Immunomodulatory activities of the somatostatin analogue BIM 23014c: effects on murine lymphocyte proliferation and natural killer activity

Regulatory Peptides, 32 (1991) 129-139

129

Elsevier REGPEP 00990

Immunomodulatory activities of the somatostatin analogue BIM 23014c" effects on murine lymphocyte proliferation and natural killer activity Albert Agro, Ireneusz P a d o l and Andrzej M. Stanisz Intestinal Diseases Research Unit, Department of Pathology, McMaster University,Hamilton (Canada)

(Received 23 May 1990; revised version received 17 October 1990; accepted 18 October 1990) K e y words." Somatostatin; Lymphocyte; Immunomodulation; BIM 23014c

Summary We have examined the effect of somatostatin and its octapeptide analogue BIM 23014c on concanavalin A-induced lymphocyte proliferation and target-specific natural killer activity both in vitro and in vivo. Using Peyer's patches and spleen as a source of lymphocytes, we found that both peptides modulated immunity in a dose-dependent manner. Comparatively, there was no significant difference between the activity of somatostatin or BIM 23014c in the modulation of immunity. Proliferation, both in vitro and in vivo, was significantly inhibited by both peptides in each organ with a higher specificity towards the Peyer's patch lymphocytes. Natural killer activity was also inhibited in both organs in vivo and in vitro. Thus, not only did somatostatin and BIM 23014c have similar effects on proliferation and natural killer activity, but their effect was organ specific. Preliminary data suggest that BIM 23014c works via the same receptor as somatostatin, therefore intimating that these two peptides are both clinically and immunologically similar.

Introduction Recent evidence is supportive of a communication network existing between the neuroendocrine system and the immune system [ 1 ]. The immunoregulatory properties

Correspondence: A.M. Stanisz, Intestinal Diseases Research Unit, Department of Pathology, McMaster University, Room HSC-3N5C, 1200 Main Street W., Hamilton, Ontario L8N 3Z5, Canada

0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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of neurally-derived products, including hormones [2-6] and peptides [7,8], have been demonstrated both in vitro and vivo. The work of Felten and colleagues, showing that lymphoid tissues are innervated, gives confirmation to this relationship [9]. The experimental focal point of the majority of work into this neuroimmune network has centered around the immunoregulatory activity ofneuropeptides including members of the tachykinin family such as substance P (SP); other peptides including vasoactive intestinal peptide (VIP) and somatostatin (SOM). The activity of these peptides both in vitro and in vivo has been recently reviewed [10-12]. It appears that the diverse immunomodulatory activities of these neuropeptides is mediated by specific receptors on various immune cells [ 13]. SP and VIP receptors have been demonstrated on human and murine lymphocytes [ 14-16], SOM receptors also have been demonstrated on human lymphocyte cell lines and murine lymphocyte populations [ 17-20]. SOM is a tetradecapeptide originally isolated from bovine hypothalamus and is widely distributed among sensory fibres and neuroendocrine cells of the pancreas and intestine [21]. Recent reports have suggested that SOM is present in various immune cells including macrophage and eosinophils from the murine schistosome granuloma [22], as well as in mast cells [23]. We have studied the immunomodulatory role of SOM in the murine system and have determined that it possesses inhibitory activity with regard to lymphocyte proliferation, isotype-specific immunoglobulin synthesis in both Peyer's patch and splenic lymphocytes [7]. SOM and its various analogues, have recently been used as a therapeutic agent in the treatment of various gastrointestinal tract disorders including, VIP- and growth hormone-secreting tumors (acromegaly) and in the treatment of hypermotility symptoms associated with bacterial infections in AIDS patients [24-26]. Its success has been limited due to its relative serum instability and short half-life. Current treatment has utilized various analogues of SOM including SMS 201-995, which are more stable with a longer half-life [27]. The synthetic peptide BIM 23014c is an eight amino acid analogue of SOM with only two amino acid residues common to both. In clinical trials, BIM has been used with some success in the treatment of diarrhea, as well as inhibiting growth hormone release [28]. As various chronic gastrointestinal tract pathologies (Crohn's and Colitis) intimately involve the immune system, the effects of such agents on immune function should be established. Having validated the immunomodulatory activities of SOM, we here discern the immune-mediating properties of the synthetic peptide BIM 23014c and determine that these properties are very similar to those of SOM. The functional assays chosen for analysis, natural killer activity and proliferation, represent important aspects of mucosal and systemic immunology, which may be altered by such therapeutic agents.

Materials and Methods

Female BALB/c mice, 6-8 weeks of age were used. They were maintained at room temperature in a 12 h light/dark cycle and received standard mouse chow and water ad libitum. Animals.

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Reagents. SOM was purchased from Peninsula Laboratories (Belmont, CA) and BIM was kindly donated by The Institute Henri-Beaufour, Les Ulis-Cedex, France. lZSI-labelled SOM (125I-[Tyro~l ]-SOM, specific activity 1800-2000 Ci/mmol) was purchased from Amersham Canada (Oakville, Ontario). 51Cr used for cytotoxic assays was purchased from New England Nuclear (Boston, MA). Cell isolation. Animals were killed by cervical dislocation. Spleens and Peyer's patches from each group of mice were aseptically removed and lymphocytes were teased out as described in Ref. 7. Cells were washed in RPMI 1640 supplemented with 10~o fetal bovine serum (FBS) and Hepes (10 mM (pH 7.4), GIBCO, Grand Island, NY). Proliferation assays. Lymphocytes were counted to a final concentration of 2.106 cells/ml. Cultures were set in 96-well plates in RPMI 1640 media supplemented with 10~o FBS, gentamicin (0.05/~g/ml), t-glutamine (10 mM) and concanavalin A (Con A) at 1.0/~g/ml. Various concentrations of Con A were attempted, with this being the best sub-optimal concentration. The effects of BIM 23014c and S OM at each concentration were inhibitory (data not shown). 1.0 #g/ml gave slightly sub-maximal proliferation responses in spleen and Peyer's patch lymphocytes. Cells were exposed to various dilutions of either SOM or BIM 23014c ranging from 1 . 1 0 - 1 1 M to 1.10 -5 M. Cultures were incubated in 5~o CO2 in air at 37 °C for 48 h after which [3H]thymidine (1 #Ci/well; specific activity 6.7 mCi/nM, New England Nuclear, Boston, MA) was added for an additional 24 h. The cells were then harvested using a Titrek cell harvester and [3H ]thymidine uptake was measured using scintillation counting (Beckman Instruments, Fullerton, CA). The results are presented as a percentage of change from the control assay and are calculated as follows:

[(cpm's of lymphocytes with neuropeptides) (cpm's of lymphocyte without neuropeptide)] cpm's of lymphocyte without neuropeptides

× 100

Statistical analysis of the results entailed Student's t-test. The mean ( + S.E.M.) are reported from four experiments with twelve replicates in each. In vivo pump assays. Proliferation assays were also run using tissues from animals receiving SOM or BIM 23014c via a mini-osmotic pump as described previously [29]. Briefly, Alzet (2001) mini-osmotic pumps (Alza Corp., Palo Alto, CA) were inserted subcutaneously into BALB/c mice lightly anaesthetized with diethyl ether. Each pump contained a fixed concentration of either SOM, BIM 23014c or saline and was in place for 7 days. The delivery of peptide was constant for each day (30 #l/day at a concentration of 10-5 M); serum concentrations ofpeptide remained constant for the 1 week period and were 3- to 4-times higher than in untreated animals. After 7 days, lymphocytes from spleen and Peyer's patch populations were assayed in proliferation assays with or without exogenous stimulation with SOM or BIM 23014c.

132 Natural killer assays. Target cells consisted of the tumour cell line YAC-1. These were pre-incubated with 51Cr for 2 h, after which co-incubation with splenic or Peyer's patch lymphocytes processed from in vivo stimulated animals (receiving SOM or BIM 23014c via mini-osmotic pump) followed for an additional 6 h. For in vitro studies, the same protocol was used only the cells were co-incubated with 10-8 M SOM or BIM 23014c during the final 6 h. In an attempt to determine whether the cells responsible for this activity were classical T cells (CD 8 + ) or N K cells, anti-Thyl.2 (T cell industries, Boston MA.) depletion was used. Natural killer (NK) activity was measured as 5~Cr release above spontaneous release. Total release was assessed by co-incubation with the detergent nonidet P40 (NP40-, Sigma, St. Louis, MO). The data are expressed as percent killing versus control which is arrived at by the following:

measured 5~Cr - spontaneous 5~Cr total 51Cr - spontaneous 51Cr

x 100

Results Lymphocyte proliferation assay The effect of SOM on in vitro proliferation assays has already been documented by this group and has demonstrated that SOM inhibits lymphocyte proliferation [7]. The effect of SOM in this assay was compared to that of BIM 23014c to determine if BIM 23014c could mimic the action of SOM as a suppressive agent to lymphocyte proliferation. Fig. la depicts results with Con A-activated splenic lymphocytes exposed to various concentrations of BIM 23014c and SOM for 72 h. BIM 23014c was effective in suppressing the uptake of [3H]thymidine in lymphocytes derived from spleen (P < 0.05) over concentrations ranging from 10 -7 M to 10 -5 M. These effects were similar to those seen with SOM at the same concentrations. The effects of both SOM and BIM 23014c on Peyer's patch lymphocytes shows that the two peptides work in a similar yet elevated manner in this population oflymphocytes (Fig. lb). Both peptides, at concentrations of 10- 8 M to 10 - 5 M showed significant inhibition (P < 0.05) of [3H]thymidine uptake. There was no significant difference between BIM 23014c and SOM treatment. In vivo pumps and proliferation with B I M 23014c and S O M Results from the isolation of cells after 7 days with BIM 23014c or SOM and subsequent plating in 96-well plates in a proliferation assay are shown in Fig. 2. Splenic lymphocytes, subjected to SOM in vivo only demonstrate an obvious reduction (50 ~ ) in the incorporation of [3H]thymidine. This same cell population exposed to SOM in vivo and again in vitro, demonstrated a further reduction in [3H]thymidine incorporation (85~o). BIM 23014c ability to alter [3H]thymidine incorporation in vivo and again in vitro is demonstrated here as well. It is evident that BIM 23014c works in the same magnitude as SOM. There is no significant difference between the activities of SOM and BIM 23014c in vitro or ex vivo. In Peyer's patch, SOM and BIM 23014c

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Fig. 1. Proliferation of splenic (la) and Peyer's patch (ib) lymphocytes was measured via [3H]thymidine incorporation in vitro. Cells were exposed to various concentrations of somatostatin (-O-) or Bim ( . A . ) for 48 h and counted. Graphs represent the results or 4 experiments with 12 replicates at each point. The shaded areas represent the variation in the 12 replicates in control proliferation. Significance is only expressed when both treatments were significantly different from controls. *P < 0.05. Peyer's patch control cpm's: 5636 + 421. Spleen control cpm's: 75,391 + 6026.

administered via in vivo pumps, elicited a suppression in proliferation of 65 % and 72 %, respectively. With the addition of BIM 23014c and SOM in vitro, the inhibition is again further enhanced below in vivo values (BIM 23014c, 95% and SOM, 93%). These results reconfnm our in vitro findings of BIM 23014c ability to mimic SOM as an inhibitor of [3H]thymidine uptake with a preference for Peyer's patch lymphocytes.

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Natural killer activity Figs. 3a and b depict the results obtained for NK activity in the spleen and Peyer's patches of in vivo stimulated BALB/c lymphocytes. There is a measurable decrease in NK activity of splenic lymphocytes exposed to BIM 23014c and SOM in vivo (Fig. 3a; P < 0.05 at 1 : 100). Interestingly, the inhibition ofcytotoxic activity by SOM and BIM 23014c was significantly higher in Peyer's patches then in the spleen. This decrease was significant for all ratio s between effector and target (YAC-1) cell for both peptides tested in vivo (Fig. 3b; P < 0.05). Fig. 4 confirms the in vivo findings by demonstrating that SOM and BIM 23014c act similarly by significantly inhibiting the cytotoxic activity in vitro. The cytotoxicity was not altered by Thyl.2 depletion (data not shown), therefore NK cells are the most likely cells responsible for the cytotoxic action witnessed here. Data for Peyer's patches are not shown, but SOM and BIM 23014c did inhibit NK activity though not to a significant degree.

Discussion

Somatostatin, the gastrointestinal neuropeptide originally isolated from bovine hypothalamic extracts [21 ] has only recently been implemented as a possible immunoregulatory agent [7,15-17,22]. The ability of this peptide to suppress the proliferation of gut derived lymphoid cells expands the existing knowledge regarding somatostatin's role as a gastrointestinal tract peptide. BIM 23014c, the low-molecular-weight analogue

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of SOM has been looked at under two parameters of immunoregulation; the ability to suppress the proliferation of activated lymphocytes in 72-h cultures and the ability to alter the NK activities of two populations of lymphocytes. It has been shown here that BIM 23014c does in fact mimic the suppressive actions of somatostatin in vitro and in vivo. To our knowledge, this is the first report investigating the immunoregulatory role of an analogue to its naturally occurring peptide. Clinically, BIM 23014c and other SOM analogues including SMS 201-995 have been compared to SOM in the treatment of various carcinomas and intestinal tract pathologies [27,28,30]. More recently, SOM and SMS 201-995 have been utilized in clinical trials on rheumatoid arthritis patients in an attempt to inhibit the release of mediators contributing to the inflammation involved in the disease [ 31]. It has been shown that both are effective in the treatment of this disease due to their ability to eliminate the inflammation and pain associated with this disorder [31 ]. The binding properties of SOM have been determined on various murine and human lymphocyte cell lines [ 17,18,20 ]. The fact that B IM 23014c shares only two amino acid residues with SOM may suggest that they act via different mechanisms. However, our unpublished observations suggest that SOM and BIM 23014c do act through a similar receptor on lymphocytes. Our previous findings have demonstrated that SOM preferentially manipulates mucosal immune tissue in an inhibitory fashion [7]. The results here are supportive of this hypothesis as Peyer's patch proliferation and in vitro NK activity are affected to

137

a greater degree than spleen by BIM 23014c and SOM. This may not be surprising, since receptor numbers for SOM are higher on Peyer's patch lymphocytes then splenocytes [ 18 ]. As well, the innervation of the intestinal tract and particularly immunocompetent regions (mesenteric lymph nodes, Peyer's patches) by neuropeptide-containing sensory fibres have been show to contain SOM [32]. Though SOM-containing nerves within the spleen have not been confirmed, sensory fibres are present, but in a much lower magnitude than in other regions [9]. It is of interest to see that BIM 23014c and SOM act to down-regulate the NK activity of both spleen and Peyer's patch-derived lymphocytes during in vivo and in vitro stimulation. The NK activities demonstrated here are relatively low, but are significant and repeatable. The ability for SOM to work in a stimulatory manner must not be dismissed, since others have reported that SOM can also enhance cell proliferation in vitro [33]. Though our results do not demonstrate that SOM has a stimulatory effect, it may be necessary to expand our knowledge of its actions, since dual functional effects (both stimulatory and inhibitory) for SOM in immunity have previously been reported [34]. Although the exact mechanism of the above mentioned effects is not clear, it is probable that SOM and BIM 23014c are regulating immunity in an identical fashion. Whether this is a direct interaction or encompasses the inhibition or augmentation of various cytokines is presently being investigated. Having demonstrated that SOM affects proliferation after 48 h, it may be important to determine the effects which this peptide and its analogue have on the manipulation of such factors. The significance of these observations, in light of recent clinical advancements in the treatment of various GI disorders using SOM and its analogues, must be emphasized and incorporated into the conceptual framework of immunomodulation.

Acknowledgements We wish to thank Drs. P. Braquet and J. M. Mencia-Huerta for their helpful support and discussion. This work was supported by MRC-Canada, Canadian Arthritis Society and Henri Beaufour Institute, France.

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