells. Peripheral blood mononuclear
cells (PBMC) were isolated from buffy coats (Sanquin Blood Bank, Rotterdam,
The Netherlands) by Ficoll (density 1.077 g/ml; Pharmacia, Uppsala, Sweden)
gradient centrifugation. Subsequently, monocytes were isolated from PBMC using
a Percoll (density 1.063 g/ml; Pharmacia) density gradient centrifugation as
described in detail previously
(27). Isolated monocytes were
frozen in 10% DMSO medium and stored in -80°C until use.
Cell culture. A frozen suspension of monocytes was thawed rapidly
in a water bath at 37°C, and viability was evaluated by Trypan blue
exclusion (Life Technologies, Grand Island, NY). Cell viability always
exceeded 95%. Cells were seeded at a density of 0.5 x 106
cells/cm2 in a volume of 1 ml/well in 24-well Nuncleon plates
(Nalge Nunc International) in RPMI 1640+ supplemented with 10%
heat-inactivated (30 min for 56°C) FCS (Life Technologies),
L-glutamine (2 mM; Life Technologies), and penicillin (1,000 U/ml;
Yamanouchi Pharma, Leiderdorp, The Netherlands). Plates were then incubated
for 90 min at 37°C to allow adherence of the monocytes to the plate.
Thereafter, plates were washed to remove potential contaminating T and B
cells. To generate macrophages, fresh medium was added containing granulocyte
macrophage colony-stimulating factor (GM-CSF, 500 U/ml; Novartis Pharma,
Arnhem, The Netherlands). The cells were cultured for 6 days, with medium
refreshment at day 3. To generate dendritic cells, the isolated
monocytes were incubated for 6 days with GM-CSF (500 U/ml) and interleukin
(IL)-4 (1,000 U/ml), with a medium refreshment at day 3. This method
of the generation of macrophages and dendritic cells has been described
previously in detail (47).
To study the regulation of sst2, SS, and CST mRNA expression,
monocytes, 6-day macrophages, or 6-day dendritic cells were incubated during
24 h without or with LPS (2 µg/ml; Sigma Aldrich, Zwijndrecht, The
Netherlands). To study the time course of mRNA expression during
differentiation of monocytes into macrophages and dendritic cells, cells were
collected at day 1, 2, 3, 4, 5, or 6 of culture without or
with a prior 24-h incubation with LPS (2 µg/ml). A schematic overview of
the experimental setup is shown in Fig.
1. At the end of each incubation period, cells were collected and
used for both PCR and fluorescence-activated cell sorter (FACS) analysis.

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Fig. 1. Experimental setup of monocyte culture and activation. Isolated monocytes
were allowed to differentiate into macrophages and dendritic cells in 6-day
cultures. Arrows indicate the different time points at which cells were
collected for RT-PCR analysis. In the experiments in which cells were
incubated with lipopolysaccharide (LPS), arrows indicate the time points at
which cells were collected after a prior incubation for 24 h with LPS. GM-CSF,
granulocyte macrophage colony-stimulating factor; IL, interleukin.
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FACS analysis. By FACS analysis, evaluating the CD-marker pattern
of the cells, the phenotypes of the cultured cells were confirmed. Cells were
collected using cell scrapers and were centrifuged at 300 g for 5
min. Supernatant was removed, and 25 µl of the diluted monoclonal antibody
were added to each cell pellet. The monoclonal antibodies we used included
FITC- and phycoerythrin (PE)-labeled antibodies purchased from
Becton-Dickinson (San Jose, CA), CD1a-PE (diluted 1:250), CD14-PE (1:400),
CD26-PE (1:120), CD68-FITC (1:10), CD71-FITC (1:10), CD80-PE (1:50), CD86-FITC
(1:100), and human leukocyte antigen class II-PE (1:20). Cells were incubated
for 15 min at room temperature. Cells were then washed two times with PBS-0.5%
BSA and resuspended in 200 µl PBS-0.5% BSA followed by FACS analysis on a
FACSCalibur-FACS (Becton-Dickinson).
RT-PCR studies. RT-PCR was performed as described previously
(25). Briefly,
poly(A)+ mRNA was isolated using Dynabeads oligo(dT)25
(Dynal, Oslo, Norway) from tissue samples and cells. cDNA was synthesized
using the poly(A)+ mRNA, which was eluted from the beads in 40
µl H2O for 10 min at 65°C using
oligo(dT)1218 primer (Life Technologies). One-twentieth of
the cDNA library was used for each amplification by PCR using primer sets
specific for human SS, sst15, CST, and
hypoxanthine-phosphoribosyltransferase (HPRT) as a control (see
Table 1). The primer set used
to detect CST mRNA was adapted from Ejeskar et al.
(22). As positive controls for
SS, CST, and HPRT, cDNA of human brain RNA (Invitrogen, Groningen, The
Netherlands) was used. As positive control for sst15, DNA of
a B-lymphoblastoid BSM cell line (an epstein-barr virus-transformed B cell
line) was used. The PCR reaction was carried out in a DNA thermal cycler with
a heated lid (Applied Biosystems, Nieuwerkerk a/d Ijssel, 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 60°C, and extension for 1 min at 72°C. After a final extension
for 10 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. The identity of the products was confirmed by direct sequencing using
an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) according to the
manufacturer's protocol.
Quantitative PCR. To quantify sst2 and CST mRNAs, a
quantitative PCR was performed by TaqMan Gold nuclease assay (Perkin
Elmer, Foster City, CA) and the ABI PRISM 7700 sequence Detection System
(Perkin Elmer) for real-time amplifications, according to the manufacturer's
protocol. Quantitative real-time PCR (Q-PCR) was performed for sst2
only, because no expression of the other sst subtypes was detected in the
cells we investigated. The primer sequences that were used included:
sst2 forward, 5'-ATGCCAAGATGAAGACCATCAC-3';
sst2 reverse, 5'-TGAACTGATTGATGCCATCCA-3'; CST forward,
5'-GGAGAGAAGCTCCAGTCAGC-3'; CST reverse,
5'-GGTCCACTCAAACCACCAA-3'; HPRT forward,
5'-TGCTTTCCTTGGTCAGGCAGTAT-3'; and HPRT reverse,
5'-TCAAATCCAACAAAGTCTGGCTTATATC-3'. The probe sequences that were
used included: sst2,
5'-FAM-TGGCTCTGGTCCACTGGCCCTTTG-TAMRA-3'; CST,
5'-FAM-TATGCTCGCTGTCTCGGCCG-TAMRA-3'; and HPRT,
5'-FAM-CAAGCTTGCGACCTTGACCATCTTTGGA-TAMRA-3'.
The relative amount of sst2 mRNA was determined using a standard
curve generated from known amounts of human genomic DNA. For the RT reaction,
50 ng/µl total RNA was used in every reaction. For determination of HPRT
mRNA, a standard curve was generated of cDNA obtained from a DU45 (prostate
cancer) cell line. The amount of sst2 mRNA was calculated relative
to the amount of HPRT and is given in arbitrary units.
Statistical analysis. Data were analyzed statistically using SPSS
for Windows, release 9.0 (SPSS, Chicago, IL). The differences in expression
levels of sst2 and CST mRNA in the stimulation experiments were
determined using one-way ANOVA. When significant overall effects were obtained
by ANOVA, multiple comparisons were made using the Newman-Keuls test.
Synthesis of octreotate-FITC conjugate. The peptide was prepared
on solid support by standard automated fluorenylmethoxycarbonyl (Fmoc)
procedures (6). The synthesis
was performed on a 25-µmol scale starting with Fmoc threonine preloaded
Wang resin. To avoid inadvertent labeling of the
-K5 amino
group that is necessary for receptor binding, this amino acid was protected as
Fmoc Lys(ivDde)-OH, where ivDde is
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl
(1). Activation of the carboxyl
group and coupling of subsequent amino acids (75 µmol) were performed in
situ by using N-hydroxylbenzotriazole (2 M) and 2-(1-H
benzotriazole-1-yl)-1,1,1,3-tetramethyluronium hexafluorophosphate (2 M).
Intramolecular cyclization of the acetamidomethyl-protected dithiol groups of
cysteine was accomplished by adding thallium trifluoroacetate (23 mg, 42
µmol) in anhydrous dimethylformamide (DMF; 1 ml). Cleavage of the peptide
from the resin and concomitant removal of the side-chain protecting groups
were performed with 85% trifluoroacetate, 5% H2O, 5% phenol, and 5%
thioanisole for 4 h at room temperature. The crude peptide was precipitated
with cold t-butylmethyl ether and lyophilized in
H2O/acetonitrile (3:2). Without further purification, FITC (12 mg,
28 µmol) and NaHCO3 (10 mg, 120 µmol) were added to a
solution of the crude peptide in 2 ml DMF and stirred for 12 h. The mixture
was filtered, and the filtrate was treated with a 1-ml solution of 2%
hydrazine in DMF for 20 min to remove the ivDde. The resulting mixture was
poured in cold methyl tertiary butyl ether to precipitate the conjugate, which
was lyophilized and purified by HPLC and characterized by liquid
chromatography-mass spectrometry. HPLC purity of the final compound was
>99.5%.
By autoradiography, using rat brain tissue, binding affinity of the
octreotate-FITC compound was evaluated. Octreotate-FITC displaced binding of
[125I-Tyr3]octreotide with relative high affinity
(IC50 = 5.5 x 10-8 M).
Visualization of sst2 receptors on human
macrophages. Monocytes were cultured for 6 days on round coverslips
(diameter 24 mm; Omnilabo) in the presence of GM-CSF to obtain macrophages or
in the presence of both GM-CSF and IL-4 to obtain dendritic cells. After 6
days, cells were incubated with 20 nM octreotate-FITC, which binds to
sst2 selectively
(43), to determine binding to
sst2 receptors and internalization of the receptor-ligand complex
using confocal microscopy (LSM 410; Carl Zeiss Instruments, Jena, Germany). A
x40/1.4 numerical aperture objective lens was used. FITC was excited
with a 488-nm Ar laser, and fluorescence emission was detected using a 515-
and 540-nm bandpass filter. Images were typically taken at a x4 zoom. As
a positive control, a stably sst2-transfected cell line (CC2B) was
used. To determine specificity of the binding of octreotate-FITC, binding was
displaced by excess unlabeled octreotide.
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RESULTS
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By FACS analysis, the phenotypes of the isolated monocytes, cultured
macrophages, and dendritic cells were confirmed. In
Table 2, the mean percentages
of positively stained cells are shown. Monocytes showed low expression of CD80
compared with macrophages (at day 6) and dendritic cells (at day
6). CD80 is upregulated in macrophages, with a peak at day 3 of
culture, and declined thereafter, whereas expression of CD80 in dendritic
cells was upregulated and remained stable
(47). Dendritic cells express
lower CD68, CD71, and CD86 compared with macrophages. This has been described
previously (18), although also
higher expression levels of CD71 expression have been reported
(55). Dendritic cells show a
high expression of CD1a, which is known as a dendritic cell marker
(45), together with the
finding that addition of IL-4 to culture results in cells becoming nonadhesive
(47).
By RT-PCR, as presented in Fig.
2, top, a selective expression of sst2 mRNA
was found in monocytes and in in vitro cultured macrophages and dendritic
cells. Activation of these cells with 2 µg/ml LPS did not result in a
different expression pattern of the sst subtypes
(Fig. 2, bottom). At
lower concentrations of LPS (range 10 ng/ml-1 µg/ml), sst2 mRNA
was expressed selectively as well (data not shown). The expression of SS mRNA
itself could not be detected in any of these cell types, neither in the basal
nor in the LPS-activated state (Fig.
3). On the other hand, we detected the expression of the mRNA
encoding a recently discovered SS-like peptide, i.e., CST, in monocytes,
macrophages, and dendritic cells. As a control, human thymic tissue was used,
which expresses both SS and CST mRNA (Fig.
3). To evaluate the quantitative expression of the different mRNAs
found by RT-PCR, Q-PCR was used for detection of sst2 and CST mRNA
levels. We detected relative low expression levels of sst2 mRNA in
cells under basal conditions (macrophages and dendritic cells showed an
10-fold higher sst2 mRNA expression compared with monocytes,
P < 0.001), whereas sst2 mRNA expression was
significantly upregulated when cells were stimulated with 2 µg/ml LPS
(Fig. 4). In experiments using
increasing concentrations of LPS (range 10 ng/ml-2 µg/ml), 2 µg/ml LPS
was shown to give maximal induction of sst2 mRNA expression (data
not shown). As shown, LPS induced an
40 (P < 0.0001)-, 200
(P < 0.0001)-, and 20 (P < 0.0001)-fold increase in
sst2 expression in activated monocytes, macrophages, and dendritic
cells, respectively. Whereas RT-PCR demonstrated that SS mRNA was absent after
stimulation of the cells with LPS, expression of the CST mRNA as a possible
alternative ligand for sst2 in these cells was upregulated upon LPS
activation (Table 3). CST mRNA
was upregulated significantly by
2 (P = 0.002)-, 3 (P
< 0.0001)-, and 10 (P < 0.0001)-fold in activated monocytes,
macrophages, and dendritic cells, respectively. To further investigate the
upregulation of both sst2 and CST mRNA by LPS stimulation in
monocytes and mature macrophages and dendritic cells, expression and possible
regulation of both mRNA levels were studied during differentiation of
monocytes into its derived cells. Cells were collected from day 1 to
6 of culture to measure sst2 and CST mRNA levels. During
differentiation of monocytes into macrophages and dendritic cells, cells were
incubated without or with LPS for 24 h at days 1, 2, 3, 4, 5, or
6 of culture. The expression levels of both sst2 and CST
mRNA were measured during this time-dependent differentiation of monocytes
into macrophages and dendritic cells. In unstimulated differentiating cells,
using Q-PCR, no expression of sst2 mRNA could be detected in
macrophages and dendritic cells until day 6 (data not shown). In the
LPS stimulation experiments, sst2 mRNA expression was detected in
dendritic cells only at days 5 (P < 0.0001) and
6, whereas sst2 mRNA expression in macrophages was low and
relatively constant at days 14, with a significant increase at
days 5 and 6 (P < 0.0001 vs. day 4).
This is shown in Fig. 5. In
contrast to the time-dependent basal sst2 mRNA expression, we could
detect sst2 mRNA expression in LPS-stimulated macrophages at
days 15 of culture, although it was very low. However, in
LPS-stimulated dendritic cells, we could not detect sst2 mRNA
expression at days 14. This finding is in accordance with the
much lower expression of sst2 mRNA in dendritic cells in both basal
(data not shown) and LPS-stimulated conditions
(Fig. 5) when compared with
macrophages and suggests that sst2 mRNA expression levels in LPS
stimulated dendritic cells are below assay detection limits, rather than
dendritic cells do not respond to the LPS stimulation. In unstimulated cells,
expression of CST mRNA in macrophages increased after day 1 and
reached its maximum already at day 2 (P = 0.01;
Fig. 6). Dendritic cells showed
maximal expression of CST mRNA already at day 1. LPS-induced CST mRNA
expression during differentiation of macrophages was already maximal at
day 1 of culture. In LPS-activated dendritic cells, CST mRNA
expression was low at day 1, two-thirds of maximum at day 2
(P < 0.01), and reached its maximum at day 3
(Fig. 6, P < 0.01
compared with CST mRNA expression at day 1).

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Fig. 2. Somatostatin receptor (sst) subtype mRNA expression in monocytes, 6-day
macrophages, and 6-day dendritic cells. Poly(A)+ mRNA was prepared
from human monocytes, macrophages, and dendritic cells cultured in vitro. cDNA
was synthesized and amplified using primers specific for the 5 different sst
subtypes. Top: sst mRNA expression in unstimulated monocytes, 6-day
macrophages, and 6-day dendritic cells. Bottom: sst mRNA expression
in monocytes, 6-day macrophages, and 6-day dendritic cells after a 24-h
stimulation with 2 µg/ml LPS. M, 100-bp DNA ladder.
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Fig. 3. Expression of somatostatin (SS) and cortistatin (CST) mRNA in monocytes,
6-day macrophages, and 6-day dendritic cells. Experiment was repeated 4 times.
Top: expression in unstimulated cells. Bottom: expression in
cells after a 24-h incubation with 2 µg/ml LPS. Human thymic tissue was
used as a positive control for both SS and CST mRNA expression. M, 100-bp DNA
ladder.
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Fig. 4. Regulation of sst2 mRNA expression in monocytes, macrophages,
and dendritic cells. Monocytes were allowed to differentiate into macrophages
or dendritic cells and were incubated for 24 h with LPS at day 6 of
culture. Bars represent sst2 mRNA expression in unstimulated (-)
and LPS-stimulated (+) cells, given in arbitrary units, relative to a
generated standard curve from a Jurkat (T cell) cell line and adjusted for
hypoxanthine-phosphoribosyltransferase (HPRT) expression. P <
0.0001 compared with cells in unactivated state (*) and with
monocytes (#). Results are the means of 3 independent experiments using cells
from 3 different healthy donors.
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Fig. 5. Time-dependent increased sst2 mRNA expression in monocytes
during differentiation into macrophages and dendritic cells after stimulation
with LPS (2 µg/ml) at the different time points. Bars represent
sst2 mRNA expression in macrophages (A) and dendritic
cells (B) at day 1, 2, 3, 4, 5, or 6 of culture
after a prior incubation for 24 h with LPS, given in arbitrary units, relative
to a generated standard curve from a Jurkat (T cell) cell line and adjusted
for HPRT expression. *P < 0.0001 compared with
expression of sst2 mRNA at day 4. +P
< 0.01 compared with expression of sst2 at day 5.
Results are the means of 3 independent experiments using cells from 3
different healthy donors.
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Fig. 6. CST mRNA expression in monocytes during differentiation into macrophages
and dendritic cells. All bars represent CST mRNA expression given in arbitrary
units generated to a standard curve from a RAJI (B) cell line as results of
quantitative RT-PCR using 50 ng/µl total RNA in the RT reaction/sample.
A: CST mRNA expression in unstimulated macrophages. P = 0.01
compared with expression at previous day (*) and compared with
expression at day 1 (+). B: CST mRNA expression in
macrophages at day 1, 2, 3, 4, 5,or 6 of culture after a
prior incubation for 24 h with LPS. C: CST mRNA expression in
unstimulated dendritic cells. D: CST mRNA expression in dendritic
cells at day 1, 2, 3, 4, 5, or 6 of culture after a prior
incubation for 24 h with LPS. P < 0.01 compared with expression at
previous day (*) and with expression at day 1 (+). Results
are means from 2 experiments.
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To demonstrate that sst2 receptors were actually expressed on
the cultured cells, macrophages and dendritic cells were incubated with the
FITC-labeled octreotate and visualized using a LSM410 confocal laser
microscope (Carl Zeiss). Unstimulated macrophages and dendritic cells did not
show a positive signal when incubated with octreotate-FITC (data not shown).
On the other hand, LPS-stimulated macrophages showed binding of the
fluorescent SS analog, as shown by the spots in
Fig. 7B. During the
first minutes of incubation, a positive signal was detected at the cell
membrane (data not shown). After the experiment in time, positive signals were
also found inside the cells because of internalization of the receptor-ligand
complex. When macrophages were incubated with both octreotate-FITC and a high
amount of unlabeled octreotide (10-6 M), no fluorescent
signal could be detected (Fig.
7D), demonstrating specificity of binding of
octreotate-FITC to the sst2 receptors. LPS-stimulated dendritic
cells did not stain positive when incubated with octreotate-FITC. This is in
concordance with the data found with Q-PCR in which we described an
10-fold lower expression level of sst2 mRNA in LPS-stimulated
dendritic cells.

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Fig. 7. Visualization of sst2 receptors on LPS-stimulated macrophages.
LPS-stimulated macrophages were incubated with FITC-labeled octreotate, a
sst2-selective analog, and visualized by confocal microscopy.
A and B: visualization of macrophages incubated with 20 nM
FITC-octreotate. A: transmitted light image (arrows indicate
macrophages). B: fluorescence signal of octreotate-FITC accumulated
inside the macrophages, showing membrane binding and subsequent
internalization of the labeled compound. C and D:
macrophages incubated with FITC-octreotate and an excess of unlabeled
octreotide. C: transmitted light image. D: in the
fluorescence channel, no signal was detected in the cells. Scale bar, 20
µm.
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DISCUSSION
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SS-14 and SS-28 are neuropeptides that play an important role in inhibition
of hormone release. SS also functions as a neurotransmitter, immunomodulator,
and suppressor of angiogenesis and cell proliferation
(15,
16,
34,
3941).
Neuropeptides are involved in the interactions that exist between the human
neuroendocrine and immune system
(32,
48). A role might be ascribed
to SS and its receptors in this network as well. The ssts have been
demonstrated in various endocrine and lymphoid tissues by classical
ligand-binding studies (31,
42,
44). The role of SS and its
receptors in the human immune system is still unclear. In contrast to the
human immune system, the role of SS and sst in the murine immune system has
been studied extensively by Weinstock and Elliott
(57). In their studies, the
expression and role of SS and sst in a model of mice infected with a
Schistosoma Mansoni infection were evaluated. In granulomas formed after this
Schistosoma infection, T lymphocytes expressed sst2
(11), whereas macrophages in
these granulomas expressed SS mRNA
(56). Stimulation of murine
splenic macrophages with IFN-
induced upregulation of the SS mRNA
within 4 h (23). Treatment of
Schistosoma-infected mice with SS resulted in granuloma growth inhibition by
decreasing the IFN-
production by T cells. Moreover, in rat lymphoid
tissues, SS is expressed (2)
and rat T and B lymphocytes from the thymus and spleen synthesize and secrete
SS (3). In the human immune
system, the role of SS and sst and the regulation of their expression still
remain unclear. It is not known whether SS plays a regulatory role in the
human immune system and whether this is comparable to the situation in the
experimental rodent models. Cells of the monocyte lineage, i.e., monocytes,
macrophages, and dendritic cells, are known to be an important component in
the human immune system (8,
17,
29,
54).
Therefore, in the present study, we investigated by RT-PCR the expression
and regulation of the mRNA levels encoding the five known sst and SS in cells
of the human monocyte lineage. We detected expression of only sst2
mRNA in monocytes, macrophages, and dendritic cells. Freshly Ficoll
density-gradient isolated monocytes showed no expression of sst2
mRNA (Ligtenauer-Kaligis EGR, Dalm VASH, Oomen SPMA, Mooij DM, van Hagan PM,
Lamberts SWJ, and Hofland LJ, unpublished observations). When monocytes were
subsequently isolated from PBMC by Percoll density-gradient centrifugation,
cells were already activated and expressed sst2 mRNA, explaining
the expression of sst2 mRNA in our Percoll-Ficoll isolated
monocytes. In addition, LPS-activated macrophages and dendritic cells were
studied. LPS was chosen as an activating factor because it is known as an
activator of dendritic cells, since it induced maturation and migration
(21,
46), and LPS effectively
activates macrophages and induces tumoricidal activity
(5,
14). When cells were activated
with LPS, no expression of other sst subtypes could be detected, suggesting a
selective role for sst2 in these cells. Because SS itself has a
very short half-life, it is expected that SS to which the sst-bearing cells
respond is produced locally, probably by the sst-positive cells itself.
However, despite expression of sst2, no expression of the SS mRNA
itself could be detected in any of the cell samples tested. Even after
activation of the cells with LPS, no SS mRNA could be detected, ruling out the
possible autocrine role of SS in the human immune system, in contrast to the
rodent immune systems. Interestingly, we found the expression of the mRNA
encoding CST, a recently discovered SS-like peptide in all three cell types
that were evaluated. CST is a 17-amino-acid peptide that shows structural
resemblance to SS (20) and has
high binding affinity to the five known ssts
(26). In one preliminary
report, the expression of CST mRNA has been demonstrated in different human
tissues, including leukocytes
(22). Previously, we
demonstrated a selective expression of CST mRNA in cells and tissues of the
human immune system, while no expression of SS mRNA was detected
(19).
In the present study, we mainly focused on the expression and regulation of
the five ssts in cells of the monocyte lineage. As shown, only the ssts mRNA
could be detected by RT-PCR, in both activated and nonactivated cells. A
previous study showed that sst2 expression in leukocytes was
upregulated after phytohemagglutinin stimulation
(52). In this study, however,
the regulation of sst2 expression in cells of the monocyte lineage
was not addressed. To investigate this regulation, monocytes were cultured in
macrophages or dendritic cells, as schematically shown in
Fig. 1, and expression levels
of sst2 mRNA were measured using Q-PCR. Under basal conditions,
expression of sst2 was very low. Macrophages and dendritic cells
expressed
10-fold higher sst2 mRNA levels than monocytes. In
LPS-stimulated cells, expression of sst2 mRNA was upregulated
significantly, suggesting a potential role of the sst2 in more
mature and activated cells. We demonstrated that sst2 mRNA is
upregulated relatively late during culture. In this respect, the dramatic rise
in sst2 mRNA between days 4 and 5 of culture is
obvious. We observed this dramatic rise during maturation of monocytes into
dendritic cells, as well as into macrophages. Therefore, we hypothesize that
sst2 expression only reaches significant levels after full
maturation of monocytes, irrespective of the direction of differentiation. In
the murine immune system, it has been demonstrated that sst2 plays
an important role in granuloma formation in Schistosoma-infected mice and that
SS treatment inhibited granuloma growth through binding to sst2 on
T cells and a subsequent decline of IFN-
production by these T cells
(11,
24). The question is now
addressed whether sst2 also plays a role in the human immune
system. In previous studies, sst2 expression was detected in
granulomatous diseases as sarcoid granulomas and rheumatoid arthritis
(51,
53). Recently, in a
preliminary study, 10 patients with rheumatoid arthritis have been treated
with the long-acting SS analog, octreotide. Significant clinical improvement
was found in these patients
(33). In one study, two
patients with sarcoidosis were treated with octreotide, an
sst2-selective SS analog
(50). One patient responded
very well, whereas the second showed no clinical response. These results, in
combination with the expression of sst in cells of the monocyte lineage,
indicate that SS or its analog octreotide might have clinical significance in
diseases affecting the human immune system, acting via the
sst2-expressing macrophages or dendritic cells. As we demonstrated
expression of sst2 mRNA in cells of the monocyte lineage and no
expression of SS mRNA itself, we also investigated the expression of the mRNA
encoding CST as a possible autocrine ligand for the sst2, which has
been proposed for SS in the rodent immune system
(11,
24,
56,
57). As previously reported
(19), we found CST mRNA
expression in all cell samples. Therefore, we evaluated the regulation of CST
mRNA during differentiation of monocytes and by LPS stimulation.
Monocytes showed low expression levels of CST mRNA, which were considerably
upregulated in macrophages and dendritic cells. LPS activation of these cells
resulted in a significant upregulation of the CST mRNA levels as well. This
upregulation during differentiation and by LPS activation suggests a possible
role for CST in these human immune cells, especially in the mature cells and
in cells in the activated state. CST mRNA expression was also measured during
differentiation of monocytes under basal and LPS-stimulated conditions.
Overall, CST mRNA expression was upregulated earlier during differentiation,
both in basal and LPS-stimulated conditions, than sst2 mRNA
expression. Whereas an autocrine SS-sst2 regulatory pathway has
been described in the murine immune system, the absence of SS and presence of
CST mRNA suggest a more likely autocrine CST-sst2 pathway in the
human immune system. However, different lymphoid organs are innervated by
nerves of the sympathetic nervous system and by sensory nerves containing
neuropeptides such as SS (49).
SS from these nerves might interact with the sst present in the human immune
tissue, explaining the absence of SS itself in the immune cells. Moreover, CST
is expressed in neurons as well. It may not be ruled out that CST as well can
reach the sst via this route. The presence of CST mRNA in the immune cells
itself, however, suggests an autocrine role of CST and the sst in addition to
a possible paracrine role of CST from the neurons. The presence of functional
sst2-binding sites on membranes of the cells, both activated and
nonactivated, was investigated by confocal laser microscopy using the new
compound, FITC-labeled octreotate. By autoradiography, we confirmed that
FITC-octreotate was able to displace binding of
[125I-Tyr3]octreotide to sst2 in rat brain
tissue with relatively high affinity. In LPS-stimulated dendritic cells, no
binding of FITC-octreotate to the sst2 could be visualized. These
findings are in concordance with the relatively low expression levels of
sst2 mRNA in these cells. On the other hand, on LPS-stimulated
macrophages, expression of the sst2 protein was visualized, and the
binding of FITC-octreotate was displaced successfully when cells were also
incubated with an excess amount of unlabeled octreotide, demonstrating
specificity of binding. Thus sst2 receptor levels on unstimulated
cells and in LPS-activated dendritic cells are probably below the detection
level of our assay. Unfortunately, at this moment, we are unable to detect the
protein for CST in the cells expressing CST mRNA. However, the expression of
the mRNA and, moreover, the regulation of this expression during
differentiation and by activation suggest that CST indeed may play a role in
the human immune system. Further functional studies should be performed to
elucidate the possible effects of CST on these cells.
In conclusion, we investigated the mRNA expression of the five known ssts
in cells of the monocyte lineage, as well as its endogenous ligand SS and an
alternative ligand CST. In monocytes, macrophages, and dendritic cells, only
expresion of sst2 mRNA could be detected, whereas no SS mRNA was
expressed. Interestingly, CST mRNA was expressed in all cells.
When cells were stimulated with LPS, sst2 expression was
upregulated significantly, pointing to a possible role of at least these
receptors in the human immune system. These receptors might show interaction
with CST in the immune system, but it is also possible that SS reaches the
place of inflammation by nerve endings, in which SS is produced. During
differentiation of monocytes into macrophages and dendritic cells, both
sst2 and CST mRNA levels were upregulated as well, pointing to a
more important role of the sst2-CST system in more mature cells.
Interestingly, it seemed that CST mRNA was upregulated earlier during
differentiation than sst2 mRNA. Finally, the expression of
sst2 has been visualized on membranes of LPS-activated macrophages
but not on unstimulated cells and LPS-activated dendritic cells, in
concordance with the lower expression levels of sst2 mRNA found
with QPCR. The functional significance of the expression of CST and
sst2 in these cells needs to be further investigated.
 |
DISCLOSURES
|
---|
This research project was supported by Grant 90343092 from
the Dutch Organization for Scientific Research (Nederlandse organisatie voor
Wetenschappelijk Onderzoek).
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: L. J. Hofland, Dept. of
Internal Medicine, Rm. Bd 240, Erasmus MC, Dr. Molewaterplein 40, 3015 GD,
Rotterdam, The Netherlands (E-mail:
l.hofland{at}erasmusmc.nl).
Submitted 3 February 2003
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.
 |
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