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Somatostatin Receptor Genes Are Expressed in Lymphocytes from Retroorbital Tissues in Graves’ Disease

Istituto di Endocrinologia, Seconda Università di Napoli (D.P., A.N., A.B., A.A.S.), and Istituto di Scienze Oftalmologiche, Università Federico II, (G.B., P.V.) Naples, Italy

Address all correspondence and requests for reprints to: Dr. Antonio A. Sinisi, Istituto di Endocrinologia, Seconda Università di Napoli, Building 16, Via Pansini 5, 80131 Naples, Italy. E-mail: antonio.sinisi{at}unina2.it.

Abstract

The radiolabeled somatostatin (SST) analog octreotide accumulates within the orbits of active Graves’ ophthalmopathy (GO), and octreotide and lanreotide have been proposed to treat this disorder. It is still unclear which retroorbital cells the SST analogs target. Lymphocytic infiltration of retroorbital tissues is a peculiarity of GO, and labeled octreotide could accumulate at specific sites on retroorbital-activated lymphocytes. The accumulation of radiolabeled analogs is due to the interaction with specific cell surface SST receptors. Five subtypes of somatostatin receptors (SST1–5), member of the G protein-coupled, seven-transmembrane superfamily, are described. It still unknown which SST subtype is expressed in retroorbital activated lymphocytes. The aim of this study was to evaluate the expression of SST1–5 genes in lymphocytes recovered from retroorbital tissues obtained from patients with GO undergoing orbital decompression. Cultured phytohemagglutinin-stimulated lymphocytes from retroorbital blood samples, drawn during orbital surgery in five patients with GO and in two control patients without autoimmune or thyroid diseases and without orbital inflammatory conditions, were also studied. RT-PCR of total RNA extracted from lymphocytes was performed using primers for SST1–5 and, as internal control, for glyceraldehyde-3-phosphate dehydrogenase. All SSTs transcripts were found in lymphocytes both from GO retroorbital tissues and blood samples. The levels of expression of SST1, -2, and -4 mRNA were higher than those of the SST3 and -5 transcripts. In the lymphocytes from control subjects, the SST subtypes with high affinity for octreotide were barely found. The presence, even if at different concentrations, of all SST1–5 receptors in retroorbital lymphocytes from GO shows that they are targeted by SST analogs and could explain the effects described in GO patients treated with SST analogs.

GRAVES’ OPHTHALMOPATHY (GO), the ocular feature of Graves’ disease, is generally considered an autoimmune process affecting the orbital tissue (1). Immune-suppressive drugs and/or orbital radiotherapy are used to control GO, but they often do not provide satisfactory results (2, 3, 4). There are few clinical reports showing that octreotide and lanreotide, two long-acting somatostatin (SST) analogs, have beneficial effects on GO, but their mechanism of action remains unclear (5, 6, 7, 8). Octreotide-specific binding in the retroorbital tissue of patients with GO during the active phase of the disease has been suggested by [¹¹¹In]diethylenetriamine pentaacetic acid-D-Phe¹-octreotide scintigraphy (Octreoscan) (9, 10, 11). Different studies proposed that orbital uptake of labeled octreotide can be used to evaluate the stage of GO (12, 13, 14). Some researchers suggested that octreoscan positivity predicts the efficacy of treatment with SST analogs, although some patients with negative octreoscan still have beneficial effects to octreotide (14). This is probably because SST receptor imaging in vivo does not distinguish the analog-binding cells (15). In fact, labeled octreotide could bind to infiltrating immune cells or orbital components or both. Several studies demonstrate that the presence of SST receptors, often in high density on neuroendocrine tumors and in different malignant and nonmalignant tissues, is proving useful for their localization and allowing the development of various novel receptor-mediated treatment modalities (16). SST acts on target tissues through specific membrane receptors, coded by five genes (SST1–5), cloned, and characterized (17). As the pathogenesis of GO seems to be multifactorial SST analogs could act at different levels (1). A recent study demonstrated that SST and its receptor genes are expressed on primary fibroblast cultures from retro-orbital tissues of GO patients (18). Retroorbital fibroblasts specifically bind SST-14 and respond in vitro to the SST analog octreotide by inhibiting cAMP production and cell growth and inducing apoptosis. Consequently, it appears that the uptake of labeled octreotide in the orbits of GO subjects in vivo may be due to specific binding to connective tissue elements and that octreotide interact directly with retroorbital fibroblasts. SSTs have been demonstrated on activated human peripheral lymphocytes by binding and molecular studies (19), but their expression has not been investigated in retroorbital lymphocytes. The aim of this study was to evaluate the expression of the SST1–5 genes by RT coupled with PCR amplification on lymphocytes recovered from surgical retroorbital tissues of patients with GO and from blood samples obtained by direct aspiration from the orbits during surgery.

Materials and Methods

Origin of tissues and cell cultures

Retroorbital connective tissue was obtained from 14 patients (10 women and 4 men; aged 35–72 yr; mean, 51.5 yr) during orbital decompression surgery for severe GO. Four patients did not present significant congestive signs and symptoms, the other had severe inflammatory GO (activity score, 4–7). The diagnosis was based on endocrinological and ophthalmological criteria, including laboratory determination of hormones and antibodies and imaging study of orbits. All were euthyroid (six not taking medication, five receiving methimazole therapy, three taking L-T₄ supplementation for hypothyroidism); nine had been previously treated with steroids and were off therapy for over 6 months; five had untreated GO, including two of recent onset. Informed consent was obtained from patients to use orbital tissue samples for in vitro study. Lymphocyte cultures from heparinized blood samples, collected during surgery by direct aspiration from the orbit in five patients with GO and in two control patients without any history of autoimmune or thyroid diseases and without orbital inflammatory conditions who had undergone eye surgery for trauma or strabism were set up adding blood drops to 199 medium with phytohemagglutinin, l-glutamine, 10% fetal bovine serum, and antibiotics (Life Technologies, Inc., Milan, Italy). Single-cell preparations were made by mincing retroorbital tissues and lysing red blood cells in ammonium chloride (0.15 M), potassium carbonate (1 nM), and EDTA (0.1 nM) buffer (pH 7.3) lysing buffer using standard techniques (20). Lymphocytes were isolated using a modification of a previously described procedure (20). Briefly, retroorbital tissues from GO patients were cut into 2- to 5-mm segments and stirred at 37 C for 45 min in DMEM containing 50 U/ml type IV collagenase (Sigma, St. Louis, MO), 0.5 mg/ml dispase grade II (Roche, Indianapolis, IN), 0.01% gentamicin (Life Technologies, Inc.), and 5% fetal calf serum. After collecting the cell suspension in the supernatant, the enzymatic treatment was repeated. The supernatants were filtered through nylon wool, and enriched lymphocytes were centrifuged through a discontinuous 44%/70% Percoll (Amersham Pharmacia Biotech, Alameda, CA) gradient for 18 min at 800 x g at 4 C. Cells at the interface between the 70% and 44% layers were collected and washed. Approximately 95% of the lymphocytes were viable, as measured by trypan blue exclusion. The recovered lymphocytes were stored at -80 C or immediately used for RNA extraction.

RT-PCR analysis

Total RNA was isolated from lymphocytes using TRIzol (Invitrogen, Milan, Italy). Residual DNA was removed by ribonuclease-free deoxyribonuclease I treatment (Promega Corp., Florence, Italy). RT-PCR was carried out as previously described (21). RNAs were reversely transcribed using 5 µg of total RNA after annealing with 0.2 nM oligo (deoxythymidine) for priming of cDNA in the presence of reverse transcriptase (Superscript-BRL-Invitrogen 200 U, Milan, Italy) at 37 C for 1.5 h. The reaction was stopped by incubation at 95 C for 5 min. To obtain a negative control for the amplification reaction, RNA transcription was performed without adding reverse transcriptase. The amplification reaction was carried out in a DNA thermal cycler (Perkin-Elmer/Cetus, Milan, Italy) using 600 ng cDNA and 5'/3' end oligonucleotides for SST1, SST2, SST3, SST4, and SST5. To evaluate variability in the expression of SSTs a semiquantitative PCR was performed in which these genes were amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using a technique previously described (22). Briefly, before performing semiquantitative PCR the number of cycles was chosen in the middle of the exponential phase of the reaction, separately for each gene type. To establish the number of cycles, GAPDH was amplified at 15, 22, 32, and 40 PCR cycles; SST1, SST2, SST3, SST4, and SST5 were subjected to 25, 32, and 40 amplification cycles in separate experiments (data not shown). Then, PCR conditions were as previously described, and the reaction consisted of 32 cycles of amplification for SSTs and of 22 cycles for GAPDH. The specific primers for GAPDH were added to the PCR reaction after the first 10 cycles. The levels of mRNAs, quantified by densitometry scanning of the amplification products electrophoresed on agarose gels, are expressed as ratio between the density of each gene product and that of coamplified GAPDH. We used oligonucleotide sequences for SST1–5 and GAPDH as previously described (18, 21). The PCR products were analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide using 100-bp DNA ladder (Invitrogen, Milan, Italy) as size marker and by comparing their size with the size expected from the gene sequence.

Results

SST1–5 genes are expressed at different levels in lymphocytes deriving from GO retroorbital samples

SST1–5 transcripts were found in all lymphocyte samples isolated from GO retroorbital tissues and in cultured lymphocytes from GO orbital blood samples (Figs. 1 and 2). The SST expression levels in all the subjects studied are presented in Table 1. The level of the transcripts of SST1, -2, and -4 was higher compared with SST3 and 5 mRNA (Fig. 1, lower panel) in lymphocytes from GO retroorbital tissues (Table 1). We found a similar expression pattern of SST1–5 transcripts in cultured lymphocytes from GO orbital blood samples (Fig. 2, lower panel, and Table 1). The SST expression resulted not related to GO activity. Only SST2, -3, and -4 were detectable at low level of expression in lymphocytes from control retroorbital blood samples (Table 1). Treatment with deoxyribonuclease and coamplification of the GAPDH gene containing introns excluded genomic DNA contamination. Moreover, we found no products in control amplifications performed in absence of cDNA (negative control; data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1. Expression of sst1–5 and GAPDH in lymphocyte cultures from retroorbital tissue samples of patients with GO. Upper panel, sst1–5 and GAPDH RT-PCR products, separated on a 1.5% agarose gel, in a representative sample from retroorbital tissue of a patient with GO. M, 100-bp DNA size marker. sst1 (334 bp), sst2 (461 bp), sst3 (221 bp), sst4 (247 bp), sst5 (223 bp), and GAPDH (876 bp) transcripts are present in retroorbital tissue samples of patients with GO. Lower panel, Quantification of relative sst1–5 mRNAs. The sst1–5 bands were normalized to GAPDH mRNA control. Each column represents the mean ± SE of three separate experiments.

View larger version (43K): [in this window] [in a new window]   Figure 2. Expression of sst1–5 and GAPDH in lymphocyte cultures from orbital blood samples of patients with GO. Upper panel, sst1–5 and GAPDH RT-PCR products, separated on a 1.5% agarose gel, in a representative sample from orbital blood of a patient with GO. M, 100-bp DNA size marker. sst1 (334 bp), sst2 (461 bp), sst3 (221 bp), sst4 (247 bp), sst5 (223 bp), and GAPDH (876 bp) transcripts are present in lymphocyte culture from orbital blood sample from GO. Lower panel, Quantification of relative sst1–5. The sst1–5 bands were normalized to the GAPDH mRNA control. Each column represents the mean ± SE of three separate experiments.

The present study shows that all SSTs genes are expressed in retroorbital lymphocytes from GO patients. The presence of binding sites for SST on retroorbital tissues of patients with GO has been suggested by uptake of [¹¹¹In]diethylenetriamine pentaacetic acid-D-Phe¹-octreotide into the orbits (5, 6, 7, 8, 9), but it cannot distinguish between the binding on tissue cells and that on other elements, such as blood vessels and immune cells (15, 23, 24). Octreoscan positivity in GO has been previously explained as a result of accumulation of radiolabeled octreotide in infiltrating immune cells, as in other pathological processes, including granulomatous and autoimmune diseases such as sarcoidosis, Crohn’s disease, and rheumatoid arthritis (15, 19, 23, 24). In a previous study we have demonstrated that cultured fibroblasts from retroorbital tissues of patients with GO express functional SSTs (18, 25). Our present and previous data showed that those orbital hot spots seen in vivo indeed represent true SST receptors localized on retroorbital connective tissue and in infiltrating lymphocytes. The retroorbital lymphocytes investigated express all SST receptor subtypes, even if at different expression levels. In particular, we demonstrated a higher expression level of SST2 subtype that has high affinity for the somatostatin analogs currently used in clinical practice (26).

The natural SST peptide and its stable analogs negatively regulate the growth and activity of several normal and pathological cells through specific receptor subtypes and receptor-coupled intracellular signals (17, 18). Both receptor subtypes (SST2 and SST5), which are known to bind SST synthetic analogs and are required for the negative mitogenic signal, are expressed in GO retroorbital cells. Moreover, the level of expression of SST5 is quite low compared with that of SST1 and SST4, suggesting that novel SST analogs specific for SST1 and SST4 might achieve better results in GO patients. Thus, our molecular data offer the basis for a possible direct effect of SST and its synthetic analogs on retroorbital cells. It has been suggested that the uptake of labeled SST analog in GO may be an index of activity of eye disease and could identify the patients who might benefit from medical treatment (10, 11, 12, 13).

The different orbital accumulation of radioactivity between patients with apparently active and inactive disease might be explained by taking into account the variable lymphocyte infiltration between active and inactive stages. The difference effects of SST analog treatment on GO could be interpreted by the different levels of SST1 and -2 expression compared with SST5 that we found by our RT-PCR studies. Barely detectable expression of SST subtypes with high affinity for octreotide in lymphocytes from subjects without autoimmune eye diseases further support this conclusion. Moreover, it must be considered, as suggested by our previous data, that qualitative and/or quantitative differences in the expression of SST receptors on connective tissue can also contribute. In particular, our data showed that all primary cultures of retroorbital fibroblasts of GO patients expressed the SST gene transcript and one or more SSTs that have a high affinity for the 2 analogs (class 1 SST). The SST2 transcript was found in 9, SST3 in 5, and SST5 in 8 of 10 GO cell cultures. SST2 was detected in all 6, and SST3 in 4 of the 6 control cell cultures. SST4 was absent from all samples, and SST1 was found only in 6 of the 10 GO samples (18). Some clinical reports based on orbit positive scan showed that octreotide or lanreotide has beneficial effects on the clinical course of GO (6, 7, 8), and one of the action mechanisms could be explained by the inhibitory effects of SST analog on growth of primary cultures of fibroblasts deriving from retroorbital tissue of GO patients (18).

SST analogs may act inhibiting the immune system (19, 27). It is known that SST suppresses IGF-I activity (28) and exerts a direct inhibition of the release of cytokines, such as interferon-, IL-1, and TNF. Macrophages, dendritic cells, and infiltrating activated lymphocytes produce cytokines that seem to have a key role in starting and continuing the complex cascade of reactions occurring in the retroorbital space of GO also by inducing glycosaminoglycan (GAG) synthesis in orbital preadipocytes and fibroblasts (2, 29). SST could act at the level of these proinflammatory molecules. Our results suggest that SST analogs could inhibit immune cells infiltrating retroorbital tissues through specific cell surface receptors. Therefore, the beneficial effects on GO described in GO patients treated with octreotide or lanreotide may be due to the direct action of the analogs on retroorbital cells together with the effect mediated by inhibition of the local immune system.

Acknowledgments

We are grateful to Dr. Joseph Sepe for editing this manuscript.

Footnotes

This work was supported in part by grants from Progetti di Interesse Nazionale (PRIN) 2000 (to A.B.).

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GO, Graves’ ophthalmopathy; SST, somatostatin.

Received May 22, 2002.

Accepted August 13, 2002.

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