Inhibitory effect of somatostatin-14 and some analogues on human natural killer cell activity

Peptides, Vol. 15, No. 6, pp. 1033-1036, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All fightsreserved 0196-9781/94$6.00 + .00

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Inhibitory Effect of Somatostatin-14 and Some Analogues on Human Natural Killer Cell Activity M. C. S I R I A N N I , * ' B. ANNIBALE,i" S. F A I S t A N D G. D E L L E FAVEi"

*Cattedra di Allergologia ed Immunologia Clinica and ?~I Cattedra di Gastroenterologia, University of Rome "La Sapienza," 00185 Rome, Italy Received 8 N o v e m b e r 1993 SIRIANNI, M. C., B. ANNIBALE, S. FAIS AND G. DELLE FAVE. Inhibitory effect ofsomatostatin-14 and some analogues on human natural killer cell activity. PEPTIDES 15(6) 1033-1036, 1994.--The effect of somatostatin-14 (SST) and the SST analogues SMS and RC160 on human natural killer (NK) activity mediated by large granular lymphocytes (LGL), as well as on IL-2- and/or anti-CDl6 monoclonal antibody (mAb)-induced activation of these cells, was investigated.The NK activity of LGL was studied by the release of S~Cr by the erythroleukemia-derived cell line K562, whereas S'Cr release by the P815 murine mastocytoma-derived cell line, for which lysis was redirected by the use of an anti-CDl6 mAb, was used to study the cytolytic potential of these cells. IL-2 was used at the final concentration of 100 IU and was incubated overnight with LGL. SST and the analogues, added to these systems at final doses ranging from 10-12 to 10-s M, were inhibitory of the NK cell activity to K562, with a dose-response curve starting from 10-8 M and reaching a significant level at 10-6 M. On the contrary, no effect was observed on the redirected killing assay to P815 and on the IL-2-induced activation of NK cells. These results provide additional evidence for the immunomodulatory action of somatostatin. Somatostatin

Somatostatin analogues

Immunomodulatory action

THE presence of somatostatin-14 (SST) receptors on human mononuclear leukocytes (4), on cell lines of lymphoid origin (10), on peripheral blood lymphocytes (PBL), as well as on intestinal lamina propria lymphocytes (LPL) (6) and on leukemia and plasmocytoma-derived cell lines (13), has already been demonstrated, and SST has been found to be synthesized in lymphoid organs (2). A possible involvement of SST in immunomodulatory actions was then suggested (11), and some patients, suffering from somatostatin-secreting tumors, presenting immunological abnormalities (3) similar to those reported by some in vitro studies (6,15), were reported. To date, there have been no reports on the effects of SST on natural killer (NK) cell activity of human PBL, and NK cells are operative in protection against tumor and viral spreading, because they preferentially kill sensitive tumors and some virus-infected target cells (7). The aim of the present study was to investigate whether SST was able to modulate NK cell activity of PBL derived from norreal donors. Our results demonstrated a diminished NK cell activity to tumor targets induced by SST, which was not due to a decreased response to N K cell stimulation by IL-2 or antiCD16 monoclonal antibodies (mAb). METHOD

Isolation of PBL Blood samples from 10 normal donors (five males and five females, aged 25--43 years) were collected by venipuncture and

Cytotoxicity

blood was heparinized (10 IU/ml) (Liquemin, Roche, Rome, Italy). Peripheral blood lymphocytes were separated on FicollHypaque, washed twice with Hank's balanced salt solution (HBSS), and resuspended in serum-free RPMI 1640 (Gibco, Grand Island, NY) at a concentration of 106/ml for immediate use. To obtain an NK cell-enriched population, large granular lymphocytes (LGL) were further separated, as previously described (14), by centrifugation on a discontinuous seven-layer Percoll density gradient (Pharmacia, Uppsala, Sweden). The first two layers, enriched in LGL, were used. The LGL viability was always over 95%, as judged by trypan blue dye exclusion test. The rate of purification was 70-80%, as assessed by May Grumwald-Giemsa staining. The cells were phenotypically CD16/ CD56 positive (75%), CD3 negative (5%), as assessed by the use of mAb and immunofluorescence. The remaining cells were CD4+ (10%) and CD8+ (10%). Cells were phenotyped by the use of anti-CD16, anti-CD56, anti-CD4, and anti-CD8 mAbs of the Leu series (Becton-Dickinson, Mountain View, CA).

NK Activity of LGL A previously described 5'Cr release assay against the human erytroleukemia cell line K562 was used (14). Target cells (106) were labeled for 1 h at 37°C with 100 t~Ci of Na2S~CrO4 (Amersham Corp., Searle, England), washed three times, and resuspended in RPMI 1640 medium. Labeled targets were mixed

' Requests for reprints should be addressed to M. C. Sirianni, Cattedra di Allergologiaed Immunologia Clinica, Universita'di Roma "La Sapienza," viale dell'Universita' 37, 00185 Roma, Italy.

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SIRIANNI ET AL.

80

Italy). All peptides were resuspended in RPMI 1640 medium and incubated overnight, in plastic vessels and in a 5% CO2 humidified chamber, with LGL in a dose ranging from 10-5 to 10 ~2M. Cytotoxic tests were then performed. In some additional experiments, SST-incubated cells were washed three times with RPMI 1640 medium before testing in the NK assay. Control cultures containing medium alone obtained a final volume in the wells corresponding to that of the neuropeptide-treated cultures.

70 60 UJ

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IL-2 Stimulation

20 10-

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I

I

I

I

I

I

I

I

-12

-11

-10

-9

-8

-7

-6

-5

SST-14 LOg (M)

FIG. 1. Effect of SST-14 on NK activity of human LGL, tested at the E:T ratios of 10:1 (closed circles), 5:1 (open ~uares), and 2.5:1 (open triangles). Each point represents the mean percent ~Cr release + SD of five experiments.C is the value obtained in untreated cultures (controls).

with effector cells in V-shaped microtiter wells (Linbro, CN) to give final effector:target (E:T) ratios of 10:1, 5:1, and 2.5:1. Spontaneous release of 5~Cr by target cells was determined by adding to the targets medium alone and maximal release by lysing targets with Nonidet P40. Plates were then centrifuged and incubated in a humidified chamber of 5% CO2 for 4 h at 37°C, to avoid recycling. Plates were then centrifuged again at 500 rpm for 5 min and 100 #1 ofsupernatant was removed and counted in an automatic LKB gamma counter. Percentage of lysis of triplicate wells was determined by the formula: (experimental cpm - spontaneous cpm/maximum cpm - spontaneous cpm) × 100.

The LGL were resuspended in RPMI 1640 medium and incubated overnight in the presence of recombinant IL-2 (rIL-2) (Cctus Corporation, Emeryville, CA) at a final concentration of 100 IU/ml (final dilution in the culture). Cultures were set up in V-shaped microtiter wells (Linbro, CN) and maintained in a 5% CO2 humidified chamber. SST, at various concentrations, was added to some IL-2-treated cultures at the beginning of the incubation period. Cells were then assayed for cytotoxicity.

Statistical Analysis Analysis of variance (ANOVA) was performed by the ANOVA test. A value of p < 0.05 was considered statistically significant. Later a multiple comparison of variance was performed by the Newman-Keuls test. RESULTS

Effect of SST and Analogues on NK Activity SST and analogues did not affect LGL as well as K562 viability, as assessed by the trypan blue dye exclusion test (data not shown). Figure 1 shows the effect of SST on NK activity of human LGL, as assessed in a 5tCr release assay from the tumor cell line K562. SST was able to inhibit the NK cell activity at all the E:T ratios tested. The inhibitory effect was detectable starting from a concentration of 10-s M a n d becoming significant

Redirected Killing Assay The ability of an anti-CD 16 mAb to trigger the cytolytic activity of LGL was assessed in a 4-h 5~Cr release assay in which target cells were represented by the murine mastocytoma cell line P815, which expresses Fcgamma-R for IgGl, IgG2a, and lgG2b on the surface (9). In this assay, 5 × 103 labeled targets were added to each well containing 5 × 103 or 5 X 104 effector cells (for final E:T ratios of 1:1 and 10:1 and a final volume of 200 #l/well) in the presence of 1 #g of the anti-CD 16 mAb Kdl (ascitic fluid), bearing an IgG2a isotype (9). PHA (Wellcome, Rome, Italy), which among circulating PBL is able to stimulate the cytolytic potential of both NK cells and activated T cells (9), was used in this assay at a final dilution of 1:1000 (v/v). After 4-h incubation

go

60 tu uq, ~: 40 t3 7, o~ 20

a t 3 7 ° C , 100 # l o f t h e s u p e r n a t a n t w a s c o l l e c t e d

from each well and counted in the gamma counter. Percentage of lysis was calculated as above.

Peptides SST- 14, a naturally occurring peptide, and two somatostatin analogues, selected on the basis of their wide therapeutic use in gastrointestinal as well as in neoplastic diseases (12), were used in the present study. Synthetic SST-14 and its analogue RC 160 were purchased by Bachem (Switzerland). The other analogue SMS 201-995 was kindly provided by Dr. G. Camboni (Sandoz,

0

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, -12

, -11

, -10

, -9

, -a

, -7

, -6

, -5

(Log M)

FIG. 2. Effect of SST-14 (closed circles), SMS 201-995 (open squares),

and RC160 (open triangles)on NK cell activity of human LGL to K562 (E:T ratio 10:1). Each point repr'~ents the mean percent ~'Cr release _+ SD of 10 experiments. C is the value obtained in untreated cultures (controls).

SOMATOSTATIN AND NATURAL KILLER ACTIVITY

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tration of 100 IU. Some tests carried out with concentrations of rlL-2 ranging from 10 to 50 IU did not show significant differences vs. the results obtained with the highest cytokine dose (data not shown). SST was used at the different concentrations reported in the table.

TABLE 1 EFFECT OF SST-14 AND ANALOGUES ON THE CDI6 REDIRECTED KILLING OF HUMAN LGL Lysis (%) P815 +

None SST (-5 M) SMS(-5M) RC160 (-5 M)

DISCUSSION

P815

PHA

Anti-CD16

3+ 1 2 + 0.5 2+1 3 + 0.6

50+7 48 _+ 8 52---7 53 + 8

61 + 6 63 ___8 65+7 66 _ 9

The table describes the results of the redirected killing, triggered by either PHA and anti-CD16 mAb, of human LGL in the absence (none) or presence of SST and its analogues, used at the final dilution/well of 10-5 M. Results are given as percentage (%) of lysis and are the mean -!-_SD of five experiments, for an E:T ratio of 1:1. The target cell line in this assay is the P815 cell line.

at 10 -6 M ( F = 3.09, p = 0.039 for the E:T ratio 10:1; F = 5.77, p = 0.003 for the E:T ratio 5:1; F = 4.83, p = 0.007 for the E: T ratio 2.5:1). The difference was statistically signifcant vs. control (untreated) cultures and vs. cultures set up in the presence of SST at concentrations of 10 -12 and 10 -1° M. There was no difference between SST 10 -s and 10 -6 M. Figure 2 shows the dose-response effect obtained when incubating L G L (E:T ratio 10:1) with SST and its analogues. The decline of N K activity started at SST and analogue concentrations of 10 -9 M , became more evident at 10 -8 M, and was highly statistically different from control cultures at 10 -6 M ( F = 5.51, p = 0.009). The same results were obtained at the other E:T ratios tested and when using SST-incubated cells, washed before the assay as effectors (data not shown).

Effect of SST and Analogues on the Redirected Killing of NK Cells To assess the cytolytic potential of C D 16+ N K lymphocytes, a redirected killing assay in which N K cells were stimulated by an a n t i - C D l 6 m A b was used. A suitable F c g a m m a receptorpositive source of target cells in this type of assay is represented by the murine mastocytoma cell line termed P815. This cell line is relatively resistant to circulating N K cells and, as shown in Table 1, 5~Cr-labeled P815 cells were not killed by normal L G L (3% iysis at an E:T ratio of 1:1 both in the presence or absence of neuropeptides); however, when anti-CD 16 mAbs were added to the test, activation o f C D 16-positive L G L resulted in significantly increased percentages of lysis ( F --- 220.9, p = 0.000). Similar increments of cytolytic activity were observed when the redirected killing assays were performed in the presence of PHA. The same results were obtained for the E ' T ratio 10:1,and SST did not induce modulation of surface C D I 6 (data not shown).

Effect of SST and Analogues on the IL-2-Induced Activation of LGL To assess if the inhibitory effect of SST and analogues on N K activity to t u m o r targets could be due to a negative modulation o f the IL-2-induced cytotoxicity, L G L were cocultured in the presence of rlL-2 and SST. IL-2 was able to significantly increase the cytotoxic response to K562 ( F = 14.76, p = 0.005) and the addition of SST did not result in any decrease of the cytotoxic response (Table 2). rlL-2 was used at a final concen-

In the present study, a decrease of h u m a n natural killer activity to t u m o r target cells induced by SST and two analogues is reported. This is not accompanied either by decreased cytolytic potential of N K cells, as assessed by the use of anti-CD 16 mAbs, or by decreased response to in vitro N K activation by IL-2. The effect of SST on the i m m u n e system is still poorly elucidated, even if somatostatin and some analogues are widely used in the treatment of several gastrointestinal disorders, including diarrhea associated with the acquired immunodeficiency syndrome, and in the treatment o f neoplastic diseases (5,8,12). Some studies have demonstrated several immunological abnormalities in the human system, induced by SST, including a decreased in vitro immunoglobulin production as well as a diminished nitrogen-induced expression of receptors for IL-2, in a system of PBL, stimulated by optimal concentrations of lectins (6,15). Some of these effects were explained by the existence of SST receptors on PBL, for which affinity, however, was lower than that reported for intestinal lamina propria lymphocytes (6). There is no report regarding the effect of SST on N K activity of human PBL, even if an inhibitory effect of SST and its octapeptide analogue BIM 23014c on murine N K activity in vivo and in vitro has already been described (1), which was corrected by the depletion of N K cells with appropriate antisera. Our data on the h u m a n system show the same results, by the use of an enriched population of N K cells and also by amplifing them. The decreased activity is not due to a decreased cytolytic potential of the cells, as assessed by a killing assay redirected by an antiC D 16 mAb and is not accompanied by a diminished activation by IL-2. N K cells bear the low-attinity receptor for the Fc portion of IgG, clustered as CD16, able to induce their cytolytic machinery (9). Thus, redirected killing in the presence of anti-CD 16 mAbs, through cross-linking of the Fcgamma-receptor, present both on the effector cell and on some target cells, induces the

TABLE 2 EFFECT OF SST-14 ON THE IL-2-INDUCED ACTIVATION OF NK CELLS Lysis (%) None IL-2 IL-2 + IL-2 + IL-2 + IL-2 + IL-2 +

SST (-5 M) SST (-6 M) SST (-8 M) SST (-10 M) SST(-12M)

60 + 10 82 +__ 8 80 _+ 9 83 + 8 85 _ 9 84 + 10 83 + 9

LGL were incubated overnight without stimuli (none), in the presence ofrlL-2 (IL2, final concentration 100 IU), and in the presence of alL-2 plus SST at the different concentrations reported in the table. Results are given as percentage of iysis of the K562 target cell line, for an E:T ratio of 10:1. Resuits are the mean _+SD of five experiments.

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SIRIANNI ET AL.

functional program of CD16+ NK cells, mainly by triggering the phosphoinositide turnover, the subsequent intracellular Ca ++ increase, the cytoskeleton polarization, and finally the release oflytic enzymes to the target [reviewed in ( 16)]. SST was unable to interfere with either CD 16-triggered lysis or with IL-2-induced activation, thus suggesting that a putative SST receptor does not share similarities with CD16 or the receptor for IL-2, described on NK cells (16). N K killing is, as already stated, the consequence of an extremely complex series of events that requires an initial adhesion between the effector NK cell and its target (16). The possibility that SST may interfere with some cell-adhesion molecules expressed on N K cells, such as CD56 and CD1 lb/CD18, as well as the integrin complex, should also be ruled out. Our results showing that SST does not interfere with the CD 16 functional program may also support this possibility, and this observation could be a point for future research. Modulation of N K cells by cytokines, such as IL-2 and interferon(s), and/or

mAbs of the IgG isotype, leads to the activation of several intracellular mechanisms, which result, on one side, in lysis of the target cell and, on the other, in the transcription of mRNAs for cytokine secretion (16). Our results, demonstrating that CDl 6 triggering of the cytolytic machinery of NK cells as well as activation by IL-2 is unaffected by SST treatment, suggest that SST may interfere selectively with one of these mechanisms, resulting in the inhibition of NK killing but not of NK triggering by appropriate stimuli. ACKNOWLEDGEMENTS We thank Prof. A. Moretta, University of Genoa, for the Kdl mAb and Ms. A. Constantine for the revision of the manuscript. This work has been supported by grants No. 02.12.01.10 and 02.12.01.28 [60% University of Rome, 40% MURST (Neuroimmunology Project)] and by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC) to M.C.S.

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9. Moretta, A.; Ciccone, E.; Pantaleo, G.; et al. Surface molecules involved in the activation and regulation of T or natural killer lymphocytes in humans. Immunol. Rev. 111:144-175; 1989. 10. Nakamum, H.; Koike, T.; Hiruma, K.; Sato, T.; Tomioka, H.; Yoshida, S. Identification of lymphoid cell lines beating receptors for somatostatin. Immunology 62:655-658; 1987. 11. Pawlikowski, M.; Zelazowski, P.; Stepien, H.; Schally, A. V. Somatostatin and its analog enhance the formation of human leukocyte migration inhibiting factor. Further evidence for immunomodulatory action of somatostatin. Peptides 8:951-952; 1987. 12. Schally, A. V. Oncological applications of somatostatin analogues. Cancer Res. 48:6977-6985; 1988. 13. Sciechitano, P.; Dazin, P.; Bienenstock, J.; Payan, D. G.; Stanisz, A. M. The mufine IgA-sect~ng plasmocytoma MOPC-315 expresses somatostatin receptors. J. Immunol. 141:937-941; 1988. 14. Sirianni, M. C.; Annibale, B.; Tagliaferri, F.; et al. Modulation of human natural killer activity by vasoactive intestinal peptide (VIP) family. VIP, glucagon and GHRF specifically inhibit NK activity. Regul. Pept. 38:79-87; 1992. 15. Stanisz, A. M.; Befus, D. A.; Bienenstock, J. Differential effects of vasoactive intestinal peptide, substance P and somatostatin on immunoglobulin synthesis and proliferation by lymphocytes from Peyer's patches, mesenteric lymph nodes and spleen. J. Immunol. 136:152-156; 1986. 16. Trinchieri, G. Biology of natural killer cells. Adv. Immunol. 47: 187-376; 1989.