Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells

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Regulatory Peptides 60 (1995) 159-166

Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells M6nica Del Rio, M6nica De la Fuente * Departamento de Fisiologla Animal, Facultad de C. Biol6gicas, Universidad Complutense, Madrid, Spain

Received 5 April 1995; revised 10 August 1995; accepted 14 August 1995

Abstract

Bombesin and the two mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) and neuromedin C, at physiological concentrations have been previously shown to stimulate significantly in vitro the antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activities in BALB/c mouse leukocytes from axillary nodes, spleen and thymus. In the present work we have shown that adherent cells are required in leukocyte samples for stimulation of cytotoxicity by the neuropeptides, which suggests that this effect may be mediated by those cells. Here we demonstrate the specificity of the effects by reversing them in the presence of the bombesin-antagonist (Leu 13-~ CH 2NH.Leu14).BN, and by detecting specific receptors for GRP on macrophages of high and low affinity. Using the same binding technics, no receptors for this neuropeptide were found in non-adherent leukocytes. Keywords: Bombesin; Gastrin-releasing peptide; Neuromedin C; Cytotoxicity; Leukocyte

1. Introduction

The existence of a functional connection between the nervous and immune systems is supported by the observation of neuropeptide receptors in immune cells and the resulting effects on the function of these cells [1]. Bombesin-related peptides, a group of neuropep-

* Corresponding author. Tel.: +34 1 3944989; Fax: +34 1 3944935.

tides found in several neuronal groups in the central and peripheral nervous system [2-5] have a large variety of biological effects, including sensorial transmission, thermoregulation, release of pituitary and gastroenteropancreatic hormones, gastric and pancreatic secretions, gastrointestinal motility, food intake and satiety [6,7]. Bombesin, a tetradecapeptide, was originally isolated from the skin of the European frog Bombina bombina [8]. Gastrin-releasing peptide (GRP), a 27-amino acid peptide isolated from porcine gut [9], shares a similar C-terminal decapeptide with bombesin and reproduces several effects of bombesin in mammals [10]. An additional

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bombesin-related peptide found in mammals is the C-terminal decapeptide of GRP, neuromedin C, which was isolated from porcine spinal cord [11]. Bombesin and GRP are of special interest as mitogenic factors in selected cells, including murine Swiss 3T3 fibroblasts [12] and small-cell lung cancer cells [13]. Moreover, receptors for bombesin-related peptides have been observed in human and murine cells [14]. In relation to the effect of these peptides on immune function, we have found a stimulation by bombesin, GRP and neuromedin C of phagocytic processes in murine peritoneal macrophages [15]. These neuropeptides are potent chemoattractants for phagocytic cells [16,17] and lymphocytes [17]. Bombesin has been shown to produce a significant increase in rat intestinal IgA and IgG antibody secretions [18]. Moreover, it inhibits IL-2 induced proliferation of CTLL-2 cells [19] and augments slightly the alloantigen induced lymphoproliferative response in mice [20]. As regards the effects of bombesin on lymphoproliferative function, we have recently shown that this neuropeptide, in addition to GRP and neuromedin C, exerts a light stimulating effect on proliferation but significantly decreases the proliferative response in presence of the mitogen Concanavalin A [21 ]. The cytotoxic capacity of immune cells, principally antibody dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity, plays a key role in immune surveillance against neoplastic growth. There are some data implicating the nervous system in NK activity modulation [22]. Some neuropeptides, such as VIP [23,24], NPY [25] and substance P [26,27] have been shown to affect NK activity. Recently, we have found that the cytotoxic capacity of murine leukocytes is stimulated in the presence of bombesin-related peptides [28] in agreement with the studies of Van Tol et al. in humans [29]. The role of antigen presenting cells, adherent accessory cells such as macrophages, seems to be crucial in the modulation of lymphoproliferation by the neuropeptides studied [21] and therefore, the aim of the present work is to investigate whether the stimulation of cytotoxic activities by bombesin-related peptides is also mediated by those adherent cells. In addition, we want to determine the specificity of the

effect and the presence of receptors for GRP on the inmunocompetent cells studied~

2. Materials and methods

2.1. Animals Male B A L B / c mice (Mus musculus) (Iffa Credo, France) aged 15 + 2 weeks were maintained at a constant temperature (22 + 2°C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France) on a 12-h light/dark cycle and fed Sander Mus (Panlab, Barcelona, Spain) and water ad libitum. Mice were checked periodically to verify their pathogen-free condition.

2.2. Neuropeptides Bombesin, gastrin-releasing peptide and neuromedin C, as well as bombesin-receptor antagonist, (Leula-~bCH2NH Leul4)-bombesin and NPY, were obtained from Sigma Chemicals (St. Louis, MO). They were all dissolved in dimethyl sulfoxide (DMSO; Merck) to micromolar stock solutions which were stored at -25°C. Dilutions to final concentrations were made in phosphate-buffered saline each day of experimentation.

2.3. Collection of leukocytes Leukocyte suspensions were obtained from axillary nodes, spleen and thymus of mice. Organs were removed aseptically, freed of fat, minced with scissors and gently pressed through a mesh screen (Sigma) to obtain a cell suspension. The cellular suspensions of three mice were pooled and centrifuged in a gradient of Ficoll-Hypaque (Sigma) with a density of 1.070. The halos were resuspended in RPMI 1640 enriched with L-glutamine (Gibco Canada, Burlington, Ontario) and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco) and gentamicin (100 /zg/ml, Gibco), washed and the cell viability measured using the trypan blue exclusion test, showing a viability of 98%.

M. Del Rio, M. De la Fuente / Regulatory Peptides 60 (1995) 159-166

2.4. Obtainment of non-adherent leukocytes

Leukocyte suspensions, obtained as mentioned above, were incubated 30 min at 37°C in a nylon/wool column prewashed with RPMI medium supplemented with 10% FCS. The cells were eluted with 6 ml of the same medium without pressing the wool and washed three times. 2.5. Peritoneal macrophage isolation

Peritoneal suspensions obtained by intraperitoneal injection of 4 ml of Hank's medium were adjusted to 5 . 1 0 6 macrophages/ml, dispensed into Eppendorf tubes and maintained during 45 min at 37°C to allow the attachment of adherent cells, then the supernatants were decanted to remove non-adherent cells and debris and the remaining cells were incubated with trypsin 0.5% during 1 h to eliminate lymphocytes. Then, macrophages were gently washed three times, and allowed to recuperate receptors during an overnight incubation at 37°C. 2.6. ADCC and N K assays

An enzymatic colorimetric assay was used for cytolysis measurements of target cells (Cytotox 96 TM Promega, Boerhinger Ingelheim) based on the determination of LDH using tetrazolium salts. This technology has been demonstrated to provide identical values (within experimental error) to those obtained by parallel 51Cr release assays of our own and of other authors [30]. Cells of the hemopoietic tumor cell lines K562 were used as targets in the ADCC assay. Murine YAC-1 cells were used as targets in the NK assay. The cells were maintained in complete medium (RPMI-1640 plus 10% FCS, Gibco). Target cells were seeded in 96-well U-bottom culture plates (Costar) at l 0 4 cells / w e l l in 1640 RPMI without phenol red. In ADCC assays, tumoral suspensions were incubated for 15 min with an anti-CD15 monoclonal antibody (Oncor, Gaithesburg, MD) at different dilutions: 1:200; 1:500 and 1:1000. Effector cells (leukocytes from axillary nodes, spleen or thymus or non-adherent leukocytes populations) were added at 105 cells/well, and the neuropeptides were used at concentrations from 10-8 to l 0 -12 M. The effector/target rate used, 10/1, al-

161

lowed us to observe similar results to the ones obtained previously. The plates were centrifuged at 250 g for 4 min to facilitate cell contacts and then they were incubated for 4 h at 37°C. After the incubation, LDH enzymatic activity was measured in 50 /~l/well of the supernatants by addition of the enzyme substrate and absorbance recording at 490 nm. Four kinds of control measurement were performed: a target spontaneous release, a target maximum release, an effector spontaneous release and a volume correction control to adjust the volume change caused by the addition of lysis solution to maximum release control wells. To determine the percentage of target cells killed, the following equation was used: %lysis = ( E - ES) - T S / M - TS) x 100 where E is the mean of absorbances in the presence of effector cells; ES the mean of absorbances of effector cells incubated alone; TS the mean of absorbances in target cells incubated with medium alone; and M mean of maximum absorbances after incubating target cells with lysis solution. In order to determine the specificity of the neuropeptides action, some experiments included an antagonist of bombesin receptor, (LeuI3-~bCH2NH LeuI4)-bombesin, which had been already shown for us to revert other effects of these neuropeptides [ 17] at equimolecular concentration with GRP, 2.7. Binding studies

Binding studies were performed following the method by Segura et al. [31]. Peritoneal macrophages or T-enriched populations (1.5. 10 6 cells/ml) were resuspended in 0.5 ml of 35 mM Tris-HC1 buffer (pH 7.5) containing 50 mM NaCl, 1.4% ( w / v ) bovine serum albumin and 45 pM 125I-GRP alone or with increasing concentrations of unlabeled GRP, bombesin, neuromedin C or NPY (from 10-12 M to 10 -7 M). After 90 min of incubation at 15°C, the macrophage-bound peptide was separated by centrifugation (2000 g, 5 min) and the cell sediment was washed three times. The radioactivity of the cell sediment was measured in a LKB gamma-counter. The specific binding to macrophages was calculated subtracting the non-specific binding, which was determined from binding of tracer in the presence of

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unlabeled GRP at 10 -6 M concentration. This nonspecific binding was 4.0 + 0.8% of the total radioactivity added. The degradation of ~25I-GRP was quantified by precipitation in 10% trichloroacetic acid, being only about 10%.

2.8. Statistical analysis All values are expressed as the mean + S.D. of the number of experiments, performed in triplicate, as indicated in the corresponding tables and figures. The data were evaluated statistically by one way analysis of variance (ANOVA) and by the Scheffe F-test between each two groups, P < 0.05 being the minimum significant level.

50,

40'

30'

20'

10'

0

control

8

9

10

11

12

control

8

9

10

11

12

10

11

12

50-

3. Results 40

The stimulation of cytotoxic activities by bombesin-related peptides was previously demonstrated in our laboratory [28]. The different methodology chosen in this occasion has allowed us to obtain the same results using the appropriate effector-target relation with the additional benefit of been a non-radioactive technic. The effect of GRP on NK activity of leukocytes from axillary nodes, spleen and thymus from B A L B / c mice is shown in Fig. 1 (white cOlumns) compared to the results obtained on NK activity of adherent cell depleted populations in the presence of the neuropeptide (dotted columns). Similar results were obtained in the presence of bombesin and neuromedin C (data not shown). Only in axillary nodes the effect of GRP on total leukocyte population was significantly higher (37 + 3) than the stimulatory effects of bombesin (31 + 5) and neuromedin C (29 + 5) on the same populations. The higher stimulation of NK is obtained with the 10-~o M concentration of bombesin, GRP and neuromedin C in the three lymphoid organs studied. Although some organs show an increment in NK lysis with the 10 -8 M or 10 -~2 M concentrations, in general a dose-response modulation is observed without any effect at the higher or lower concentrations. In this way, the results obtained with the concentrations 10 -9, 10-1° and 10- 11 M of bombesin, GRP and neuromedin C are significantly reduced with the 10 -s and 10 -~2 M concentrations.

30

20

10

0

50' b 40,

30. 20,

10"

o

~ conlro!

8

9

Fig. 1. NK lysis percentages of total leukocyte suspensions (white columns) and suspensions depleted of adherent cells (dotted columns) from axillary nodes, spleen and thymus of B A L B / c mice incubated with the concentrations of GRP indicated in x-axis (from 10 -8 to 10- ~2 M). Each value is the m e a n + S . D , of eight experiments performed in triplicate, a p < 0.001;b p < 0.01; c p _< 0.05, with respect to the control total leukocyte population. • P < 0 . 0 5 ; ** P < 0 . 0 1 ; * * * P < 0 . 0 0 1 , with respect to the value of total leukocyte population.

M. Del Rio, M. De la Fuente/ Regulatory Peptides 60 (1995) 159-166

163

Table 2 Effect of 1 h preincubation with 3 M concentrations of GRP on NK cytotoxicity percentages of leukocytes from axillary nodes, spleen and thymus of BALB/c mice, using YAC-1 as target cells

The stimulation of NK cytotoxicity observed at the concentrations from 10 -9 to 10 -t2 M of the three neuropeptides was completely lost when we eliminated the adherent cells from the cellular suspensions. These peptides also stimulated the ADCC of leukocytes from the three lymphoid organs studied using different monoclonal antibody dilutions (1:200; 1:500 and 1:1000). The higher values were obtained with a 1:500 monoclonal antibody dilution, and 10-l0 M GRP showed the strongest capacity in stimulating the ADCC of leukocytes, as we have observed in previous experiments [28]. With respect to samples depleted of adherent cells, we observed no effect of the neuropeptides (Table 1). Previously, we had proven the effects of the neuropeptides incubating the cell samples in their presence during the four hours of the cytotoxic experiments. Presently, we performed some assays doing preincubations to determine whether the presence of the neuropeptides was necessary. When leukocytes from axillary nodes, spleen and thymus were

Concentration(M) Axillary nodes Spleen

0 10-8 10-10 10-12

Thymus

P

NP

P

NP

P

NP

225:1 235:3 395:4 315-5

215-2 255-2 375-3 275-5

205-1 315-1 37+7 265-3

215:3 325-4 425:5 305-6

175:3 225:3 315:7 215:3

175:2 225:2 305:3 225:6

Each value is the mean 5- S.D. of six experiments performed in triplicate. P, preincubated samples; NP, non-preincubated.

incubated with three concentrations of GRP (10 -8, 10 -1° and 10 -12 M) for 1 h previous to the NK cytotoxic assay, and washed afterwards, the effect of the neuropeptide persisted unchanged compared to the samples incubated with GRP during the whole assay (Table 2). In order to demonstrate the specificity of the stimulation of cytotoxic capacities of leukocytes by these neuropeptides, we included the bombesin an-

Table 1 Effect of bombesin, GRP and neuromedin C on antibody-dependent cellular cytotoxicity (ADCC) percentage of axillary nodes, spleen and thymus leukocytes (L) and non-adherent leukocytes (NAL) from BALB/c mice using K-562 cells as targets. The anti-CDl5 dilution was 1:500 and the effector/target relation was 10:1 AxiUary nodes

Spleen

Thymus

L

NAL

L

NAL

L

NAL

Controls Bombesin (M)

19 -I- 1

16 + 3

19 -t- 1

17 5:1

17 + 2

15 5:1

10 -8 10 -9 10 - t ° 10 -I= 10 -12 GRP (M) 10 -8 10 -9 10 - I ° 10 -Ik 10 -12

21 5:3 275:2 b 29+ 1 ~ 265-3 b 205-2

15+ 1 16+2 155- 1 165-1 165:2

235:2 255:2 c 265:5 b 255:3 c 23 5:4

195:5 195:4 185:5 195:5 21 5:5

225:2 24+2 b 265-2 a 245-3 c 21 5:1

18+3 155:3 145:4 14+4 175:2

23 5- 1 285-3 b 32+4 ~ 295:5 ~ 23 5:5

14 5- 4 155-3 175:2 165-2 175- 1

25 5:6 285:5 b 31 5:7 ~ 275:7 c 23 ::t:4

17 5- 2 195:2 205:3 185:3 205:1

23 5:2 265:3 b 295-3 ~ 26+4 ° 20+ 2

13 5:2 13 5:4 15 5:2 15+2 145:4

195- 1 265-3 b 29+ 1 a 2 7 5 - 5 ~' 21 5-5

16+3 15-1-5 135-4 145:3 145:2

23-t-7 26-t-6 c 29-t-4 ~ 285:6t' 255:7

165:3 17:t:3 18:1:4 185:2 165: 3

175-1 235:2 ¢ 265:2 a 2 4 + 2 t, 205:1

145:2 15_+2 15 + 4 135: 3 145:2

Neuromedin C (M) 10 -8 10 -9 10 - I ° 10 - t l 10 -12

Each value is the mean 5: S.D. of six experiments performed in triplicate. ~ P < 0.001; b P < 0.01; c p < 0.05 with respect to control samples.

M. Del Rio, M. De la Fuente / Regulatory Peptides 60 (1995) 159-166

164

100.

%NK 40"

GRP Neuromed|na C BombesIna NPY

80.

i

¢

30

"6

20

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li ill 0

8

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GRP concentration

60.

,=

0

12

11

10

9

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7

Neuropeptlde concentration (4og M) 11

12

(-log M)

Fig. 2. NK lysis percentages of leukocytes obtained from spleen of B A L B / c mice incubated with the concentrations of GRP indicated in the x-axis (from 10 - s to 10-12 M) with or without the addition of a BN-antagonist at equimolecular concentrations (hatched or white columns, respectively). Each value is the mean +S.D. of six experiments performed in triplicate, a P_<0.001; b p < 0.01; c p < 0.05 respect to control; * P < 0.05; * " P0.01; • * * P ~ 0.001, respect to the concentration of GRP without the antagonist.

tagonist (Leu13-~CH2NH-Leu 14) in some assays of NK cytotoxicity of leukocytes from spleen, axillary nodes and thymus. The stimulatory effect of GRP in spleen was reverted in presence of its antagonist, as it can be seen in Fig. 2. Similar results were found in the three organs with the three neuropeptides. Since the stimulatory effect of the neuropeptides on NK and ADCC was mediated through adherent cells, binding assays were performed to determine the presence of specific receptors for GRP in membranes of adherent and non-adherent leukocytes. Peritoneal macrophages were chosen as representants of the adherent population for their good availability and non-adherent leukocytes were obtained from thymus. Both populations were incubated separately with GRP concentrations ranging from 10 - 1 2 to 10 -7 M in the presence of a fixed concentration (45 pM) of 125I-GRP. A competitive binding to mice macrophages was found as shown in Fig. 3. The Scatchard analysis suggested the existence of two different types of GRP binding sites, one with high affinity (K d = 1.1 + 0.07) and low binding capacity ( 6 . 0 _ 1.0 fmol/106 cells) and another with lower affinity ( K d = 79.0 + 8.6 nM) and higher binding capacity (703.3 + 32.2 fmol/106 cells) (Fig. 4). The specificity of the GRP receptors in peritoneal

Fig. 3. Competitive inhibition of 1251-GRP binding t o murine peritoneal macrophages by unlabeled GRP, bombesin or neuromedin C, as well as by unlabeled NPY as negative control. Each point represents the mean + S.D. of five experiments performed in triplicate.

macrophages was determined by specific binding of ~25I-GRP in the presence of bombesin and neuromedin C (Fig. 3). Both peptides were able to inhibit tracer binding but with lower potency than GRP. A negative control, neuropeptide Y (NPY), was unable to inhibit to any extent the 125I-GRP binding. In contrast to what we found in macrophages, non-adherent leukocytes did not show the presence of specific receptors in their membranes. The percentages of binding of 125I-GRP remained invariable in the presence of different concentrations of unlabeled neuropeptides (data not shown). 12

B/F 10

8 6 4'

2'

0

•

, 2

-

,4

-

, 6

-

, 8

=

, 10

B (pmol)

Fig. 4. Scatchard plot of the specific binding data of a25I-GRP binding to murine peritoneal macrophages by unlabeled GRP. Each point represents the mean + S.D. of five experiments performed in triplicate.

M. Del Rio, M. De la Fuente / Regulatory Peptides 60 (1995) 159-166

4. Discussion The results confirm our previous work showing that physiological concentrations of bombesin, GRP and neuromedin C stimulate ADCC and NK activities of resting leukocytes in vitro [28], even though, in this occasion, we used a different non-radioactive technic. The range of concentrations was extended to 10 -8 M and 10 -12 M, and a dose-response curve can be observed. Thus, the effects seen with 10 - l ° M of bombesin, GRP and neuromedin C were maintained with the 10 -9 M and 10-11 M concentrations of the peptides while at the lower concentration (10-12 M) this effect was significantly reduced and the same happened with the higher concentration (10 -8 M) probably due to a process of down-regulation, as it was suggested for other neuropeptides such as VIP [32]. This decreased response to the higher neuropeptide concentrations has been also observed in the modulation of other aspects of the immune response, like the phagocytic process [15,17]. When leukocyte populations were depleted of adherent cells, the effect of the neuropeptides disappeared. The controls without neuropeptides remained practically unaffected by the elimination of adherent cells, what could be expected since adherent cells are not necessary in order to observe a complete cytotoxic response. Thus, these adherent cells seem directly involved with the transmission of neuropeptide stimulatory signals to the cytolytic effector cells. NK activity, which is crucial in host defense, is stimulated by many different compounds including cytokines and bacterial products [33]. Moreover, other neuropeptides regulate NK and ADCC activities. Thus, endorphins and enkephalins increase these cytolytic functions [34,35], VIP inhibits NK cell activity in vitro [23,24], and NPY and substance P stimulate it [25-27]. The specificity of the stimulatory action of BN-related peptides on cytotoxic cells has been disclosed using a BN-antagonist shown to be highly specific [36]. We have already observed the reversion, in the presence of this antagonist, of other effects of GRP and bombesin on peritoneal leukocytes, such as the chemoattractant capacity [17]. Binding experiments corroborate the idea of specificity revealing the existence of receptors for GRP of two type of affinity on macrophages as we had previously shown for bombesin [21]. Apparently, non-adherent leukocytes

165

do not express receptors for these neuropeptides or at least, they are not similar in affinity and number to those in macrophages, as they have not been detected by the binding technics used. These results support the hypothesis of a possible role for adherent cells in transmitting the neuropeptides signal to cytotoxic non-adherent cells. The requirement of adherent cells to mediate the effects of bombesin-related peptides was also observed in vitro in the modulation of the proliferative response of lymphocytes by these peptides [21]. We are presently carrying out studies to investigate the possible secretion by these cells of a mediator factor which would be responsible for the modulatory effects of these neuropeptides on lymphocytes or NK cells. These results on the effects of neurotransmitters and hormones on immune cells should be considered in order to clarify the mechanisms involved in nervous and endocrine system modulation of immune function in the organism in vivo.

Acknowledgements We thank Dr. J. Miquel and Dr. A. Hernanz for their critical reviews of this manuscript. This work has been supported by the Grant No. C239/91 from the Comunidad de Madrid, and by the FISss Grant No. 0697/92 from Ministerio de Sanidad y Seguridad Social.

References [1] Blalock, J.E., Molecular basis for bidirectional communications between the immune and neuroendocrine systems, Physiol. Rev., 69 (1989) 1-9. [2] Dockray, G.J., Vaillant, C. and Walsh, J.H., The neuronal origin of bombesin-likeimmunoreactivityin the rat gastrointestinal tract, Neuroscience, 4 (1979) 1561-1568. [3] Roth, K.A., Evans, C.J., Lorenz, R.G., Weber, E., Barchas, J.D. and Chang, J-K., Identification of gastrin releasing peptide-related substances in guinea pig and rat brain, Biochem. Biophys. Res. Commun., 112 (1983) 528-536. [4] Shaw, C., Thim, L. and Conlon, J.M., Primary structure and tissue distribution of guinea pig gastrin-releasing peptide, J. Neurochem., 49 (1987) 1348-1354. [5] Hernanz, A., Characterization and distribution of bombesinlike peptides in the rat brain and gastrointestinal tract, Biochem. Cell Biol., 69 (1990) 1142-1145. [6] Lezoche,E., Basso,N. and Spcranza,V., Actions of bombesin in man. In: S.R. Bloom and J.M. Polak (Eds.), Gut Hormones, Churchill Livingstone,Edinburgh, 198l, pp. 419-439.

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[7] Mc Coy, J.G. and Avery, D.D., Bombesin: Potential integrative peptide for feeding and satiety, Peptides, 11 (1990) 595-607. [8] Anastasi, A., Erspamer, V. and Bucci, M., Isolation and structure of bombesin and alytesin: two anallogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia, 27 (1971) 166-167. [9] McDonald, T.J., Jomvali, H., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S.R. and Mutt, V., Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue, Biochem. Biophys. Res. Commun., 90 (1979) 227-233. [10] Panula, P., Histochemistry and function of bombesin-like peptides, Med. Biol., 64 (1989) 177-192. [1 l] Minamino, N., Kangawa, K. and Matsuo, H., Neuromedin C: a bombesin-like peptide identified in porcine spinal cord, Biochem. Biophys. Res. Commun., 119 (1984) 14-20.14. [12] Zachary, I. and Rozengurt, E., Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking, J. Biol. Chem., 262 (1987) 3947-3950. [13] Weber, S., Zuckerman, J.E., Bostwick, D.G., Bensch, K.G., Sikic, B.I. and Raffin, T.A., Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro, J. Clin. Invest., 75 (1985) 306-309. [14] Kris, R.M., Hazan, R., Villines, J., Moody, T.W. and Schlessinger, J., Identification of the bombesin receptor on murine and human cells by cross-linking experiments, J. Biol. Chem., 262 (1987) 11215-11220. [15] De la Fuente, M., Del Rio, M., Ferrandez, M.D. and Hernanz, A., Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C, Immunology, 73 (1991) 205-211. [16] Ruff, M., Schiffman, E., Terranova, V. and Pert, C.B., Neuropeptides are chemoattractants for human tumor cells and monocytes: A possible mechanism for metastasis, Clin. Immunol. Immunopathol., 37 (1985) 387-396. [17] Del Rio, M. and De la Fuente, M., Chemoattractant capacity of bombesin, gastxin-releasing peptide and neuromedin C is mediated through PKC activation in murine peritoneal leukocytes, Regul. Pept., 49 (1994) 185-193. [18] Jin, G-F., Guo, Y-S. and Houston, C.W., Bombesin: an activator of specific Aeromonas antibody secretion in rat intestine, Dig. Dis. Sci. 34 (1989) 1708-1712. [19] Fink, R., Ehrhardt, R. and Dancygier, H., Bombesin and its analogues inhibit interleukin-2 induced proliferation of CTLL-2 cells, Regul. Pept. 23 (1988) 323-330. [20] Krco, CJ., Gores, A. and Go, V.L.W., Gastrointestinal regulatory peptides modulate mouse lymphocyte functions under serum-free conditions in vitro, Immunol. Invest., 15 (1986) 103-111. [21] Del Rio, M., Hernanz A. and De la Fuente, M., Bombesin, gastrin-releasing peptide and neuromedin C modulate murine lymphocyte proliferation through adherent accessory cells and activate protein kinase C, Peptides, 15 (1994) 15-22. [22] Arora, P.K., Neuromodulation of natural killer cell activity. In E.J. Goetzl and N.H. Spector (Eds.), Neuroimmune Networks: Physiology and Diseases. Alan R. Liss, New York, 1989, pp. 105-111.

[23] Rola-Peszczynski, M.R., Bolduc, D. and St.-Pierre, S., The effect of vasoactive intestinal peptide on human natural killer cell function, J. Immunol., 135 (1985) 2569-2573. [24] Yiangou, Y., Serrano, R., Bloom, S.R., Pefia, J. and Festenstein, H., Effects of preprovasoactive intestinal peptide-derived peptides on the murine immune response, J. Neuroimmunol., 29 (1990) 65-72. [25] Nair, M.P.N., Schwartz, S.A., Wu, K. and Kronfol, Z., Effect of neuropeptide y on natural killer activity of normal human lymphocytes, Brain Behav. lmmun., 7 (1993) 70-78. [26] Biennenstock, J., Croitoru, K., Ernst, P.B. and Stanisz, A.M., Nerves and neuropeptides in the regulation of mucosal immunity, Plenum Press, New York, 1989, pp. 19-26. [27] Croitoru, K., Ernst, P.B., Bienenstock, J., Padol, I. and Stanisz, A.M., Selective modulation of natural killer activity of murine intestinal intraepithelial leukocytes by the neuropeptide substance P, Immunology, 71 (1990) 196-201. [28] De la Fuente, M., Del Rio, M. and Hemanz, A., Stimulation of natural killer and antibody-dependent cellular cytotoxicity activities in mouse leukocytes by bombesin, gastrin-releasing peptide and neuromedin C: involvement of cyclic AMP, inositol 1,4,5-trisphosphate and protein kinase C, J. Neuroimmunol., 48 (1993) 143-150. [29] Van Tol, E.A.F., Eizo kraemer, C.V., Verspaget, H.W., Masclee, A.M. and Lamers, C.B.H.W., intravenous administration of bombesin in man stimulates natural killer cell activity against tumor cells, Neuropeptides, 18 ( 1991) 15- 2 I. [30] Decker, T. and Lohmann-Matthes, M.L., A quick and simple method for the quantitation of lactate dehydrogenase release in measurments of cellular cytotoxicity and tumor necrosis factor (TNF) activity, J. Immunol. Methods, 115 (1988) 61-70. [31] Segura, J.J., Guerrero, J.M., Gobema, R. and Calvo, J.R., Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages, Regul. Pept. 33 (1991) 133-143. [32] Ottaway, C.A., Receptors for vasoactive intestinal peptide on murine lymphocytes turn over rapidly, J. Neuroimmunol., 38 (1992) 241-254. [33] Conti, P., Dempsey, R.A., Reale, M., Barbacane, R.C., Panara, M.R., Bongrazio, M. and Mier, J.W., Activation of human natural killer cells by lypopolysaccharide and generation of interleukin-lalfa, beta, tumor necrosis factor and interleukin-6. Effect of IL-1 receptor agonist, Immunology, 73 (1991) 450-456. [34] Kayu, N., Allen, J. and Morley, U.J.E., Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity, Life Sci., 35 (1984) 53-59. [35] Froelich, C.J. and Bankhurts, A.D., The effect of /3-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity. Life Sci., 35 (1984) 261-266. [36] Coy, D.H., Heinzerian, P., Jiang, N-Y., Sasaki, Y., Taylor, J., Moreau, J-P., Wolfrey, W.T., Gardner, J.D. and Jensen, R.T., Probing peptide backbone function in bombesin: a reduced peptide bond analogue with potent and specific receptor antagonist activity, J. Biol. Chem., 263 (1988) 5056-5060.