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Originally published online as doi:10.1189/jlb.0606417 on December 5, 2006

Published online before print December 5, 2006
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(Journal of Leukocyte Biology. 2007;81:845-855.)
© 2007 by Society for Leukocyte Biology

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

Chiara Armani*, Elisabetta Catalani{dagger}, Alberto Balbarini*, Paola Bagnoli{ddagger} and Davide Cervia{dagger},{ddagger},1

* Cardiac Thoracic and Vascular Department, University of Pisa, Pisa, Italy;
{dagger} Department of Environmental Sciences, University of Tuscia, Viterbo, Italy; and
{ddagger} Department of Biology, Unit of General Physiology, University of Pisa, Pisa, Italy

1 Correspondence: Department of Environmental Sciences, University of Tuscia, Largo dell’Università snc, Blocco D, 01100 Viterbo, Italy. E-mail: d.cervia{at}unitus.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Somatostatin (SRIF)-14 is recognized as an important mediator between the nervous and the immune system, although the functional role of its receptors (sst1–sst5) is poorly understood in humans. In our study, we demonstrate that human macrophages, differentiated from PBMC-derived monocytes, express sst1 and sst2 mRNAs. sst1 and sst2 are mostly localized at the cell surface and display active binding sites. In particular, sst1/sst2 activation results in a weak internalization of sst1, and the sst2 internalization appears more efficient. At the functional level, the activation of SRIF receptors by the multiligand analogs SOM230 and KE108, but not by SRIF-14 or cortistatin-14, reduces macrophage viability. Their effects are mimicked by the selective activation of sst1 and sst2 using CH-275 and SMS 201-995/L-779,976, respectively. Further, sst1- and sst2-mediated effects are reversed by the sst1 antagonist SRA-880 or the sst2 antagonist CYN 154806, respectively. CH-275, SMS 201-995, and L-779,976, but not SRIF-14, decrease mRNA expression and secretion of the MCP-1. In addition, SRIF-14, CH-275, SMS 201-995, and L-779,976 decrease IL-8 secretion, and they do not affect IL-8 mRNA expression. In contrast, SRIF-14 and sst1/sst2 agonists do not affect the secretion of matrix metalloproteinase-9. Collectively, our results suggest that the SRIF system, through sst1 and sst2, exerts mainly an immunosuppressive effect in human macrophages and may, therefore, represent a therapeutic window that can be exploited for the development of new strategies in pharmacological therapy of inflammation.

Key Words: neuropeptides • monocytes • trafficking • cell viability • chemokines • metalloproteinases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The complex network of communication between the nervous and the immune system has become the subject of intense research in recent years. To maintain the function of the neuroimmune circuitry, cells in both systems produce diverse chemical messengers, which are involved in the regulation of immunological and inflammatory responses [1 ]. In particular, cytokines and neuropeptides, which exist in the nervous and immune system, are potential mediators of cross-talk between the two systems. There is evidence to suggest that the circulating neuropeptide somatostatin (SRIF)-14, acting through its specific G protein-coupled receptors (sst1–5) [2 , 3 ], is one such mediator and may represent an important regulator of the immune response [4 , 5 ]. For example, SRIF-14 has potent, modulatory actions on the release of Igs and cytokines. Furthermore, the peptide is also involved in inhibition of chemotaxis, phagocytic activity, and the NK cell activity of immune cells. In this respect, the synthetic SRIF analog TT232 has recently become a therapeutic candidate with anti-inflammatory indications and is currently under evaluation in clinical trials [6 ].

Monocytes and macrophages are recognized as important components of innate and acquired immunity. In rodent cells of the monocyte lineage, the inhibitory role of SRIF-14 has been studied extensively [7 8 9 10 11 12 13 14 ]. However, in human monocytes, the role of SRIF-14 is less well-understood, and reported results have been controversial [5 , 15 ]. Human monocytes derived from PBMC do not express mRNA for SRIF-14 [16 17 18 ], and they do express the mRNA for cortistatin [16 , 17 ], a SRIF-14-like peptide, which may serve as an alternative ligand for SRIF receptors in the immune system [19 , 20 ]. The presence of SRIF receptors in monocytes has been suggested from binding studies [4 , 5 ]. In addition, sst2 mRNA has been observed in PBMC-derived monocytes [17 ]; however, this expression was undetectable under basal conditions and required cell activation to up-regulate the mRNA to a level that could be detected [15 ]. Circulating monocytes are versatile precursor cells with the ability to differentiate into various forms of specialized macrophages [21 , 22 ], a process that is influenced profoundly by the cytokine milieu. The functional activation of macrophages can occur by "classical" or "alternative" activators, resulting in functionally polarized cells [23 , 24 ]. The activation of macrophages by LPS is considered a classical route of activation and leads to macrophages that have immunostimulatory properties, cytotoxic function against tumor cells, and bactericidal activity, thus defending the host against pathogenic infections. In particular, these macrophages play physiological roles by producing proinflammatory mediators such as cytokines (ILs and chemotactic molecules) and metalloproteinases or by antigen recognition, capture, clearance, and transport [22 , 25 ]. In addition, they recruit hemopoietic cells to local sites of inflammation and immunity and regulate their activities. However, when macrophage activation persists over time, pathologic conditions and tissue injury may occur, influencing the onset and the progression of several inflammatory diseases and tumors [23 , 26 ]. In recent years, an important role of activated macrophages in neurodegeneration (i.e., axonal loss occurring in nervous system disorders) has also been proposed [27 ]. In view of these findings, there is increasing interest in defining new strategies aimed at reducing the unwanted side-effects of the persistent activation of macrophages and their release of proinflammatory mediators.

As discussed previously for monocytes, human macrophages differentiated from PBMC-derived monocytes have been shown recently to express cortistatin mRNA but not SRIF-14 mRNA [16 , 17 ]. In addition, they seem to express sst2 mRNA and the related protein [17 ], although the functional significance of this expression pattern is still unknown. We therefore investigated in human macrophages differentiated from PBMC-derived monocytes the mRNA expression of SRIF receptors, the expression level of distinct SRIF receptor proteins and their trafficking in the presence of SRIF analogs, and the pharmacology and functional role of distinct SRIF receptors. In particular, we assessed the role of specific SRIF receptors on cell viability and their effects on the expression and/or the secretion of different proinflammatory mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of human PBMC-derived monocytes
PBMCs were isolated from 20–40 ml blood from 17 different healthy donors. Immediately after blood withdrawal and centrifugation, the buffy coats were underlayered with Lymphoprep density gradient (density, 1.077 g/ml) and centrifuged at 400 g for 30 min at 25°C. Using published protocols [28 29 30 ], PBMCs, in a broad band below the interface, were collected using a siliconized pasteur pipette and washed with PBS. The final pellet was resuspended in culture medium (RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 50 UI/ml penicillin, 50 µg/ml streptomycin, and 10 mM HEPES) before seeding the cells in six-/96-well plates and in 250 mm coverslips at a density of 5 x 106/105 or 106 cells/well, respectively. Plates were then incubated for 24 h at 37°C, 5% CO2, in a humidified atmosphere to allow adherence of the monocytes to the plate. Cell monolayers were then washed with PBS to remove potential contaminating T and B cells.

Macrophage cell culture
To generate macrophages, culture medium was supplemented with 50 ng/ml GM-CSF [31 , 32 ] for 10 days with medium refreshment at Day 3. Cultured cells, before and after macrophage differentiation, were inspected routinely by phase-contrast microscopy using an inverted microscope (Helmut Hund, Wetzlar, Germany) equipped with a 10x objective and a Nikon Coolpix 4500 digital camera. After 10 days, cells were incubated for 24 h with RPMI 1640 supplemented with 0.2% BSA in the absence or presence of 2 µg/ml LPS, a specific activating factor of macrophages [23 , 24 ]. Where indicated, SRIF-14 and different synthetic SRIF analogs were also added.

FACS analysis
The phenotype of the cultured cells was confirmed by FACS analysis. Macrophages were scraped from six-well plates and centrifuged at 400 g for 5 min. Supernatant was removed, and FITC-labeled mAb, i.e., CD14-FITC and CD64-FITC, were added to each cell pellet at the final concentration of 10 µg/ml in PBS (30 min, 4°C). Cells were then washed and resuspended in PBS followed by FACS analysis on a FACSCalibur FACS (Becton Dickinson, San Jose, CA, USA).

Semiquantitative RT-PCR
Total cellular RNA was extracted from macrophages, which were cultured in six-well plates for 24 h with the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommended procedure. cDNA was synthesized from 1 µg total RNA using a first-strand cDNA synthesis kit (Roche, Nutley, NJ, USA). Primer pairs for SRIF receptors, the MCP-1 and IL-8 (Table 1 ), were designed to hybridize to unique regions of the respective gene sequence. Primers for GAPDH (Table 1) , a presumably stable mRNA, were used as an internal standard (housekeeping gene). The PCR reactions were carried out using 5 µl cDNA in a 25-µl (SRIF receptors) or 50-µl (MCP-1/IL-8) total volume of PCR buffer [670 mM Tris-HCl, pH 8.8, 160 mM (NH4)2SO4, 0.1% Tween-20], containing 1.5 mM MgCl2 (2.5 mM for MCP-1/IL-8; 5 mM for sst5), 0.2 mM deoxy-unspecified nucleoside 5'-triphosphate (dNTP; 1.6 mM for sst1), 5% DMSO (sst5 only), and 1 µM appropriate primers (0.6 µM for IL-8). Taq polymerase was also added: 1.25 U for SRIF receptors, 2.5 U for MCP-1, and 1 U for IL-8. SRIF receptors, MCP-1 and IL-8, were coamplified with GAPDH. The amplification reaction was carried out in a MWG thermocycler (Ebersberg, Germany) for 35 cycles (SRIF receptors) or 30 cycles (MCP-1/IL-8). Each cycle consisted of denaturation for 45 s (SRIF receptors) or 60 s (MCP-1/IL-8) at 94°C, annealing for 45 s (SRIF receptors) or 60 s (MCP-1/IL-8) at 60°C (SRIF receptors) or 55°C (MCP-1/IL-8), and an extension for 45 s (SRIF receptors) or 60 s (MCP-1/IL-8) at 72°C. A final extension step at 72°C for 5 min (SRIF receptors) or 7 min (MCP-1/IL-8) terminated the amplification. For each amplification, two types of controls were performed: RT-PCR mixture with no RT to control for genomic DNA contamination and PCR mixture with no cDNA template to check for possible external contamination. A 20-µl sample of the PCR reaction was electrophoresed on a 3% agarose gel by the use of the Bio-Rad Sub-Cell GT system and stained with GelStar (BMA, Rockland, ME, USA). After migration, bands corresponding to the amplified products were analyzed with the Gel Doc 2000 system equipped with Quantity One software (Bio-Rad, Hercules, CA, USA). Semiquantitative analysis of the PCR products was performed by normalizing the OD of the bands with respect to intensity of the OD for GAPDH in the same lane (mRNA/GAPDH mRNA).


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  		Table 1. Primers Used for RT-PCR Analysis

 
MTT assay
Macrophage viability was assessed from the reduction of MTT [33 ], which is a water-soluble tetrazolium salt that is reduced by the dehydrogenase activity of viable cells to a purple-colored, water-insoluble formazan salt. Using published protocols [34 ], macrophages were cultured in 96-well plates for 24 h to have six wells per experimental condition. After removing the medium, cells were rinsed with PBS before the addition of PBS and 10 µl MTT (5 mg/ml stock solution). Plates were then incubated at 37°C, 5% CO2, in a humidified atmosphere for 2-4 h. The assay was stopped by replacement of the MTT-containing medium with DMSO. Formazan salts were dissolved in DMSO by gentle shaking for 5 min at room temperature. Absorbance at {lambda} 570 nm was quantified spectrophotometrically by the Ultramark microplate systems (Bio-Rad).

Immunocytochemistry
Macrophages were cultured in 250 mm coverslips for 24 h to have three wells per experimental condition. Using published protocols [35 , 36 ], the cells were fixed in 4% paraformaldehyde for 10 min at room temperature before washing with 0.1 M PB. The polyclonal antibodies directed against sst1 and the sst2A isoform of sst2 [37 , 38 ] were diluted 1:100 in PB containing 0.1% Triton X-100 overnight at 4°C. Cells were then washed in PB and incubated in the presence of the appropriate secondary antibodies conjugated with Alexa Fluor 488 fluorescent dyes at a dilution of 1:200 in PB containing 0.1% Triton X-100 for 1 h at room temperature. Subsequently, the cells were washed in PB, mounted on glass slides, and coverslipped in a PB-glycerin mixture. sst1 and sst2A immunoreactivities [sst1- and sst2A-immunoreactivity (IR), respectively] were examined with a confocal laser-scanning microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) using Pl-Fluotar 100x (1.4 NA) oil objectives. Generally, 10–12 optical sections were taken with a z-axis resolution of 1 µm through the immunolabeled cells. The images from the confocal microscope were sized and optimized for contrast and brightness using Adobe Photoshop (Adobe Systems, Mountain View, CA, USA). Final images were saved at a minimum of 300 dpi. Specificity of the immunostaining was assessed by the omission of the primary antibody and the use of preimmune serum instead of the primary antibody. sst1- and sst2A-IR was abolished in these control conditions (n=3; data not shown).

ELISA assay
An ELISA assay was used to measure MCP-1 and IL-8 secreted by macrophages, cultured for 24 h in six-well plates. The conditioned supernatants were collected at –80°C or used directly. Sandwich ELISA with human MCP-1 and IL-8 cytosets (Biosource International, Camarillo, CA, USA) was used according to the manufacturer’s recommended procedure. Chemokine concentrations were quantified spectrophotometrically by the Ultramark microplate systems (Bio-Rad) at {lambda} 450 nm (MCP-1) or 680 nm (IL-8). Three replicate samples were included in each experiment. The ELISA assay was highly specific for each chemokine, and no cross-reactivity with other cytokines was detected (n=3, data not shown).

Zymogram protease assay
Using published protocols [39 ], matrix metalloproteinase (MMP)-9 secretion by macrophages cultured for 24 h in six-well plates was measured by SDS-PAGE zymography, which enables the detection of the zymogenic and the activated forms of MMP-9. Briefly, 20 µl samples of conditioned supernatants were electrophoresed at 4°C by the Bio-Rad Mini-PROTEAN 3 system on 7.5% polyacrylamide gels containing 10% SDS and gelatine (1 mg/ml) as substrate under nonreducing conditions and without boiling. After electrophoresis, SDS was removed from gels by washing (2x15 min) with 2.5% Triton X-100 at room temperature. Gels were then incubated overnight at 37°C with gentle shaking in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 10 mM CaCl2, and 1 µM ZnCl2 to activate the ability of MMPs to digest the substrate. Gels were stained with 0.25% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic acid and then destained in the same solution without Coomassie brilliant blue. The clear bands that were obtained against the blue background indicated the presence of MMP-9 in comparison with the molecular weight markers and standard positive control (Calbiochem, San Diego, CA, USA), which were run on each gel. Data acquisition was performed by the Gel Doc 2000 system (Bio-Rad).

Statistical analysis
Upon verification of normal distribution, statistical significance of data was evaluated using ANOVA followed by multiple comparison Tukey post-test. GraphPad Prism software package (Graph Software, San Diego, CA, USA) was used. Differences with P < 0.05 were considered significant. The nonlinear regression curve analysis of the concentration-effect responses was performed using GraphPad Prism. Where specified, results are given as means ± SEM of the indicated n values.

Chemicals
Lymphoprep was purchased from Axis Shield (Oslo, Norway). RPMI 1640, FBS, and L-glutamine were purchased from Cambrex (Walkersville, MD, USA). GM-CSF was purchased from Listarfish (Peprotech, Rocky Hill, NJ, USA). PBS and penicillin/streptomycin were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). CD14-FITC and CD64-FITC were purchased from Immunotech (Marseille, France). The forward and reverse primers, Taq polymerase, dNTPs, and agarose gel were purchased from Eurobio (Les Ulis, France). SRIF-14, cortistatin-14, and KE108 were purchased from Bachem (Bubendorf, Switzerland). SOM230, SMS 201-995, D-Tyr8 CYN 154806 (CYN), and SRA-880 were a gift of Prof. Daniel Hoyer and Dr. Herbert Schmid (Novartis Pharma, Basel, Switzerland). CH-275 was purchased from Neosystem (Strasbourg, France), and L-779,976 was kindly provided by Merck Research Laboratories (Rahway, NJ, USA). The sst1 and the sst2A antibodies were kindly provided by Prof. Stefan Schulz (Otto-von-Guericke University, Magdeburg, Germany). The secondary antibodies conjugated with Alexa Fluor 488 fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). Where not specified, chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of SRIF receptor mRNAs in human macrophages
Macrophage differentiation was induced by treatment of cultured PBMC-derived monocytes with 50 ng/ml GM-CSF [31 , 32 ]. After a 10-day incubation, cultures appeared as a homogenous set of cells with the characteristic fried egg-like morphology of standard macrophages, whereas the presence of elongated fibroblast-like macrophages reported by Zhao et al. [21 ] was negligible (data not shown). Monocyte/macrophages are APCs with characteristic cell-surface molecules such as CD14 and Fc{gamma}RI (or CD64) [25 ]. CD14 is a GPI-linked glycoprotein, which acts as a high-affinity receptor for complexes of LPS and LPS-binding protein, and CD64 is a glycoprotein defined by its ability to bind certain isotypes of monomeric Ig. In human PBMC-derived cells treated with GM-CSF, FACS analysis revealed that the percentage of cells that immunostained positively for CD14 and CD64 was 82 ± 10% and 68 ± 6%, respectively (n=3), thus confirming their monocyte/macrophage phenotypes.

Human macrophages are exquisitely sensitive to LPS [23 , 24 ] and respond by the induction of many genes [40 ], including those encoding for specific SRIF receptors [17 ]. As shown in Figure 1A , semiquantitive RT-PCR demonstrated that under basal conditions, macrophages express mRNA for sst1 (375 bp) and sst2 (672 bp), and no bands were observed at the expected size of the sst3,4,5 amplicons (n=3; data not shown). Furthermore, treatment of the macrophages with 2 µg/ml LPS for 24 h led to a ca. 50% increase in sst1 mRNA, whereas the mRNA levels of sst2 were unaltered (Fig. 1B) . To demonstrate that sst1 and sst2 proteins were actually expressed by cultured cells, immunocytochemistry with sst1 or sst2A antibodies was performed, and the respective fluorescent signals were visualized by confocal microscopy. LPS-activated macrophages showed punctate sst1- and sst2A-IR, which was essentially localized at the cell surface, where it formed a continuous ring (see Go Fig. 3 ).


Figure 1
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  		Figure 1. RT-PCR analysis of sst1 and sst2 expression in macrophages differentiated from human PBMC-derived monocytes. Macrophages were incubated in the absence or presence of 2 µg/ml LPS for 24 h. GAPDH mRNA was used as an internal standard. (A) Expression pattern of sst1 and sst2 mRNA from one donor, as representative of the three donors used. (B) Semiquantitative analysis of the PCR products performed by normalizing the OD of the bands for sst1 and sst2 mRNA with respect to intensity of the OD of the bands for GAPDH in the same lane. Each histogram represents the mean ± SEM of data from three experiments. *, P < 0.001, sst1 + LPS versus sst1 – LPS (ANOVA followed by Tukey test).

 

Figure 2
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  		Figure 2. Effects of SRIF-14, cortistatin-14, or different synthetic SRIF analogs on viability of macrophages differentiated from human PBMC-derived monocytes. Macrophages were treated with 2 µg/ml LPS for 24 h in the absence or presence of ligands before MTT assay. (A) Effects of increasing concentrations of SRIF-14 and cortistatin-14. (B) Effects of the multiligand analogs SOM230 and KE108. (C) Effects of the sst1 agonist CH-275 or the sst2 agonists SMS 201-995/L-779,976 (1 µM) in the absence or presence of the sst1 antagonist SRA-880 or the sst2 antagonist CYN (1 µM), respectively. Each histogram represents the mean ± SEM of data from three independent experiments run in sextuplicate. *, P < 0.01, and **, P < 0.001, versus control (ANOVA followed by Tukey test). Data are expressed by setting the absorbance of the reduced MTT ({lambda} 570 nm) in control conditions as 100%.

 

Figure 3
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  		Figure 3. Confocal images depicting immunocytochemical localization and trafficking of sst1 and the sst2A isoform of sst2 in macrophages differentiated from human PBMC-derived monocytes. Macrophages were treated with 2 µg/ml LPS for 24 h in the absence or presence of SRIF-14, the sst1 agonist CH-275, or the sst2 agonist L-779,976 (1 µM). Images were from one donor, as representative of the three donors used. Original scale bar = 30 µm.

 
Pharmacological and functional properties of SRIF receptors in human macrophages: effects of SRIF analogs on cell viability
To examine the metabolic effects of SRIF receptors expressed in LPS-activated macrophages, cell viability was measured by MTT assay after 24 h treatment with SRIF-14, the cognate peptide cortistatin-14, or different synthetic SRIF analogs, which share the functions of the natural peptide. As shown in Figure 2A , increasing concentrations of SRIF-14 or cortistatin-14 had no significant effect on basal absorbance (which reflects the amount of viable cells) of LPS-activated macrophages. Because of their long-acting properties in vitro and in vivo, low molecular weight/metabolically stable analogs are more suitable for clinical application or experimental investigations than SRIF-14 [2 , 41 ]. Among synthetic ligands, we used the peptidyl multiligand analog SOM230, which binds with high affinity to sst1,2,3,5 [42 43 44 ], and the peptidyl multiligand analog KE108, which binds with high affinity to all five SRIF receptors [44 , 45 ]. The sst1-selective peptidyl agonist CH-275 [35 , 41 , 46 , 47 ], the sst2-preferring peptiydyl agonist SMS 201-995 (also known as sandostatin or octreotide) [38 , 41 , 46 , 48 , 49 ], and the sst2-selective nonpeptidyl agonist L-779,976 [46 , 49 50 51 ] were also used in the absence or presence of the sst1-selective nonpeptydil antagonist SRA-880 [46 , 52 , 53 ] and the sst2-selective peptydil antagonist CYN [36 , 38 , 48 , 49 , 54 ]. All compounds were applied at 1 µM, which from previous studies, is a concentration giving maximal receptor occupancy [2 , 41 ]. This concentration was also used in previous studies investigating the effects of SRIF analogs in cells of the immune system, including macrophages [13 , 16 ]. As shown in Figure 2B , the application of SOM230 or KE108 significantly decreased basal absorbance of LPS-activated macrophages by 58 ± 6% and 42 ± 12%, respectively. When specific SRIF receptor agonists or antagonists were used (Fig. 2C) , the application of CH-275 decreased basal absorbance significantly by 78 ± 5%, and this effect was reversed completely in the presence of SRA-880. In addition, the application of SMS 201-995 or L-779,976 decreased basal absorbance significantly by 48 ± 5% and 68 ± 5%, respectively, and this effect was reversed completely in the presence of CYN. The application of SRA-880 or CYN alone had no effect on basal absorbance (n=3; data not shown).

The interactions of SRIF analogs with their receptors may result in a receptor-dependent internalization, resulting in receptor desensitization [55 ]. Therefore, we studied the trafficking of sst1 and sst2 in LPS-activated macrophages after 24 h treatment with different SRIF analogs (1 µM). As shown in Figure 3 , immunocytochemistry by confocal microscopy revealed a punctuate, weak sst1-IR, which emerged inside cells after treatment with SRIF-14 and CH-275, besides surface sst1-IR. After cell treatment with SRIF-14 and L-779,976, sst2A-IR surface-labeling had almost disappeared completely. The sst2A-IR increased in intensity and translocated from the cell surface to intracellular, endosome-like compartments.

Pharmacological and functional properties of SRIF receptors in human macrophages: effects of SRIF analogs on the production of proinflammatory mediators
Macrophages function as scavenger cells and immune mediator cells in addition to producing cytokines, the key chemical substances in an inflammatory event. Among the cytokines, chemokines (chemotactic cytokines) are small, heparin-binding proteins, which direct the migration of circulating cells to sites of inflammation or injury [56 ]. In immune cells, SRIF-14 has a broad spectrum of activity, including immunomodulatory effects [4 ]. Using semiquantitative RT-PCR and ELISA assay, we investigated whether the activation of sst1 by CH-275 or sst2 by SMS 201-995 or L-779,976 might influence the mRNA expression/secretion of different chemokines in LPS-activated macrophages. The effects of 1 µM SRIF analogs after 24 h treatment were then compared with those obtained after SRIF-14 application. The largest family of chemokines is known as the CC family, as the first two of the four conserved cysteine residues that are characteristic of chemokines are adjacent to each other [56 ]. In contrast, the CXC family of chemokines have a single amino acid residue between the first two canonical cysteines. Within the CC family, the most well-characterized chemokine is MCP-1 (also known as CCL2). As shown in Figure 4A , cell treatment with CH-275, SMS 201-995, or L-779,976 decreased the RT-PCR-amplified product significantly at 233 bp, which corresponds to MCP-1 mRNA. In particular, the OD of MCP-1 mRNA in the presence of CH-275, SMS 201-995, and L-779,976 was ca. 43%, 38%, and 44% lower than in basal conditions (n=3; P<0.001). In contrast, the application of SRIF-14 did not modify the expression of MCP-1 mRNA. As shown in Figure 4B , treatment with CH-275, SMS 201-995, or L-779,976 decreased basal secretion of MCP-1 significantly by 30 ± 5%, 24 ± 4%, and 36 ± 3%, respectively, and the application of SRIF-14 was devoid of effects. Another key substance in the inflammatory event is IL-8 (also known as CXCL8), the prototypical member of the CXC family of chemokines. As shown in Figure 5A , the RT-PCR-amplified product at 283 bp, corresponding to IL-8 mRNA, was unaffected by cell treatment with SRIF-14, CH-275, SMS 201-995, or L-779,976. In contrast, the application of SRIF-14, CH-275, SMS 201-995 or L-779,976 decreased basal secretion of IL-8 significantly by 18 ± 4%, 14 ± 4%, 17 ± 3%, and 16 ± 4%, respectively (Fig. 5B) .


Figure 4
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  		Figure 4. Effects of SRIF-14 or different synthetic SRIF analogs on MCP-1 production by macrophages differentiated from human PBMC-derived monocytes. Macrophages were treated with 2 µg/ml LPS for 24 h in the absence or presence of SRIF compounds before assays. (A) RT-PCR analysis of the MCP-1 mRNA expression in cells under control conditions or treated with SRIF-14, the sst1 agonist CH-275, or the sst2 agonists SMS 201-995/L-779,976 (1 µM). Results were from one donor, as representative of the three donors used. GAPDH mRNA was used as an internal standard. (B) ELISA assay of the MCP-1 content in the supernatants of cells under control conditions or treated with SRIF-14, the sst1 agonist CH-275, or the sst2 agonists SMS 201-995/L-779,976 (1 µM). Each histogram represents the mean ± SEM of data from three independent experiments run in triplicate. *, P < 0.05, and **, P < 0.01, versus control (ANOVA followed by Tukey test). Data are expressed by setting the content in control conditions as 100%.

 

Figure 5
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  		Figure 5. Effects of SRIF-14 or different synthetic SRIF analogs on IL-8 production by macrophages differentiated from human PBMC-derived monocytes. Macrophages were treated with 2 µg/ml LPS for 24 h in the absence or presence of SRIF compounds before assays. (A) RT-PCR analysis of the IL-8 mRNA expression in cells under control conditions or treated with SRIF-14, the sst1 agonist CH-275, or the sst2 agonists SMS 201-995/L-779,976 (1 µM). Results were from one donor, as representative of the three donors used. GAPDH mRNA was used as an internal standard. (B) ELISA assay of the IL-8 content in the supernatants of cells under control conditions or treated with SRIF-14, the sst1 agonist CH-275, or the sst2 agonists SMS 201-995/L-779,976 (1 µM). Each histogram represents the mean ± SEM of data from three independent experiments run in triplicate. *, P < 0.05, and **, P < 0.01, versus control (ANOVA followed by Tukey test). Data are expressed by setting the content in control conditions as 100%.

 
MMPs, which have a zinc ion in their active site, are a class of secreted enzymes with major functions in the degradation and remodeling of all components of the extracellular matrix. MMP-9 (also known as gelatinase B) is a major secretory product of macrophages and plays an important role in inflammatory processes [57 58 59 ]. In LPS-activated macrophages, we used zymography to measure the secretion of MMP-9 24 h after the application of SRIF analogs. Using this method, which enables the detection of the zymogenic and the activated forms of MMP-9, only the latent form of the enzyme (proMMP-9, 92 kDa band) could be observed (Fig. 6 ), as reported recently in human PBMC-derived monocytes [39 ]. In particular, LPS-treated macrophages exhibited a distinct band corresponding to proMMP-9 which was unaffected by cell treatment with 1 µM SRIF-14, CH-275, SMS 201-995, or L-779,976, suggesting that activation of SRIF receptors did not lead to secretion of MMP-9.


Figure 6
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  		Figure 6. Effects of SRIF-14 or different synthetic SRIF analogs on proMMP-9 secretion by macrophages differentiated from human PBMC-derived monocytes. The supernatants of cells treated with 2 µg/ml LPS for 24 h in the absence (control) or presence of SRIF-14, the sst1 agonist CH-275, or the sst2 agonists SMS 201-995/L-779,976 (1 µM) were collected before zymography. Results were from one donor, as representative of the three donors used. PC, Positive control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neuropeptides are involved in regulating the interactions between the nervous and the immune system [1 ], and the neuropeptide SRIF-14 appears to play an important role in this network [4 , 5 ]. Cells of the monocyte lineage, i.e., macrophages, are known to be important components of the human immune circuitry and are involved in the modulation of immunological and inflammatory responses. Although the inhibitory role of SRIF-14 in cells of the monocyte lineage has been studied extensively in rodents [7 8 9 10 11 12 13 14 ], little is known about whether these findings can be extended to human monocytes. In this article, we report for the first time the expression and functional effects of distinct SRIF receptors in human macrophages differentiated from PBMC-derived monocytes. In this respect, advances in the understanding of the immunological responses to neuropeptides may lead to the development of new strategies in pharmacological therapy of inflammation.

sst1 and sst2 are expressed in human macrophages
Of the five known SRIF receptors, only sst2 mRNA was detected previously in human macrophages differentiated from PBMC-derived monocytes [17 ]. In addition, sst2 protein was expressed on plasma membranes of LPS-activated cells, a specific activating factor with immunostimulatory and tumoricidal/bactericidal activity [23 , 24 ], but not on unstimulated cells [17 ]. In contrast, we demonstrated the expression of sst1 and sst2 mRNAs in LPS-activated and unstimulated cells, suggesting a potential, functional role for these receptors in human macrophages. In addition, we demonstrated by immunocytochemistry that sst1 and sst2 proteins were localized mainly at the cell surface of LPS-activated macrophages. A similar expression pattern was found in murine macrophages [13 ]. In our experimental protocol, we used cells that had been subjected to differentiating conditions for 10 days to obtain a homogenous population of differentiated macrophages. This is in contrast to the 6-day incubation used by Dalm et al. [17 ], and therefore, it is reasonable to assume the discrepancy between that study and ours is because SRIF receptor expression reaches significant levels only after full maturation of human macrophages, as hypothesized previously [17 ]. Indeed, up-regulation of sst1 mRNA expression seems to become evident relatively late during cell differentiation, indicating that prolonged differentiation times result in mature macrophages, which display appropriate levels of specific SRIF receptors. In addition, sst1 seems to have a major functional role in activated macrophages, as we found that sst1 mRNA, but not sst2 mRNA, becomes up-regulated significantly after cell treatment with LPS. In contrast to our findings, Dalm et al. [17 ] reported that sst2 mRNA was up-regulated by LPS. Futher explanations for the differences between our study and that of Dalm et al. [17 ] include different experimental parameters as well as the use of a different monocyte isolation technique, which could influence the activity and subsequently, SRIF receptor expression [15 , 17 ]. Alternatively, the possibility that the expression of SRIF receptors in macrophages (specifically, the coexpression of sst1 and sst2) may change with respect to different donors cannot be excluded. The cause of this variability is unclear but has been shown to occur in a wide range of tissues [60 , 61 ].

Functional properties of sst1 and sst2 in human macrophages
In vitro culture of human PBMCs has been used previously to demonstrate the effects of SRIF-14 and its analogs on different immune functions of these cells. However, these studies have produced conflicting results about the effects of the SRIF system on cell proliferation/viability and cytokine production [5 , 15 ]. A major finding of the present work was the inhibitory role of the SRIF system on the functional activity of human macrophages differentiated from PBMC-derived monocytes. We demonstrated that the activation of SRIF receptors by the long-acting, multiligand, synthetic analogs SOM230 and KE108 reduced viability of LPS-activated cells consistently. In contrast, the activation of SRIF receptors by the native peptides SRIF-14 or cortistatin-14, a SRIF-14-like peptide that binds to the human SRIF receptors with similar high affinity to that of SRIF itself [62 ], did not affect cell viability at any concentrations tested. However, SRIF receptor reserve and coupling efficiency may depend on the nature of the ligand and the receptor at play. For instance, when a given SRIF receptor is activated by the natural peptide or alternatively, by a synthetic ligand, the functional effect may be different. Indeed, SRIF receptor coupling at a given function can be influenced strongly by the ligand used, and compelling evidence suggests the existence of multiple ligand-specific SRIF receptor conformations, which may couple differentially to the effectors [2 , 3 ]. Alternatively, SRIF receptor desensitization may account for the lack of effects of native peptides in our system (see below). In human macrophages differentiated from PBMC-derived monocytes, cortistatin-14, but not SRIF-14, was suggested to represent a potential endogenous ligand for SRIF receptors [16 , 17 ]. In addition, cortistatin-14 was proposed recently as an alternative ligand for SRIF receptors in the immune system and as a new immunomodulatory factor with the capacity to deactivate the inflammatory response [19 ]. Recent studies reported a possible therapeutic action of cortistatin-14 in inflammatory bowel disease and septic shock, as assessed in murine models of colitis or endotoxemia [20 , 63 ].

The capacity of SRIF receptors, including sst1 and sst2, to regulate cell proliferation/viability negatively through indirect and direct mechanisms has been reported repeatedly in many systems [3 , 64 ]. As SOM230 and KE108 are able to activate sst1 and sst2, it is unclear whether activation of one or both receptor subtypes leads to the inhibition of macrophage viability. However, the finding that LPS-activated macrophage viability was inhibited by the sst1 agonist CH-275 and the sst2 agonists SMS 201-995/L-779,976 indicates that sst1 and sst2 are equally functional in these cells. The fact that the sst1 antagonist SRA-880 blocked the effects of CH-275 and that the sst2 antagonist CYN blocked the effects of SMS 201-995 and L-779,976 further confirms that the effects of CH-275 and SMS 201-995/L-779,976 were mediated through sst1 and sst2, respectively. In different types of immune cells, the modulation of cell viability by SRIF-14 is complex and may occur through distinct receptor subtypes. For example, the observed actions of SRIF-14 on lymphocyte proliferation are multimodal: inhibitory and stimulatory effects have been reported depending on the SRIF-14 concentration, the way in which the immune cell activation was achieved, and the cell phenotype [4 , 64 ].

The interactions of SRIF analogs with their receptors may result in a receptor-dependent internalization of receptor-ligand complexes. The internalization process was shown to vary according to the receptor subtypes involved, the ligand used, and the cell phenotype, and it may play a role in receptor desensitization [36 , 55 , 65 ]. We found that the continuous activation (24 h) of sst1 by SRIF-14 or CH-275 resulted in a weak internalization of sst1-IR in LPS-activated macrophages, and the sst2A-IR internalization induced by SRIF-14 or L-779,976 appeared more efficient. In the same experimental model, the internalization of the FITC-labeled octreotate-sst2 complex has been demonstrated recently [17 ]. Our results demonstrate further the presence of functional sst1- and/or sst2-binding sites for SRIF-14, CH-275, and L-779,976 in macrophages. Of all SRIF receptor subtypes, sst2 is the one that was found to be the most efficiently internalized by SRIF analogs [36 , 55 , 65 , 66 ], including L-779,976 [65 , 67 ]. In contrast, the internalization of sst1 was suggested to be species-specific [68 ], although the cloned human sst1 seems to internalize poorly [69 , 70 ]. Taken together, our results do not support the hypothesis that the lack of effects of SRIF-14 (and cortistatin-14) on macrophage viability can be ascribable to the more efficient receptor down-regulation by SRIF-14 as compared with its synthetic analogs. Conversely, our experiments were performed after 24 h treatment with a relatively high concentration of ligands, and the possibility that the internalization of the SRIF-14-sst1/sst2 complex displays a more efficient time-course cannot be excluded.

In our study, the sst1/sst2-mediated inhibition of viability in human macrophages differentiated from PBMC-derived monocytes paralleled the inhibition of chemokine production. In particular, we demonstrated that the activation of sst1 by CH-275 and of sst2 by SMS 201-995/L-779,976 decreased mRNA expression and secretion of MCP-1 by LPS-activated cells, suggesting a down-regulation of de novo synthesis of this molecule and not of preformed molecule secretion. In line with its effect on cell viability, SRIF-14 had no effect on MCP-1 production. Recently, SMS 201-995 was found to inhibit the LPS-induced mRNA expression and secretion of MCP-1 by rat Kupffer cells, the resident macrophages of the liver [10 ]. MCP-1 is a potent mononuclear cell chemoattractant, which has a critical role in vascular and nonvascular diseases, and efforts are under way to develop potent and specific modulators of this and related chemokines. MCP-1 has potent effects on monocytes, memory T cells, and basophils bearing the chemokine receptor CCR-2. It has been implicated as a key player in the recruitment of monocytes from the blood into early atherosclerotic lesions, the development of intimal hyperplasia after angioplasty, as well as in vasculogenesis, and in aspects of thrombosis [56 , 71 ]. Enhanced MCP-1 expression has been detected in macrophages, endothelial cells, and vascular smooth muscle cells in the atheromatous plaque. Activation of macrophages by MCP-1 also appears to be involved in the vulnerability of the plaque and in restenotic lesions [72 ].

Chemotaxis of neutrophils, the signature cell of the acute inflammatory response, may also be evoked by IL-8, which is produced by a wide variety of cell types, including virtually all of the nucleated cells within the body. However, among these cells, monocytes and macrophages typically represent the principal cellular source [73 ]. IL-8 is important in the regulation of the acute inflammatory response. It is synthesized rapidly at sites of local inflammation, where it fulfills its function to recruit and activate acute inflammatory cells [56 , 71 ]. In particular, IL-8 attracts polymorphonuclear leukocytes and has been implicated in acute pulmonary inflammation. IL-8 also activates monocytes and may direct their recruitment to vascular lesions. In humans, SRIF-14 and/or SMS 201-995 have been found to exert an immunomodulatory effect in LPS-activated PBMCs by suppressing IL-8 production [74 , 75 ]. Accordingly, our study showed that SRIF-14 decreased IL-8 secretion significantly in LPS-activated macrophages, although its effect appeared marginal. This is in contrast to previous studies, which failed to detect an effect of SMS 201-995 on the secretion of IL-8 by rat macrophage Kupffer cells activated with LPS [10 ]. We also found that SRIF-14 (and its synthetic analogs) did not affect IL-8 mRNA expression, suggesting the coupling of SRIF receptors to transduction pathways, which may not interact with nuclear regulatory factors of the IL-8 gene but rather with biochemical events triggering exocytosis of IL-8. Calcium and activated G proteins are known to regulate the exocytosis of different factors, i.e., chemokines, synergistically from the activated macrophages, although a Ca2+-independent component may also be involved [76 ]. SRIF receptor coupling to Ca2+ homeostasis is well established [2 ] as well as the fact that SRIF receptors may regulate exocytosis through mechanisms dependent and independent on Ca2+ [2 , 35 , 51 ]. Whether the SRIF system couples to Ca2+ homeostasis in human macrophages remains to be established. In our study, we demonstrated that the selective activation of sst1 by CH-275 and of sst2 by SMS 201-995/L-779,976 decreased IL-8 secretion in LPS-activated macrophages to a similar extent than SRIF-14, indicating that sst1 and sst2 are equally responsible for the inhibitory effects of SRIF-14. The fact that in our system, SRIF-14 had a detectable effect on IL-8 but not on MCP-1 secretion or cell viability suggests that sst1/sst2-induced inhibition of IL-8 secretion involves highly efficient coupling between receptor and effector and that a small degree of G protein activation is required to obtain a significant inhibition (see also above). Conversely, we also showed that the immunoregulatory role of the SRIF system in human macrophages did not involve MMP-9, a major secretory product of macrophages, which constitutes a prototypical example of the regulation of immune functions by proteolysis [57 58 59 ]. Indeed, the application of SRIF-14 and SRIF receptor-selective agonists did not affect the secretion of proMMP-9 by LPS-activated macrophages. In contrast, MMP-9 production stimulated by LPS was suppressed by SMS 201-995 in rat macrophage Kupffer cells [12 ]. In particular, the fact that 24 h incubation with CH-275 or L-779,976 induced a marked decrease in macrophage viability, as assessed by MTT assay (an index of the metabolic activity of cells), without affecting the secretion of proMMP-9, suggests that proMMP-9 secretion is immediate and rapid after LPS activation, in agreement with previous results reporting the secretion of different cytokines in rat alveolar macrophages [77 ]. Conversely, the effects of different stimuli on the metabolic activity of macrophages and the secretion of proinflammatory mediators are clearly not superimposable [78 , 79 ]. It may also be the case that the secretion of proinflammatory mediators is increased after the inhibition of the metabolic activity of cells [80 , 81 ].

Remarks
Collectively, our results suggest that although the SRIF system has diverse effects on immune cells, it exerts mainly an immunosuppressive effect in human macrophages differentiated from PBMC-derived monocytes. In particular, we have demonstrated that SRIF-14 effects are mediated by sst1 and sst2, which are expressed and display active binding sites on the cell surface. Conversely, the coupling efficiency of the native peptides SRIF-14 and cortistatin-14 to sst1 and sst2 is elusive, and the activation of sst1 and sst2 by synthetic ligands is more relevant. In particular, the findings that the activation of sst1 and sst2 similarly inhibited macrophage viability and chemokine production, i.e., MCP-1 and IL-8, without affecting MMP-9 secretion suggest a redundancy of SRIF receptor function, as both receptors are capable of producing similar responses alone. In agreement with our results, sst1 and sst2 are functionally active in murine macrophages [13 ]. Further investigation is needed to explore the pharmacological significance and the possible immunoregulatory and therapeutic effects of sst1/sst2 agonists in inflammatory disorders in vivo.


   ACKNOWLEDGEMENTS
 
Funding support for this work was provided by the Cardio-Thoracic Department (University of Pisa, Italy). We are grateful to Dr. C. Cerri and Dr. A. Celi (Cardio-Thoracic Department, University of Pisa) for the excellent assistance in the ELISA assay and for providing access to the microplate reader. We thank Dr. G. Gambellini (Department of Environmental Sciences, University of Tuscia, Italy) for the assistance in confocal microscopy. We are greatful to Dr. M. C. Iorio (Unit of Immunohematology, Hospital of Cisanello, AOP, Pisa, Italy) for the excellent assistance in the FACS analysis. We thank Dr. C. Nunn (Department of Physiology and Pharmacology, University of Western Ontario, Canada) for revising the manuscript.

Received June 28, 2006; revised September 20, 2006; accepted November 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shepherd, A. J., Downing, J. E., Miyan, J. A. (2005) Without nerves, immunology remains incomplete—in vivo veritas Immunology 116,145-163[CrossRef][Medline]
  2. Cervia, D., Nunn, C., Bagnoli, P. (2005) Multiple signaling transduction mechanisms differentially coupled to somatostatin receptor subtypes: a current view Curr. Enzyme Inhibition 1,265-279
  3. Olias, G., Viollet, C., Kusserow, H., Epelbaum, J., Meyerhof, W. (2004) Regulation and function of somatostatin receptors J. Neurochem. 89,1057-1091[CrossRef][Medline]
  4. Krantic, S. (2000) Peptides as regulators of the immune system: emphasis on somatostatin Peptides 21,1941-1964[CrossRef][Medline]
  5. Lichtenauer-Kaligis, E. G., van Hagen, P. M., Lamberts, S. W., Hofland, L. J. (2000) Somatostatin receptor subtypes in human immune cells Eur. J. Endocrinol. 143(Suppl. 1),S21-S25[Free Full Text]
  6. Szokoloczi, O., Schwab, R., Petak, I., Orfi, L., Pap, A., Eberle, A. N., Szuts, T., Keril, G. (2005) TT232, a novel signal transduction inhibitory compound in the therapy of cancer and inflammatory diseases J. Recept. Signal Transduct. Res. 25,217-235[CrossRef][Medline]
  7. Ryu, S. Y., Jeong, K. S., Yoon, W. K., Park, S. J., Kang, B. N., Kim, S. H., Park, B. K., Cho, S. W. (2000) Somatostatin and substance P induced in vivo by lipopolysaccharide and in peritoneal macrophages stimulated with lipopolysaccharide or interferon-{gamma} have differential effects on murine cytokine production Neuroimmunomodulation 8,25-30[CrossRef][Medline]
  8. Weinstock, J. V., Elliott, D. (2000) The somatostatin immunoregulatory circuit present at sites of chronic inflammation Eur. J. Endocrinol. 143(Suppl. 1),S15-S19[Abstract/Free Full Text]
  9. Valatas, V., Kolios, G., Manousou, P., Xidakis, C., Notas, G., Ljumovic, D., Kouroumalis, E. A. (2004) Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide Regul. Pept. 120,215-225[CrossRef][Medline]
  10. Valatas, V., Kolios, G., Manousou, P., Notas, G., Xidakis, C., Diamantis, I., Kouroumalis, E. (2004) Octreotide regulates CC but not CXC LPS-induced chemokine secretion in rat Kupffer cells Br. J. Pharmacol. 141,477-487[CrossRef][Medline]
  11. Kang, B. N., Jeong, K. S., Park, S. J., Kim, S. J., Kim, T. H., Kim, H. J., Ryu, S. Y. (2001) Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages Regul. Pept. 101,43-49[CrossRef][Medline]
  12. Xidakis, C., Ljumovic, D., Manousou, P., Notas, G., Valatas, V., Kolios, G., Kouroumalis, E. (2005) Production of pro- and anti-fibrotic agents by rat Kupffer cells; the effect of octreotide Dig. Dis. Sci. 50,935-941[CrossRef][Medline]
  13. Perez, J., Viollet, C., Doublier, S., Videau, C., Epelbaum, J., Baud, L. (2003) Somatostatin binds to murine macrophages through two distinct subsets of receptors J. Neuroimmunol. 138,38-44[CrossRef][Medline]
  14. Lang, A., Sakhnini, E., Fidder, H. H., Maor, Y., Bar-Meir, S., Chowers, Y. (2005) Somatostatin inhibits pro-inflammatory cytokine secretion from rat hepatic stellate cells Liver Int. 25,808-816[CrossRef][Medline]
  15. Lichtenauer-Kaligis, E. G., Dalm, V. A., Oomen, S. P., Mooij, D. M., van Hagen, P. M., Lamberts, S. W., Hofland, L. J. (2004) Differential expression of somatostatin receptor subtypes in human peripheral blood mononuclear cell subsets Eur. J. Endocrinol. 150,565-577[Abstract]
  16. Dalm, V. A., van Hagen, P. M., van Koetsveld, P. M., Langerak, A. W., van der Lely, A. J., Lamberts, S. W., Hofland, L. J. (2003) Cortistatin rather than somatostatin as a potential endogenous ligand for somatostatin receptors in the human immune system J. Clin. Endocrinol. Metab. 88,270-276[Abstract/Free Full Text]
  17. Dalm, V. A., van Hagen, P. M., van Koetsveld, P. M., Achilefu, S., Houtsmuller, A. B., Pols, D. H., van der Lely, A. J., Lamberts, S. W., Hofland, L. J. (2003) Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells Am. J. Physiol. Endocrinol. Metab. 285,E344-E353[Abstract/Free Full Text]
  18. Seboek, D., Linscheid, P., Zulewski, H., Langer, I., Christ-Crain, M., Keller, U., Muller, B. (2004) Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation J. Clin. Endocrinol. Metab. 89,4833-4839[Abstract/Free Full Text]
  19. Li, M., Yan, S., Fisher, W. E., Chen, C., Yao, Q. (2005) New roles of a neuropeptide cortistatin in the immune system and cancer World J. Surg. 29,354-356[CrossRef][Medline]
  20. Gonzalez-Rey, E., Chorny, A., Robledo, G., Delgado, M. (2006) Cortistatin, a new antiinflammatory peptide with therapeutic effect on lethal endotoxemia J. Exp. Med. 203,563-571[Abstract/Free Full Text]
  21. Zhao, Y., Glesne, D., Huberman, E. (2003) A human peripheral blood monocyte-derived subset acts as pluripotent stem cells Proc. Natl. Acad. Sci. USA 100,2426-2431[Abstract/Free Full Text]
  22. Gordon, S. (1998) The role of the macrophage in immune regulation Res. Immunol. 149,685-688[CrossRef][Medline]
  23. Gordon, S. (2003) Alternative activation of macrophages Nat. Rev. Immunol. 3,23-35[CrossRef][Medline]
  24. Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., Locati, M. (2004) The chemokine system in diverse forms of macrophage activation and polarization Trends Immunol. 25,677-686[CrossRef][Medline]
  25. Aderem, A., Underhill, D. M. (1999) Mechanisms of phagocytosis in macrophages Annu. Rev. Immunol. 17,593-623[CrossRef][Medline]
  26. Lewis, C. E., Pollard, J. W. (2006) Distinct role of macrophages in different tumor microenvironments Cancer Res. 66,605-612[Abstract/Free Full Text]
  27. Hendriks, J. J., Teunissen, C. E., de Vries, H. E., Dijkstra, C. D. (2005) Macrophages and neurodegeneration Brain Res. Brain Res. Rev. 48,185-195[CrossRef][Medline]
  28. Hashimoto, S., Yamada, M., Motoyoshi, K., Akagawa, K. S. (1997) Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10 Blood 89,315-321[Abstract/Free Full Text]
  29. Bellosta, S., Via, D., Canavesi, M., Pfister, P., Fumagalli, R., Paoletti, R., Bernini, F. (1998) HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages Arterioscler. Thromb. Vasc. Biol. 18,1671-1678[Abstract/Free Full Text]
  30. Aikawa, M., Rabkin, E., Sugiyama, S., Voglic, S. J., Fukumoto, Y., Furukawa, Y., Shiomi, M., Schoen, F. J., Libby, P. (2001) An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro Circulation 103,276-283
  31. Fleetwood, A. J., Cook, A. D., Hamilton, J. A. (2005) Functions of granulocyte-macrophage colony-stimulating factor Crit. Rev. Immunol. 25,405-428[CrossRef][Medline]
  32. Santin, A. D., Hermonat, P. L., Ravaggi, A., Chiriva-Internati, M., Cannon, M. J., Hiserodt, J. C., Pecorelli, S., Parham, G. P. (1999) Expression of surface antigens during the differentiation of human dendritic cells vs. macrophages from blood monocytes in vitro Immunobiology 200,187-204[Medline]
  33. Molinari, B. L., Tasat, D. R., Palmieri, M. A., Cabrini, R. L. (2005) Kinetics of MTT-formazan exocytosis in phagocytic and non-phagocytic cells Micron 36,177-183[CrossRef][Medline]
  34. Cervia, D., Martini, D., Garcia-Gil, M., Di Giuseppe, G., Guella, G., Dini, F., Bagnoli, P. (2006) Cytotoxic effects and apoptotic signaling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumor cells Apoptosis 11,829-843[CrossRef][Medline]
  35. Cervia, D., Petrucci, C., Bluet-Pajot, M. T., Epelbaum, J., Bagnoli, P. (2002) Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations Neuroendocrinology 76,99-110[CrossRef][Medline]
  36. Petrucci, C., Cervia, D., Buzzi, M., Biondi, C., Bagnoli, P. (2000) Somatostatin-induced control of cytosolic-free calcium in pituitary tumor cells Br. J. Pharmacol. 129,471-484[CrossRef][Medline]
  37. Schulz, S., Handel, M., Schreff, M., Schmidt, H., Hollt, V. (2000) Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies J. Physiol. (Paris) 94,259-264[CrossRef][Medline]
  38. Cammalleri, M., Cervia, D., Dal Monte, M., Martini, D., Langenegger, D., Fehlmann, D., Feuerbach, D., Pavan, B., Hoyer, D., Bagnoli, P. (2006) Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission Eur. J. Neurosci. 23,2404-2422[CrossRef][Medline]
  39. Rahat, M. A., Marom, B., Bitterman, H., Weiss-Cerem, L., Kinarty, A., Lahat, N. (2006) Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association J. Leukoc. Biol. 79,706-718[Abstract/Free Full Text]
  40. Guha, M., Mackman, N. (2001) LPS induction of gene expression in human monocytes Cell. Signal. 13,85-94[CrossRef][Medline]
  41. Weckbecker, G., Lewis, I., Albert, R., Schmid, H. A., Hoyer, D., Bruns, C. (2003) Opportunities in somatostatin research: biological, chemical and therapeutic aspects Nat. Rev. Drug Discov. 2,999-1017[CrossRef][Medline]
  42. Lewis, I., Bauer, W., Albert, R., Chandramouli, N., Pless, J., Weckbecker, G., Bruns, C. (2003) A novel somatostatin mimic with broad somatotropin release inhibitory factor receptor binding and superior therapeutic potential J. Med. Chem. 46,2334-2344[CrossRef][Medline]
  43. Van der Hoek, J., Waaijers, M., van Koetsveld, P. M., Sprij-Mooij, D., Feelders, R. A., Schmid, H. A., Schoeffter, P., Hoyer, D., Cervia, D., Taylor, J. E., Culler, M. D., Lamberts, S. W., Hofland, F. J. (2005) Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells Am. J. Physiol. Endocrinol. Metab. 289,E278-E287[Abstract/Free Full Text]
  44. Cervia, D., Langenegger, D., Schuepbach, E., Cammalleri, M., Schoeffter, P., Schmid, H. A., Bagnoli, P., Hoyer, D. (2005) Binding and functional properties of the novel somatostatin analogue KE 108 at native mouse somatostatin receptors Neuropharmacology 48,881-893[CrossRef][Medline]
  45. Reubi, J. C., Eisenwiener, K. P., Rink, H., Waser, B., Macke, H. R. (2002) A new peptidic somatostatin agonist with high affinity to all five somatostatin receptors Eur. J. Pharmacol. 456,45-49[CrossRef][Medline]
  46. Cammalleri, M., Cervia, D., Langenegger, D., Liu, Y., Dal Monte, M., Hoyer, D., Bagnoli, P. (2004) Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices Eur. J. Neurosci. 20,2711-2721[CrossRef][Medline]
  47. Cervia, D., Fiorini, S., Pavan, B., Biondi, C., Bagnoli, P. (2002) Somatostatin (SRIF) modulates distinct signaling pathways in rat pituitary tumor cells; negative coupling of SRIF receptor subtypes 1 and 2 to arachidonic acid release Naunyn Schmiedebergs Arch. Pharmacol. 365,200-209[CrossRef][Medline]
  48. Cervia, D., Fehlmann, D., Hoyer, D. (2003) Native somatostatin sst2 and sst5 receptors functionally coupled to G(i/o)-protein, but not to the serum response element in AtT-20 mouse tumor corticotrophs Naunyn Schmiedebergs Arch. Pharmacol. 367,578-587[CrossRef][Medline]
  49. Cervia, D., Nunn, C., Fehlmann, D., Langenegger, D., Schuepbach, E., Hoyer, D. (2003) Pharmacological characterization of native somatostatin receptors in AtT-20 mouse tumor corticotrophs Br. J. Pharmacol. 139,109-121[CrossRef][Medline]
  50. Rohrer, S. P., Birzin, E. T., Mosley, R. T., Berk, S. C., Hutchins, S. M., Shen, D. M., Xiong, Y., Hayes, E. C., Parmar, R. M., Foor, F., et al (1998) Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry Science 282,737-740[Abstract/Free Full Text]
  51. Cervia, D., Zizzari, P., Pavan, B., Schuepbach, E., Langenegger, D., Hoyer, D., Biondi, C., Epelbaum, J., Bagnoli, P. (2003) Biological activity of somatostatin receptors in GC rat tumor somatotrophs: evidence with sst1–sst5 receptor-selective nonpeptidyl agonists Neuropharmacology 44,672-685[CrossRef][Medline]
  52. Hoyer, D., Nunn, C., Hannon, J., Schoeffter, P., Feuerbach, D., Schuepbach, E., Langenegger, D., Bouhelal, R., Hurth, K., Neumann, P., Troxler, T., Pfaeffli, P. (2004) SRA880, in vitro characterization of the first non-peptide somatostatin sst(1) receptor antagonist Neurosci. Lett. 361,132-135[CrossRef][Medline]
  53. Pavan, B., Fiorini, S., Dal Monte, M., Lunghi, L., Biondi, C., Bagnoli, P., Cervia, D. (2004) Somatostatin coupling to adenylyl cyclase activity in the mouse retina Naunyn Schmiedebergs Arch. Pharmacol. 370,91-98[Medline]
  54. Nunn, C., Schoeffter, P., Langenegger, D., Hoyer, D. (2003) Functional characterization of the putative somatostatin sst2 receptor antagonist CYN 154806 Naunyn Schmiedebergs Arch. Pharmacol. 367,1-9[Medline]
  55. Hofland, L. J., Lamberts, S. W. (2003) The pathophysiological consequences of somatostatin receptor internalization and resistance Endocr. Rev. 24,28-47[Abstract/Free Full Text]
  56. Charo, I. F., Ransohoff, R. M. (2006) The many roles of chemokines and chemokine receptors in inflammation N. Engl. J. Med. 354,610-621[Free Full Text]
  57. Opdenakker, G., Van den Steen, P. E., Van Damme, J. (2001) Gelatinase B: a tuner and amplifier of immune functions Trends Immunol. 22,571-579[CrossRef][Medline]
  58. St-Pierre, Y., Van Themsche, C., Esteve, P. O. (2003) Emerging features in the regulation of MMP-9 gene expression for the development of novel molecular targets and therapeutic strategies Curr. Drug Targets Inflamm. Allergy 2,206-215[CrossRef][Medline]
  59. Milner, J. M., Cawston, T. E. (2005) Matrix metalloproteinase knockout studies and the potential use of matrix metalloproteinase inhibitors in the rheumatic diseases Curr. Drug Targets Inflamm. Allergy 4,363-375[CrossRef][Medline]
  60. Adams, R. L., Adams, I. P., Lindow, S. W., Zhong, W., Atkin, S. L. (2005) Somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium Br. J. Cancer 92,1493-1498[CrossRef][Medline]
  61. Reubi, J. C., Waser, B., Schaer, J. C., Laissue, J. A. (2001) Somatostatin receptor sst1–sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands Eur. J. Nucl. Med. 28,836-846[CrossRef][Medline]
  62. Siehler, S., Seuwen, K., Hoyer, D. (1998) [125I]Tyr10-cortistatin14 labels all five somatostatin receptors Naunyn Schmiedebergs Arch. Pharmacol. 357,483-489[CrossRef][Medline]
  63. Gonzalez-Rey, E., Varela, N., Sheibanie, A. F., Chorny, A., Ganea, D., Delgado, M. (2006) Cortistatin, an antiinflammatory peptide with therapeutic action in inflammatory bowel disease Proc. Natl. Acad. Sci. USA 103,4228-4233[Abstract/Free Full Text]
  64. Krantic, S., Goddard, I., Saveanu, A., Giannetti, N., Fombonne, J., Cardoso, A., Jaquet, P., Enjalbert, A. (2004) Novel modalities of somatostatin actions Eur. J. Endocrinol. 151,643-655[Abstract]
  65. Cescato, R., Schulz, S., Waser, B., Eltschinger, V., Rivier, J. E., Wester, H. J., Culler, M., Ginj, M., Liu, Q., Schonbrunn, A., Reubi, J. C. (2006) Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists J. Nucl. Med. 47,502-511[Abstract/Free Full Text]