Intravenous administration of bombesin in man stimulates natural killer cell activity against tumour cells

Neuropeptides (1991) 18, 15-21 0 Longman Group UK Ltd 1991

Intravenous Administration of Bombesin in Man Stimulates Natural Killer Cell Activity Against Tumour Cells E. A. F. VAN TOL, C. V. EL20 KRAEMER, H. W. VERSPAGET, Ad A. M. MASCLEE and C. B. H. W. LAMERS Department

of Gastroenterology

and Hepatology,

University Hospital, Leiden, The Netherlands

Abstract-Peptides from both the nervous and endocrine system have been shown to influence immune functions. This study describes the stimulatory effect of bombesin on natural killer cell activity of peripheral blood mononuclear cells. The stimulation of cytotoxicity by bombesin in vivo was much higher than found in vitro. In vitro studies with bombesin and gastrin revealed that the stimulatory effect of bombesin in vivo can for a major part be attributed to other stimulator-y mediators which are released by BBS. These results indicate that neuropeptide release might rapidly interfere, both directly and indirectly, with natural killer activity of peripheral blood mononuclear cells.

been shown to influence the immuno-surveillance dealing with tumour development and infection Neurotransmitters, opioid peptides and hormones (7-11). can interfere with both humoral and cellular The pathway by which the brain regulates immune functions (see for reveiws l-4). Previous immune function is designated as neuro-immunestudies in the field of neuroendocrine immunology axis. Indeed, the autonomic innervation of lymrevealed that lesions in certain brain regions could phoid organs as thymus, spleen, and bone marrow alter the immune response (5, 6). Moreover, provides a direct route for the central nervous psychological and physiological stress factors have system to regulate responses of the immune system (12, 13). Neuropeptides present in peripheral nerves and in peptidergic neurons which are Date received 3 April 1990 abundant in the gastrointestinal tract, have also Date accepted 16 July 1990 been found to affect lymphoid cell function in vitro Correspondence: Dr. H. W. Verspaget, M.Sc., Ph.D. Dept. (14). Activated lymphocytes are able to secrete Gastroenterology and Hepatology University Hospital, Buildpeptides and lymphokines which not only have ing 1, C4-PO12 Rijnsburgenveg 10 2333 AA Leiden The activity but may act as Netherlands. Telephone: 031 71 262680, Telefax: 031 71 local immuno-regulatory feedback signals for certain brain centres. Binding 142588. Introduction

15

16 of IL-l to brain cells for instance has already been reported (15). These observations indicate that the nervous-, endocrine-, and immune system share bi-directional communicatory pathways. Although a considerable number of hormones and neuropeptides are known to bind to mononuclear leucocytes by means of specific high-affinity receptors (16), thereby affecting immunological functions, this has not yet been described for bombesin. Bombesin (BBS) and its mammalian equivalent gastrin-releasing-peptide (GRP) act not only as powerful stimuli for the release of many gastrointestinal peptides (17) but influence gastrointestinal function also by a direct action. BBS is mainly found in the brain and peptidergic neurons of the myenteric plexus of the gut, where it acts as neurotransmitter under physiological circumstances. Plasma BBS levels are extremely low and hardly measurable, but increased concentrations of BBS have been found in patients with intestinal carcinoids (18-19)) endocrine tumours (20-21), acute idiopathic urticaria (22), and in some patients with oat cell carcinoma (23), tumour cells with APUD characteristics which have been found to secrete BBS in vitro (24). In vitro studies with BBS have demonstrated that this neuropeptide is able to modulate mitogen responses of murine lymphocytes (25) and to stimulate human NK-cell activity against tumour cells in vitro (26). Moreover, infusion of BBS has been found to antibody specific anti-bacterium stimulate secretion in the rat intestine (27). However, it is not known whether bombesin affects immune function in man in vivo. Therefore we have investigated the effect of intravenous administration of BBS on spontaneous cell mediated cytotoxicity against tumour cells in healthy volunteers. Materials and Methods Subjects

Six normal healthy volunteers aged 22-37 years, four males and two females participated in a single-blind study with in vivo administration of bombesin. The protocol had been approved by the local ethical committee and informed consent was obtained from each person. At another instance

NEUROPEPTIDES

blood from these persons was obtained in order to isolate peripheral blood mononuclear cells for short BBS and gastrin in vitro preincubation studies. For in vitro analysis of the effect of gastrin peripheral blood was obtained from 12 healthy volunteers consisting of six females and six males of a similar age group. In vivo experiments

Experiments started at 8.30a.m. and participants appeared after overnight fast. They were requested to avoid physical exercise as much as possible. Each person was studied twice, in random order, (1) during intravenous infusion of a 0.9% saline solution and (2) during bombesin infusion. In both experiments a continuous infusion of 0.9% saline solution was administered into the cubital vein of one arm whereas blood samples were drawn from the vein in the opposite arm. From a pilot study it became apparent that NK-cell activity showed considerable fluctuations within the first hour after introduction of i.v. catheters. Therefore in this study the subjects were given rest for lh after introduction of the i.v. catheters and saline infusion. The infusion of saline was continued for 3 h after the start of the experiment. In the BBS experiment, at t = 90min, BBS was administered i.v. for 20min in a dose of 3pmoYkg.min-’ . This solution could not be distinguished visually from physiological saline, also changed at t = 90, and syringes on the infusion pump were regularly changed. Blood samples

Blood samples (10ml) were drawn from the cubital vein at t = 60,75,90, 110, 120, 150, 180min after the start of the experiment. Immediately after sampling the blood samples were divided in 5ml heparin tubes for mononuclear cell isolation and 5ml EDTA tubes on ice (O’C) for the measurements of plasma gastrin by a sensitive and specific radioimunoassay (28). Effector cells

Peripheral blood mononuclear cells were obtained by density gradient centrifugation on Ficoll-isopaque at room temperature, cells were washed and resuspended in RPMI-1640 culture medium with

BOMBESIN AND NATURAL

17

KILLER CELL ACTIVITY

additives. Viability of the cells, as determined by Trypan-Blue dye exclusion was consistently above 98%. Immuno~uorescence studies with monoclonal antibody NKH-1A (anti-CD56; Coulter Immunology, USA) were performed on mononuclear cell cytospins fixed in methanol.

10 -min pre-incubation experiments, comparable with the time of maximal plasma levels after BBS infusion. In each assay, the influence of peptides on spontaneous release of 51Cr label from target cells was evaluated. Statistical analysis

Target cells

The human NK-cell sensitive erythroleukemia cell line K-562 was used as target in the cytotoxicity assay. These cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and always harvested when in their growing phase. K-562 cells were cultured in RPM1 1640 medium with 20mM Hepes-buffer (Gibco; Paisley, Scotland) supplemented with 10% fetal calf serum (FCS; Gibco), penicillin (lOOU/ml; Gibco), streptomycin (100 &ml; Gibco) gentamycin (50 @ml; Flow Laboratories; Irvine, Scotland), amphoteri~in B (2Spg/ml; Gibco) and glutamine (2mM; Flow Laboratories). Cytotoxicity assay

Target cells were harvested and labelled with sodium-‘i chromate (‘iCr, lOO@i/S X lo6 cells) for lh at 37°C. After washing, the cells were resuspended at 1 x lo5 cells/ml. Viable cells were quantitated by Trypan-Blue dye exclusion. The cytotoxicity assay was performed at a 50: 1 effector to target (E:T) ratio for all incubations for 4 and 18h. Cellular cytotoxicity was calculated by the following formula: cytotoxicity (%) = experimental reiease - spontaneous release

x 100

maximal release - spontaneous release Peptides

For the in vivo experiments 3pmoYkg.min’. BBS solution (Hoechst , Amsterdam, Holland) was given. Lyophilised gastrin 17-I (Sigma, St Louis, USA) and BBS (Sigma) were freshly dissolved in culture medium without additives for the in vitro studies. Cytotoxicity assays were performed in the presence of 10-16, 10-14, 10-l’, lo-“, lo-*, 10v6M gas&in for the co-incubation experiments and 1 x -I’M BBS or 6 x 10-‘lM gas&in for the

All data are expressed as mean t standard error of the mean (SEM). The statistical significances of the differences were evaluated with the paired Student’s t-test. Friedman’s analysis of variance for repeated measurements was used for analysis of gastrin co-incubation experiments.

Results Intravenous administration of BBS significantly stimulated NK-cell activity of peripheral blood mononuclear cells. Figure 1A shows the alterations in cytotoxicity of PBMC in both 4 and 18h assays. All individuals responded to intravenous administration of BBS but the maximum response was not always seen at the same timepoint and the relative increase in cytotoxicity compared with the mean basal levels varied markedly. Apart from this observation two other phenomena were seen. First, stimulation of NK-cell activity was followed by a steep dectine in c~otoxicity resulting in a tendency to even lower levels compared with the values before the start of infusion. These latter differences however, were found to be not statistically significant. Second, two individuals showed increased cytotoxicity just before the beginning of the BBS infusion compared to their preceding basal levels. This finds expression in the elevated level of cytotoxicity just before the beginning of the BBS infusion. Nevertheless, the mean stimulatory effect of bombesin infusion, expressed as percentage relative increase was 41 * 13% (p 0.028) in the 4h and 12 rt 4% (p = 0.04) in the 18h assay. No alterations in cytotoxicity were found during the continuous physiological saline infusion (Fig. 1B). Although physiological saline infusion showed a stable cytotoxicity in the overall experiments, it was learned from the pilot experiments to wait 1 h after positioning of iv. catheters and start of saline infusion to achieve stabilisation of

18

NEUROPEI‘TIDES

2% (p = 0.022) and 6 + 2% (p = 0.036) in respectively 4 and 18h assays (Fig. 2). All individuals showed physiological responsivity to the BBS infusion as monitored by plasma gastrin levels (Fig. 3). Bombesin infusion caused a very rapid increase of plasma gastrin levels in all individuals. Gastrin, among other hormones released by bombesin, could thus be one of the important candidates for stimulation of the NKactivity. However, no effects on cytotoxicity were seen in the 4 and 18h co-incubation experiments with different gastrin concentrations (Fig. 4). When mononuclear cells from individuals of the in vivo experiments were pre-incubated with a gastrin concentration comparable with the level reached in plasma, only small relative increases in cytotoxicity of approximately 4% were seen in both 4 and 18h assays (Fig. 2). Discussion

1 t

Fig. l(A)

Mean percentual cytotoxcity of PMBC against K-562 target cells in the bombesin infusion experiment (3pmoV kg.min-’ for 20min). Significant differences in comparison to the mean of the basal levels at t = 60,75 and 9Omin are denoted by asterisks (N = 6, (*) p = 0.04, (**) p = 0.028). BBS infusion period is indicated by the hatched area. (B) Mean percentual cytotoxicity of PBMC against K-562 targets. Control experiment for detecting the influence of continuous physiological saline infusion on cytotoxicity (N = 6).

Modulation of immunological responses by peptides from the nervous and endocrine system have been described in various experimental models. Gastrointestinal peptides have been found to affect both humoral and cellular immune responses (29). In the present study the effect of bombesin infusion on the NK-cell activity was

12 -

IO -

8-

61

4

NK-cell activity. These observed effects are illustrated by a decline in Figure 1B between 60 and 75min followed by stabilisation of cytotoxicity. To approach the experimental design of in vivo stimulation by BBS, in vitro pre-incubation studies with cells from the same individuals were performed with lO_l’M BBS for 10min which resulted in relative increase in cytotoxicity of 9 +

24

0

Fig. 2 Mean percentage relative increase of cytotoxicity of PBMC against K-562 targets. Pre-incubation of PBMC with 1 X 10-i”M BBS or 6 = lo-“M Gastrin for 10min. Same individuals used as in the BBS and physiological saline infusion experiments (N = 6. (*) p = 0.036, (**) p = 0.022).

19

BOMBESIN AND NATURAL KILLER CELL ACTIVITY GASTRlh

(PM)

80 i

Fig. 3 Mean gastrin plasma levels of the individu~s tested for PBMC cytotoxicity during BBS or control saline infusion experiments. BBS infusion period is indicated by the hatched area (N = 6).

studied by regularly sampling before and after the infusion. Preliminary studies with physiological saline infusion showed strong fluctuations in NKcell activity shortly after the introduction of iv. catheters. Although Malec et al (30) in their peptide infusion experiment found that NR-cell activity measured only before and after saline infusion was not changed, it seems to be very impo~ant in these kind of experiments to protect individuals from psychological and physical stress factors which have been found to interfere with immune responses (7,8, 11). In this study a 20-min BBS infusion significantly stimulated NK-activity against tumour cells even 10min after the end of the bombesin infusion. Previous experiments in humans with bombesin infusion have shown that maximal plasma BBS levels are reached between 10 and 20min after starting the infusion and decrease immediately to basal levels within 10 min after stopping the infusion (31). These observations are in accordance with a very rapid stimulating effect of bombesin on cells with NK-cell activity since stimulation indeed is highest when the maximal plasma levels are reached. The effect of BBS on NK cell activity is still present 10 min after stopping the BBS infusion. Whether this represents circulating cells which had been stimulated during the BBS infusion period, mobilisation of

cells with NK activity from the bone marrow or the influence of other mediators which are released by BBS, was unclear. As we have pub~shed earlier, incubation of PBMC with BBS resulted in significant increase of cytotoxicity against K-562 target cells (26). Although the relative stimulation with BBS in vitro was in the same order of magnitude as in the short pre-incubation experiments (+ 10%). it was much lower than observed in the in vivo BBS infusion experiments. These observations support the idea that apart from a direct effect of BBS, (1) other mediators induced by BBS infusion may also be involved or (2) increase mobilisation of cells with NK-cell activity to the peripheral blood occcurs. It is known, for instance, that adrenalin is released by bombesin infusion and that this stress hormone rapidly increases mobilisation of NKcells from the bone marrow although this was not reflected in the cell count of the various blood samples in our study. Moreover, immunofluoresence studies in two of the individuals revealed no alteration in the number of NK-cells during the BBS infusion experiments at t = 60,110 and 180min. Because BBS is a powerful stimulator of the secretion of several gut hormones these may be candidates to participate in stimulation of NK-cell activity. Similar kinds of observations, where %Cytotoxiclty 1OOT

0 4h 0

lOE-16

tOE-14

10512

10x510

Concentration GASTRIN

to&6

18h

,oE-6

’

(M)

Fig. 4 Effect of different concentrations gastrin on spontaneous ceil mediated cytotoxicity of PBMC in co-incubation experiments against K-562 target cells after 4 and 18h (N = 12).

20 involvement of other peptides is suggested, have been found in BBS stimulated specific antibody secretion in the rat intestine (27). In our study gastrin levels in plasma, measured to monitor the physiological response to BBS infusion, increased significantly. However, gastrin hardly changed NK-cell activity in the in vitro pre- and co-incubation experiments and apparently does not play a role in the in vivo stimulation after BBS infusion. The differences between in vitro pre- and co-incubation experiments with BBS on one hand and the relatively high stimulation by this peptide in the infusion experiments on the other hand, do point to a contribution of other mediators in stimulating cytotoxicity. Although the mechanisms of in vivo stimulation of NK-cell activity by BBS are not fully elucidated, this study indicates that BBS is able to stimulate peripheral cellular cytotoxi~ty against K-562 tumor cells. This modulatory role for BBS may reflect a mechanism for the nervous system to intervene with immune responsiveness of lymphoid cells in the circulation. Moreover, this may be important in tissues where abundant peptidergic innervation is observed and high peptide concentrations can be reached, e.g. lymphoid tissue and the gut (32,33). Acknowledgements We are indebted to Marij Mieremet-Ooms and Annie van der Zon for their careful technical assistance. We also thank Loes Niepoth and Nelia Koek for typing the manuscript.

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