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Somatostatin Through Its Specific Receptor Inhibits Spontaneous and TNF-- and Bacteria-Induced IL-8 and IL-1ß Secretion from Intestinal Epithelial Cells¹

Yehuda Chowers^3,*, Liora Cahalon, Maor Lahav^*, Hagai Schor, Ruth Tal^*, Simon Bar-Meir^* and Mia Levite

^* Department of Gastroenterology, Chaim Sheba Medical Center, Tel-HaShomer, Israel; and Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-- and Salmonella-induced secretion of IL-8 and IL-1ß derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF--induced secretion of IL-8 and IL-1ß. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1ß after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1ß similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1ß. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal epithelial cells function as the first line of defense between the microbe-rich lumen and the sterile cellular compartment. Functionally this is achieved by forming a physical barrier and by actively secreting proinflammatory cytokines such as IL-1ß, IL-1, and TNF-, as well as chemokines, such as IL-8 and monocyte chemoattractant protein-1, which, in turn, can induce a defensive inflammatory response. These proinflammatory mediators may be secreted by intestinal epithelial cells after both autocrine stimulation, such as stimulation by TNF-, and external stimulation, such as bacterial invasion of the cells (1, 2, 3, 4, 5), suggesting an ongoing role for epithelial cells in the mucosal inflammatory process.

The secretion of proinflammatory mediators by intestinal epithelial cells must be tightly regulated, because it is a two-edged sword. On the one hand, such secretion is needed for combating the inflammation, but on the other hand, if this response is sustained and uncontrolled, it may lead to chronic inflammatory bowel disease. For example, several studies have suggested the involvement of proinflammatory mediators and their respective receptors in intestinal pathology by showing increased secretion of IL-8 and an increased ratio of IL-1 to IL-1R antagonist in the mucosa of ulcerative colitis patients (6, 7, 8, 9, 10). To date, little is known regarding the mechanisms by which the secretion of epithelial cytokines and chemokines is controlled. Unveiling these mechanisms is important for understanding mucosal homeostasis under normal conditions as well as for treatment of intestinal diseases that result from an uncontrolled mucosal inflammatory response.

Mechanisms that are expected to control intestinal inflammatory responses should act rapidly and be specific in their effect. Conceivably, they could be mediated by the nervous system through neuropeptides delivered to the epithelial cells by nerve endings. A candidate neuropeptide that may be involved in such neuronal regulation is somatostatin, a 14-aa cyclic peptide that is released from nerve endings that reach the intestine and thus may affect various cells within the mucosa. Within the intestine, somatostatin is also secreted from nonneuronal cells distributed throughout the length of the gastrointestinal tract, specifically in the antrum and duodenum, as well as in the small intestine and colon (11, 12, 13, 14). The idea that various cells within the intestine can respond to somatostatin is supported by studies showing that various cell types found throughout the intestine, including epithelial cells, expressed somatostatin receptors (15, 16, 17).

Within the intestinal tissue, somatostatin has been shown to exert potent inhibitory effects on nonimmune intestinal functions such as intestinal motility, hormone secretion, and the regulation of mesenteric blood flow (18). Moreover, somatostatin can markedly affect immune functions such as the proliferation of lymphoid cells and the production of Ig (19, 20). In contrast to many reports of its inhibitory effects, somatostatin activates the ß₁ integrin function of T cells (i.e., the integrin-mediated adhesion to extracellular matrix components (21)), through its specific receptors. Furthermore, somatostatin directly triggers Th1 and Th2 mouse T cell lines to secrete both typical and atypical ("forbidden") cytokines (22).

In view of these potent immunomodulatory effects of somatostatin, its abundance in the intestinal tract, and its proximity to epithelial cells that express surface somatostatin receptors, we postulated that it can regulate proinflammatory cytokine secretion from intestinal epithelial cells. In this study we demonstrate that somatostatin indeed markedly inhibits both spontaneous and TNF-- and bacteria-induced secretion of IL-8 and IL-1ß from intestinal epithelial cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

TNF- was obtained from Roche (Indianapolis, IN). Somatostatin-14 and cyclo-somatostatin were obtained from Sigma (St. Louis, MO). Octreotide was obtained from Sandoz (Basel, Switzerland).

Cell lines and culture

HT-29 (ATCC HTB38) and Caco-2 (ATCC HTB27) cells were obtained from American Type Tissue Culture Collection (Manassas, VA). The cells were maintained in culture (at 37°C, 5% CO₂ incubation) in DMEM (Biological Industries Kibbutz, Beit Haemek, Israel) supplemented with 10% FCS (Bet-Haemek), 1% glutamine, 1% penicillin, and streptomycin (Bet Haemek). Caco-2 cells were also supplemented with Neut-mix F12 (Life Technologies, Wien, Austria). Routine testing of cultures for Mycoplasma infection was negative. All incubations were conducted at 37°C.

TNF--induced cytokine secretion

HT-29 test cells were grown as confluent monolayers in 24-well tissue culture plates. After the cells reached confluence, the culture medium was replaced by fresh medium and supplemented with either somatostatin or octreotide. After 30-min incubation, TNF- was added at a concentration of 200 ng/ml, and the cells were returned to the incubator for 24-h incubation. The cells were then harvested, the supernatants were collected, and the concentrations of IL-1ß and IL-8 were determined by ELISA. In experiments including cyclo-somatostatin, the cells were preincubated with this antagonist for 30 min before adding somatostatin or octreotide. For RNA extraction, cells were grown in 10-cm tissue culture dishes until they reached confluence. The medium was then replaced by fresh medium and supplemented with somatostatin and TNF- as previously described. After 2 h of incubation, the cells were harvested, and their RNA was extracted.

Salmonella-induced cytokine secretion

HT-29 cells were grown as confluent monolayers. Either somatostatin or octreotide (10^–8 M) was added for 1 h. In experiments in which cyclo-somatostatin was added, the cells were preincubated with this antagonist for 30 min. Subsequently, 10⁷ CFU of Salmonella type D, obtained from clinical isolates, was added to the culture for 4 h of incubation (37°C, 5% CO₂). The cells were then extensively washed with culture medium containing 50 ng/ml gentamicin. Subsequently, the cells were incubated overnight, after which the supernatant was collected and assayed for IL-8 and IL-1ß concentrations. Test cells were lysed using distilled water, and the lysate was plated for bacterial CFU quantitation.

Determination of cytokine secretion and cell viability

IL-8 was measured by ELISA as previously described (1). Briefly, 96-well plates were coated with polyclonal goat anti-human IL-8 Abs (R&D Systems, Minneapolis, MN) as capturing Abs. After having been incubated with the tested supernatants and washed, polyclonal rabbit anti-human IL-8-detecting Abs (Endogen, Boston, MA) were added. Alkaline phosphatase-labeled mouse anti-rabbit IgG (Sigma) was used as a second Ab. Quantification of bound Abs was conducted using p-nitrophenylphosphate (Sigma). IL-1ß was measured by ELISA using a commercially available kit (Genzyme, Cambridge, MA), according to the manufacturer’s protocol. Three replicate samples were included in each experiment. Cell viability was determined using the MTT method (23).

RNA extraction and RNA protection assay

RNA extraction was performed using the Tri-Reagent kit (MRC, Cincinnati, OH), and mRNA levels were measured by the RiboQuant multiprobe RNA protection assay (PharMingen, San Diego, CA), following the manufacturer’s instructions.

Briefly, antisense RNA probes were transcribed using the cDNA template sets human cytokine kit 5 and human cytokine kit 2. For transcription, 1 µl of the template was incubated (1 h at 37°C) in a mixture containing 1 µl of RNasin, 1 µl of the nucleotide pool, 2 µl of DTT, 4 µl of 5x transcription buffer, 10 µl of [-³²P]UTP, and 1 µl of T7 RNA polymerase. All reagents were supplied by the manufacturer. The reaction was terminated by adding DNase. Labeled RNA probes were extracted using phenol/chloroform/isoamyl alcohol and were precipitated using ethanol. The level of [-³²P]UTP incorporated was determined using a scintillation counter.

For hybridization, 20 µg of RNA was precipitated by ethanol, and the pellet was dried using a vacuum evaporator centrifuge. The RNA samples were then resuspended in an 8-µl hybridization buffer (80% formamide, 1 mM EDTA, 400 mM NaCl, and 40 mM Prpes (pre-eazine-N,N'-bis-ethanesulfonic acid)) at 56°C, mixed with 2 µl of probe prepared as previously described, heated to 90°C, and then incubated at 56°C for 12 h.

For RNase digestion, 6 µl of a mixture containing RNase A and RNase T1 was added and reacted for 45 min at 30°C. After digestion, the samples were mixed with proteinase K and an appropriate buffer (PharMingen) for 15 min at 37°C, after which they were extracted by phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The samples were then air-dried, resuspended in loading buffer, and size-separated using PAGE. Appropriate bands representing IL-8, IL-1ß, and GAPDH RNA were measured using a phosphorimager.

Statistical analysis

All statistical analysis was performed using an unpaired, two-tailed, t test; p values larger than 0.05 were not considered significant. Error bars represent the variation among three individual wells used in the ELISA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Somatostatin inhibits the spontaneous IL-8 and IL-1ß secretion from two different intestinal cell lines

Intestinal epithelial cell lines such as HT-29 are known to spontaneously secrete IL-8 and express IL-1ß mRNA (1). Therefore, we tested the effect of somatostatin on such proinflammatory cytokine secretion and found, as shown in Fig. 1A, that at a concentration as low as 10^–9 M, somatostatin markedly inhibits IL-8 secretion from HT-29 cells. This inhibitory effect was dose dependent, ranging from 0.75 x 10^–9 to 1.2 x 10^–8 M, reaching 87.4% inhibition at a concentration of 3 x 10^–9 M. Fig. 1B shows that somatostatin also markedly inhibited the spontaneous secretion of IL-1ß from these cells. Notably, the dose-response relationship of the IL-1ß inhibition resembled that of the IL-8 inhibition, reaching >90% inhibition at 3 x 10^–9 M. These results show that at physiological concentrations, within the nanomolar range, somatostatin markedly inhibits the spontaneous secretion of two proinflammatory mediators, IL-8 and IL-1ß, from intestinal epithelial cells.

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Somatostatin suppresses the secretion of IL-8 and IL-1ß from HT-29 cells. HT-29 cells were grown until confluence was reached and then were incubated in the presence of somatostatin overnight. Supernatants were collected and assayed by ELISA for IL-8 (A) and IL-1ß (B) concentrations. The reduction of IL-8 and IL-1ß concentrations from baseline was significant at a somatostatin concentration of 0.75 nM (p < 0.0001). One experiment representative of three is shown.

View larger version (9K): [in this window] [in a new window]   FIGURE 2. Somatostatin suppresses IL-8 secretion from Caco-2 cells. Caco-2 cells were grown until confluence was reached and then incubated in the presence of somatostatin (10–8 M) overnight. Culture supernatants were then collected and assayed for IL-8 concentrations. The reduction of IL-8 concentrations after incubation with somatostatin was significant (p < 0.0001). One experiment representative of three is shown.

The effects of somatostatin can be mediated by up to five receptor subtypes, termed somatostatin receptor (SSTR)³ 1–5. These receptors are diversely distributed on different cell types (24). Octreotide is a synthetic somatostatin octapeptide analogue that binds with high affinity to receptor subtypes 2 and 5 and with low affinity to receptor subtype 3 (18). Therefore, we used octreotide to investigate whether the inhibitory effect of somatostatin on proinflammatory cytokine secretion of intestinal epithelial cell is mediated by its interaction with its receptors and, if so, by which subtype. Fig. 3 shows that, when applied to the HT-29 epithelial cell line, octreotide markedly suppressed IL-8 and IL-1ß secretion. A similar effect of octreotide was noted using Caco-2 cells (data not shown). Like somatostatin, the octreotide-induced inhibitory effect was dose dependent and occurred within a similar dose range. These findings suggest that the inhibitory effects of somatostatin on spontaneous IL-8 and IL-1ß secretion result from its interaction with its specific receptors, primarily subtypes 2 and/or 5. To further support the idea that somatostatin directly interacts with its specific receptors to inhibit IL-8 and IL-1ß secretion, we used the somatostatin antagonist cyclo-[7-aminoheptanoyl-phe-trp-lys-thr(bzl)], termed cyclo-somatostatin (25, 26). Fig. 4 shows that cyclo-somatostatin blocked the somatostatin-induced inhibition of IL-1ß secretion after coincubation at an equimolar ratio. Cyclo-somatostatin also blocks the effects of somatostatin on IL-8 secretion and the effect of octreotide on IL-1ß and IL-8 secretion (data not shown). Note that these effects were not correlated with an inhibition of cell proliferation or viability, because neither somatostatin nor octreotide, at the same concentrations that blocked secretion of the proinflammatory mediators, affected cell viability, as consistently verified by the use of an MTT assay (OD: medium, 0.202 ± 0.004; somatostatin, 0.219 ± 0.02; octreotide, 0.227 ± 0.02; cyclo-somatostatin, 0.23 ± 0.02; see also Table I). Taken together, these results demonstrate that somatostatin directly binds to its specific receptors of subtypes 2 and/or 5 on the surface of intestinal epithelial cells and thereby causes a marked inhibition of spontaneous IL-1ß and IL-8 secretion.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Octreotide has effects similar to those of somatostatin on IL-8 and IL-1ß secretion from HT-29 cells. HT-29 cells were grown until confluence was reached and then were incubated in the presence of octreotide overnight. Supernatants were collected and assayed for IL-8 (A) and IL-1ß (B) concentrations. The reduction in IL-8 concentration was significant at 0.15 nM somatostatin (p < 0.049), and that in IL-1ß (p < 0.0001) was significant at 1.25 nM somatostatin. One experiment representative of three is shown.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. The inhibitory effect of somatostatin on IL-8 and IL-1ß secretion is mediated through its specific receptor. HT-29 cells were grown until confluence was reached and then preincubated with cyclo-somatostatin (10–8 M) for 30 min, after which somatostatin (10–8 M) was added for an overnight incubation. The culture supernatants were collected and assayed for IL-1ß concentrations. The reduction in IL-1ß concentration from baseline was significant (p < 0.0001), whereas after treatment with cyclo-somatostatin, the IL-1ß concentration was not significantly different from baseline (p = 0.54). One experiment representative of three is shown.

Somatostatin and octreotide inhibit IL-8 and IL-1ß secretion triggered by TNF- stimulation

Intestinal epithelial cells secrete proinflammatory mediators at high concentrations after TNF- stimulation (1, 2, 3, 4, 5). The important role played by TNF- in mucosal inflammation is suggested by the increased mucosal TNF- concentrations in Crohn’s disease (27, 28, 29, 30), as well as by the beneficial effect of neutralizing TNF- in the treatment of this disease (31, 32, 33, 34). Accordingly, we investigated whether somatostatin can inhibit not only the spontaneous, but also the TNF--evoked, cytokine secretion.

TNF- stimulated the secretion of IL-8 from 108 to 162 ng/ml and IL-1ß from 1.08 to 1.96 ng/ml. As shown in Fig. 5, somatostatin inhibited the mediator secretion in a dose-dependent manner. Octreotide, the somatostatin agonist, exerted a similar inhibitory effect (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 5. Somatostatin suppresses TNF--induced IL-8 and IL-1ß secretion from HT-29 cells. HT-29 cells were grown until confluence was reached and then were treated with TNF- (200 ng/ml) for 1 h, after which somatostatin was added for overnight incubation. The supernatants were then collected and assayed for IL-8 (A) and IL-1ß (B) concentrations. The reduction from the baseline IL-8 concentration reached significance at 0.38 nM somatostatin (p < 0.0004) and that from the baseline IL-1ß concentration reached significance at 0.75 nM somatostatin (p < 0.0001). One experiment representative of three is shown.

Cytokine and chemokine secretion from epithelial cells may be triggered by exogenic stimulation such as bacterial invasion (4, 5). Therefore, we next investigated whether cytokine and chemokine secretion that was induced by bacterial invasion was also inhibited by somatostatin. HT-29 cells were pretreated with either somatostatin or octreotide, then infected with Salmonella, and IL-8 concentrations were measured after overnight incubation. As shown in Fig. 6, Salmonella invasion increased the secretion of IL-8 from a baseline level of 49.5 to 85 ng/ml. Treatment of the cells with either somatostatin or octreotide completely precluded the Salmonella-induced IL-8 secretion, resulting in concentrations even lower than the baseline. Adding the cyclo-somatostatin to the culture blocked the somatostatin or octreotide-induced inhibition, indicating that the activity of somatostatin was mediated by its specific receptors. Note that neither somatostatin nor octreotide had any effect on the extent of Salmonella invasion, because an equal number of bacteria were isolated from both untreated and somatostatin- or octreotide-treated cells, as measured by the OD of bacterial cultures from cell lysates (medium, 0.294 ± 0.001; somatostatin, 0.291 ± 0.002; octreotide, 0.292 ± 0.0007; cyclo-somatostatin, 0.292 ± 0.003; cyclo-somatostatin plus somatostatin, 0.292 ± 0.004; cyclo-somatostatin plus octreotide, 0.291 ± 0.002; Table II).

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Somatostatin and octreotide suppress IL-8 secretion induced by Salmonella invasion. HT-29 cells were grown until confluence was reached and then were treated with cyclo-somatostatin for 30 min, after which somatostatin (10–8 M) or octreotide (10–8 M) was added. After 1 h, 107 CFU Salmonella was added and coincubated with the cells for 5 h. The cells were then washed from the bacteria and remained in culture overnight in fresh medium. Subsequently, the supernatants were harvested and analyzed for IL-8 concentrations. Invasion by Salmonella induced significant elevation of the IL-8 concentration (p < 0.0002). The reduction in the IL-8 concentration after treatment with somatostatin and octreotide was significant (p < 0.0001). The effect of treatment with cyclo-somatostatin alone or with somatostatin or octreotide was not significantly different from the induced IL-8 concentrations (p > 0.36). One experiment representative of two is shown.

The inhibition of cytokine secretion may occur at many subcellular levels, such as transcription or translation. To test whether somatostatin inhibits IL-8 secretion at the transcription level, we used an RNA protection assay. Briefly, RNA was extracted from HT-29 cells that were pre-exposed to either cyclo-somatostatin plus somatostatin or somatostatin alone and then stimulated by TNF-. The cells were harvested after 2 h, because the up-regulation of IL-8 mRNA concentration after TNF- stimulation is maximal at this time (1). As shown in Fig. 7, the IL-8 mRNA concentration was markedly reduced after incubation with somatostatin. The inhibitory effect of somatostatin on IL-8 mRNA concentration was apparent for both spontaneous and TNF--stimulated IL-8 secretion and was abolished by the specific receptor antagonist cyclo-somatostatin. A similar effect was noted for IL-1ß, as shown by the quantitative phosphorimager readings of the ratios between IL-1ß and GAPDH (medium, 0.039; TNF-, 0.053; TNF- plus somatostatin, 0.024; TNF- plus cyclo-somatostatin, 0.055; TNF-, somatostatin, plus cyclo-somatostatin, 0.050). These results suggest that the direct and specific inhibitory effect of somatostatin on intestinal epithelial proinflammatory mediator secretion is directly correlated with suppression of the respective mRNA concentrations.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Somatostatin treatment down-regulates IL-8 mRNA concentrations. HT-29 cells were cultured until confluence was reached. Cells were incubated with somatostatin for 1 h, after which TNF- was added to the culture for 2 additional h of incubation. Cyclo-somatostatin was added to the culture 30 min before somatostatin. The cells were then harvested, and total RNA was extracted. IL-8 and GAPDH mRNA concentrations were measured using an RNA protection assay. The ratio between the densitometric readings of IL-8/GAPDH is indicated at the bottom. One of two experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our studies were performed using two adenocarcinoma-derived cell lines: HT-29 and Caco-2. Previous studies have shown that these two cell lines respond to TNF- stimulation and bacterial invasion in a manner similar to freshly isolated intestinal epithelial cells (5) and express a similar array of chemokine receptors (38). These similarities suggest that our findings with the HT-29 and Caco-2 cells represent immune regulatory mechanisms that are relevant to normal epithelial cells as well.

The fact that somatostatin inhibited the epithelial proinflammatory cytokine secretion triggered by both TNF- and bacterial invasion supports the idea that somatostatin may exert such a regulatory role in in vivo situations. The local immunoregulatory effect of somatostatin could, in turn, directly affect the intestinal tissue that is continuously exposed to and stimulated by the luminal flora. Furthermore, it could limit the inflammation induced in acute enteric diseases such as Salmonella infections to prevent the development of chronic inflammation and the ensuing tissue damage.

The ability of somatostatin to inhibit cytokine secretion may be of particular importance to compartmentalized tissues such as the intestinal mucosa, because this neuropeptide is produced locally by D cells throughout the gastrointestinal tract (i.e., stomach, duodenum, ileum, and colon). In addition, somatostatin is produced by enteric nerve endings innervating the intestine (11, 12, 13, 14). These diverse cellular somatostatin sources may actually control the amount, location, and timing of somatostatin secretion. Such regulated secretion could potentially determine the target effector cell population that would respond to somatostatin in normal as well as pathological situations, such as tissue injury and inflammation.

Various studies have presented seemingly different findings regarding the immunoregulatory functions of somatostatin. Peluso and co-workers (39) showed that somatostatin inhibits the secretion of TNF-, IL-1ß, and IL-6 from LPS-stimulated PBMC, whereas Komorowski and co-workers (37) found that somatostatin augmented IL-6 secretion from LPS-activated peripheral blood monocytes. In addition, somatostatin was reported to affect the cytokine receptor concentrations within the intestine, for example, to markedly inhibit IL-2R expression in intestinal mononuclear cells. Interestingly, this function was exerted at concentrations 100-1000 times lower than those needed to inhibit IL-2R in PBL (40). In contrast to its many inhibitory functions, when testing T cell responses, somatostatin directly, without any additional stimulatory molecules, stimulated the secretion of IL-2, IFN-, IL-4, and IL-10 from Th0, Th1, and Th2 cell lines and induced ß₁ integrin-mediated functions (22).

The ability of somatostatin to diversely regulate immune functions was also observed using in vivo models. For example, the somatostatin analogues BIM 23014 and octreotide reduced the volume of inflammatory exudate, the number of infiltrating leukocytes, and the expression of immunoreactive TNF- in aseptic inflammation induced by carrageenin injection (41). In a rat model of experimental arthritis, treatment with somatostatin resulted in increased local ß-endorphin concentrations and in a reduction in systemic leukocytosis, possibly linked to a corresponding reduction of IL-1ß concentrations (42). Other investigators, using granulomas induced by Schistosoma mansoni as a model system have shown that lymphocytes within the lesions express somatostatin receptors and bind somatostatin, and that such binding results in reduced secretion of IFN- (43). In addition, somatostatin reduced the amount of IFN--induced IgG2a production in a dose-dependent manner after schistosome egg Ag challenge in mice (44). Somatostatin mRNA was also shown to be expressed within macrophages in schistosoma-induced granuloma (45). In view of these findings, it has been suggested that inflammation in such granulomas is regulated by the local production of both substance P and somatostatin, and that the interaction between these molecules and the immune cells modulates IFN- production and immune activity (46). Within the intestinal tissue, treatment with the somatostatin analogue octreotide has been shown to ameliorate the inflammation in the acetic acid-induced colitis rat model (47). Finally, somatostatin also inhibited TNF--stimulated IL-8 and IL-6 production in human synovial cells obtained from rheumatoid arthritis patients (48). Taken together, these studies suggest that somatostatin exerts diverse immunoregulatory activities, and that it has the potential to either induce or inhibit cytokine secretion, depending on the cell type, its activation state (whether stimulated or not), and the specific cytokine in question. Our observation that somatostatin regulates the immune function of epithelial cells is in good agreement with these studies. The abundant expression of somatostatin in the intestine may indicate that this mediator is indeed of particular in vivo importance in this tissue.

The effects of somatostatin are mediated by five receptor subtypes (24). These receptor subtypes differ in their tissue distribution and in some of the biological consequences that result from their activation (49). The somatostatin receptor subtypes expressed by the HT-29 cells used in this study have been defined using RT-PCR. Using this method, these cells were found to express only receptor subtypes 1 and 5 (50). In the present study incubation of the cells with octreotide or somatostatin produced a similar down-regulation of secretion of proinflammatory mediators. Octreotide stimulates mainly receptor subtypes 2 and 5 (18). Because the HT-29 cells do not express receptor subtype 2, the immunoregulatory effect of somatostatin is most likely mediated by receptor subtype 5. This information may be highly relevant for the design of pharmacologic mediators for better manipulation of the intestinal immune system. Extending the knowledge regarding the mechanisms by which somatostatin regulates immune functions of epithelial cells and the roles of the different receptor subtypes may allow a specific and controlled use of somatostatin agonists in the clinical setting of intestinal inflammation.

	Footnotes

 
¹ This work was supported by a grant from the Volkswagen-Stiftung foundation to M.L.

² Address correspondence and reprint requests to Dr. Yehuda Chowers, Department of Gastroenterology, Chaim Sheba Medical Center, Tel-HaShomer 52621, Israel.

³ Abbreviation used in this paper: SSTR, somatostatin receptor.

Received for publication December 9, 1999. Accepted for publication June 20, 2000.

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